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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 models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | 0 |
"coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human AB the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [ ] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea nausea and vomiting and abdominal pain were common at presentation in COVID19 patients [ ] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto | 2 |
" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming 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 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity []in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but its raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [] therefore its necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chens in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained whats more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ¤ length length ¥ p icosandraannotated number ¤ length length ¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor whats more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] whats more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5 flora of north america vol43 chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturers instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number | 0 |
"Methods Tissue collection We obtained 113 paired NSCLC and adjacent non-tumor lung tissues from patients who underwent surgery at Jiangsu Province Hospital between 2008 and 2010 and were diagnosed with NSCLC (stages I II and III) based on histopathological evaluation. Clinicopathological characteristics including tumor-node-metastasis (TNM) staging were recorded. No local or systemic treatment was conducted in these patients before surgery. All collected tissue samples were immediately snap-frozen in liquid nitrogen and stored at 80°C until required. Our study was approved by the Research Ethics Committee of Nanjing Medical University China. Written informed consent was obtained from all patients. Cell lines Five NSCLC adenocarcinoma cell lines (A549 SPC-A1 NCI-H1975 NCI-H1299 and NCI-H1650) a NSCLC squamous carcinomas cell line (SK-MES-1) and a normal human bronchial epithelial cell line (16HBE) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai China). A549 SK-MES-1 NCI-H1975 NCI-H1299 NCI-H1650 and 16HBE cells were cultured in RPMI 1640; SPC-A1 cells were cultured in DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (FBS) 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen Carlsbad CA USA) at 37ºC/5% CO2. RNA extraction and qPCR assays Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturers instructions. Total RNA (500 ng) was reverse transcribed in a final volume of 10 ?l using random primers under standard conditions for the PrimeScript RT reagent Kit (TaKaRa Dalian China). We used the SYBR Premix Ex Taq (TaKaRa Dalian China) to determine BANCR expression levels following the manufacturers instructions. Results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific primers used are presented in Additional file 3: Table S2. The qPCR assays were conducted on an ABI 7500 and data collected with this instrument. Our qPCR results were analyzed and expressed relative to threshold cycle (CT) values and then converted to fold changes. Plasmid generation The BANCR sequence was synthesized and subcloned into the pCDNA3.1 (Invitrogen Shanghai China) vector. Ectopic expression of BANCR was achieved through pCDNA-BANCR transfection with an empty pCDNA3.1 vector used as a control. The expression levels of BANCR were detected by qPCR. Cell transfection Plasmid vectors (pCDNA3.1-BANCR and pCDNA3.1) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen Hilden Germany) and transfected into SPC-A1 or A549 cells. The siRNAs si-HDAC1 si-HDAC3 si-BANCR or si-NC were transfected into SPC-A1 or A549 cells (Additional file 3: Table S2). A549 and SPC-A1 cells were grown on six-well plates to confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. At 48 h post-transfection cells were harvested for qPCR or western blot analysis. Cell viability assays Cell viability was monitored using a Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science). The A549 cells transfected with si-BANCR (3000 cells/well) and A549 or SPC-A1 cells transfected with pCDNA-BANCR were grown in 96-well plates. Cell viability was assessed every 24 h following the manufacturers protocol. All experiments were performed in quadruplicate. For colony formation assays pCDNA-BANCR-transfected SPC-A1 or A549 cells (n?=?500) were placed in a 6-well plates and maintained in media containing 10% FBS. The medium was replaced every 4 days; after 14 days cells were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). Visible colonies were then counted. For each treatment group wells were assessed in triplicate. Flow cytometry analysis of apoptosis SPC-A1 and A549 cells were harvested at 48 h post-transfection by trypsinization. After staining with FITC-Annexin V and propidium iodide cells were analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences). Cells were discriminated into viable cells dead cells early apoptotic cells and apoptotic cells. The ratio of early apoptotic cells was compared to that for controls from each experiment. All samples were assayed in triplicate. Wound-healing assay For the wound-healing assay 3??105 cells were seeded in 6-well plates cultured overnight and transfected with pCDNA-BANCR or the control vector. Once cultures reached 85% confluency the cell layer was scratched with a sterile plastic tip and washed with culture medium then cultured for 48 h with medium containing 1% FBS. At different time points images of the plates were acquired using a microscope. The distance between the two edges of the scratch was measured using Digimizer software system. Cell migration and invasion assays For the migration assays at 48 h post-transfection 5??104 cells in serum-free media were placed into the upper chamber of an insert (8-?m pore size; Millipore). For the invasion assays 1??105 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (Sigma-Aldrich). Medium containing 10% FBS was added to the lower chamber. After incubation for 24 h the cells remaining on the upper membrane were removed with cotton wool. Cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet imaged and counted using an IX71 inverted microscope (Olympus Tokyo Japan). Experiments were independently repeated three times. Tail vein injections into athymic mice Athymic male mice (4-weeks-old) were purchased from the Animal Center of the Chinese Academy of Science (Shanghai China) and maintained in laminar flow cabinets under specific pathogen-free conditions. SPC-A1 cells transfected with pCDNA-BANCR or the empty vector were harvested from 6-well plates washed with phosphate-buffered saline (PBS) and resuspended at 2??107 cells/ml. Suspended cells (0.1 ml) were injected into the tail veins of 9 mice which were sacrificed 7 weeks after injection. The lungs were removed and photographed and visible tumors on the lung surface were counted. " | 1 |
" drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer close homolog of l1 chl1 has been identified as a tumor suppressor in most malignancies however to the best of our knowledge whether chl1 mediates chemoresistance remains unknown the present study observed that chl1 was significantly downregulated in cisplatin ddpresistant cells a549ddp and paclitaxel ptxresistant cells a549ptx compared with a549 cells when treated with or without ddp and ptx silencing of chl1 in a549 cells promoted the cell survival rate and clone formation and decreased apoptosis whereas overexpression of chl1 in a549ddp and a549ptx cells impeded the cell survival and clone formation and promoted apoptosis additionally chl1 overexpression enhanced the chemosensitivity of a549ddp cells to ddp in vivo notably the chemoresistance induced by chl1 depletion was reversed by the akt inhibitor sc66 in a549 cells the results of the present study demonstrated that chl1 enhanced sensitivity of lung cancer cells by suppressing the akt pathway which suggested that chl1 may be a potential target for overcoming chemoresistance in lung cancerintroductionlung cancer is the most common human malignancy accounting for of all cancerassociated deaths worldwide during in addition its morbidity and mortality rank the highest among all malignant tumor types worldwide according to the differentiation degree and morphological correspondence to dr rimao huang department of cardiothoracic surgery xiangya changde hospital moon avenue west of langzhou north road changde hunan pr chinaemail xyhuangrm163comkey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly classified into nonsmallcell lung cancer nsclc and smallcell lung cancer among patients with lung cancer nearly are diagnosed as nsclc which manifests with earlier diffusion and metastasis currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer for patients with advanced nsclc or those who are clinically incapacitated for surgery chemotherapy is a remarkably important treatment cisplatin ddp is widely applied in the treatment of several malignancies and it exhibits a broad spectrum of antitumor effects by inducing dna damage and hindering dna damage repair paclitaxel ptx another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division the majority of patients have a good initial response to chemotherapy agents however subsequent relapse is common and largely due to the emergence of drug resistance thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced nsclc therefore there is an urgent need to investigate the target and mechanism of chemoresistance in nsclcclose homolog of l1 chl1 is a member of the l1 family of nerve cell adhesion molecules and is located on the 3q26 locus as a nerve cell adhesion molecule chl1 serves an important role in the development regeneration and plasticity of the nervous system the absence or mutation of chl1 can trigger 3p syndrome and schizophrenia the abnormal expression of chl1 may lead to reduced working memory and social behavior mental damage and abnormal behavior chl1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers in esophageal squamous cell carcinoma escc chl1 downregulation is associated with invasion lymph node metastasis and poor overall survival functional studies revealed that chl1 has antiproliferation and antimetastasis abilities the expression of chl1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression in thyroid cancer and colonic adenocarcinoma chl1 impedes cell proliferation and invasion and acts as a tumor suppressor in lung cancer hÓ§tzel evaluated chl1 expression 0ccai chl1 enhances the chemosensitivity of lung cancer cellsin nsclc cases based on a tissue microarray and it was reported that chl1 expression is associated with t stage in adenocarcinomas as well as with metastatic lymph node status and improved survival additionally by analyzing the gene expression omnibus dataset gse21656 submitted by sun microarray results demonstrated that chl1 expression in ddpresistant h460 cells is significantly lower compared with that in parental cells suggesting that chl1 may be involved in nsclc chemoresistance however to the best of our knowledge the underlying mechanism remains unknownin the present study the expression of chl1 in ddp and ptxresistant a549 cells and the parental cells was assessed functional studies of chl1 were performed to investigate its potential role in chemoresistancematerials and methodsdata processing the human gse21656 microarray dataset was downloaded from the ncbi gene expression omnibus geo database wwwncbinlmnihgovgeo the available dataset gse21656 was based on the gpl6244 platform affymetrix human gene st array affymetrix thermo fisher scientific inc this data includes h460 cells and ddpresistant h460 cells sample and each cell has three repeats samples the online tool geo2r httpwwwncbinlmnihgovgeogeo2r was used to determine the differentially expressed genes in h460 and ddpresistant h460 cells p005 and log2foldchange¥ were set as cutoff standardscell culture the human nsclc cell line a549 the ptxresistant cell line a549ptx and the ddpresistant cells a549ddp were purchased from procell life science technology co ltd the cells were cultured in ham's f12k medium supplemented with fetal bovine serum both purchased from thermo fisher scientific inc uml penicillin and uml streptomycin cat no thermo fisher scientific inc in a Ëc humidified incubator with co2cell transfection the resistant cells a549ptx and a549ddp cells were transfected with µg chl1 recombinant expression plasmid cat no hg10143ny sino biological inc empty vector pcmv3spnha was used as the control a549 cells were transfected with pmol small interfering sirnas the sirna sequence for chl1 guangzhou ribobio co ltd were sirna 'gga gcu aau uug acc aua utt' sirna 'cag caa uau uag cga gua utt' and scrambled control 'uuc ucc gaa cgu guc acg utt' plasmids and sirnas were transfected into cells using lipofectamine® thermo fisher scientific inc following the manufacturer's instructions the time interval between transfection and subsequent experimentation was h for the rescue experiments the chl1 silenced a549 cells were treated with the akt inhibitor sc66 cat no s5313 selleck chemicals along with ddp µgml or ptx ngml both purchased from selleck chemicals for h at Ëcrna extraction and reverse transcriptionquantitative pcr rtqpcr assay total rnas were isolated using trizol reagent thermo fisher scientific inc according to the manufacturer's instructions and the mixed dnas were eliminated by dnase i new england biolabs inc firststrand cdna synthesis was conducted using the goscripttm kit promega corporation according to the manufacturer's instructions the reaction conditions for reverse transcription were Ëc for min Ëc for min and Ëc for min the sybr green realtime pcr master mix thermo fisher scientific inc was used to perform rtqpcr using a lightcycler480 system roche diagnostics gmbh the chl1 primer sequences were as follows forward 'ggc ttg gtc tct tgc ttt cc' and reverse 'atc ttc cct ccc ttt gca cg' and actin forward 'ttc ctt cct ggg cat gga gtc ' and reverse 'tct tca ttg tgc tgg gtg cc' the following thermocycling conditions were used for qpcr min at Ëc followed by cycles at Ëc for sec sec at Ëc and a final extension at Ëc for sec each reaction was conducted in triplicate relative expression levels were calculated using the δδcq method cell viability cell viability was detected by mtt assay a cell suspension µl was seeded into well plates at a density of 1x104 cellswell and incubated overnight at Ëc the concentrations of ddp used to treat a549 cells were and µgml while the concentrations of ptx used to treat a549 cells were and ngml the concentrations of ddp used to treat a549ddp cells were and µgml while the concentrations of ptx used to treat a549ptx cells were and ngml after treating with different concentrations of ddp or ptx for h at Ëc µl mtt mgml solution was added to each well and incubated for h at Ëc subsequently µl dmso was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader biotek instruments inc at nmclone formation assay a total of 1x103 cells were seeded into a mm dish in triplicate and maintained in f12k medium with or without ddp or ptx at Ëc for h a total of weeks later cells were fixed in paraformaldehyde for min at room temperature and stained with crystal violet dye at room temperature for min the rate of colony formation was calculated using the following equation number of coloniesnumber of seeded cells x100flow cytometry apoptosis was detected using a fitc annexin v apoptosis kit bd pharmingen bd biosciences according to the manufacturer's protocol cells 1x105 were collected and washed twice with pbs prior to being suspended in µl binding buffer subsequently cells were incubated with µl annexin vfitc and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a cytoflex flow cytometer beckman coulter inc data were analyzed using cytexpert software beckman coulter inc the ratio between early and late apoptosis was calculatedwestern blotting cells were collected washed twice with pbs and lysed with ripa lysis buffer thermo fisher scientific inc proteins were isolated from the cell lysis buffer and 0concology letters quantified using the piercetm¢ bca protein assay kit cat no thermo fisher scientific inc with bovine serum album as a standard equal amount of protein µg proteins were separated by sdspage gel next the proteins were transferred onto a polyvinylidene membrane thermo fisher scientific inc blocked with bsa thermo fisher scientific inc for h at Ëc and incubated overnight at Ëc with primary antibodies against chl1 cat no ap proteintech inc multidrug resistance gene mdr1 cat no ap proteintech inc multidrug resistanceassociated protein mrp cat no ig proteintech inc lowdensity lipoprotein receptorrelated protein lrp cat no ap proteintech inc phosphorylated pakt cat no ab38449 abcam and akt cat no ab227385 abcam after washing three times with pbs the membrane was incubated with horseradish peroxideconjugated goat antirabbit cat no ab6271 abcam_or rabbit antimouse cat no ab6728 abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent thermo fisher scientific inc protein expression was quantified using imagepro plus software media cybernetics incanimal experiments the animal experiments were approved by the medical ethics committee of xiangya changde hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the national institutes of health publications no a total of male balbcnu mice weekold ± g hunan sja laboratory animals center of the chinese academy of sciences were used in this study all animals were kept at the spf level laboratory at Ëc a relative humidity of a h lightdark cycle and timesh of fresh air exchange all mice were given free access to food and water the bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use a549ddp cells 1x107 transfected with empty vector and chl1 overexpression vector using lipofectamine® reagent thermo fisher scientific inc were subcutaneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group a549ddp cells transfected with empty vector and treated with µl saline solution ii vectorddp group a549ddp cells transfected with empty vector and treated with mgkg ddp iii chl1 group a549ddp cells transfected with chl1 overexpression vector and treated with µl saline solution and iv chl1ddp group a549ddp cells transfected with chl1 overexpression vector and treated with mgkg ddp ddp was administered by intraperitoneal injection every days for weeks the mice were observed daily and the tumors were measured by a vernier caliper every days the tumor volumes were calculated as length x width22 a total of weeks postinjection mice were euthanized with co2 at volume displacement rate vdr per min using a programmable logic controller barrywehmiller design group inc mice were monitored continuously and once the mice were immobile except for breathing for min the vdr was provided at for min the animals remained in the euthanasia chamber for min and were then observed for an additional min breathing and heart rate were monitored to determine deathstatistical analysis all experiments were performed in triplicate and data are presented as the mean ± standard deviation all experiments were performed at least three times paired student's ttest was performed for comparisons between two groups and oneway analysis of variance followed by tukey's multiple comparison posthoc analysis was performed for comparisons between multiple groups spss ibm corp was used to perform the analysis p005 was considered to indicate a statistically significant differenceresultschl1 is downregulated in a549ddp and a549ptxresistant cells in order to investigate the mechanism of chemoresistance in lung cancer the lung adenocarcinoma cell line a549 the ddpresistant cells a549ddp and ptxresistant cells a549ptx were used in the present study cells were exposed to different concentrations of ddp µgml and ptx ngml and mtt assay was used to detect the cell survival rate a549ddp and a549ptx cells demonstrated higher resistance to ddp and ptx compared with a549 cells fig 1a the half maximal inhibitory concentration ic50 of ddp was significantly higher in a549ddp cells ± µgml compared with a549 cells ± µgml and the ic50 of ptx was significantly higher in a549ptx cells ± ngml compared with a549 cells ± ngml fig 1b in addition the expression levels of the drugresistant markers mdr1 mrp and lrp were significantly higher in a549ddp and a549ptx cells compared with a549 cells fig 1c additionally the mrna and protein expression levels of chl1 were significantly lower in a549ddp and a549ptx cells compared with those in a549 cells fig 1d and e and this was also observed in h460 ddpresistant cells obtained from the geo dataset gse21656 fig 1f these results suggested that chl1 may be involved in regulating ddp and ptx resistance in nsclcknockdown of chl1 enhances resistance to ddp and ptx in a549 cells as chl1 was upregulated in a549 cells chl1 was silenced in a549 cells using sirnas chl1 expression was significantly reduced in the chl1 sirna groups compared with that of the scrambled control group fig 2a as sirna demonstrated the greatest interference efficiency it was selected for use in the following experiments notably chl1knockdown enhanced the resistance to ddp and ptx in a549 cells fig 2b and c colony formation assay revealed that compared with the control group chl1knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to ddp and ptx fig 2d flow cytometry results demonstrated significantly reduced apoptosis in chl1knockdown cells after ddp and ptx treatment compared with that of the control group fig 2e 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure chl1 is downregulated in ddp and ptxresistant a549 cells a cell survival of a549 and a549resistant cells a549ddp and a549ptx treated with increasing concentrations of ddp and ptx as assessed by mtt assay b the ic50 values of ddp in a549ddp and a549 cells and the ic50 values of ptx in a549ptx and a549 cells p005 vs a549 cells c western blotting demonstrated the expression of drug resistancerelated proteins mdr1 mrp and lrp in a549 cells and a549resistant cells a549ddp and a549ptx p005 vs a549 cells the protein and mrna expression levels of chl1 in a549 cells and a549resistant cells a549ddp and a549ptx were analysed by d western blotting and e reverse transcriptionquantitative pcr respectively p005 vs a549 cells f the mrna expression of chl1 in h460 and h460ddp cells in the gse21656 dataset p005 vs h460 cells chl1 close homolog of l1 ddp cisplatin ptx paclitaxel mdr1 multidrug resistance gene mrp multidrug resistanceassociated protein lrp lowdensity lipoprotein receptorrelated protein ic50 half maximal inhibitory concentration chl1 overexpression enhances the sensitivity of a549 resistant cells to ddp and ptx as chl1 is downregulated in a549ddp and a549ptx cells the present study successfully overexpressed chl1 in these cells using chl1 recombinant expression plasmids fig 3a the results demonstrated that chl1 overexpression alleviated the resistance to ddp and ptx compared with that of the control group fig 3b and c in addition chl1 overexpression inhibited colony formation in the absence or presence of ddp and ptx fig 3d additionally flow cytometry results demonstrated that restoration of chl1 expression promoted apoptosis in resistant cells following ddp and ptx treatment fig 3eto further validate the effects of chl1 overexpression on ddp or ptx sensitivity xenograft mice model experiments were performed the results demonstrated that chl1 overexpression or ddp treatment significantly impeded the tumor growth fig 3f and decreased the tumor weight fig 3g in addition chl1 overexpression further aggravated ddpmediated repression on tumor growth fig 3f and g these data suggested that chl1 overexpression suppressed tumor growth and enhanced the chemosensitivity in nsclcchl1 mediates chemosensitivity by inhibiting akt activity recently studies have confirmed that chl1 inhibits akt activity in escc and neuroblastoma cell lines thus the present study investigated whether chl1 mediates chemoresistance via the akt pathway in nsclc in a549 cells compared with the scrambled group chl1knockdown elevated the expression of paktser473 fig 4a by contrast restoring chl1 expression in a549ddp and a549ptx cells inhibited the akt phosphorylation compared with the control group fig 4a suggesting chl1 mediates chemosensitivity via the akt pathway subsequently chl1silenced a549 cells were treated with the akt inhibitor sc66 and it was demonstrated that inhibiting akt activity significantly reduced the promotive effects on cell survival fig 4b and clone formation fig 4c and the inhibitory effects on apoptosis fig 4d induced by chl1depletion these results confirmed that chl1 mediates chemosensitivity in nsclc by inhibiting the akt pathway 0concology letters figure chl1knockdown increases a549 cell resistance to ddp and ptx a western blotting was performed to validate the efficiency of transfection with chl1 sirnas p005 vs scramble mtt assays were performed to determine the survival rate of chl1knockdown a549 cells treated with b µgml ddp or c ngml ddp d colony formation assay of a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was used to detect apoptosis in a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx p005 p0001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering discussionthe results of the present study demonstrated that chl1 was significantly downregulated in a549ddp and a549ptx cells compared with a549 cells the knockdown of chl1 in a549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without ddp and ptx whereas chl1 overexpression in a549ddp and a549ptx cells inhibited cell survival and clone formation and increased apoptosis the results of the present study also demonstrated that chl1 enhances nsclc chemosensitivity through inhibition of the akt pathway these data suggested that chl1 may be a promising target to improve the efficacy of chemosensitivity in nsclcchl1 belongs to the l1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by senchenko through cellcell interactions and mediating cellcell and cellmatrix interactions chl1 has an important effect on the development regeneration and plasticity of the nervous system previous reports have demonstrated that chl1 also participates in carcinogenesis chl1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues in most tumors chl1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis for example knockdown of chl1 expression results in enhanced cervical cancer cell invasion and migration a low expression of chl1 in patients with neuroblastoma predicts a poor prognosis and enhancing chl1 expression suppresses tumor progression in contrast chl1 has been reported to promote cell proliferation metastasis and migration in human gliomas however to the best of our knowledge research on chl1 and tumor chemoresistance has rarely been reported 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure overexpression of chl1 increases the sensitivity of resistant a549 cells to ddp and ptx a western blotting was performed to detect chl1 expression in a549ddp and a549ptx cells transfected with chl1 expression plasmids p005 vs vector effect of chl1 overexpression on resistant a549 cell survival rate when treated with b µgml ddp or c ngml ptx as determined by mtt assay d colony formation assays demonstrated the number of colonies of resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was performed to assess apoptosis in resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx chl1 overexpression enhanced chemosensitivity of a549ddp cells to ddp in vivo which was demonstrated by the effect of ddp treatment or chl1 overexpression on the f growth and g weight of xenografts derived from a549ddp cells p005 p001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel the present study examined the differentially expressed genes in nsclc ddpresistant cells in a geo dataset chl1 was demonstrated to be upregulated in ddpresistant cells compared with parental cells suggesting that chl1 may be involved in nsclc chemotherapy resistance similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of chl1 in chemotherapysensitive tumor tissues is higher compared with that in drugresistant tissues suggesting that chl1 may help to predict the efficacy of chemotherapy for ovarian cancer in addition aberrant methylation of chl1 may be associated with the recurrence of colorectal cancer crc following chemotherapy azadc treatment restores flurouracil sensitivity in vitro which also suggests that chl1 may be involved in crc chemotherapy resistance the results of the present study demonstrated that chl1 was downregulated in a549ddp cells additionally as multiple drug resistance is a common characteristic another type of resistant cells a549tax cells were also used in the current study the results also demonstrated that chl1 was downregulated in a549ptx cells compared with control cells overexpression of chl1 significantly increased the sensitivity of cells resistant to ddp and ptx whereas knockdown of chl1 expression in 0concology letters figure chl1 mediates ddp and ptx sensitivity by inhibiting akt activity a western blotting was performed to detect the expression of pakt and total akt in chlsilenced and restored cell models p005 vs scramble or vector b mtt assays were performed to detect cell survival rates of a549 cells treated with chl1 sirna and akt inhibitor sc66 p005 c colony formation assays were performed in a549 cells treated with chl1 sirna and the akt inhibitor sc66 in the presence of ddp µgml or ptx ngml p005 vs sichl1 d apoptosis were measured in a549 cells treated with chl1 sirna and akt inhibitor sc66 in the presence of ddp µgml and ptx ngml p005 vs sichl1 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering p phosphorylated parent a549 cells displayed the opposite results to the best of our knowledge this study is the first study to suggest that chl1 may be involved in chemosensitivity in lung cancer the concentration of ddp used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting in a clinical trial the human initial dose was calculated from the no observed adverse effect levels noaels verified in animal experiments noael is the maximum dose level without significant adverse reactions the noael verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species in the present study the concentration of ddp used in vivo was not the noael thus it was not consistent with the concentrations used in clinical settingsakt is a serinethreonine protein kinase that is activated by phosphorylation as a key molecule of the pi3kakt signaling pathway pakt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis additionally akt activation is associated with tumor chemoresistance the results of the present study demonstrated that compared with the control groups the expression of pakt was increased in chl1knockdown a549 cells and its expression was reduced in chl1 overexpressed a549ddp and a549ptx cells when akt activity was inhibited by the akt inhibitor the sensitivity to ddp and ptx in chl1knockdown a549 cells was restored this finding suggested that chl1 enhanced the chemosensitivity of nsclc by inhibiting the akt pathway considering numerous studies have confirmed that the akt pathway mediates chemoresistance via regulation of atp binding cassette abc members the present study didn't further investigate the specific abc members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoin summary the present study demonstrated that chl1 was downregulated in resistant cells a549ddp and a549ptx and upregulation of chl1 enhanced the chemosensitivity of nsclc via inhibiting the akt pathway to the best of our knowledge this was the first study to confirm the function and 0ccai chl1 enhances the chemosensitivity of lung cancer cellsmechanism of chl1 in mediating chemosensitivity in cancer thus the development of chl1based therapeutic strategies may improve the efficacy of chemosensitivity in nsclcacknowledgementsthe authors of the present study would like to thank mr dingliang li xiangya hospital changsha china for his guidance and assistance in flow cytometric analysisfundingno funding was receivedavailability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestauthors' contributionsrh conceived and designed the present study xc bh yh and pl performed experiments and collected the data sl zz and zh analyzed and interpreted the data ml and lz analyzed the data and prepared the figure xc ml and lz drafted the initial manuscript and revised it for intellectual content all authors read and approved the final manuscriptethics approval and consent to participatethe animal experiments were approved by the medical ethics committee of xiangya changde hospital changde china approval no patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences parascandola m and xiao l tobacco and the lung cancer epidemic in china transl lung cancer res suppl s21s30 chen w cancer statistics updated cancer burden in china chin j cancer res oser mg niederst mj sequist lv and engelman ja transformation from nonsmallcell lung cancer to smallcell lung cancer molecular drivers and cells of origin lancet oncol e165e172 thatcher n faivrefinn c blackhall f anderson h and lorigan p sequential platinumbased chemotherapythoracic radiotherapy in early stage nonsmall cell lung cancer clin cancer res suppl s5051s5056 yano t okamoto t fukuyama s and maehara y therapeutic strategy for postoperative recurrence in patients with nonsmall cell lung cancer world j clin oncol fang z chen w yuan z liu x and jiang h lncrnamalat1 contributes to the cisplatinresistance of lung cancer by upregulating mrp1 and mdr1 via stat3 activation biomed pharmacother cai y dong zy and wang jy lncrna nntas1 is a major mediator of cisplatin chemoresistance in nonsmall cell lung cancer through mapkslug pathway eur rev med pharmacol sci han ml zhao yf tan ch xiong yj wang wj wu f fei y wang l and liang zq cathepsin l upregulationinduced emt phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in a549 cells acta pharmacol sin liu j meisner d kwong e wu xy and johnston mr translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer cancer res hassan wa yoshida r kudoh s kameyama h hasegawa k niimorikita k and ito t notch1 controls cell chemoresistance in small cell lung carcinoma cells thorac cancer tang h jiang l zhu c liu r wu y yan q liu m jia y chen j qin y loss of cell adhesion molecule l1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma oncogene liu h focia pj and he x homophilic adhesion mechanism of neurofascin a member of the l1 family of neural cell adhesion molecules j biol chem tassano e biancheri r denegri l porta s novara f zuffardi o gimelli g and cuoco c heterozygous deletion of chl1 gene detailed arraycgh and clinical characterization of a new case and review of the literature eur j med genet morellini f lepsveridze e kahler b dityatev a and schachner m reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule chl1 mol cell neurosci he lh ma q shi yh ge j zhao hm li sf and tong zs chl1 is involved in human breast tumorigenesis and progression biochem biophys res commun martÃnsánchez e mendaza s ulaziagarmendia a monrealsantesteban i blancoluquin i córdoba a vicentegarcÃa f pérezjanices n escors d megÃas d chl1 hypermethylation as a potential biomarker of poor prognosis in breast cancer oncotarget zhu h fang j zhang j zhao z liu l wang j xi q and gu m mir targets chl1 and controls tumor growth and invasion in papillary thyroid carcinoma biochem biophys res commun yu w zhu k wang y yu h and guo j overexpression of mir5p promotes proliferation and invasion of colon adenocarcinoma cells through targeting chl1 mol med hötzel j melling n müller j polonski a wolterseisfeld g izbicki jr karstens kf and tachezy m protein expression of close homologue of l1 chl1 is a marker for overall survival in nonsmall cell lung cancer nsclc j cancer res clin oncol sun y zheng s torossian a speirs ck sc | 0 |
" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [] an rln countof ¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1t3tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd students t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor cipreferenceage ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3rlnslymph node metastasislnr lodds ptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were à ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ¥ characteristicsunivariateormultivariateor cipreference age ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3lymph node metastasisptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ¥ deng proposed an optimal rln count of ¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncolamin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more personalized approach tocancer staging ca cancer j clin okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol tóth d bÃró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg 0c" | 0 |
inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure oï¬ce of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reï¬ect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing ï¬nancialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some ï¬gures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentiï¬ed the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspeciï¬c splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to diï¬cultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the ï¬nalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center childrens hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteï¬anked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of speciï¬c assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by ï¬rst replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was ampliï¬ed from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr ampliï¬cation ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest ï¬anking genes b mouse borcs7as3mt locus with ï¬anking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the ï¬nal borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by ï¬ushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage ï¬uid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo ï¬broblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout¢ ko medium cat no supplementedwith esqualiï¬ed fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after d in culture when the inner cell mass of the embryosreached ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualiï¬ed fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin¢ ï¬nal concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells from¼ colonies were pooled and used for rna isolation to conï¬rmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to conï¬rm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modiï¬ed bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by ampliï¬cation of the pfloxxerx plasmid previously assembled in kollers laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel puriï¬ed on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpuriï¬ed qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturers directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modiï¬ed bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene ï¬anked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasï¬anked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the ï¬rst step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene ï¬anked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psï¬ijt15neojtz17sï¬i previously assembled in kollerslaboratory see table s4 by digestion with sï¬i neb cat nor0123s the blackred homology arm was ampliï¬ed primers and using the bmq455l15 bac as a templateand the purple homology arm was ampliï¬ed primers and using the rp11753c18 bac as a template all fragments were gel puriï¬ed as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column puriï¬ed as described above theloxpï¬anked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel puriï¬edand then combined in an overlap pcr primers and the resulting pcr product was gel puriï¬ed and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modiï¬ed rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturers instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturersrecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft¢ softwareversion a portion of each of the es colonies ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientiï¬c cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientiï¬c cat no volvol triton¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturers suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a ï¬nal 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentiï¬ed and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieï¬ycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturers instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identiï¬edby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a ï¬nal extension of min the f2generationconsisting of hshs homozygotes hswt heterozygotes and wtwt homozygoteswas produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghankss balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbeccos modiï¬ed eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aï¬amedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a ï¬amedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were ï¬rst homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the speciï¬cmethod is indicated in each ï¬gure legend ddpcr is minimallyinï¬uenced by diï¬erences in the eï¬ciency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantiï¬cation of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the ï¬rst h buthad free access to puriï¬ed laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the puriï¬ed dietand diw for wk to allow for clearance of as from the bodywhich was conï¬rmed by measuring total as tas levels inurine see the results section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacriï¬ced by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were ï¬ashfrozen in dryice and stored at c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacriï¬ce ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjemstyle tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the eï¬ciencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukeys multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice students ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was | 0 |
"Two methods were used to inhibit the enzyme activity or protein expression of NQO1 an NQO1 inhibitor and NQO1 siRNA knockdown. Dicoumarol has been previously used to specifically inhibit the expression and activity of NQO1 [44]. As shown in pretreatment of cells with 100 or 250 µM sulindac (A) sulindac sulfone (B) or sulindac sulfide (C) followed by addition of 2 µM ?-lapachone for 12 h increased the cytotoxicity of ?-lapachone for both CL1-1 and CL1-5 cells and these effects were significantly reduced by addition of 10 µM dicoumarol. .0088122.g007 The increase in ?-lapachone-induced cell death caused by sulindac and its metabolites is blocked by the NQO1 inhibitor dicoumarol. CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were pretreated for 6 h with 100 or 250 µM sulindac (A) sulindac sulfone (B) or sulindac sulfide (C) with or without 10 µM dicoumarol then were incubated for a further 12 h with or without addition of 2 µM ?-lapachone then cell survival was measured by crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p<0.05. Using siRNA knockdown of NQO1 at days 1 to 3 after NQO1 siRNA transfection of CL1-1 and CL1-5 cells no change in cell growth or cell morphology was noted (Figure S6). Efficiency of knockdown in CL1-1 and CL1-5 cells was demonstrated for RNA expression by RT-PCR (A) and realtime-PCR (Figure S7) and for protein expression by western blotting (B and C) showing that NQO1 siRNA significantly downregulated NQO1 expression. As shown in D NQO1 siRNA transfection significantly inhibited the increase in NQO1 enzyme activity induced in CL1-1 cells by incubation for 6 or 24 h with 100 or 250 µM sulindac (left panel) sulindac sulfone (center panel) or sulindac sulfide (right panel). When cells transfected for 24 h with siNQO1 or control siRNA were pretreated for 6 h with sulindac or its metabolites then cotreated for 12 h with drug plus 2 µM ?-lapachone the percentage cell survival results showed results that transfection with NQO1 siRNA caused a significant decrease in the cytotoxicity of combinations of ?-lapachone with sulindac (A) sulindac sulfone (B) or sulindac sulfide (C). These results showed that NQO1 plays an important role in the increase in ?-lapachone-induced cell death caused by sulindac or its metabolites. .0088122.g008 The knockdown effects of NQO1 siRNA on NQO1 RNA protein and activity. (AC) CL1-1 cells (left) or CL1-5 cells (right) were transfected for 1 to 3 days with control siRNA (CTL) or siRNA targeting NQO1 then RNA expression was measured by PCR (A) and protein expression by Western blotting (B and C). (D) CL1-1 cells transfected for 2 days with control siRNA or NQO1 siRNA were incubated alone or with 100 or 250 µM sulindac sulindac sulfide or sulindac sulfone for 6 or 24 h then NQO1 activity was measured. * : p<0.05 ***: p<0.001 compared to the identically treated cells transfected with control siRNA. .0088122.g009 NQO1 siRNA transfection significantly inhibits the effect of sulindac and its metabolites on ?-lapachone-induced cell death. CL1-1 cells (left) or CL1-5 cells (right) were transfected with control siRNA (?) or NQO1 siRNA (+) for 24 h then were left untreated or were incubated for 6 h with 100 or 250 µM sulindac (A) sulindac sulfone (B) or sulindac sulfide (C) then 2 µM ?-lapachone or medium was added and the cells incubated for 12 h when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p<0.05 for the indicated comparison. Discussion ?-lapachone Triggers Lung Cancer Cells to Undergo Apoptosis through an Increase in Intracellular Calcium Levels Increased JNK Activation Decreased Activation of PI3K ERK and AKT and a Decrease in the MMP Cell proliferation and cell death are under complex and precise control. Normally the proteins involved in cell proliferation survival or cell death are in a remarkable balance and unbalanced survival and apoptotic signals may lead to cell death. In most cells proliferation is mainly regulated through PI3K AKT and ERK [46] and cell death is regulated through another pathway involving JNK and p38 [47] [48]. JNK has recently been reported to be an important mediator in the ?-lapachone-induced cell death of breast and prostate cancer cells [45] [49]. ?-lapachone also triggers cell death of many cancer cells by increasing calcium signaling [6] [50]. Calcium the key messenger molecule in cells plays an important role in many signaling pathways and an imbalance in intracellular calcium levels causes abnormal cell function and leads to cell death. Treatment of cells with the intracellular calcium chelator BAPTA partially inhibited ?-lapachone-induced cell death showing that calcium was involved (). Activation of the cell death signal JNK (A) and inhibition of the cell survival signals p-PI3K p-AKT and p-ERK (A and B) were also observed in ?-lapachone-mediated lung cancer cell death showing that the MAP kinase signaling pathway is involved in the anticancer effect of ?-lapachone. Although ROS could have caused the cell death induced by ?-lapachone there was no change in intracellular H2O2 levels following ?-lapachone treatment (Figure S1D). However the MMP was dramatically decreased following ?-lapachone treatment (Figure S3C) suggesting that other ROS species might be involved in the ?-lapachone-induced cell death process. NQO1 is a Key Factor in the ?-lapachone-induced Lung Cancer Cell Death High NQO1 activity and expression are seen in many human tumors including carcinoma of the liver [51] [52] colon [53] breast [52] [54] brain [55] and lung [52] and NQO1 has been shown to be an important factor in ?-lapachone-induced cell death in many kinds of cancer cells [9] [44] including breast cancer [2] glioma [56] and prostate cancer [44]. In this study we demonstrated that the cytotoxicity of ?-lapachone for three different lung cancer cell lines was positively correlated with their NQO1 expression and enzyme activity (). Inhibition of NQO1 activity using the NQO1 inhibitor dicoumarol (Figure S3) blocked the ?-lapachone-induced increase in intracellular calcium levels (Figure S3) increase in p-JNK levels (A) and decrease in p-ERK p-PI3K and p-AKT levels (A and B). These results indicated that the balance between survival and death signals in lung cancer cells was disrupted by the decrease in p-PI3K p-AKT and p-ERK levels and the increase in p-JNK levels caused by ?-lapachone treatment and that NQO1 expression and activity were involved in the activation of all these apoptotic signals. Anti-inflammatory Drugs Increase NQO1 Levels and Enzyme Activity in Lung Cancer Cells Many drugs or treatments such as cisplatin [5] heat shock [19] or radiation [57] can increase NQO1 expression or activity and facilitate the cytotoxicity of ?-lapachone for various cancer cells. However such drugs or treatments are usually harmful to normal cells as well as cancer cells so there is a need for drugs or treatments that can facilitate the anti-cancer effect of ?-lapachone while being less harmful for normal cells. Sulindac has been shown to be a potent chemo-protective agent against colorectal cancer in both human and animal models [25] while sulindac sulfide [18] and sulindac and its two metabolites [18] [28]] have been reported to upregulate the expression of carcinogen detoxification enzymes including NQO1. It is known that sulindac compounds inhibit the activity of COX-1 and COX-2 and thus block the biosynthesis of prostaglandins [58]-[60]. In in vivo studies sulindac has been shown to be reversibly reduced to sulindac sulfide which can be irreversibly oxidized to sulindac sulfone all three of which are anti-inflammatory. Since 1995 several clinical trials have established that sulindac is effective at reducing the number and size of polyps in patients with familial adenomatous polyposis a precursor to colorectal cancer (NCI-P97-0110 NCI-P00-0150 [61])." | 1 |
"high mobility group box hmgb1 is a nonhistone chromatinassociated protein widely distributed in eukaryoticcells and is involved in dna damage repair and genomic stability maintenance in response to stimulus like bacteriaor chemoradiotherapy hmgb1 can translocate to extracellular context as a danger alarmin activate the immuneresponse and participate in the regulation of inflammation and cancer progressionkeywords hmgb1 rage tlr damp inflammation cancerchromatinassociated proteinit washigh mobility group box hmgb1 is a highly conservative nucleoprotein and belongs to the group of nonhistonefirstextracted from calfthymus chromatin in andnamed for its high mobility in gel electrophoresis subsequentinvestigations found that hmgb1 couldtranslocate from the nucleus to the cytoplasm after posttranslational modifications including acetylation phosphorylation and methylation hmgb1 can be expressedat the neuron membrane as well in response to chemoradiotherapy or hypoxia hmgb1 could be transferredto the extracellular context mainly through two waysactive secretion from immunocompetent cells or passiverelease from apoptotic or necrotic cells extracellularhmgb1 transmits danger signals to surrounding cellsby interacting with its classical receptors such as thereceptor for advanced glycation end products rageand tolllike receptors tlr249indepth studies implicated that hmgb1 was a multifunctional protein involved in a variety of cellularsubcellularbiological properties depending on itslocalizationandbinding receptors fig posttranscriptional modification correspondence yizhangzzueducn1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china2state key laboratory of esophageal cancer prevention treatmentzhengzhou university zhengzhou chinafull list of author information is available at the end of the in the nucleus hmgb1 plays a key role in the processof dna replication transcription chromatin remodeling and vdj recombinationthus regulating dnadamage repair and the maintenance of genome stabilityas a dna chaperone cytoplasmic hmgb1 is involvedin immune responses by increasing autophagy inhibitingapoptosis and regulating mitochondrial function atthe membrane hmgb1 promotes axonal sprouting andneurite growth activates platelets and induces cellmigration as a typical damage associated molecular pattern damp extracellular hmgb1 is involved in manyimmune responses by promoting immune cell maturation activation and cytokine production extracellular hmgb1 can also interact with chemokines such ascxcl11 to enhance immune responses as a multifunctional protein hmgb1 exerts different biologicaleffects under different stimuli the deregulation ofhmgb1 is associated with many diseases especiallyinflammatory disorders and cancerhmgb1 in inflammationhmgb1 plays a critical role in the regulation of bothinnate and adaptive immune responses to promoteimmune response to sterile or infectious stimulus insterile inflammation during ischemiareperfusion injuryiri hmgb1 becomes disulfidebonded to increasemacrophage production of proinflammation cytokinesin a tlr4 dependent manner indicating that disulfidebonded hmgb1 can be identified as a diagnosis and the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang and zhang of hematology oncology page of fig the multifunctions of hmgb1 hmgb1 in the nucleus regulates dna damage repair and genome stability as a nonhistone chromatinassociated protein and dna chaperon in the cytoplasm or mitochondria hmgb1 increases autophagy inhibits apoptosis and regulatesmitochondria functions at the membrane hmgb1 promotes axonal sprouting and neurite growth hmgb1 can be transferred to extracellularcontext by two ways active secretion from immunocompetent cells or passive release from apoptotic or necrotic cells participating inimmune responsesin contrasttreatment biomarker for iri when initially secreted byinnate immune cells like macrophages hmgb1 is proinflammatory during the early stages of sepsis howeverthe extracellular hmgb1 could also induce immune tolerance and immunosuppression when released by othersomatic cellsintracellular hmgb1 caninduce protective autophagy and contribute to cellsurvival by delivering lipopolysaccharide lps andpromoting endocytosis hmgb1 activates the noncanonical inflammasome pathway and induces pyroptosis pyroptotic macrophage death may accelerateundesirable immune hyperactivity and immunosuppression which is a potential mechanism associatedwith late mortality from sepsis in hepatic infectious disease the release and activity of hmgb1 as acytokine could be suppressed by glycyrrhizinic acidga by binding with tlr4 hmgb1 regulatesthe hepatitis virusesinduced immunological axis providing a new therapy strategy for the treatment ofacute viral hepatitis in the clinical practice canbesecretedin severe pulmonary inflammatory diseases including covid19 hmgb1inabundance by necrotic pulmonary epithelial cells andinnate immune cells disulfidehmgb1 triggers proinflammatory cytokine release and further exacerbatessevere inflammation therefore hmgb1 mightbeofinflammationtreatmentpotentialtargetaforthethe dual effects of hmgb1 in canceraccording to the alterations of the subcellular locationsreceptors and expression levels hmgb1 is associatedwith the hallmarks of cancer proposed by hanahan andweinberg hmgb1 appears to play paradoxicalroles during the development and therapy of cancer onthe one hand hmgb1 can contribute to tumorigenesisexcessive hmgb1 production caused by chronic inflammatory response seems to be associated with tumorigenesis for example by combining with rage hmgb1plays an important role in regulating oval cells activationand inflammationassociated liver carcinogenesis in mice in established cancers hmgb1 produced by tumorcells may exacerbate inflammationrelated immunosuppression for instance previous research indicated thatlps induced the release of proinflammatory cytokinessuch as il1 il6 and tnfα in a hmgb1dependentmanner to improve colon cancer progression however the underlying mechanism of hmgb1 in the transformation ofinflammation and cancer needs to befurther studied it has been reported that hmgb1 canbe released to extracellular context by necrotic cellsunder hypoxia in growing solid tumor extracellularhmgb1 promotes the release of cytokines such as il6and il8 by activating mapk and myd88dependentnfκb pathways which in turn stimulates tumor cellsproliferation angiogenesis emt invasion and metastasis nucleusand cytoplasmic hmgb1 promotes 0cwang and zhang of hematology oncology page of autophagy and inhibits apoptosis of tumor cells to induce chemotherapy resistance on the other handhmgb1 plays a protective role in the suppression oftumor and tumor chemoradiotherapy and immunotherapy nucleus hmgb1 assists in the regulation of telomere and maintenance of genome stability loss ofhmgb1 results in the instability of genome and leads totumorigenesis thus the roles of hmgb1 in regulationof dna damage repair and cancer etiology indicate thattargeting chromosomal architectural hmgb1 may provide a new perspective for cancer therapy hmgb1located in the cytosol or mitochondria may bind toautophagy associated genes like beclin to regulate cellautophagy and mitophagy absence of hmgb1 resultsin autophagy deficiency and increased apoptosis leadingto tumorigenesis intracellular hmgb1 functions as atumor suppressor by binding tumor suppressor proteinslike rb but it remains to be studied whether hmgb1interacts with other tumor suppressors or oncoproteinsextracellular hmgb1 enhances chemotherapy efficacyby transforming tumor cells from apoptosis to senescence in addition hmgb1 can mediate immunogenic cell death during chemoradiotherapy and enhanceantitumor immunity in response to chemotherapy likeanthracycline or radiotherapy hmgb1 can be rapidlyreleased from dead cells as an alarming molecule uponrelease from necrotic cells or secreted by activated macrophages hmgb1 can recruit inflammatory cells andmediate interactions between nk cells dendritic cellsdcs and macrophages activated nk cells provide anadditional source of hmgb1 which is released into theimmunological synapse between nk cells and immaturedcs promoting the maturation of dcs and the induction of th1 response in addition hmgb1 produced from nsclc cells induced by docetaxel canstimulate t cells for antitumor immune response andimprove immunotherapy effects like cart cells therefore modulating hmgb1 may provide a potentialcombination strategy for cancer chemoradiotherapy andimmunotherapyconclusionhmgb1 is a multifunctional molecule that plays a majorrole in homeostasis as damp molecule hmgb1 playsthe complex roles in various biological processes and isinvolved in the development of many diseases such asautoimmune diseases and cancers hmgb1targetedagents including antibodies and inhibitors have shownbeneficial results in preclinicalinflammatory modelssuch as sepsis these agents await clinical developmentabbreviationshmgb1 high mobility group box rage the receptor for advancedglycation end products tlr tolllike receptors damp damage associatedmolecular pattern iri ischemiareperfusion injury lps lipopolysaccharidega glycyrrhizinic acid covid19 coronavirus disease19 mapk mitogenactivated protein kinase myd88 myeloid differential protein88 nfκb nuclear factor kappalightchainenhancer of activated b cellsemt epithelialmesenchymal transition dcs dendritic cellsacknowledgementsnot applicableauthorsâ contributionsyz designed directed and revised the manuscript sw drafted themanuscript both of the authors read and approved the final manuscriptfundingthis work was supported by the national key research and developmentprogram of china 2016yfc1303500availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1biotherapy center cancer center the first affiliated hospital ofzhengzhou university zhengzhou china 2state key laboratory ofesophageal cancer prevention treatment zhengzhou universityzhengzhou china 3henan key laboratory for tumor immunologyand biotherapy zhengzhou chinareceived july accepted august referencesgoodwin gh sanders c johns ew a new group of chromatinassociatedproteins with a high content of acidic and basic amino acids[j] febs jâpaudel yn angelopoulou e piperi c balasubramaniam vrmt othman ishaikh mf enlightening the role of high mobility group box hmgb1 ininflammation updates on receptor signalling[j] eur j pharmacol huebener p gwak gy pradere jp et al highmobility group box isdispensable for autophagy mitochondrial quality control and anfunction in vivo[j] cell metab ârivera vargas t apetoh l danger signals chemotherapy enhancers[j]immunol rev âgao q wang sm chen xf et al cancercellsecreted cxcl11 promotedcd8 t cells infiltration through docetaxelinducedrelease of hmgb1 innsclc[j] for immunotherapy of cancer andersson u tracey kj hmgb1 is a therapeutic target for sterileinflammation and infection[j] annu rev immunol âkim hm kim ym hmgb1 lps delivery vehicle for caspase11mediatedpyroptosis[j] immunity âdeng m tang y li w et al the endotoxin delivery protein hmgb1mediates caspase11dependent lethality in sepsis[j] immunity â753e747shi xd yu lj zhang yl et al glycyrrhetinic acid alleviates hepaticinflammation injury in viral hepatitis disease via a hmgb1tlr4 signalingpathway[j] int immunopharmacol saha b tornai d kodys k et al biomarkers of macrophage activation andimmune danger signals predict clinical outcomes in alcoholic hepatitis[j]hepatology baltimore md â andersson u ottestad w tracey kj extracellular hmgb1 a therapeutictarget in severe pulmonary inflammation including covid19[j] molecularmedicine cambridge mass 0cwang and zhang of hematology oncology page of hanahan d weinberg ra hallmarks of cancer the next generation[j] cellâ pusterla t nèmeth j stein i et al receptor for advanced glycationendproducts rage is a key regulator of oval cell activation andinflammationassociated liver carcinogenesis in mice[j] hepatologybaltimore md â yang y yang l jiang s et al hmgb1 mediates lipopolysaccharideinducedinflammation via interacting with gpx4 in colon cancer cells[j] cancer cellint yuan s liu z xu z et al high mobility group box hmgb1 a pivotalregulator of hematopoietic malignancies[j] j hematol oncol mukherjee a vasquez km targeting chromosomal architectural hmgbproteins could be the next frontier in cancer therapy[j] cancer res liu y dong y kong l et al abscopal effect of radiotherapy combined withimmune checkpoint inhibitors[j] j hematol oncol publisherâs notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
immunerelated genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12 Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ¥ ¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficientScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and Chinas low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25 Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ¥ ¥ ¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29 Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34 However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37 there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGFβ can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42 Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47 while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes BH In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine plenty to be done Carcinogenesis 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira ThaÃs C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can | 2 |
" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patients quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage iiiii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately of patients with crc were diagnosed with acute colonic obstruction [] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts 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 0cohta bmc surgery page of treatment for acute colonic obstruction [] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatients symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage iiiiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudents ttest and wilcoxon test for continuous variables and the Ï2 and fishers exact tests for categoricalvariables were conducted survival curves were generated using the kaplanmeier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n a claviendindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patientssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml before afterstoma creation morbidity30day complication cda iii anastomosticleakagehospital stayaclaviendindo classificationmedian range days primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplanmeier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n bts n p survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± overall suvivales vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± without obstruction [] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment []the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage iiiii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of obstructing colorectal cancers in a welldefined population dis colon rectum pubmed pmid epub engcuffy m abir f audisio ra longo we colorectal cancer presenting assurgical emergencies surg oncol pubmed pmid epub eng mcardle cs hole dj emergency presentation of colorectal cancer isassociated with poor 5year survival br j surg pubmedpmid epub eng mainar a tejero e maynar m ferral h castanedazuniga w colorectalobstruction treatment with metallic stents radiology pubmed pmid epub engzhang y shi j shi b song cy xie wf chen yx comparison of efficacybetween uncovered and covered selfexpanding metallic stents inmalignant large bowel obstruction a systematic review and metaanalysiscolor dis 2012147e367 pubmed pmid epub engtilney hs lovegrove re purkayastha s sains ps westonpetrides gk darziaw comparison of colonic stenting and open surgery for malignantlarge bowel obstruction surg endosc pubmed pmid epub engsaito s yoshida s isayama h matsuzawa t kuwai t maetani i aprospective multicenter study on selfexpandable metallic stents as a bridgeto surgery for malignant colorectal obstruction in japan efficacy and safetyin patients surg endosc pubmed pmid epub engsaida y sumiyama y nagao j uramatsu m longterm prognosis ofpreoperative bridge to surgery expandable metallic stent insertion forobstructive colorectal cancer comparison with emergency operation discolon rectum suppls44 pubmed pmid epub engtomita m saito s makimoto s yoshida s isayama h yamada t selfexpandable metallic stenting as a bridge to surgery for malignant colorectalobstruction pooled analysis of patients from two prospectivemulticenter series surg endosc pubmed pmid pubmed central pmcid pmc6342866 epub eng chen hs sheenchen sm obstruction and perforation in colorectaladenocarcinoma an analysis of prognosis and current trends surgery pubmed pmid epub eng huang x lv b zhang s meng l preoperative colonic stents versusemergency surgery for acute leftsided malignant colonic obstruction ametaanalysis j gastrointest surg pubmed pmid epub engkim hj huh jw kang ws kim ch lim sw joo ye oncologic safetyof stent as bridge to surgery compared to emergency radical surgery forleftsided colorectal cancer obstruction surg endosc pubmed pmid epub engshigeta k baba h yamafuji k kaneda h katsura h kubochi k outcomesfor patients with obstructing colorectal cancers treated with onestagesurgery using transanal drainage tubes j gastrointest surg pubmed pmid epub engvan hooft je bemelman wa oldenburg b marinelli aw lutke holzik mfgrubben mj colonic stenting versus emergency surgery for acute leftsided malignant colonic obstruction a multicentre randomised trial lancetoncol pubmed pmid epub engvan hooft je fockens p marinelli aw timmer r van berkel am bossuyt pm early closure of a multicenter randomized clinical trial of endoscopicstenting versus surgery for stage iv leftsided colorectal cancer endoscopy pubmed pmid epub engsloothaak da van den berg mw dijkgraaf mg fockens p tanis pj vanhooft je oncological outcome of malignant colonic obstruction inthe dutch stentin trial br j surg pubmed pmid epub eng gorissen kj tuynman jb fryer e wang l uberoi r jones om localrecurrence after stenting for obstructing leftsided colonic cancer br j surg pubmed pmid epub eng ormando vm palma r fugazza a repici a colonic stents for malignantbowel obstruction current status and future prospects expert rev meddevices pubmed pmid epub englauro a binetti m vaccari s cervellera m tonini v obstructing leftsidedcolonic cancer is endoscopic stenting a bridge to surgery or a bridge tonowhere digestive diseases and sciences pubmed pmid epub engschwartz lh litière s de vries e ford r gwyther s mandrekar s recist 11update and clarification from the recist committee europeanjournal of cancer oxford england p pubmed pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated fibroblasts drive the progression of metastasis throughboth paracrine and mechanical pressure on cancer tissue mol cancer res pubmed pmid pubmed central pmcidpmc4399759 epub eng nojiri t inoue m yamamoto k maeda h takeuchi y funakoshi y b maruthachalam k lash ge shenton bk han af tumour celldissemination following endoscopic stent insertion br j surg pubmed pmid epub eng matsuda a miyashita m matsumoto s sakurazawa n kawano y yamahatsuk colonic stentinduced mechanical compression may suppresscancer cell proliferation in malignant large bowel obstruction surg endosc pubmed pmid epub eng haraguchi n ikeda m miyake m yamada t sakakibara y mita e colonic stenting as a bridge to surgery for obstructive colorectal canceradvantages and disadvantages surg today pubmedpmid epub eng zhu z li b liao w lv n chen y shu x novel predictive nomogram foridentifying difficult guidewire insertion in patients with malignant colorectalobstruction and sphincterotomeassisted guidewire insertion for improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg pubmedpmid pubmed central pmcid pmc7215972 epub engpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c" | 0 |
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 investigators 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 ϲ 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 exceptional 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 statusaaaaaaaab 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 investigators 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 | 0 |
"which contains an SH2 domain and two SH3 domains and can directly bind the EGFR [36]. The results of -A demonstrate using immunoprecipitation of EGFR and immunoblotting with anti-Grb-2 antibody that EGF induces EGFR/Grb-2 complex formation which is attenuated by pretreatment of H358 cells with MNTX. Recruitment of Grb-2 to the EGFR is important for plasma membrane recruitment and consequent tyrosine phosphorylation of the scaffolding protein Grb2-associated-binding protein 1 (Gab-1) [37]. -B indicates that EGF stimulation of H358 cells induces tyrosine phosphorylation of Gab-1 (Tyr307 and Tyr627) which peaks at ?5 minutes and is attenuated by MOR inhibition with MNTX. .0091577.g003 The peripheral mu opioid receptor antagonist methylnaltrexone (MNTX) inhibits EGF-induced recruitment/activation of the linker protein Grb-2 and the scaffolding protein Gab-1 in human lung cancer cells. Panel A: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX (1 hour pre-incubation) 10 ng/ml EGF for 15 minutes or 100 nM MNTX and 10 ng/ml EGF. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates and immunoprecipitated material using anti-Grb-2 (ac) and anti-EGFR (bd) antibody. MNTX inhibits EGF-induced recruitment of Grb-2 to the EGFR. Panel B: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone 10 ng/ml EGF for 5 15 or 30 minutes or 100 nM MNTX and 10 ng/ml EGF for 515 or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY627 Gab-1 (a) anti-pY307 Gab-1 (b) and anti-actin (c) antibodies. MNTX attenuates EGF-induced Gab-1 tyrosine phosphorylation. Since tyrosine phosphorylation of Gab-1 by various tyrosine kinases including Src promotes binding to signaling molecules including Phosphatidylinositol 3-kinases (PI3Ks) which generate PIP3 and activate downstream effectors including Akt and STAT3 [37] [38] [39] [40] [41] [42] we next examined whether MOR inhibition can also influence Gab-1 binding/effector molecules. -A shows that like Gab-1 EGF challenge of H358 cells induces tyrosine phosphorylation of Src (Tyr416) the regulatory PI3K alpha subunits p85 and p55 (Tyr458/Tyr199) and the transcription factor STAT3 (Tyr705) which peak at ?5 minutes and are attenuated by MOR inhibition with MNTX in a statistically significant manner (-B)." | 1 |
Human transcription factor and protein kinase gene fusions in human cancerKari Salokas Rigbe G Weldatsadik Markku VarjosaloOncogenic gene fusions are estimated to account for upto of cancer morbidity Recently sequencelevel studies have established oncofusions throughout all tissue types However the functional implications of the identified oncofusions have often not been investigated In this study identified oncofusions from a fusion detection approach DEEPEST were analyzed in detail Of the oncofusions we found almost are expected to produce functional proteins with features from both parent genes Kinases and transcription factors were the main gene families of the protein producing fusions considering their role as initiators actors and termination points of cellular signaling pathways we focused our indepth analyses on them Domain architecture of the fusions and their wildtype interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins To understand overall oncofusion effects we performed differential expression analysis using TCGA cancer project samples Results indicated oncofusionspecific alterations in gene expression levels and lower expression levels of components of key cellular pathways in particular signal transduction and transcription regulation the sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling possibly via acquiring novel functionsAt any given moment multitudes of molecular networks are activated in cells throughout the body An important feature of these networks is highly concerted regulation of key signaling and deviation from homeostasis can result in diseases such as cancer Cancer is a complex progressive multistep disorder which stems from mutations caused by genomic instability1 The accumulation of genetic and epigenetic abnormalities ultimately leads to the transformation of normal cells into malignant derivatives Two highly enriched gene groups being mutated in the majority of cancer types are protein kinases PKs and transcription factors TFs23 PKs mediate most signal transduction events in cells by phosphorylation of specific substrates thus modifying their activity cellular localization andor association with other proteins TFs are the transistors of the cellular signaling circuits controlling the transcriptional outcome of activated signaling by binding to regulative elements of their corresponding target genes and driving or suppressing their expression Therefore it is easy to understand why mutational deregulation of these two gene groups can have such an impact on tumorigenesisIn addition to harboring activating or inactivating somatic point mutations PKs and TFs account for a large fraction of all human fusion genes involved in cancer COSMIC Catalogue of Somatic Mutations in Cancer cancersangeruk4 and dbCRID Database of Chromosome Rearrangements in Disease5 Chromosomal translocations creating fusion genes are among the most common mutation class of known cancer genes and they have long been identified as driver mutations in certain types of cancer6 Recently oncogenic fusion genes hereafter oncofusions OFs have been found in many hematological and solid tumors demonstrating that translocations are a common cause of malignancy78 Fusion mutations occur when two different gene regions fuse together via translocation Examples of consequences of chromosomal fusion to protein structure range from missense mutations to expressionchange inducing promotergene combinations to fully functional fusion proteins with neomorphic properties A classic example of gained functions is the breakpoint cluster regionAbelson tyrosineprotein kinase BCRABL1 translocation in chronic myeloid leukemia9 Alternatively a protooncogene is fused to a strong promoter and thereby the oncogenic function is upregulated due to the strong promoter of the upstream fusion partner This is common in lymphomas where oncogenes are juxtaposed to the promoters of the immunoglobulin genes10 and also in prostate cancer where ETS TF ERG is fused with TMPRSS2 regulatory Systems PathologyBiology Research Group Institute of Biotechnology HiLIFE University of Helsinki Helsinki Finland email markkuvarjosalohelsinkifiScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0csequences thus obtaining androgen receptor ARresponsive expression11 The current understanding favors the aberrant gene function model rather than promoterinduced overexpressionThe frequency of recurrent OFs varies depending on the specific type of cancer12 but identified translocations are estimated to account for up to of cancer morbidity8 Recent fusion prioritization study found that inframe transcripts were the most powerful predictor of driver fusions16 confirming the intuition that inframe transcripts are crucial to function Notably breakpoints were also observed to preferentially avoid splitting of domains Together with frameshift conservation such trends could reflect a selection on fusion proteins to maintain protein stability and evade degradation pathways17Nextgeneration sequencing NGS of genomes and transcriptomes from primary human cancer cells is constantly revealing new gene fusions that are involved in driving tumorigenesis including examples found in colorectal carcinoma bladder carcinoma breast cancer and acute lymphoblastic leukemia ALL1518 Furthermore NGS has provided enough detailed sequence information of the fusion breakpoints allowing a0us to initiate systemslevel research on human oncofusions As a result various algorithms have been developed to mine OFs from large cancer datasets such as TCGA However the concordance among the different algorithms is very low that metacaller approaches utilizing consensus calls have been employed21 which limit novel OF discoveries Recently a new statistical method DEEPEST22 was developed to overcome these limitations In this study oncofusions that involve PKs and TFs were selected from the data produced by DEEPEST applied to the TCGA datasetIn most cases it is not possible to draw definite s about the mechanisms or extent by which individual translocations contribute to cancer Predicting protein function from a sequence has proven an extremely difficult task With gene fusions the task is even more daunting However an unexpectedly large number of PKs and TFs have been found to be mutationally activated or have increased expression due to gene amplification or translocation in cancer6 The high number of PKs and TFs with relatively low individual mutational frequency suggests either that a large number of signaling pathways can contribute to cancer or that many PKs and TFs can regulate the same pathways when activated unphysiologically Some additional support for this hypothesis comes from the interconnectivity of the PKTFoncofusionsIn this study fusions predicted to produce inframe proteins were analyzed to understand the proteinlevel implications of fusion events The fusions were analyzed from the perspective of their domain architecture to understand likely modes of action of the novel proteins Furthermore known interactomes of the participating wild type proteins were used to determine possible mechanisms of action pathways of interest and possible treatment vectors for affecting as many different fusions as possible As a result multiple cellular signaling pathways were found to intersect with major subsets of these fusions and multiple individual key interactors such as NTRK1 with over and EGFR with over interacting fusions were identified as potential targets of interestMaterials and methodsfusion selection and annotation Fusions that involve protein kinase genes23 and transcription factors24 were selected from the fusions that were identified by applying DEEPEST to the whole TCGA dataset22 Of these were determined to be unique by considering Ensembl gene IDs biotypes chromosomal breakpoints AGFusion assigned fusion effects and resulting protein sequences AGFusion was used to annotate these gene fusions to the human genome assembly GRCh38 v89 from Ensembl For analysis involving gene pairs the pair entry was used in alphabetical order eg ERGTMPRSS2 instead of TMPRSS2ERG in all cases Fusions were considered protein coding if both genes contributed over amino acids to the productclinical data Clinical data for TCGA samples was obtained from the GDC data repository The data was matched to AGFusion output data based on TCGA barcode eg TCGAWBA80K using a custom inhouse python script Stage information from the clinical data was simplified where possible eg Stage IIA was changed to Stage II Entries such as Stage Stage X and III NOS were ignored Tissue entries were simplified from detailed ICDO topographical codes to more general eg C569 C56 and mapped to names accordingly Chromosomal sequence information from GRCh38 v89 was used to categorize breakpoints into chromosomal interval groupsInteractor analysis Interactors for wild type proteins of all fusion partners were obtained from IMEx consortium25 and any interactions that were not confirmed to be physical by experimental methods were discarded Interactors were added to the interactor set from each fusion while leaving out the fusion pair genes themselves Annotations for interactors were obtained using Uniprot and Reactome From Reactome mappings to all levels of pathway hierarchy were used Dijkstras algorithm26 implemented with a custom python script was then used to establish shortest paths to Reactome root nodes for each network node A weight of was used for all network edgesDomain annotation For the protein producing fusions sequences of the protein products were produced using the AGFusion tools Duplicate fusions based on fusion genes and protein sequence were discarded Domains were taken from AGFusion output and mapped to protein sequence in the wild type protein The intactness of domains was then determined by matching the WT domain sequence to the predicted fusion protein sequence and only full length intact domains were picked for further study A domain was classified as PK or TFspecific if ¥ of all its occurrences were in PK or TF proteins respectivelyData visualization Data illustrations were made with CorelDRAW Excel and inhouse python scripts using Matplotlib and Seaborn Cytoscape27 was used for creating network figuresScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cDifferential expression analysis Gene expression quantification HTSeqcounts files were downloaded from GDC data portal Samples where OFs with intact fulllength PK or TF domains were detected were grouped together based on fusion gene pairs The groups were then analyzed with DESeq228 using other samples with protein producing nonPKTF fusions as controls For each pair group differential expression analysis against an equal number of control samples picked from samples in which other proteinproducing fusions were found Analysis was repeated times for each fusion pair For the resulting significantly differentially expressed genes qvalue basemean and expected values were averaged across all runs and a fold change value calculated based on these GO annotations were then added from ensembl annotations and Reactome pathways from first mapping ensembl gene IDs to Uniprot via Ensembl BioMart and then to Reactome lowest level pathway terms via Reactome Zscore value for pathway level overunderexpression was calculated by a method used in GOplot29 ieby deducting the number of underexpressed genes from the number of overexpressed genes and dividing the result by the square root of the number of significantly changed genes FDR corrected p ¤ ResultsDetection of oncofusions from TCGA dataset reveals enrichment of PK and TF fusions In this study we focused on protein producing OF genes Translocation of chromosomal regions can result in either inframe or outofframe OFs Fig a01A To characterize the proteins produced by known OFs in the TCGA dataset which currently contains data from different cancer projects we launched an analysis to understand the potential functional space of the protein producing fusions Fig a01B and especially those that involve either a PK or a TF or both PKTF fusionsThe DEEPEST dataset included fusions detected from cancer samples Of these were unique fusions Fig a01C upper panel Among the unique OFs were predicted to retain frame and also produce potentially functional proteins where both genes contributed over inframe amino acids Fig a01B Supplementary table a0S1 The limit of amino acids was the length of the shortest nonrepeat domain present in the fused proteins Examining the resulting protein producing OF set we noticed an abundance of those involving PK or TF Indeed these fusions constituted protein producing OFs Fig a01C lower panel Generally the proportion of PKTF fusions a0was under except in the PKTFfusion prone cancers acute myeloid leukemia cholangiocarcinoma thyroid carcinoma and thymoma The number of OFs per sample varied across cancer types The types most prone to protein producing fusions were sarcoma SARC with an average of protein producing fusions per sample esophageal carcinoma ESCA fusions uterine corpus endometrial carcinoma UCEC stomach adenocarcinoma STAD breast invasive carcinoma BRCA uterine carcinosarcoma UCS and ovarian serous cystadenocarcinoma OV Due to the prevalence of PK and TF genes in the fusions we next investigated if they are enriched in particular cancers While in most cancers PKTF fusions made up around of all protein producing OFs the percentage reached in acute myeloid leukemia LAML samples in thymoma THYM in thyroid carcinoma THCA and and in kidney renal papillary cell carcinoma KIRP and cholangiocarcinoma CHOL respectively Fig a01C Acute myeloid leukemia is well known as an OFprone cancer30 However aside from the four fusions detected between ABL1 and BCR the high percentage was mostly TFdriven with KMT2A RUNX1 and RARA being found in and fusions respectively This is in contrast to the peak in THCA which is driven by BRAF fusions fusions of RET of NTRK1 and of NTRK3 among other protein kinasesReading frame retention is common in pK and tf oncofusions The fusions consisted of unique gene pairs and individual genes of the pairs did not have any protein producing fusions The top protein producing fusion was RPS6KB1VMP1 with unique protein producing fusions in the dataset all the others having less than Fig a02A There were fusion gene pairs that were predicted to produce protein in at least fusions in fusions in and in fusion Out of the fusion gene pairs that produced or more unique proteins were PKTF fusionsTo better understand the behavior of prolific gene pairs we next mapped tissue annotations from TCGA to fusions of each gene pair based on barcodes from samples where a fusion of the gene pair was present In contrast to RPS6KB1VMP1 and ITGB6RBMS1 which were seen in samples from different cancers pairs were seen in samples of only one cancer type Out of these cancerspecific fusions were predicted to produce or more unique proteins with ERGTMPRSS2 predicted to produce different unique proteins supplementary table a0S2 PKTF fusions featured different PK or TF genes ERG being the most common TF and ERRB2 the most common PK supplementary table a0S3 Between and percent of oncofusions in each cancer project were unique highest being sarcoma with unique gene pair combinations and thyroid carcinoma the lowest with supplementary table a0S4 Protein producing fusions followed a similar theme unique protein producing fusions making up between and of all oncofusions in each given cancer project supplementary table a0S4We next looked in more detail what cancer stages PK and TF fusions were detected in The most prominent group was stage II breast invasive carcinoma which also had the most samples in the data set Fig a02B In total of the PK and TF fusions were found in stage I samples in stage II in stage III and in stage IV On average samples had PKTF fusions per sample However in some cancers PK or TF fusions are enriched towards the more severe stages Discounting stage groups with less than samples groups had more than fusions per sample ESCA stage III samples in particular had PKTF fusions per sample while STAD and BRCA stage IV samples had and respectively and STAD stage I had Supplementary table a0S5 The distribution of protein producing OFs mirrored that of PKTF fusions quite closely supplementary figure S1A In terms of chromosomal breakpoint locations those in the PKTF fusions varied compared Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cFusion gene Fusion gene PromoterProtein coding regionetis noisuFTranslocationChr Chr Chr Chr Inframe fusionOutofframe fusionTCGA DEEPEST dataset gene fusionsAGFusionEnsemblGRCh38TCGAclinical dataUniprotKinases Manning et al TFs Lambert et al et al Gene expressionquantiï¬cation dataprotein producing uniqueoncofusions Protein codingProtein kinase or Transcription factoroncofusionsPfamdomainsIMEx consortium interactor databaseCOSMICcancer genecensusReactomepathwaysClinical overviewDomain analysisInteraction analysis Pathway analysisDifferential expressionanalysisSamplesOncofusions per sampleABCselpmas fo rebmuNCCAACLBACRBCSECLOHCDAOCCBLDACSEMBGCSNHHCKICRKIPRKILMALGGLCHILDAULCSULOSEMVODAAPGPCPDARPDAERCRASMCKSDATSTCGTACHTMYHTCECUSCUMVUnietorp ni FT ro KP fo noitroporPsnoisufocno gnicudorpelpmas rep snoisufocno fo rebmuNACCBLCABRCACESCCHOLCOADDLBCESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diffuselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinoma Uterine carcinosarcomaUCSUVM Uveal melanomaScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Schematic illustration of the gene fusions workflow and the number of gene fusions in human cancer A Schematic description of gene fusions formation Fusions are formed mainly via balanced and unbalanced chromosomal rearrangements such as translocations deletions inversions and insertions This usually leads to formation of a fusion gene with the ² end of Gene and ² end of Gene If the fusion occurs between two protein coding genes depending on whether the reading frame is violated and where exactly the fusion occurs a fusion protein may be transcribed with features and domains from both partners Other possible outcomes include full or truncated ² gene under the control of the promoter of the ² gene B Workflow used in this study Analysis progressed from the total set of fusions discovered by the DEEPEST method22 and moved towards more specific kinase TF containing protein producing oncofusions We started with TCGA databased fusion set from Dehghannasiri et a0al for which we generated protein sequences with AGFusion Domains were added by matching sequence to Uniprot proteins annotated with Pfam domains after which nonunique entries were dropped Fusions were classified as protein producing if both gene fragments were predicted to produce AA of protein sequence From this set the two most prominent protein groups were protein kinases and transcription factors and thus we focused further analysis on the unique protein kinase or transcription factor containing fusions using the full protein producing fusion set for comparison Known interactions for wild type fusion proteins were obtained from IMEx consortium and used for estimating maximal foreseeable effect on signaling pathways from Reactome Finally TCGA gene expression quantification data was used to probe observable effects of kinaseTF fusions using other protein producing fusions as background C Top Breakdown of samples and fusion mutations by TCGA project Largest single contributor of samples with fusions was TCGA breast invasive carcinoma project BRCA which had the highest number of samples and identified fusion mutations Bottom Proportion of protein producing fusions that include PK or TF genesto all protein producing fusion mutations but the prominent role of PKTF fusions is illustrated by overlapping hotspots supplementary figure S2Intact inframe domains are commonly retained in OFs To understand the contribution of each OF to the overall development or survival of cancerous cells the functional consequences of any given mutation and its impact on the pathways the proteins are involved in must be understood To this end we analyzed all identified unique protein producing fusions and the fulllength inframe domains of the fusion proteinsWhile AGFusion does predict protein sequence for each fusion partner and corresponding conserved or lost domains a domain is counted as conserved already if only amino acids are included in the sequence To adapt this to the study of fulllength domains we first mapped the Pfam identifiers of the domains to sequences in the wild type proteins from Uniprot The domains were then defined as conserved only if the full sequence was present in the fusion protein This resulted in conserved domains in all protein producing fusions Over of these domains are in PKTF fusions which account for of all protein producing fusions supplementary tables S1 S6 suggesting overall domain count strongly favors PK and TF genes perhaps indicating that these fusions produce more functional proteins in comparison to all protein producing fusionsThe most conserved domain was the protein tyrosine kinase domain Fig a03A supplementary table a0S6 which was conserved in fusions This was followed by the PH domain a common domain in intracellular signaling proteins and proteins of the cytoskeleton and the protein kinase domain To assess retention of nonobvious PK or TF domains we classified domains as PK or TF specific if over of the copies were found in PK or TF halves of the fusion proteins This resulted in copies of different TFspecific domains predicted to exist in the fusions compared to copies of PKspecific domains Most common TF domains were zinc finger C2H2 type KRAB and HLH DNA binding domains present in and copies respectively Many TF domains such as KRAB are involved in both transcriptional activation and repression depending on the molecular contextOn average protein producing fusions in samples of most cancer projects tended to have close to intact full length domain per protein producing OF PKTF fusions on average had more intact domains in all except for projects Fig a03B On average fusions in all projects tended to have between and intact domains while PKTF fusions featured a slightly higher average Although some cancers do appear to have particularly many domains this is mostly due to low count of fusions detected in the project Exception seems to be acute myeloid leukemia with detected protein producing fusions of which contain either a PK or a TF Most striking differences being seen in mesothelioma thyroid carcinoma rectum adenocarcinoma and uveal melanoma with and more retained domains on average in PKTF fusions compared to protein producing fusions respectivelyOn the cancer project level thyroid carcinoma had the highest percentage of PK domains of all domains identified in the samples of the project supplementary table a0S7 supplementary Figure S3 which totaled to only exceeded by breast invasive carcinoma with PK specific domains of all BRCA domains and lung adenocarcinoma LUAD with Proportion of TF domains varied less Kidney renal papillary cell carcinoma had of its intact domains in the TFspecific set followed by acute myeloid leukemia with and rectum adenocarcinoma and prostate adenocarcinoma both at Aside from prostate adenocarcinoma these projects had samples in the TCGA datasetinteractors of fusion partners can point to impact of ofs To understand what kind of implications the functional changes of lost conserved PK or TF specific domains in new combinations could have for the Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cNonprotein producing fusionProtein producing fusionLMEKLASMBRBGTIPMVBKSPRTXNURXNURHPMRYLKNWCREATMKLLEPXOFDNCCNPTPCDCCMUPNAKNICCATRFGFSSRPMTGREPAGPBBREARARLMPGRPTPTRCBLBAKRTNVTEBTBZASARFNZPBPSLEHYTTLDAMIUCMCCSAHDTMBMTPALDNYMZAYEDHSABRLBRSMTONCSRAYMUPSGRHPCMProtein kinaseTranscription factorProtein kinase and transcription factorAsnoisufocno fo rebmuNBIJNNAMDKCCMNBDDZDPONEPIRTSLYCHALAGTSfroCIV IIIIIIACCBLCABRCACESC CHOLCOADDLBC ESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diï¬uselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinomaUCS Uterine carcinosarcomaUVM Uveal melanomaFigure a0 Clinical characterization of protein producing oncofusions by cancer stage A The most common protein producing gene pairs in oncofusions In total protein producing fusions were comprised of unique gene pairs predicted to produce one or more unique protein products The most common pair was RPS6KB1VMP1 with over unique proteins followed by ITGB6RBMS2 and ALKEML4 with each and LYRM9P3H4 and RUNX1RUNX1T1 at Kinase and TF fusions were common in top protein producing gene pairs illustrated by blue shading for the presence of a protein kinase in gene pair red for TF and orange for both B Sunburst diagram of project and stage distribution of PKTF oncofusions The innermost layer represents the number of fusions in each project The layers radiating out are the proportion of fusions detected in Stage I II III and IV samples in order from in to out Total numbers of fusions from each stage is marked under the stage indicatorsScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cProtein kinase or TF containing oncofusionsProtein producing oncofusionsAsniamod fo rebmuNniamodHS staeper niryknAniamod III epyt nitcenorbiFniamodC taeper atebG niamodDW niamod HC ygolomohninoplaC fitomnoitingocer ANRregnï¬DHPniamodZDP epyt HC regnï¬ cniZniamod lanimret Cdevresnoc esacileHfitom ahpla eliretS niamodMAS xobBARKniamodHS niamodZOPBTBniamod HS tnairaV niamodnirehdaC niamodgnidnib lorecylglycaidsretse lobrohP niamodgnidnibANDxilehpoolxileH niamodFEGohR niamodPAGohRtaeper nirtcepSnietorp tropsnart noIniamod nilubolgonummIA ssalc niamod rotpecer LDLniamodomorBniamodAHF taeper hcir enicueL niamod tesVnilubolgonummI niamod epyt nidnopsobmorhTniamodMIL niamodWW niamod ekilFGEriap niamoddnahFEniamod lartnec MREF niamodFGEgnidnibmuiclaC esacilehxobHAEDDAEDniamodstEniamodoemoHfitom ahpla eliretS niamodMAS etis yrotibihni aX rotcaFnoitalugaoCrotpecer enomroh raelcun foDBLniamodYRPS niamod htaeDniamodXP niamod HMretropsnart CBAtaeper editpepocirtarteTniamod FASDGlaR noitaicossa saRylimaf nispodohr rotpecer enarbmemsnartniamodHP esanik enisoryt nietorPniamod esanik nietorPniamod tesI nilubolgonummIBnoisufocno gnidoc nietorp rep sniamoDProtein producing oncofusionsProtein kinase or TF containing oncofusionsCsnoisufocnoFT ro esanik nietorp gnitcaretni fo rebmuNLMALOSEMACHTLOHCDAERMVUPRKIMYHTMBGGGLGPCPTCGTDARPCSULDAOCMCKSCSECACLBDAULCRASCSNHCRKIACSECHILACRBVOCCADATSDAAPSCUCECUCBLDHCKIFound in COSMIC cancer gene censusPK oncofusionsTF oncofusionsPKTF oncofusionsOther protein producing oncofusions Percent of unique interactors in COSMIC canger gene census KRTNGAHWYOPXCBURGERFGEAPSHBAHWYRACADHAPSHPECHTRISALERMCMQAHWYFNRXDDNUJLCDCLBVUREAHWYCRSAPRCDCCDAMSMDMCADHPSLMPACPPPNFSAPRROTMBERCDAMSAPSHDNACCADHPCVCDKRPTATSACRBDNCCBDDABUIMBANCPARAROXBFUPNRNHFigure a0 Domain analysis of protein producing fusions A Intact fulllength domains identified in unique protein producing fusions In total intact domains were detected The protein tyrosine kinase domain was the most prevalent with identifications In addition protein kinase domain was detected with copies each Kinase or TF specific domains included and unique domains respectively copies of kinasespecific domains were seen and of TF specific Kinase domains focused more on the two top kinase domains whereas TF domains were a much more evenly distributed group the top TFspecific domain C2H2 type zinc finger having copies B The number of domains per protein coding oncofusion in the TCGA projects C Most common interactors of protein kinaseTF fusions Yaxis describes numbers of unique protein producing fusions where one or both of the fusion partner WT genes interact with the protein Top right inset Proportion of interactors found in COSMIC cancer gene census is higher in both protein kinase and transcription factor fusions and most common in fusions between protein kinase and transcription factor genesScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cATranscriptional regulationpathwaysRNA Polymerase II transcriptionGeneric transcription pathwaySignaling by Rho GTPasesMAPK family signaling cascadeGPCR signalingSignaling by NotchTGFβ signalingSignal transductionTranscriptional regulationby TP53Hedgehog pathwaySignaling by Receptor tyrosine kinasesIntegrin signalingIntracellular signaling bysecond messengersWnt signalingDeath receptor signalingNode proteins interact withTF fusionsKinase fusionsBCytokine Signaling in Immune systemDevelopmental BiologyAxon guidanceMetabolismPIP3 activates AKT signalingIntracellular signaling by second messengersPTEN RegulationRegulation of PTEN gene transcriptionOxidative Stress Induced SenescenceHDMs demethylate histonesSUMOylation of transcription cofactorsTranscriptional Regulation by E2F6SUMOylation of chromatin anization proteinsTranscriptional regulation by RUNX1Chromatin anizationChromatin modifying enzymesCellular SenescenceSUMO E3 ligases SUMOylate target proteinsSUMOylationInterferon alphabeta signalingEPHEphrin signalingPhospholipid metabolismEPHephrin mediated repulsion of cellsEPHAmediated growth cone collapsePI MetabolismSynthesis of PIPs at the plasma membraneInterferon SignalingMetabolism of lipidsOncogenic MAPK signalingSignaling by moderate kinase activity BRAF mutantsParadoxical activation of RAF signaling by kinase inactive BRAFSignaling downstream of RAS mutantsrRNA modiï¬cation in the nucleus and cytosolRHO GTPases Activate ForminsMajor pathway of rRNA processing in the nucleolus and cytosolrRNA processing in the nucleus and cytosolrRNA processingProcessing of Capped IntronContaining PremRNAmRNA SplicingmRNA Splicing Major PathwayMacroautophagyAutophagyToll Like Receptor TLR3 CascadeMyD88independent TLR4 cascade TRIFTICAM1mediated TLR4 signaling Tolllike Receptor CascadesToll Like Receptor TLR4 CascadeSignaling by TGFbeta family membersSignaling by Nuclear ReceptorsTCF dependent signaling in response to WNTSignaling by WNTSignaling by | 2 |
"Toxicity and tumor markers were not associated with response. Grade 2 or greater skin rash and low pMAPK were associated with improved survival. Conclusions Overall survival was similar in this trial compared to first-line chemotherapy in this unselected patient population. Dose escalation to the development of grade 2 skin rash was associated with improved survival in this patient population. Bioinformatics Bioinformatics bioinformatics bioinfo Bioinformatics 1367-4803 1367-4811 Oxford University Press 25161229 4147902 10.1093/bioinformatics/btu449 btu449 Eccb 2014 Proceedings Papers Committee Original Papers Pathways and Molecular Networks Personalized identification of altered pathways in cancer using accumulated normal tissue data Ahn TaeJin 1 2 3 Lee Eunjin 1 2 Huh Nam 1 * Park Taesung 3 4 * 1Samsung Advanced Institute of Technology130 Suwon-si Gyeonggi-do 443-803 Korea 2Samsung Genome Institute Seoul 135-710 Korea 3Interdisciplinary Program in Bioinformatics and 4Department of Statistics Seoul National University Seoul South Korea *To whom correspondence should be addressed. 01 9 2014 22 8 2014 22 8 2014 30 17 i422 i429 The Author 2014. Published by Oxford University Press. 2014 This is an Open Access distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons./licenses/by-nc/4.0/) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited. For commercial re-use please contact journals.permissionsoup.com Motivation: Identifying altered pathways in an individual is important for understanding disease mechanisms and for the future application of custom therapeutic decisions. Existing pathway analysis techniques are mainly focused on discovering altered pathways between normal and cancer groups and are not suitable for identifying the pathway aberrance that may occur in an individual sample. A simple way to identify individuals pathway aberrance is to compare normal and tumor data from the same individual. However the matched normal data from the same individual are often unavailable in clinical situation. Therefore we suggest a new approach for the personalized identification of altered pathways making special use of accumulated normal data in cases when a patients matched normal data are unavailable. The philosophy behind our method is to quantify the aberrance of an individual sample's pathway by comparing it with accumulated normal samples. We propose and examine personalized extensions of pathway statistics overrepresentation analysis and functional class scoring to generate individualized pathway aberrance score. Results: Collected microarray data of normal tissue of lung and colon mucosa are served as reference to investigate a number of cancer individuals of lung adenocarcinoma (LUAD) and colon cancer respectively. Our method concurrently captures known facts of cancer survival pathways and identifies the pathway aberrances that represent cancer differentiation status and survival. It also provides more improved validation rate of survival-related pathways than when a single cancer sample is interpreted in the context of cancer-only cohort. In addition our method is useful in classifying unknown samples into cancer or normal groups. Particularly we identified amino acid synthesis and interconversion pathway is a good indicator of LUAD (Area Under the Curve (AUC) 0.982 at independent validation). Clinical importance of the method is providing pathway interpretation of single cancer even though its matched normal data are unavailable. Availability and implementation: The method was implemented using the R software available at our Web site: http://bibs.snu.ac.kr/ipas. Contact: tsparkstat.snu.ac.kr or namhuhsamsung.com Supplementary information: Supplementary data are available at Bioinformatics online. 1 INTRODUCTIONCancer arises from normal cells and can evolve to become malignant metastatic and/or resistant to therapy. The analysis of altered pathways in an individual cancer patient may help to understand the disease status and suggest customized anticancer therapies.It is straightforward to compare the molecular profile of an individuals tumor and normal cells to discover molecular aberrances specific to his/her cancer. However it may not be feasible in the current clinical practice environment to perform a metastatic tumor biopsy at the time of treatment resistance in patients with advanced cancer (Dancey et al. 2012). A case study of custom-tailored medicine based on an individuals genome and transcriptome highlights this limitation (Jones et al. 2010). A patients tumor had metastasized to the lung after surgery at the primary site. A biopsy from his lung tumor was analyzed by mutation and transcription profiling; however the patients normal lung tissue was not biopsied. Because there was no matched normal tissue messenger RNA (mRNA) expression in the patients own blood and information collected from various normal tissues were used to identify differentially expressed genes (DEGs). The results of pathway analysis based on DEGs integrated copy number variation and mutation information led the doctor to change the patients drug treatment and the disease was stabilized for 3 months.Although the personalized interpretation of pathways can be demanding most current pathway analyses have been developed to investigate deregulated pathways between two phenotype groups. Khatri et al. (2012) classified these methods into three types: overrepresentation analysis (ORA) functional class scoring (FCS) and a pathway topology (PT)-based approach.ORA approaches typically apply an arbitrary threshold value (e.g. fold change >2 or P < 0.05) on gene expression to assess whether the number of genes beyond threshold are significantly over- or underrepresented in the given pathway. There are two drawbacks to ORA. First it uses only the most significant genes and discards others thus resulting in information loss for marginally significant genes (Breitling et al. 2004). Second it considers only the number of genes and does not consider the magnitude of expression changes leading to information loss regarding the importance of genes (e.g. a gene with a fold change of 2.01 and a gene with a fold change of 4 are considered equally). Unlike ORA FCS methods do not discard genes with an arbitrary threshold but use all available genes which is an improvement over ORA (Tian et al. 2005). PT methods are essentially based on FCS methods with the addition that they consider network topology information. They compensate for the common limitation of ORA and FCS in reporting false-positive gene sets due to sets of overlapping genes. In our we focus on ORA and FCS methods extending and implementing each for personalized pathway analysis.There are two exceptional studies examining individualized pathway analysis (Drier et al. 2013; Vaske et al. 2010). PARADIGM is a tool that infers a pathway status by using known functional structures. The method models the functional structure of pathway as a set of interconnected variables where the variables are omic objects such as DNA mRNA and protein where the interaction between variables describes the functional status of a pathway. PARADIGM may perform better with multiple omics as it uses known functional relationships between a gene or inter-gene DNA and protein. Hence it might not perform well with single layer omic data such as from mRNA microarrays.Drier et al. (2013) proposed a personal pathway deregulation score (PDS) which represents the distance of a single cancer sample from the median of normal samples on the principal curve. To calculate PDS they reduced the dimensions by principal component analysis and found the best principal curve using entire cohort samples containing both normal and/or different stages of cancers. Driers method performs better than PARADIGM in the mRNA only datasets of brain and colon cancers. Calculating PDS requires data dependent preprocessing steps including selecting the number of principal components to be used and filtering out noisy gene data to obtain optimized principal curves. PDS fully uses whole cohort data to interpret an individuals pathway which can be a drawback in that it requires a number of cohort data to extract principal curve to interpret a single patient data. It has a limitation to interpret a single sample such as a patients recurrent tumor that is not accompanied with cohort dataset to extract the principal curve.Our proposed method is based on the comparison of one cancer sample with many accumulated normal samples (we use nRef to refer to the accumulated normal samples) that is different from the previous studies in following sense. The proposed method is suitable to adopt single-layer omics data and expendable to interpret a patient in the context of many published or user-defined pathway gene sets. PARADIGM has less freedom in terms of data and gene sets as it prefers multi-layered omics data and requires predefined functional structure among omics objects. Unlike PDS which extracts the principal curve from entire cohort data our method does not assume an individual sample belongs to a cohort. We introduce using accumulated normal tissue data as a reference. This is a simple and biologically intuitive guideline in such a case to interpret a single sample that lack cohort data.Our method provides a series of analysis steps which consists of four parts: data processing gene-level statistics individualized pathway aberrance score (iPAS) and a significance test. To discover the most feasible method for iPAS we extend existing pathway analysis techniques namely ORA and FCS to properly reflect the nature of testing one cancer to many normal samples.To demonstrate that iPAS captures biologically and clinically relevant information in a sensible valid and useful manner we apply it to samples of lung and colon adenocarcinoma. We show that our representation generates clinically relevant stratifications and outcome predictors which would not have been achieved when the same data are analyzed by the conventional method that does not use accumulated normal data.Our empirical study suggests two different strategies depending on the biological question that iPAS is focused on. In the case of cancer diagnosis a method that uses the inter-gene correlation structure of the accumulated normal samples performs best. In the case of cancer prognosis a simple averaging of all member genes standardized gene expression values performs best.2 METHODS AND MATERIALS2.1 Gene expression dataWe built nRef by the manual curation of data obtained from NCBI GEO (Barrett et al. 2012). Microarray data of adjacent normal tissues obtained from patients undergoing surgery were selected to serve as the nRef. Data from biopsied samples primary cultures of normal tissues and post-mortem donors were not included in the nRef. We collected 120 nRef for lung 60 from GSE19804 (Lu et al. 2011) 27 from GSE7670 (Su et al. 2007) and 33 from GSE10072 (Landi et al. 2008). Samples came from individuals with variable smoking histories and different ethnic backgrounds. We collected 101 nRef for colon concentrating on normal mucosa tissue samples from six datasets available at GEO. To evaluate the effectiveness of our method in survival analysis we used Beers data of 442 lung adenocarcinomas (LUADs) (Beer et al. 2002) to discover survival-related pathways and validated the associations of 61 LUAD samples of GSE8894 (Lee et al. 2008). The pathway based identification of LUAD were tested on 120 cancers and 120 normal samples of GSE19804 GSE7670 and GSE10071. Further validation was conducted with 48 cancers and 35 normal samples collected from GSE19188 (Hou et al. 2010) and GSE31547. For patient stratification by colon cancer differentiation status we used 566 microarrays of GSE39582 (Marisa et al. 2013) which provided in a separate manner 443 for discovery 123 for validation. GSE17536 (Smith et al. 2010) was also used for validation.2.2 Pathway dataInformation from gene sets representing biological pathways were obtained from REACTOME (Croft et al. 2011) which are also provided in the Molecular Signature Database (Subramanian et al. 2005). Pathways with small number of genes are more easily understood by human experts. We decided to filter out pathways of which gene set size is >97. The cutoff covers at least 80% of contents of each public pathway resources. Of 674 pathways in REACTOME 583 pathways (86.7%) remained after filtering by the gene set size.2.3 Individualized analysis using the nRefThe aim of our approach is to identify altered pathways in an individual by making use of the nRef. A schematic diagram of our method of individualized pathway analysis is described in Figure 1 and the following sections describe each step. Fig. 1.Schematic description of individualized pathway analysis using accumulated normal data (nRef). An individuals tumor data are normalized with the nRef. Gene expression is standardized by mean and SD of the nRef. The iPAS is calculated from standardized gene expression values in the pathway. Null distribution calculated from the nRef provides significance2.3.1 Data preprocessing and gene-level statisticsExpression level was defined by using the robust multichip average (Irizarry et al. 2003). For datasets using different microarrays only those with probes in common from Affymetrix U133A to Affymetrix U133Plus 2.0 were used for further analysis. For individual tumor cases we performed quantile normalization (Bolstad et al. 2003) after combining the single tumor microarray with all nRef samples. In cases of genes with multiple probes gene expression level was summarized by averaging probe-level expression. Individual tumor sample gene expression was standardized using the mean and standard deviation of the reference.2.3.2 Pathway-level statistics and significance testWe introduce five methods as candidates for iPAS. Each method is a modification of existing pathway analysis techniques enabling us to test an individual tumor samples pathway aberrance by using the nRef. A summary is provided in Table 1. Table 1.Modification of existing pathway analysis methods for iPASNote. Significance can be obtained against the null distribution generated from normal samples. All the collected normal samples for the nRef are one by one compared with the nRef to yield statistics of the null distribution. A statistic from a single cancer case is compared with this null distribution to yield P-value.Average ZStandardizing the gene expression by mean and standard deviation from datasets is often used in microarray analysis. A vector Z = (z1 z2 ¦ zn) denotes the expression status of a pathway where zi symbolizes the standardized expression value of i-th gene where the number of genes belonging to the pathway is n. In typical settings standardization is performed using the mean and SD of a given dataset mostly the cancer-only cohort data thus y¯/n indicates how much the given samples overall pathway gene expression deviates from the center of the cancer samples. We made the simple modification Z = (z1 z2 ¦ zn) where zi is derived from mean and SD of the nRef. In this case y'¯/n gives the samples deviation from the nRef. We believe this modification is biologically valid because every cancer starts its malignancy from normal cell. Thus the clinical characteristics of a single cancer can be captured by measuring the difference of it against common characteristic of normal cells which is represented by the nRef in our study.Fisher exact testWe generated a 2 à 2 contingency table for a given pathway (S) and DEGs (D) for the test. For individualized interpretation we define D by the ranking of z-value which is standardized gene expression for the mean and SD of the nRef. For each individual sample 5% (highest 2.5% and lowest 2.5%) of the total genes are defined as D. We applied a two-tailed test to detect alteration of pathways due to enrichment or depletion of differential genes. The result of this statistic can be interpreted as how many DEGs are enriched in the given pathway where the expression difference refers to how much a patients gene expression deviates from the nRef.Gene set enrichment analysisWe adopted the original version of gene set enrichment analysis (GSEA) proposed by Subramanian et al. (2005). Typically inputs for GSEA are generated by testing whole cohort samples using phenotype label; t-statistic correlation coefficients and fold changes are usually used. In the personalized analysis setting we use the z-value as an input for the GSEA algorithm which is standardized gene expression by mean and SD of the nRef. The GSEA output enrichment score reflects the degree to which a gene set in the pathway is overrepresented at the extremes (low or high) of the entire ranked list of z-values from a single patient.Non-parametric quadratic testGene expression in a pathway of a tumor sample is represented by vector Z = (z1 z2 ¦ zn) where zi is standardized expression level of i-th gene by mean and SD of the nRef where n is the number of genes belonged to the pathway. A pathway characteristic of an individual patients pathway can be represented by the averaged Euclidean distance (ZTZ/n). This gives the distance of a single patient from the center of the nRef due to the square of standardized expression difference and thus does not reflect increased or decreased expression only the extent of the expression difference. Genes in the pathway are usually functionally correlated; therefore use of the correlation structure of the normal samples may increase sensitivity enough to capture the aberrance of a single cancer case. We also consider the averaged Mahalanobis (ZTSZ/n) distance which uses the covariance matrix calculated from the nRef. This value describes the statistical distance from the center of normal samples taking into account correlation among normal samples. The covariance matrix S is calculated for each pathway from the nRef.3 RESULTS3.1 Pathway-based identification and validation of cancer survivalTo assess whether our method can sensitively detect pathway aberrances that are associated with a patients clinical outcome a known survival pathway that showed strong association with patient survival from Beers data was tested. Bryant et al. (2010) reported that the cell cycle stimulatory pathway of 51 genes is significantly associated with patient survival (Cox proportional hazards model P = 0.000113). In that study pathway gene expression was represented as an average of z-values where the z-value indicates the standardized expression level by the mean and SD of all cancer samples. The high-risk group was defined as those in which pathway expressions were >0 and the pathway showed poor prognostic outcome. The association was significant with or without adjusted clinical covariates and thus the pathway alone is a strong indicator of cancer prognosis. This finding was also validated in the Japanese LUAD cohort (n = 87 survival data are not provided to public) in Bryants study. As studies have shown a clear association between the cell cycle pathway and cancer in terms of driving cancer proliferation we considered this pathway as a pathway that should be detected. All of the methods proposed as candidates for iPAS showed significant associations of the cell cycle stimulatory pathway from Beers data (Table 2). The same pathway analyzed using GSE8894 (n = 61) data yielded significant associations in all proposed methods with the marginal exception of Mahalanobis (P = 0.0549). Table 2.Survival analysis of cell cycle stimulatory pathway reported by Bryant et al. (2010)DatasetPathway statisticsCoefficientP-valueBeer (N = 432) Bryant et al. Overall survivalAverage Za0.370.00011Beer (N = 442) Overall survivalAverage Zb0.620.00003Fisher0.500.00068GSEA0.650.00001Euclidean0.650.00001Mahalanobis0.670.00001GSE8894 (N = 61) Recurrent free survivalAverage Zb0.900.01163Fisher0.910.01076GSEA0.780.02899Euclidean0.870.01544Mahalanobis0.680.05485aDerived from mean and SD of all cancer samples in the dataset bDerived by mean and SD of the nRef.Prognostic gene expression signatures for Stages II and III colon cancers have been reported in seven papers yielding 207 genes in total (Bandres et al. 2007; Barrier et al.2006 2007; Eschrich et al. 2005; Kopetz and Abbruzzese 2009; Lin et al. 2007; Wang et al. 2004). The genes are enriched in 32 REACTOME pathways (False Discovery Rate (FDR) < 0.05 pathway size < 96). We assumed the 32 pathways were valid as ground truth to be identified and analyzed in the colon cancer dataset GSE39585 (Stages II and III were only considered). Average Z provided best performer (sensitivity = 0.88) with 28 pathways deemed as significant. GSEA Fisher Euclidean and Mahalanobis gave the following values0.780.66 0.06 and 0.03 respectively.These results satisfied us that our approach captures the fundamental knowledge of cancer thus it is reasonably considered as iPAS.To investigate which of the candidates for iPAS most robustly reflect phenotype association we evaluated the proposed methods by determining whether survival-associated pathways are validated in datasets never used for discovery using LUAD and colon cancer [LUAD: Beers set n = 442 for discovery GSE8894 (n = 61) GSE3141 (n = 58) for validation; colon cancer: GSE39582d (n = 443) for discovery GSE39582v (n = 123) and GSE17536 (n = 109) for validation logrank P < 0.05 comparing tumors in the top 50th percentile of aberrance scores to those in the bottom 50th percentile]. Validation rates varied depending on the dataset and these were possibly affected by the small sample size compared with that of the discovery set. In these cases we were not able to determine a superior method that outperformed the others. Average Z gave the highest validation rate in three of four dataset with validation rates of GSE8894 (43.6% 92/211) GSE3141 (13.3% 28/211) and GSE17536 (10.7% 24/224). When validation rates from four datasets are averaged Average Z gave the highest validation rate (21.9% Fig. 2 blue bars). Pathways validated as significantly associated with patient survival for each cancer are listed in the Supplementary Materials (Supplementary Tables S1 and S2). Fig. 2.Averaged validation rate of discovered survival-related pathway at four datasets. Proposed approach using nRef (blue) versus conventional approach that standardizes individual sample by mean and SD of entire cohort dataset (red)We also investigated the validation rate of iPAS candidates under the conditions where the same data are not standardized by the nRef but instead standardized by the mean and SD of the cohort dataset which consists of only cancers (Fig. 2 red bars). It is noteworthy that use of the nRef increased the validation rate for every iPAS candidate investigated. This implies that the strategy of using accumulated normal samples as a reference is beneficial in terms of pathway-based survival analysis.3.2 Identification of clinical importanceCluster analysis of using Average Z as the iPAS method on Beers data identified 12 pathway clusters (denoted by 1?12 in Fig. 3) and 3 sample clusters (S2?S4; S1 is from the nRef; Fig. 3). Sample clusters S2 and S4 represent well the differentiation status of LUAD (Fisher exact test P < 4.65 à 10?15). Well-differentiated adenocarcinoma resembles the normal glandular structure; therefore it is a reasonable result that cluster S2 is close to the nRef. The survival outcome of S2 and S4 are significantly different (P < 0.0028) and this assures us that unbiased clustering-based iPAS has enough sensitivity to capture clinically important associations. This finding is concordant with prior knowledge that well-differentiated LUAD patients are likely to have better prognosis (Barletta et al. 2010). Pathway cluster P9 is distinguished as commonly upregulated in tumor samples. The pathways are transfer RNA aminoacylation amino acid or purine synthesis DNA elongation and the extension of telomeres. Fig. 3.Clustered iPAS of LUAD dataset. Pathways (n = 583) and samples (n = 442) are clustered according to iPAS. Normal samples are clustered at left (S1). Tumors (S2?S4) deviate from normal in both up- and downregulated directions (darker red and blue respectively). Sample clusters are well-representing histopathological differentiation status (S2: for well-differentiated LUAD P < 4.65 à 10?15) and overall survivalUnbiased pathway-based clustering of colon cancer data also captures clinically important associations by revealing sample clusters that are survival related (S2 and S3 P = 0.0037 Supplementary Fig. S1). It is important to note that iPAS is not only sensitive enough to identify clinically meaningful substructure of patients but also reveals common characteristics of a cancer at the same time. For example pathways commonly up- or downregulated in all cancer samples for example P9 or P2 would have not been discovered if the analysis had been performed by a conventional approach that does not make use of nRef (Supplementary Fig. S2).3.3 Pathway-based identification of cancerCancer develops unique mechanisms for malignancy. Therefore it is reasonable to believe that identifying the unique molecular aberrances of cancer will aid in cancer diagnosis. Our empirical study of iPAS-based clustering of LUAD revealed several pathways commonly up- or downregulated in all of the cancer samples. Further analysis was performed to determine whether iPAS could be successfully used in the accurate identification of cancer. We tested this in a simple unsupervised way by judging whether an unknown sample is significantly different against the nRef as a tumor if not as normal. We performed a 5-fold cross-validation one hundred times with the LUAD dataset which consisted of 120 cancers and 120 normal samples. Microarray data from the normal samples was randomly divided into five groups and four of the five served as the reference group. The remaining group was used as the true normal set for the test of pathway-based identification of cancer. To build true cancer set for the test the same number of cancer sample was randomly picked. We considered 583 pathways in REACTOME giving 293 500 (583 pathways à 5-fold à 100 repeats) AUCs and accuracy values. We averaged AUCs and accuracies from the five candidate methods for iPAS and used this as a representative AUC and accuracy of a given pathway.By ranking the pathways by AUC top pathways that marked averagely high performance by all iPAS candidates are listed (Supplementary Table S3). The amino acid synthesis and interconversion and transamination pathway showed the highest classification performance. Unsurprisingly this pathway was one of the commonly upregulated pathways in the analysis of the Beers data (Fig. 3 pathway cluster P9). Among the tested iPAS candidates for this pathway Mahalanobis yielded the highest AUC (0.980) while Average Z gave 0.936 and Fishers exact test gave the lowest value (0.914). The standardized gene expression pattern for this pathway differed between tumor and normal. Many of the genes deviated from mean of the nRef by more than two orders of sigma contributing to its best performance out of all iPAS candidate methods including ORA method like Fishers exact test (Fig. 4a). Fig. 4.(a) Expression pattern of genes in the pathway. Each line represents sample. (gray: normal red: tumor). Dashed line represents expression value deviated 1.96? from the mean expression value of normal tissues. (b). Performance of classification of cancer by amino acid synthesis and interconversion and transamination. AUC of 0.980 has marked in discovery set (95% confidence interval provided as error bar) independent validation set results AUC of 0.982 (Validation 1: normal samples in validation set served as reference) and 0.982 (Validation 2: normal samples in discovery set served as reference)We also analyzed the influence of using the subset of normal samples as nRef. We compared the pathway-based cancer identification results using the full set of normal samples (n = 120) against 100 different runs using 75% (n = 90) 50% (n = 60) of randomly chosen normal samples. Among the pathways that marked averagely high performance in the identification of cancer the best and the second best pathways are considered amino acid synthesis and interconversion and transamination and unwind of DNA respectively. The result shows little loss of performance even though only a half of normal samples were used for the test (Fig. 5a and b). Fig. 5.Performance of pathway-based identification of cancer (AUC) when only a subset of normal samples are served as nRef. (a) amino acid synthesis and interconversion and transamination (b) unwind of DNA3.4 Validation of the discovered pathwayThe amino acid synthesis and interconversion and transamination pathway consists of 17 genes involved in three major reactions as it is described at REACTOME. The pathways are responsible for (i) synthesis of three amino acids (aspartate asparagine glutamate) (ii) the synthesis of glucose under fasting conditions by using carbon atoms from these four amino acids and (iii) conversion of amino acids to their corresponding alpha-keto acids coupled to their conversion to glutamate which is the first step in the catabolism of most amino acids.This function makes sense in terms of the glutamine addiction of cancer cells. The nutrients glucose and glutamine are specifically required by cancer cells as metabolites for growth and for production of adenosine triphosphate (Munoz-Pinedo et al. 2012). Myc and p53 have been revealed to be associated with this addiction by upregulating glutamine synthesis in cancer cells. Thus our finding is in accordance with prior knowledge regarding the upregulation of glutamine synthetase.We further validated our findings with an independent set that were not used in the discovery set. We collected two more LUAD gene expression datasets with normal data at GEO (GSE19188 GSE31547). Aggregated datasets of 48 microarrays from tumor tissues and 35 microarrays from normal tissues were used for independent validation. The pathway was also altered in a cancer-specific way in a validation set yielding an AUC of 0.982 by Mahalanobis-based iPAS (Fig. 4b Validation 1). We also assessed the same validation set in a different manner by using the nRef from the discovery set. Normal sample microarrays from the discovery sets (GSE10082 GSE7670 GSE10072) served as the nRef to classify samples in the independent validation set. The resulting AUC was 0.982 by the Mahalanobis method (Fig. 4b Validation 2).In our experiments using LUAD samples the Mahalanobis distance which used a pre-calculated covariance matrix from the nRef gave the best performance. The usage of covariance matrix empowers Mahalanobis to consider a cancer sample as an outlier delivering higher accuracy in terms of pathway-based identification of cancer than other methods. One caution of using Mahalanobis method is that it requires a large number of normal samples to guarantee the estimation of covariance matrix. For a small sample size a structured covariance matrix would be desirable to avoid the estimation issue.The biological role of this identified pathway is to supply nutrients and energy to cancer cells. " | 1 |
" mirnas regulate a multitude of cellular processes and their aberrant regulation is linked to humancancer however the role of mir4255p in lung cancer lca is still largely unclear here we explored the role ofmir4255p during lca tumorigenesismethods cell proliferation was evaluated by cell counting kit8 and colony formation assay western blot and realtime pcr were accordingly used to detect the relevant proteins mirna and gene expression luciferase reporterassays were used to illustrate the interaction between mir4255p and ptenresults we demonstrate that mir4255p is overexpressed in lca tissue and enhances the proliferative and colonyformation capacity of the lca cell lines a549 and ncih1299 through predictive binding assays pten wasidentified as a direct gene target and its exogenous expression inhibited the procancer effects of mir4255pthrough its ability to downregulate pten mir4255p activated the pi3kakt axis we conclude that mir4255p promotes lca tumorigenesis through ptenpi3kakt signalingkeywords mir4255p lung cancer pten pi3kakt signaling pathways lung cancer lca is a leading cause of cancer relatedmortality across the globe lca is prevalent in males and asymptomatic during early disease stages as manyas in every cases are at an advanced stage iii or ivwhen diagnosed and the 5year survival rates remainlow particularly for those with metastatic lca improved lca therapeutics is thus urgently requiredmicrornas mirnas regulate many cell processesincluding differentiation metabolism and tumorigenesis[] emerging evidence suggests that mirnas are keyplayers during lca tumorigenesis [] the aberrantexpression of mir4255p is linked to hepatocellular carcinoma hcc gastric cancer gca and colorectal correspondence kjhhev163comcardiothoracic surgery the fourth affiliated hospital zhejiang universityschool of medicine shangchen road no1 of yiwu zhejiang chinacancer crc [] here we report the upregulationof mir4255p in lca and highlight its contribution tolca development we further identify pten as a novelmir4255p target that is inhibited in lca to promoteptenpi3kakt signalingmethodspatient specimenslca samples n and adjacent healthy tissue at least cm from the resection margin were collected from thefourth affiliated hospital zhejiang university school ofmedicine the study was fully supported by the institutional review board of the fourth affiliated hospitalzhejiang university school of medicine no2015009all participants provided consent for sample analysisand anything about their identities will not be includedin the data 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 0czhou bmc pulmonary medicine page of lca cell lines cell culture and cell transfectionhuman lung cancer cell lines a549 ncih1299 ncih460 hcc827 and normal human lung epithelial cellline beas2b were obtained from the cell bank of thechinese academy of sciences shanghai china a549 hcc827 cells were cultured in dmem plus fbsand penstrep ncih1299 ncih460 cells weregrown in rpmi1640 plus fbs at °c in co2beas2bs were cultured in clonetics¢ mediathe mir4255p mimics and negative control mirnanc were chemically synthesized by shanghai genepharma co ltd songjiang shanghai china lipofectamine invitrogen eugene or usa was usedfor transfection according to the manufacturers protocol pi3k activity was assayed as previously described pi3k inhibitor ly294002 was obtained from abcamto analyze the effects of mir4255p on pi3kakt indicated lca cells were cultured in the presence or absence ofthe workingconcentrations of ly294002 were μm for experiments with ly294002 treatmentsindicated lca cellswere pretreated with ly294002 for h prior to exposure to proteasome inhibitorsthe drugs for h at °ccell growth assayslca viability was assessed using cell counting kit8from shanghai haling biotechnology co ltd shanghai china as per the manufacturers protocols brieflyafter incubating the transfected cells for one full daythey were collected after trypsinization and seeded cellswell into 96well plates ten microliters ofcck8 solution were added per well and kept for h at °c the absorbance of the mixture was estimated in amicroplate readerinchercules usa at nmfrom biorad laboratoriescolony formation assaythe colony formation assays were performed as previous each group of treated cells per well wasseeded into cm culture dish and cultured for weeksfinally colonies were stained using crystal violet andthe number of cell colonies was countedqrtpcr analysistotal rna was isolated by trizol® reagent from invitrogen thermo fisher scientific inc and a nanodropnanodrop technologies thermo fisher scientific incwas used to estimate its quality and concentration theexpression of mir4255p was done by reverse transcription using the mirx¢ mirna firststrand synthesis kitfrom takara biotechnology co ltd dalian chinaand quantitative evaluation of the synthesized cdnawas done by quantitative pcr rtqpcr using themirx¢ mirna qrtpcr tb green® kit from takaraforwardbiotechnology as an endogenous control the small nuclear rna u6 normalized the expression of mir4255pthe δδcq system was used to evaluate all genes expressions and the primer sequences were shown as fol² ggggagttaglows mir4255pgattaggtc3² reverse ² tgcgtgtcgtggagtc3² u6 forward ²ctcgct tcggcagcaca² reverse ²aac gct tca cga att tgc gt3²pten forward ² tggattcgacttagacttgacc ² reverse ² aggatattgtgcaactctgcaa² gapdh forward ²catcaccatcttccaggagcg3² reverse tgaccttgcccacagcctt3²to getdual luciferase assaysthe design and synthesis of pten fragments containingbinding sites for wt wildtype and mut mutant onmir4255p was done by shanghai genepharma thesewere cloned into the target expression vector pmirglo dualluciferase from promega corporation wiusathe reporter plasmids wtpten andmutpten one night prior to transfection seeding ofcells confluence was done in plates with wells transfection of these cells was done with reporterplasmids harboring wt or mut in the presence ofmirnc or mir4255p mimic post h of transfectionluciferase activity of the cells was estimated as per instructions using the dualluciferase reporter assay system from promega corporation promega fitchburgwi usa the data normalization was done by the activity of renilla luciferasewbcell lysates ripa lysates were resolved on sds pageand transferred to pvdf membranes membraneswere blocked for h in milk plus tbstincubated with primary antibodies against pten dilution cat no ab170941 abcam pi3k dilution cat no ab32089 abcam pakt dilution cat no ab81283 abcam akt dilution cat no ab32505 abcam pakt dilution cat no ab81283 abcam actin dilution cat no ab8226 abcam at °c overnights andlabeled with hrpconjugated secondarys for h atroom temperature bands werevisualized usingchemiluminescent hrp substrate and analyzed usingimage lab tm softwarestatistical analysesdata analysis was performed using spss treatmentgroups were compared using a oneway anova pvalues were taken as significant experiments wereperformed on at least three occasions data representthe mean ± sd 0czhou bmc pulmonary medicine page of resultsmir4255p is upregulated in lcawe compared mir4255p levels in paired lcaand normal lung tissue samples by qrtpcr analysismir4255p was upregulatedspecimensfig 1a and expressed to high levels in a549 ncih1299 ncih460 and hcc827 cells compared tonormal human lung epithelial cellline beas2bfig 1b consistent with previous findings in othercancer types previous results indicated that mir4255p is upregulated in lcain lcamir4255p enhances proliferation and inhibits apoptosisin lca cellsto dissect the role of mir4255p in lca its expressionwas manipulated using mir4255p mimics figure 2acshows that mir4255p upregulation enhanced cell survival meanwhile enhanced cell colony formation abilityfig 2d and e taken together above results indicatedmir4255p is thus an oncogene in lca cellsmir4255p targets ptenfrom targetscan pten was identified as a predictedmir4255p target fig 3a in pten ²utr reporterassays mir4255p suppressed wt pten expressionfig 3bc but had little effect on mutated pten ²utr fragments fig 3bc the levels of pten werelower in cells transfected with mir4255p mimics fig3de mir4255p also negatively related pten mrnalevels in lca tissue p r2 fig 3f thesedata implicate pten as a cellular target of mir4255pmir4255p promotes lca via ptento define whether mir4255p regulate pten in lcawe firstly overexpression pten in lca fig 4a wbanalysis demonstrated that mir4255p reduces ptenlevels which could be recovered by exogenous expression fig 4b cell viability assays showed that mir4255p enhances lca proliferation which could be reversedby pten transfection fig 4cd suggesting mir4255pmediates its activities via pten this indicates that mir4255p targets pten to mediate its protumor effectsmir4255p activates ptenpi3kakt signalingit has been reported ptenpi3kakt signaling wasclosely related to cell proliferation and apoptosis [] next we explored the effect of mirna4255p onptenpi3kakt signaling as shown in fig 5aincomparison to nc groups pten was down regulated inresponse to mir4255p mimics whilst pi3k and paktlevels increased in addition nsclc cells were treatedwith the pi3kakt inhibitor ly294002 or ly294002 mir4255p mimics mimic fig 5b kinase activity assays further showed that pi3k activity in nsclc transfected with ly294002 was significantly lower than thattransfected with mimic control p and pi3k activity in nsclc cells transfected with both ly294002and mir4255p mimic was significantly higher than thattransfected with ly294002 p take together thisimplicates ptenpi3kakt signaling in the protumorigenic effects of mir4255pdiscussionthe poor prognosis of lca highlights the need for urgent therapeutic strategies mirnas are novel targets forfig mir4255p in highly expressed in lca a qrtpcr of mir4255p b qrtpcr of a549 ncih1299 ncih460 hcc827 and beas2b cellsdata mean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca a mir4255p expression in a549 and ncih1299 cells b c cck8 assays in mir4255p mimic transfectedcells d e colonyforming assay in mir4255p mimic transfected cells the raw data of all colony formation experiments was listed insupplemental table data mean ± sd p p p fig mir4255p targets pten in lca a predicted binding of mir4255p and pten b c relative luciferase activity of ptenwt ptenmut inlca cells expressing mir4255p mimic d pten mrna levels are significantly lower after transfection with mir4255p mimic e pten expressionassessed by wb fulllength blotsgels are presented in supplementary figure f mir4255p and pten negatively correlate in lca tissue datamean ± sd p p p 0czhou bmc pulmonary medicine page of fig mir4255p promotes lca growth by targeting pten a qrtpcr of pten in indicated lca cell lines b wb analysis of pten expression fulllength blotsgels are presented in supplementary figure c d cck8 analysis of cell viability in the indicated cell lines with empty vector mir4255p mimic andor pten in a549ncih1299 cells data mean ± sd p p p fig effects of mir4255p on ptenpi3kakt a wb analysis of pten pi3k pakt and akt in lca cells fulllength blotsgels are presented insupplementary figure b the pi3k kinase activity was determined in nsclc cells transfected with ly294002 was significantly lower than thattransfected with mimic control and the pi3k kinase activity in nsclc transfected with both ly294002 and mir4255p mimic was significantlyhigher than that transfected with ly294002 data mean ± sd p p p 0czhou bmc pulmonary medicine page of cancer therapies and their dysregulation occurs in lcatissue [] duan showed that mir203 bindsto zeb2 to suppress emt yuan showed thatmir30a inhibits eya2 migration and invasion andli showed that mir1304 inhibits lca cell divisionthrough heme oxygenase1 the cellular roles ofmir4255p in lca are poorly understoodin thepresent work we further explore the underlying mechanisms of mir4255pinduced lca cell progressionin the present study we confirmed that mir4255p isoverexpressed in lca cell lines and tissues implicating arole in lca tumorigenesis upregulating mir4255plevels enhanced the cell survival and colony formationability of lca cells in vitro implicating it as a novel lcaoncogene in the mechanism using the algorithms targetscan website tools we identified pten as the potential target of mir4255p furthermore we performedluciferase reporter assays and the results showed thatmir4255p may directly target pten3utr the resultof qrtpcr and western blot also confirmed that overexpression of mir4255p could suppress the expressionlevel of pten all the above suggested that pten was apotentialtarget of mir4255p moreovermir4255p also negatively related pten mrna levelsin lca tissue rescue experiment indicated that exogenous pten expression inhibited the procancer effects ofmir4255p pten was downregulated in lca tissuepten is a tumor suppressor with wellcharacterizedphosphatase activity pi3kakt promotes cell cycleprogression inhibits apoptosis and is known to be overactive in a multitude of human cancers [ ] ptencan suppress pi3kakt signaling and thus displays anticancer effects upon assessment of the molecularmechanisms of mir4255p in lca cells its procancereffects were found to be mediated through manipulationof the ptenpi3kakt signaling axisfunctionalin we highlight mir4255p as an oncogenein lca that promotes an oncogene phenotype by inhibiting pten these findings enhance our knowledge of therole of mir4255p and reveal new therapeutic strategiesfor the diagnosis and treatment of lca angiogenesisand metastasis biological experiments will clarify thefunctions and roles of mir4255p in lcasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12890020012610additional file supplement figure s1 uncropped images of blotsand gels related to fig supplement figure s2 uncropped imagesof blots and gels related to fig supplement figure s3 uncroppedimages of blots and gels related to fig table s1 the raw data of allcolony formation experimentsabbreviationslca lung cancer pten gene of phosphate and tension homology deletedon chromsome ten pi3k phosphatidylinositol 3kinase nsclc nonsmallcell lung cancer gca gastric cancer crc colorectal cancerhcc hepatocellular carcinoma emt epithelialmesenchymal transitionacknowledgementsnot applicableauthors contributionsjsz zsy syc and qf did patient recruitment and assessment andperformed the experiments jhy and cjh were responsible for statisticalanalysis jsz zsy and syc interpreted the data and wrote the manuscriptall authors contributed to the subsequent drafts and approved the finalversionfundingthis research was funded by zhejiang provincial natural science foundationof chinaexploration program lq19h020010 core talent program ofdepartment of health of zhejiang province 2013rca018 zhejiangprovincial natural science foundation of chinageneral programly15ho2004 the funders had no role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participateethics approval for this study was obtained from the fourth affiliatedhospital zhejiang university school of medicine no2015009 all patientsgave written informed consent for participation in the studyconsent for publicationnot applicablecompeting interestsall authors declare no conflicts of interestreceived march accepted august referenceschen w zheng r zuo t zeng h zhang s he j national cancer incidenceand mortality in china chin j cancer res aberle dr berg cd black wc the national lung screening trialoverview and study design radiology ni js zheng h huang zp microrna1973p acts as a prognosticmarker and inhibits cell invasion in hepatocellular carcinoma oncol lettzhang tj wang yx yang dq downregulation of mir186 correlateswith poor survival in de novo acute myeloid leukemia clin lab wang b teng y liu q microrna153 regulates nrf2 expression and isassociated with breast carcinogenesis clin lab lin h huang zp liu j mir4943p promotes pi3kakt pathwayhyperactivation and human hepatocellular carcinoma progression bytargeting pten sci rep yang j li j le y zhou c zhang s gong z pfklmir128 axis regulatesglycolysis by inhibiting akt phosphorylation and predicts poor survival inlung cancer am j cancer res wang r chen x xu t mir326 regulates cell proliferation andmigration in lung cancer by targeting phox2a and is regulated by hotairam j cancer res qin q wei f zhang j wang x li b mir134 inhibits nonsmall cell lungcancer growth by targeting the epidermal growth factor receptor j cellmol med hu h xu z li c mir145 and mir203 represses tgfbetainducedepithelialmesenchymal transition and invasion by inhibiting smad3 in nonsmall cell lung cancer cells lung cancer 0czhou bmc pulmonary medicine page of fang f song t zhang t cui y zhang g xiong q mir4255p promotesinvasion and metastasis of hepatocellular carcinoma cells through scaimediated dysregulation of multiple signaling pathways oncotarget cristobal i madozgurpide j rojo f garciafoncillas j potential therapeuticvalue of mir4255p in metastatic colorectal cancer j cell mol med zhang z wen m guo j clinical value of mir4255p detection and itsassociation with cell proliferation and apoptosis of gastric cancer pathol respract huang z xing s liu m deng w wang y huang z huang y huang x wuc guo x pan x jiang j feng f li t mir26a5p enhances cellsproliferation invasion and apoptosis resistance of fibroblastlikesynoviocytes in rheumatoid arthritis by regulating ptenpi3kakt pathwaybiosci rep meng j liu gj song jy chen l wang ah gao xx wang zj preliminaryresults indicate resveratrol affects proliferation and apoptosis of leukemiacells by regulating ptenpi3kakt pathway eur rev med pharmacol sciliu hy zhang yy zhu bl feng fz yan h zhang hy zhou b mir21regulates the proliferation and apoptosis of ovarian cancer cells throughptenpi3kakt eur rev med pharmacol sci yu g wang c song x liu s zhang y fan l yang y huang y song jformaldehyde induces the apoptosis of bmcs of balbc mice via the ptenpi3kakt signal transduction pathway mol med rep sun xh wang x zhang y hui j exosomes of bonemarrow stromal cellsinhibit cardiomyocyte apoptosis under ischemic and hypoxic conditions viamir4865p targeting the ptenpi3kakt signaling pathway thromb res mou x liu s mir485 inhibits metastasis and emt of lung adenocarcinomaby targeting flot2 biochem biophys res commun su tj ku wh chen hy oncogenic mir137 contributes to cisplatinresistance via repressing casp3 in lung adenocarcinoma am j cancer resliu y wang f xu p mir590 accelerates lung adenocarcinoma migrationand invasion through directly suppressing functional target olfm4 biomedpharmacother yang l luo p song q fei x dnmt1mir200agolm1 signaling pathwayregulates lung adenocarcinoma cells proliferation biomed pharmacother duan x fu z gao l direct interaction between mir203 and zeb2suppresses epithelialmesenchymal transition signaling and reduces lungadenocarcinoma chemoresistance acta biochim biophys sin shanghai yuan y zheng s li q overexpression of mir30a in lungadenocarcinoma a549 cell line inhibits migration and invasion via targetingeya2 acta biochim biophys sin shanghai li cg pu mf li cz microrna1304 suppresses human nonsmall celllung cancer cell growth in vitro by targeting heme oxygenase1 actapharmacol sin li dm sun h ptenmmac1tep1 suppresses the tumorigenicity andinduces g1 cell cycle arrest in human glioblastoma cells proc natl acad sciu s a bellacosa a chan to ahmed nn akt activation by growth factors is amultiplestep process the role of the ph domain oncogene li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep qin y huo z song x chen x tian x wang x mir106a regulates cellproliferation and apoptosis of colon cancer cells through targeting theptenpi3kakt signaling pathway oncol lett publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
poorest prognoses of all malignancies with little improvement in clinical outcome over the past years Pancreatic ductal adenocarcinoma is responsible for the vast majority of pancreatic cancer cases and is characterised by the presence of a dense stroma that impacts therapeutic efficacy and drives protumorigenic programs More specifically the inflammatory nature of the tumour microenvironment is thoughtto underlie the loss of antitumour immunity and development of resistance to current treatments Inflammatory pathways are largely mediated by the expression of and signallingthrough cytokines chemokines and other cellular messengers In recent years there hasbeen much attention focused on dual targeting of cancer cells and the tumour microenvironment Here we review our current understanding of the role of IL6 and the broader IL6cytokine family in pancreatic cancer including their contribution to pancreatic inflammationand various roles in pancreatic cancer pathogenesis We also summarise potential opportunities for therapeutic targeting of these pathways as an avenue towards combating poorpatient outcomesIntroductionPancreatic 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 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 oncogenichit 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 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 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 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 signiï¬cant 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 cells 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 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 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 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 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 dysfunctionea | 2 |
"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 cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant 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 hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l 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 ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj 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 cprds 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 ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu | 0 |
despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has conï¬rmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modiï¬cations nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these ï¬ndingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and eï¬cient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our ï¬ndings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratiï¬ed according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as proï¬les for each hcc patient as previouslydescribed li y zhu each as eventwas quantiï¬ed using the percent spliced in psi value rangingfrom to a commonly used method to reï¬ect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict ï¬lters average psi ¥ percentage of samples withpsi ¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentiï¬cation of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ¤ and log2fc ¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterproï¬ler package version yu the parent genes of deas events were importedinto the string database and used to determine proteinprotein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identiï¬ed by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artiï¬cial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplanmeier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii à lito divide the patients into the high or lowrisk group kaplanmeier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coeï¬cients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at ¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspeciï¬c reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin ampliï¬ed usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by ï¬ow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief à cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was ï¬lled with a culturemedium containing fbs incubation was carried out for h at ¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere ï¬xed with paraformaldehyde stained with crystalviolet and ï¬nally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identiï¬ed deas n the tcga hcc cohortin the current study was stratiï¬ed by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplanmeier curves asdescribed by xiong 2018aresultslandscape of as event proï¬les in hccto systematically characterize the as events and their clinicalsigniï¬cance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identiï¬ed asevents from genes based on their splicing patterns theseas events could be roughly classiï¬ed into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ¥ percentage of sampleswith psi ¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent proï¬les in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentiï¬cation of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as proï¬les of pairednormal and tumor tissues were ï¬nally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents ï¬ltered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes ï¬ltered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas proï¬les were used to identify deas eventually deaswere identiï¬ed from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the ï¬ltered as and deaswas inconsistent the es events accounted for of ï¬lteredas but only of deas however the proportion of apevents rose from of ï¬ltered as to of deasfigure 2b these statistical ï¬ndings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identiï¬ed deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identiï¬ed the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identiï¬ed deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2ac speciï¬c go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identiï¬cation of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identiï¬ed in hcc the blue and the red points represent the deas withstatistical signiï¬cance logfc ¥ adj p b proportions of different as types among ï¬ltered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could inï¬uence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we ï¬rst identiï¬ed sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionproï¬les of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoeï¬cient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapat1393e1593e4948e9941e2008e4387e8636e1393e4574e2519e1528e2202e6860e1522e2379e7699e1780e1843e4211e2609e1994e8164e1036e7765e1089e3569e1065e2135e5986e1038e1611e1110e2586e7736e2117e1072e4524e1151e1829e8136ebetween sfs and speciï¬c as events were illustrated using dotplots figures 5bg for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplanmeier curves for bothos and dfs these results conï¬rmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic signiï¬cance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure proteinprotein interaction ppi analysis of the identiï¬ed differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratiï¬edto form significant kaplanmeier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were ï¬ve and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these ï¬ndings conï¬rm that deas events possessnot only an important biological meaning but also a potentialclinical signiï¬canceconsidering the prognostic value of the aboveidentiï¬ed asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coeï¬cient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coeï¬cients to be less than a ï¬xed value certaincoeï¬cients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coeï¬cients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our ï¬ndings of significant heterogeneity among deas atan individual level which could reï¬ect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown signiï¬cance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height ampliï¬cation is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was veriï¬ed using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classiï¬cation overlap among clustersi iii and iv figures 8bc kaplanmeier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratiï¬cation of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratiï¬cation betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure speciï¬c regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength bg representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and conï¬denceintervals horizontal lines | 0 |
" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming 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 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity []in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but its raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [] therefore its necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chens in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained whats more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ¤ length length ¥ p icosandraannotated number ¤ length length ¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor whats more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] whats more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5 flora of north america vol43 chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturers instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number | 0 |
the periparturient period is one of the most challenging periods in dairy cows and encompasses the a0wk prior to and a0wk after parturition the nutrient requirements of dairy cows change greatly during this time largely due to the exponential growth of the gravid uterus and fetus followed by the demands of lactation nrc inflammation oxidative stress and adipose tissue mobilization lead to a reduction in dry matter intake dmi during the periparturient period this reduction in dmi leads to a negative nutrient balance nnb with a shortfall in the nutrient availability for the cow and fetus ingvartsen and andersen additionally this reduction in dmi also increases the risk of metabolic ketosis fatty liver milk fever and immunerelated disorders the risk of these diseases poor reproduction and low efficiency is greatly impacted by the degree and length of time during which these systems metabolism and immune response remain out of balance loor et a0al 2013a much of the research over the last decade have examined these biological interactions to identify the mechanisms behind the metabolic physiologic and immune adaptations that occur during the periparturient period loor et a0al 2013a 2013b roche et a0al bradford et a0al it is now known that nutrients such as amino acids aa serve functional roles outside of their use as building blocks for proteins and have immunomodulatory properties and interact through common biochemical pathways eg 1carbon metabolism figure a0 this concept has been well explored in nonruminant species li et a0 al sikalidis with nutritional management during the periparturient period continuing to be an active area of research it is important to develop a system understanding the potential immunometabolic role that dietary aa may play during this period thus the objective of this review is to provide an overview of physiological adaptations during the periparturient period the immune system and methods to assess immune function and oxidative stress this will be followed by a more specific discussion of the immunometabolic roles of specific aa and their potential effects in dairy cow during the periparturient period the potential effects of enhanced aa supply during the preweaning period will also be discussed brieflybiological adaptations in the transition cowa brief overviewduring the nnb associated with the periparturient period biological mechanisms coordinate the mobilization of body reserves in order to support fetal growth and milk production bauman and currie insulin concentrations are reduced and the response of hormonesensitive lipase in adipose tissue eg low insulin high growth hormone and catecholamines or high glucocorticoid concentrations is greater to facilitate lipid mobilization this periparturient period is also characterized by a state of inflammation encompassing an increase in hepatic production of positive acutephase proteins app such as haptoglobin and serum amyloid a a0saa and a decrease in the production ingvartsen the authors published by oxford university press on behalf of the american society of animal sciencethis 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 spermissionsoupcoms175 0cs176 of animal science vol no suppl abbreviationsaa akt app asct2 bcaa bcat bcka bhmt bmec bsa cbs csad cth dmi gator1 gcl gclc gpx gsh gsr gss il inos kegg klh lps mpo mtor mtorc1 mtr nfe2l2 nfκb nnb nos pc pmn pmnl pmtor rns rom ros rpm saa sahh sam samtor sell shmt slc1a1 slc1a5 sod stat tag th tlr tnfα vldl amino acidsprotein kinase bacutephase proteinsalanineserinecysteine transporter branchedchain amino acidsbranchedchain amino transferasesbranchedchain αketoacidsbetaine homocysteine methyltransferasebovine mammary epithelial cellsbovine serum albumincystathionine synthasecysteine sulfinic acid decarboxylasecystathionine gammalyasedry matter intakegtpaseactivating protein activity towards rags glutamate cysteine ligaseglutamate cysteine ligase catalyticglutathione peroxidaseglutathioneglutathione reductaseglutathione synthaseinterleukininducible noskyoto encyclopedia of genes and genomeskeyhole limpet hemocyaninlipopolysaccharidemyeloperoxidasemechanistic target of rapamycinmtor complex 5methyltetrahydrofolatehomocysteine methyltransferasenuclear factor erythroid 2like2nuclear factor kappa bnegative nutrient balancenitric oxide synthasephosphatidylcholinepolymorphonuclear neutrophilspolymorphonuclear leukocytesphosphorylated mtorreactive nitrogen speciesreactive oxygen metabolitesreactive oxygen speciesrumenprotected metserum amyloid asadenosyl homocysteine hydrolasesadenosyl methioninesadenosylmethionine sensor upstream of mtorlselectinserine hydoxymethyltransferasesolute carrier family member solute carrier family member superoxide dismutasesignal transducer and activator of transcriptiontriacyglycerolthelpertolllike receptorstumor necrosis factorαverylowdensity lipoproteinsof negative app such as albumin bertoni et a0 al it has been well established that these responses are mediated by the proinflammatory cytokines interleukin il6 il1 and tumor necrosis factorα tnfα kindt et a0al additionally oxidative stress also occurs during this period and is driven by the imbalance between the production of reactive oxygen metabolites rom reactive nitrogen species rns and the neutralizing capacity of antioxidant mechanisms in tissues and blood some of the wellestablished cellular antioxidants include glutathione gsh taurine superoxide dismutase sod and vitamins a a0and e bernabucci et a0al when oxidative stress overwhelms cellular antioxidant capacity rom induce an inflammatory response that is controlled via changes in mrna abundance of transcription regulators eg signal transducer and activator of transcription [stat3] nuclear factorkappa b [nfκb] the increase in oxidative stress and inflammation during this period is also negatively associated with a reduction in liver functionality and measurement of app can provide a useful tool to assess liver function as well as inflammation bertoni and trevisi during the periparturient period aa metabolism is also altered with moderate carcass protein losses reported even when animals are fed to their predicted metabolizable protein requirements bell et a0 al additionally circulating aa concentrations change dramatically with favorable circulating profiles of many aa not being restored until d postpartum zhou et a0al 2016b furthermore the total concentration of aa in plasma reaches a nadir around day postpartum zhou et a0al 2016b which corresponds to a peak in total disease incidence during early lactation ingvartsen this decrease in circulating aa is likely associated with the increased use of aa for gluconeogenesis as well as for hepatic production of app thus it is important to investigate how supplemental aa during the periparturient period may modulate metabolism and immune responses to promote production and reduce susceptibility to diseasegeneral overview of the immune a0systemthe immune system consists of both the adaptive and innate immune responses which are linked together through signaling molecules such as cytokines the innate immune system is the first line of defense and includes physical barriers such as epithelial cell layers that express tight cell junctions and the mucus layer of the respiratory gastrointestinal and genitourinary tracts chaplin cells involved in the innate immune response include macrophages polymorphonuclear neutrophils pmn dendritic cells mast cells eosinophils natural killer cells and natural killer t cells turvey and broide the focus of the present review is on the effects of aa on phagocytic cells that is cells that engulf and kill such as macrophages and pmn macrophages not only phagocytose invading pathogens but also produce cytokines such as ils and tnfα which initiate innate and adaptive immune responses and recruit pmn to the site of infection chaplin the adaptive immune response consists of t lymphocytes b lymphocytes and humoral factors marshall et a0 al there are two types of t lymphocytes cytotoxic t cells cd8 cells and thelper th cells cd4 cells marshall et a0al cytotoxic t cells detect and eliminate infected cells while th cells produce ils and interferonγ ingvartsen and moyes the b lymphocytes are cells that produce antibodies that bind to antigens on the surface of pathogens to mark them for destruction marshall et a0al 0ccoleman et a0al s177aa immune function and oxidative a0stressduring the periparturient period the metabolic status is associated with the inflammatory regulation of peripheral blood mononuclear cells with a more pronounced inflammatory response in those cells during the nnb immediately postpartum agrawal et a0 al mann et a0 al this period is also associated with altered signaling of nutrientsensing kinases in immune cells such as the protein kinase b akt and mechanistic target of rapamycin mtor pathway which may modulate cytokine production mann et a0al aa can directly and indirectly alter the immune system besides being used in energy metabolism reactions and the synthesis of proteins aa are critical for the synthesis of other functional molecules such as the antioxidants gsh and taurine no histamine and hydrogen peroxide li et a0 al thus this section of the review will focus on the role that aa play in modulating immune function and oxidative stress in dairy cows during the periparturient period a a0special focus will be placed on aa involved in 1carbon metabolism as this represents an interconnected route through which aa could impact molecular events such as epigenetic regulation protein synthesis via mtor energy metabolism and antioxidant synthesis a a0summary of studies investigating the effects of aa on immune function and oxidative stress in dairy cows is provided in table a0 a a0summary model of how aa might alter immune function and oxidant status is depicted in figure a0methioninemethionine not only is essential for protein synthesis but also serves as a functional nutrient that is needed for the production of the antioxidants gsh and taurine atmaca and provision of methyl groups finkelstein these features are exemplified by the central role of met in 1carbon metabolism figure a0 in these pathways met is used to synthesize sadenosyl methionine sam which can be used to methylate dna and to support phosphatidylcholine pc and carnitine synthesis for fatty acid metabolism vance et a0al during the periparturient period triacyglycerol tag accumulate in the liver leading to mitochondrial dysfunction inflammation and reduced liver function li et a0al du et a0al pc is the main phospholipid component of verylowdensity lipoproteins vldl vance thus enhancing pc synthesis through greater met supply may help improve vldl synthesis and reduce hepatic tag accumulationas part of 1carbon metabolism homocysteine can be remethylated to met using betaine or folate as methyl donors bhmt and via betaine homocysteine methyltransferase 5methyltetrahydrofolatehomocysteine methyltransferase mtr respectively homocysteine is also used to synthesize cystathionine via cystathionine synthase cbs in the first reaction of the transsulfuration pathway banerjee et a0 al cystathionine is used to make cys which is utilized to synthesize the antioxidants taurine or gsh cbs is allosterically figure interrelationships among components of the 1carbon metabolism pathway methionine cycle folate cycle and transsulfuration pathway 5mthf 5methyltetrahydrofolate amd1 adenosylmethionine decarboxylase arg arginase b12 cobalamin b2 riboflavin b6 pyridoxal ²phosphate cbs cystathionine betasynthase dcsam decarboxylated sadenosylmethionine dhfr dihydrofolate reductase dnmt dna methyltransferase dtmp thymidine monophosphate dump deoxyuridine monophosphate ftcd formimidoyltransferase cyclodeaminase gnmt glycine nmethyltransferase mat methionine adenosyltransferase mthfr methylenetetrahydrofolate reductase mtrr 5methyltetrahydrofolatehomocysteine methyltransferase reductase odc1 ornithine decarboxylase pemt phosphatidylethanolamine nmethyltransferase sahh sadenosylhomocysteine hydrolase shmt serine hydroxymethyltransferase sms spermine synthase srm spermidine synthase tdh threonine dehydrogenase thf tetrahydrofolate tyms thymidylate synthetase 0cs178 of animal science vol no suppl activated by sam banerjee et a0 al therefore enhanced sam production with increased met supply can help enhance the flux of the transsulfuration pathway increasing taurine and gsh production to help reduce oxidative a0stressin dairy cattle low levels of serum met postpartum are associated with severe hepatic lipidosis shibano and kawamura and other than his met is the only aa for which net uptake by the liver increased pre and postpartum larsen and kristensen therefore enhancing postruminal met supply during the periparturient period is of interest to increase circulating concentrations of met for its functional roles in the body the work from dalbach et a0 al demonstrated that rumenprotected met rpm can be used to increase serum concentrations of met in the first 2wk postpartum which will enhance the availability of met for protein synthesis and metabolism via the 1carbon pathways in terms of production supplementation of met during the peripartal period concomitantly increases milk yield milk protein and milk fat soon after calving ordway et a0 al osorio et a0 al these responses are in large part driven by enhancing met availability and by the additional flux of met through the met cycle in the liver which consequently increases the production of downstream compounds such as sam pc gsh and taurine additionally work feeding rpm during the periparturient period has detected positive responses in maintaining consistent rates of dmi prepartum last d and faster and greater rates of dmi during the first to d after calving osorio et a0al zhou et a0 al 2016c batistel et a0 al the same milk production response was also observed when met was supplemented postpartum as the isopropyl ester of 2hydroxy4methylthiobutanoic acid stpierre and sylvester as described earlier the transient inflammatorylike status around parturition appears to be a normal aspect of the adaptations to lactation as cows approach parturition those with greater but still subclinical concentrations of circulating cytokines have greater inflammation and oxidative stress and lower liver function along with lower milk yield and lower postpartum dmi bertoni et a0 al in addition to their function in the immune system cytokines interferons and tnfα also elicit pathophysiological effects leading to sickness table summary of studies in dairy cows investigating the effects of supplemental aa on immune function oxidative stress and inflammation tissuecellstreatmentmain outcomeplasmarpm for d prepartum and d postpartumimprovements in plasma biomarkers indicating reduced oxidative stress and inflammation and enhanced liver function increased neutrophil phagocytosis and oxidative burstreferencebatistel et a0al plasmaabomasal infusion of glu for first infusions of gln increased the abundance of cd4 tcells on day doepel et a0al d postpartumpostpartum and increased the abundance of monocytesmammary rpm for d prepartum and d methionine supply upregulated expression of genes involved in han et a0al glandplasmapostpartumantioxidant metabolism and increased activation of nfe2l2intravenous infusions of gln for glutamine infusion decreased plasma haptoglobin and increased jafari et a0al d postcalvinglpsbinding protein and saa subcutaneous rpm for d prepartum and d enhanced met supply increased mrna and protein abundance of liang et a0al 2019aadiposepmnlpostpartumenzymes related to gsh metabolismincubation with met andor supplemental met coupled with adequate choline enhanced gene lopreiato et a0al cholineexpression of pathogen recognition mechanisms methionine ameliorated the increased inflammation and oxidative stress observed when cells were incubated without choline plasma and protected gln for d postpartum increased total blood protein and albumin decreased plasma nemati et a0al milkwhole bloodrpm for d prepartum and d aspartate aminotransferase and milk somatic cell countincreased whole blood neutrophil phagocytosis on day osorio et a0al 2013apostpartumpostpartum with supplemental metliverrpm for d prepartum and d methionine supply altered flux through 1carbon metabolism via osorio et a0al 2014aliver and plasmaplasmapostpartumchanges in mrna to support antioxidant and met synthesisrpm for d prepartum and d methionine increased liver gsh and decreased concentrations of osorio et a0al 2014bpostpartum plasma biomarkers of inflammationrpm for d during midlactation increased proliferative ability of peripheral blood t lymphocytes soder and holden with supplemental metplasmarpm for d prepartum and increased antioxidant capacity of plasma and cd4cd8 t sun et a0al postpartumlymphocyte ratio with met supplywhole bloodrpm for d prepartum and d methionine damped the hyperresponse of il1 during an lps vailatiriboni et a0al plasmajugular infusion of arg and lps in arginine alleviated lpstriggered inflammation by decreasing il6 zhao et a0al 2018apostpartumchallenge through improvements in oxidative stressserumjugular infusion of arg and lps in infusion of arg promoted antioxidant mechanisms during lpszhao et a0al 2018bmidlactation cowsinducible nos and lpsbinding proteinmidlactation cowsrpm for d prepartum and d postpartumrpm for d prepartum and d triggered inflammation by increasing total antioxidant capacity and gsh peroxidase activity and decreasing malondialdehyde increased hepatic gsh and improved plasma biomarkers of liver function and inflammation with met neutrophil phagocytosis capacity and oxidative burst were also increased with metenhanced met supply increased mrna expression of genes zhou et a0al 2016azhou et a0al 2017bpostpartumassociated with pc and antioxidant synthesisrpm for d prepartum and d decreased expression of genes related to inflammation and zhou et a0al 2018bliver and plasmaliver pmnlpostpartumoxidative stress 0ccoleman et a0al s179figure the theoretical model of cellular aa utilization amp adenosine monophosphate atp adenosine triphosphate ctp cytidine triphosphate dttp deoxythymidine triphosphate gmp guanosine monophosphate imp inosine monophosphate no nitric oxide r5p ribose 5phosphate tca cycle tricarboxylic acid cycle ump uridine monophosphate utp uridine5²triphosphatebehaviors whose primary manifestation is satiety larson and dunn an example of this behavior in dairy cows is the reduction in dmi around calving in mice these cytokines have been shown to reduce meal size and duration as well as decrease meal frequency and prolong intermeal intervals platasalamán furthermore cytokines directly affect the hypothalamus il1 and ifn act directly and specifically on the glucosesensitive neurons in the brain satiety and hunger sites platasalamán in addition to increases in dmi and milk production rpm supplementation during the periparturient period has been associated with positive health responses across four studies osorio et a0al 2014b sun et a0al zhou et a0al 2016a batistel et a0 al there have been consistent improvements in the concentrations of plasma biomarkers of inflammation where il1 and haptoglobin have decreased and albumin has increased summarized in table a0 improvements in biomarkers of oxidative stress have also been observed with enhanced met supply during the periparturient period in the study by batistel et a0al plasma concentrations of ferricreducing antioxidant power carotene tocopherol and total and reduced gsh were increased with rpm while rom were lower sun et a0al also observed an improvement in blood antioxidant status with rpm increasing total antioxidant capacity glutathione peroxidase gpx activity and vitamin e a0a a0similar effect was observed in liver tissue by osorio et a0 al 2014b where cows fed rpm had greater hepatic concentrations of total and reduced a0gshfrom a mechanistic standpoint changes in the mrna abundance of sadenosyl homocysteine hydrolase sahh mtr sod1 glutamate cysteine ligase catalytic gclc subunit and dna methyltransferase 3a suggested alterations in flux through 1carbon metabolism osorio et a0al 2014a importantly sahh the enzyme that makes homocysteine from sadenosylhomocysteine was upregulated with met which would support a supply of homocysteine to be used for antioxidant and met synthesis furthermore in the study by zhou et a0al 2016a greater met supply compared with rumenprotected choline increased antioxidant concentration in liver tissue despite a lower concentration of pc those responses were due to the greater abundance of phosphatidylethanolamine methyltransferase the enzyme that utilizes sam and phosphatidylethanolamine to make pc and cbs zhou et a0al 2017b additionally enhanced met supply during the periparturient period was observed to increase mrna and protein abundance of enzymes related to gsh metabolism in subcutaneous adipose tissue suggesting greater activation of those pathways liang et a0al 2019a a a0greater dietary supply of choline did not change the mrna abundance of bhmt and mtr in cows zhou et a0al 2017ba positive effect of met supplementation on mammary gland antioxidant mechanisms was observed by han et a0 al mrna abundance of gpx1 gclc glutamate cysteine ligase gcl modifier subunit malic enzyme ferrochelatase and ferritin heavy chain and genes involved in gsh and iron metabolism were upregulated protein abundance of phosphorylated nuclear factor erythroid 2like2 nfe2l2total nfe2l2 was also increased han et a0al nfe2l2is a regulator of transcription of antioxidant genes hence an increase in phosphorylated nfe2l2 suggested greater activation of antioxidant systems and is likely one of the mechanisms behind the changes in mrna abundance lastly across studies there has also been a consistent improvement in the concentrations of plasma biomarkers of liver function such as increases in paraoxonase and cholesterol with rpm osorio et a0al 2014b zhou et a0al 2016a batistel et a0 al which is likely linked to the reduction in inflammation and oxidative stress thus the consistent changes across studies in metabolites and plasma biomarkers as well as mrna abundance across tissues indicate that enhanced met supply during the periparturient period reduces oxidative stress and inflammation however more work is needed to verify the exact mechanisms behind the observed changes 0cs180 of animal science vol no suppl enhanced postruminal supply in the form of rpm during the periparturient period has been associated with improvements in immune cell function when rpm was provided for d prepartum and d postpartum whole blood neutrophil phagocytosis was increased compared with control cows at d postpartum osorio et a0 al in the study by zhou et a0 al 2016c rpm supplementation from day prepartum to day postpartum increased neutrophil phagocytosis capacity and oxidative burst activity zhou et a0 al 2016a this same improvement in neutrophil immune function was observed when rpm was supplied from day prepartum to day postpartum batistel et a0 al furthermore an increase in the proliferative ability of peripheral blood t lymphocytes was observed when rpm was supplemented to midlactation cows soder and holden zhou et a0 al 2018b isolated polymorphonuclear leukocytes pmnl and observed lower abundance of genes related to inflammation il1b tlr2 nfκb and stat3 and oxidative stress cbs gpx1 glutathione synthase [gss] and sod2 as well as an increase in plasma taurine with supplemental met suggesting a better redox and inflammatory status of those cells additionally those same cows were used for an ex vivo whole blood challenge with lipopolysaccharide lps to further investigate immune cell responses during this challenge a table summary of additional beneficial effects of feeding rpm during the transition period and early lactationbiomarkerresponse12sitebiological functionmetabolism carnitine cholesterolonecarbon metabolism cystathionine betasynthase activity cystathionine cystine homocysteineinflammation il1beta haptoglobin albumin oxidative stress romliveroxidation of fatty acidsplasmalipoprotein metabolismliverplasmaplasmaplasmaantioxidant synthesish2s biosynthesis redox statusredox statusmethylation reactionsplasmaproinflammatory cytokineplasmainflammation signalplasmaacutephase responseplasmaperoxides gsh taurine antioxidant capacity paraoxonasesuperoxide ohradicalsliver blood antioxidantantioxidantplasmaplasmatotal antioxidants in bloodplasmaantioxidant enzyme beneficial increase beneficial decrease no change in concentration2relative to a control or rumenprotected choline supplemented diet osorio et a0al 2014b zhou et a0al 2016a sun et a0al batistel et a0al vailatiriboni et a0al hyperresponse in il1 was observed around parturition which likely arose from oxidative stress vailatiriboni et a0 al however rpm supplementation dampened this hyperresponse likely through improvements in oxidative stress vailatiriboni et a0al the recent work by lopreiato et a0 al investigated the effects of incubating bovine pmnl with met andor choline and observed that supplemental met coupled with adequate choline enhanced gene expression of tlr2 and lselectin sell which are pathogen recognition mechanisms in the same experiment cells incubated without choline had greater mrna abundance of il1b il6 il10 and myeloperoxidase mpo glutathione reductase gsr gss cystathionine gammalyase cth and cysteine sulfinic acid decarboxylase csad indicating greater inflammation and oxidative stress this effect however was ameliorated by supplementing additional met lopreiato et a0 al thus the increased dmi and milk production observed when feeding rpm can be partly explained by a reduction in inflammation as it directly at the hepatic level and by dampening the immune cell overresponse and indirectly reducing oxidative stress decreases circulating proinflammatory cytokinesenhanced met supply during the periparturient period has also been associated with changes in mtor signaling mtor is a serinethreonine kinase that plays a central role in integrating environmental cues from growth factors nutrients particularly aa and energy powell et a0al in dairy cattle mtor has traditionally been studied in the context of its role in regulating protein synthesis however work from humans and nonruminant species has indicated that mtor is an important regulator of immune responses powell et a0 al jones and pearce when activated by aa mtor directs an activation of anabolic metabolism which allows growth proliferation and development this makes the activation of mtor in immune cells particularly important for maintaining proliferation and without proper activation cells may enter periods of growth arrest jones and pearce methionine specifically may interact with mtor via the production of sam specifically sam can bind to sadenosylmethionine sensor upstream of mtor samtor a protein that inhibits mtor complex mtorc1 by interacting with gtpaseactivating protein activity towards rags gator1 gu et a0al when sam binds to samtor it inhibits the association of samtor and gator1 allowing mtorc1 to be activated gu et a0al to our knowledge there is only one study investigating the expression of mtor signaling proteins in immune cells in dairy cattle and work is also lacking in nonruminant species in periparturient cows the activation of aktmtor signaling in immune cells was reduced postpartum compared with prepartum mann et a0al importantly agrawal et a0al also identified the expression of aa transporters and the kyoto encyclopedia of genes and genomes kegg pathways related to 1carbon metabolism and mtor in pmnl from peripartal cows a a0 list of these transporters and kegg pathways related to aa and 1carbon metabolism are summarized in table a0 together these studies support the potential importance of aa for nutrient signaling in immune a0cellsrecent work indicating that enhanced met supply activates mtor signaling in the mammary gland supports its role in enhancing protein synthesis for example in vitro enhancing met supply to immortalized bovine mammary epithelial cells bmec by varying the ratio of lys to met increased the concentration and utilization of essential aa particularly branchedchain aa bcaa dong et a0 al this change 0cwas potentially mediated by alterations in aa transporters controlled by mtor dong et a0al other studies with bmec have also revealed a potential for greater mtor activation when met supply is enhanced nan et a0al sala ma et a0al in vivo the effects of supplemental met during the periparturient period on mtor signaling were explored using cows from the study by batistel et a0al in the mammary gland cows receiving rpm had lower protein abundance of total mtor and phosphorylated mtor pmtor compared with control cows on day postpartum but the ratio of pmtortotal mtor was not different suggesting that there was no difference in mtor activation between treatments ma et a0 al however changes in aa transporters and insulin signaling indicated that insulin sensitivity of the mammary gland was enhanced with supplemental met ma et a0al in subcutaneous adipose tissue the mrna and protein abundance of some aa transporters and pmtor were upregulated with enhanced met supply while changes in insulin signaling and plasma glucose also indicated that met helped improve insulin sensitivity liang et a0 al 2019b thus enhancing met supply during the periparturient period may lead to mtor activation in immune cells as well as improved nutrient uptake which could help to support proliferation and development additional work in ruminants and nonruminants is needed to understand whether met modulates mtor signaling in immune cellscysteinecysteine can be synthesized endogenously from homocysteine as described earlier and is needed to synthesize gsh and taurine gsh is synthesized via two enzymes gcl and gss lushchak to synthesize taurine cysteine sulfinic acid is first synthesized from cysteine by cysteine dioxygenase and can then be utilized by csad to produce taurine park et a0al dietary cys has been explored as a way to improve health in nonruminant species and humans under a variety of conditions including type2 diabetes cardiovascular disease and liver disease to name a few yin et a0 al across these studies increased dietary cys increased the concentrati | 0 |
"Using fluorescence microscopy we identified apoptotic cells by the presence of highly condensed or fragmented nuclei. Apoptotic cells were counted in 5 different fields under microscopic observation. Western blot analysis The detailed protocol for the Western blot analysis is described in Method S1. It was performed under conventional conditions using the following antibodies: rabbit anti-human REIC/Dkk-3 antibody raised in our laboratory [11]; rabbit anti-human GRP78/BiP (GRP78) (ab21685; Abcam Cambridge MA); rabbit anti-human SAPK/JNK (#9252) and rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185; #9251) (Cell Signaling Technology Beverly MA); rabbit anti-human coxsackievirus and adenovirus receptor (CAR) (HPA030411; Atlas antibodies Stockholm Sweden); and mouse anti-actin (MAB1501; Millipore Billerica MA). The following secondary antibodies were used: goat anti-rabbit or anti-mouse IgG-conjugated horseradish peroxidase (Santa Cruz Biotechnology Santa Cruz CA). To detect the specific signals the membranes were examined using ECL plus Western Blotting Detection Reagents (Amersham Biosciences UK Limited Buckinghamshire UK). In addition the band intensities for GRP78 CAR and actin representing their expression levels were measured using ImageQuant TL software (GE Healthcare Bioscience) and quantified by GRP78 or CAR/actin ratio. Tumor growth assay in vivo A549 cells (5106 in 50 µL of phosphate buffered saline [PBS]) mixed with 50 µL of Matrigel (BD Biosciences San Jose CA) were subcutaneously injected into the right flank of adult female BALB/c nu/nu mice (CLEA Japan Tokyo Japan). The tumor volume was calculated using the empirical formula V?=?1/2[(the shortest diameter)2(the longest diameter)]. When the tumors had reached approximately 50100 mm3 mice (n?=?15) were randomly divided into 3 treatment groups: (a) PBS; (b) Ad-LacZ; and (c) Ad-REIC. Viruses (1109 pfu) in 100 µL of serum-free medium were administered intratumorally. At the end of experiments mice were sacrificed after 24-days after the viral injection and tumors were harvested measured and photographed. Statistical analyses All data were analyzed using STATA ver.12 (STATA Corp. College Station TX). Fisher's exact test was applied when appropriate. For a comparison of induction of apoptosis between Ad-REIC-treated and Ad-LacZ-treated A549 cells a Cochran-Mantel-Haenszel statistics was applied for comparing. Repeated measurement ANOVA was applied for the comparison of xenotransplanted NSCLC tumor sizes among PBS Ad-LacZ and Ad-REIC. P<0.05 was considered significant. All tests were two-sided. Results Effect of Ad-REIC on NSCLC cell lines We examined the inhibition of cell viability using Ad-REIC and an MTS assay. In 13 (52%) of 25 NSCLC cell lines Ad-REIC treatment at 20 MOI inhibited the cell viability (40%60% inhibition) compared with Ad-LacZ treatment ( ). These cell lines were regarded as highly sensitive to Ad-REIC. In contrast 12 cell lines (48%) were not inhibited by Ad-REIC treatment at 20 MOI and were regarded as resistant cells. OUMS-24 was not inhibited at 20 or 200 MOI of Ad-REIC. Of note Ad-REIC treatment at 100 and 200 MOI improved the inhibition of cell viability (100 MOI: 15%59% inhibition 200 MOI: 40%78% inhibition) compared with Ad-LacZ treatment (). Thus we defined 20 MOI as a low MOI value and 200 MOI as a high MOI value. For comparison Ad-REIC treatment was also performed in the human mesothelioma cell line 211H which we previously reported to be Ad-REIC-sensitive [14]. The 211H was not inhibited at 20 MOI but was inhibited at 200 MOI of Ad-REIC (). " | 1 |
"Sulindac is a Food and Drug Administration (FDA)-approved non-steroidal anti-inflammatory drug (NSAID) for the treatment of osteoarthritis ankylosing spondylitis gout or rheumatoid arthritis [20][23]. Its anti-inflammatory activity is due to its inhibition of the synthesis of prostaglandins [24] which cause inflammation and pain in the body. Sulindac has also been found to block cyclic guanosine monophosphate-phosphodiesterase an enzyme that inhibits the normal apoptosis signaling pathway and this inhibitory effect allows the apoptotic signaling pathway to proceed unopposed resulting in apoptotic cell death and reducing the incidence of various tumors including breast esophageal stomach prostate bladder ovary and lung cancers [25] [26]. In humans sulindac is reduced to the active anti-inflammatory metabolite sulindac sulfide undergoes a 2-step reoxidation to sulindac sulfone [27] [28]. All three compounds have been shown to have chemoprotective effects. In colon cancer sulindac has been used to increase the anticancer effects of some reagents or stresses including bortezomib [4] hydrogen peroxide [29] and oxidative stress [30]. Importantly sulindac and its metabolites modulate the expression of multioxidative enzymes including glutathione S-transferases and NQO1 the latter being the key regulator of ?-lapachone-induced cell death in cancer cells [28] [31] [32] and sulindac might therefore have a synergistic anti-tumor effect with ?-lapachone. Lung cancer the major cancer worldwide is now the leading cause of cancer-related deaths [33][35]. According to a report of the Department of Health Executive Yuan ROC (Taiwan) published in 2010 the mortality rate for lung cancer is 20% topping the list of all cancer-related deaths. The cost of health care for treatment of lung disease is increasing tremendously each year and threatens to overwhelm public health services [36]. In order to get a better target therapy researchers have tried to identify key differences between lung cancer cells and normal lung cells such as mutation or overexpression of genes including EGFR ras and VEGF [37][39]. Unfortunately current chemotherapies for lung cancer lack adequate specificity efficacy and treatment heterogeneity is also a big issue [40]. There is therefore an urgent need for new therapeutic drugs or new combinations of drugs to provide more efficient lung cancer therapy. Since NQO1 overexpression has been noted in both non-small cell lung cancer (NSCLC) cell line [41] [42] ?-lapachone could be a potential therapeutic drug for lung cancers. However some lung cancer cells show lower NQO1 expression or activity and might therefore be resistant to ?-lapachone toxicity. In this study we first investigated the relationship between ?-lapachone toxicity and NQO1 levels in NSCLC cell lines then determined the signaling pathway involved in the cell death caused by high concentrations of ?-lapachone. We also used lower concentrations of ?-lapachone to explore whether sulindac and its metabolites could facilitate the anticancer effect of ?-lapachone by increasing NQO1 expression or activity in lung cancer cell lines with low NQO1 levels and checked the importance of NQO1 in this combination therapy. We found that the toxicity of ?-lapachone was related to the level of NQO1 expression or activity in lung cancer cells and that high concentrations of ?-lapachone killed cells by decreasing phosphorylation of PI3K AKT and ERK and activating JNK. In addition the cytotoxicity of low concentrations of ?-lapachone was increased by combination with sulindac and its metabolites a process involving upregulation of expression or activity of NQO1. Materials and Methods Cell Culture The human lung cancer cell lines CL1-1 CL1-5 and A549 were cultured in 5% CO2 at 37°C in RPMI 1640 medium containing 10% fetal calf serum 100 Units/ml of penicillin and 100 mg/ml of streptomycin (all from Gibco). The cell lines were gifts from Dr. PC Yang National Taiwan University Hospital [43] in whose laboratory CL1-5 cells were selected from parental CL1-1 cells for greater metastatic potential using a transwell system. Cell Viability Assays CL1-1 CL1-5 or A549 cells (1x104) were seeded for 24 h at 37°C in a 96-well culture plate then were subjected to starvation for 14 h in RPMI 1640 medium containing 2% fetal calf serum 100 Units/ml of penicillin and 100 mg/ml of streptomycin. Following 6 h pretreatment with medium or the indicated concentration of sulindac or its metabolites (all from Sigma) the cells were incubated for 12 h with or without the indicated concentration of ?-lapachone in the continued presence of sulindac or its metabolites and then cell viability was evaluated. Two cell viability assays were used. In the crystal violet staining assay the cells were fixed with 4% paraformaldehyde for 15 min stained with 0.4% crystal violet for 15 min and washed with H2O then 50% acid alcohol was used to dissolve the bound crystal violet and the OD at 550 nm measured on an ELISA reader. In the MTT assay 10 µl of MTT (0.5 mg/ml) (Sigma) was added to each well and the plates incubated at 37°C for 4 h then the formazan product was dissolved in 100 µl of DMSO at 37°C for 30 min and the OD at 570 nm measured on a microplate reader. Acridine Orange (AO) Staining Cells (5x104) cultured on cover-slides in 24-well plates were incubated for 14 h in RPMI 1640 medium containing 2% fetal calf serum preincubated with sulindac sulfide for 6h and then treated with or without ?-lapachone for 24 h then were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature (RT) and stained for 10 min with 0.5 ml of AO (10 mg/ml in PBS) (Sigma). After several PBS washes the cells were examined on an Olympus BH-2 inverted microscope equipped with a fluorescence attachment. Detection of Apoptosis and Measurement of Intracellular Calcium Levels To detect apoptosis cells (1x106) were treated for 36 or 9 h with 5 µM ?-lapachone then were washed with ice-cold PBS trypsinized with 0.05% trypsin-0.02% EDTA stained for 15 min at 37°C with Annexin V-FITC (10 µg/ml) (Strong Biotech Corporation AVK050 Taipei Taiwan) and analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson). To measure intracellular calcium levels the cells were incubated for 10 min at 37°C with 2 mM Fluo-4/AM (Molecular Probes) washed with PBS trypsinized and analyzed by FACSan flow cytometry using the FL1H parameter. Western Blot Analyses Treated cells were lysed with RIPA buffer containing 10 µg/ml of protease inhibitor (Sigma) and then the lysate was centrifuged at 10000g for 15 min at 4°C and the supernatant collected for immunoblotting. The protein concentration was measured by the Bradford assay and samples containing 20 µg of protein were separated by 10 or 12% SDSPAGE and then transferred to Immobilon-P membranes for 2 h at 200 V (Millipore) in a Trans-Blot Electrophoretic Transfer cell. The membranes were blocked for 1 h at RT with 5% skim milk in PBS-0.2% Tween 20 (PBS-T) then incubated for 2 h at RT with antibodies against NQO1 (Cell Signaling) PI3 kinase or p-PI3 kinase (Millipore) AKT or p-AKT (Epitomics) ERK p-ERK JNK or p-JNK (Cell Signalling)GAPDH (Genetex) or ?-actin (Abcam) diluted 1?1000 in 1% BSA. After washing for 30 min at RT with PBST the membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibody (Perkin-Elmer Boston MA; 1?5000 dilution in PBST) then bound antibody was detected using the ECL Western blotting reagent (Amersham) chemiluminescence being detected using a Fuji Medical X-ray film (Tokyo Japan) and quantified by gel image analyses with Image Pro software. The intensity of the band of interest was divided by that for ?-actin or GAPDH (loading controls) and this value normalized to that seen with no treatment. " | 1 |
EndocrinesyPostoperative vocal fold dysfunction in covid19 era are we still intime for a recoveryElena Bonati Elena Giovanna Bignami2 Paolo Del Rio1Received May Accepted July Springer ScienceBusiness Media LLC part of Springer Nature To the EditorThe novel coronavirus COVID19 is a highly contagious zoonosis produced by SARSCoV2 which arose inChina and spread all over the world transmitting from manto man through respiratory secretions In March itwas deï¬ned by the World Health anization WHO as apandemic to underline its spread and severityHealthcare professionals are one of the categories most atrisk of contracting the infection in particular when theiractivity involves the direct management of the patientsairways Among these categories we can count anesthetistshead and neck surgeons otolaryngologists maxillofacialsurgeons ophthalmologists and dentists For these reasonsthe latest evidencebased recommendations for otolaryngology and head and neck surgery practice suggest thathealthcare facilities should prioritize urgent and emergencyvisits and procedures until this condition stabilizes ceasingelective care []Nevertheless oncological surgical activity althoughslowed down did not stop in most hub hospitals Regardingthyroid cancer thyroid surgery is complex and the rate ofnerve damage is still considerable Immediate postoperativevocal fold rate is in our case study and decrease to after months Postoperative dysphonia can becaused by several factors other than nerve damage such astracheal intubation or scarring in the thyroid lodge It istherefore important to identify the cause of vocal corddysfunction and treat it correctly at the right time If anunilateral vocal fold paresisparalysis is diagnosedthetreatment consist in improving the speech while in case of Elena Bonatiebonati86gmailcom General Surgery Unit Department of Medicine and SurgeryParma University Hospital Parma Italy Unit of Anesthesiology Department of Medicine and SurgeryParma University Hospital Parma Italybilateral vocal fold paresisparalysis respiratory obstructionalso needs to be urgently treated Fortunately we havebroughtthe incidence of this last and most dangerouscomplication to at our Clinic since the introduction in of the routine use of intraoperative neuromonitoringduring thyroidectomyThe latest guidelines published by the Americanin March Association of Endocrine Surgeonsrecommend laryngeal examination in patients with knownor suspected new recurrent laryngeal nerve dysfunctionafter thyroidectomy for additional evaluation and possibletreatment with a speech pathologist According to theAmerican Academy of OtolaryngologyHead and NeckSurgery they assert that early referral weeks postsurgery to a laryngologistin combination with earlyintervention results in superior voice outcomes since theideal time for vocal fold augmentation is months afterthyroidectomy []A metaanalysis about therapy for vocal fold paresisparalysis after thyroidectomy concluded that the timingof therapy for unilateral vocal fold paralysis after thyroidectomy has a significant impact on the effect sizebeing significantly greater if therapy is performed within months This may be explained by progressive atrofolds and disappearance of nervephy offunction so that vocalfold movements cannot berecovered []the vocalPatients who underwent thyroid surgery from February and who had experienced a vocal fold disfunctionVFD were unable to undergo a laryngoscopy nor muchless a speech therapy according to health measuresnecessary to contain the spread of the virus This unfortunately causes a progressively reduced possibility ofrecovery increasing the speciï¬c morbidity related to surgery for thyroid cancer in this period The only indicationthat we can give to patients is the rest of the voice to avoidthe establishment of compensation mechanisms worseningthe clinical picture waiting to be able to resume the correcttreatment 0cTherapeutic diagnostic pathways in the COVID19 erahave become difï¬cult and dangerous logarithms that mustconsider the need for patient care and the possibility oftreatments delay in safety but also the risk of contagion ofthe patientsleast protection ofhealthcare personnel The hospital setup has been significantly changed and much of the economic structuraland human health resources have been dedicated to themanagement of the COVID pandemicthemselves and notIn parallel with the COVID19 emergency we areexperiencing another health emergencythe one thatinvolves the management of nonCOVID19 patients Evenin the second phase of the pandemic only urgent healthservices are provided A reanizing effort within theindividual healthcare companies is required to guaranteetreatment even for nonCOVID19 patientsMoreoverThe COVID19 pandemic highlighted the limits andweaknesses of our health system and now that the correctprotocols for the protection of healthcare personnel havebeen described allthe all healthcare companies shouldequip their staff with the appropriate materials such as N95masks hair cover protective coverall gown gloves faceshields goggles and shoe covers In the face of higherexpenses this would allow the resumption of activitiesminimizing the risk of an increase in the rate of infectionroutine health practices must be reconsidered preferring less invasive techniques in order toscreen patients who need secondlevel examination Evenif not used yet in our hospital transcutaneous laryngealultrasonography is a valid noninvasive and painlessalternative method in the assessment of vocal cords It hasbeen demonstrated in a recent prospective multicentricstudy that it has concordance with laryngoscopy in themajority of cases and so it can be a valid alternative asï¬rstline exam for vocalfold examination pre andpostoperativelyEndocrineFinally the growing use of virtual platforms for the needof social distancing could encourage their application evenin healthcare services that can be performed by teleconference such as speech therapyWe can assess that COVID19 pandemic is causingdirect morbidity and mortality and even a related one dueto missed or delayed treatment of multiple nonCOVID19diseases The delivery of the health service should beimproved and the health system itself must be modernizedto adapt to new needsCompliance with ethical standardsConï¬ict of interest The authors declare that they have no conï¬ict ofinterestPublishers note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional afï¬liationsReferences LP Kowalski A Sanabria JA Ridge WT Ng R de BreeA Rinaldo RP Takes A A Mkitie AL Carvalho CR Bradford V Paleri DM Hartl V Vander Poorten IJ Nixon C PiazzaPD Lacy JP Rodrigo O GuntinasLichius WM MendenhallA DCruz AWM Lee A Ferlito COVID19 pandemic effectsand evidencebased recommendations for otolaryngology and headand neck surgery practice Head Neck 101002hed26164 KN Patel L Yip CC Lubitz EG Grubbs BS Miller W ShenP Angelos H Chen GM Doherty TJ Fahey 3rd E KebebewVA Livolsi ND Perrier JA Sipos JA Sosa D StewardRP Tufano CR McHenry SE Carty The American Associationof Endocrine Surgeons Guidelines for the deï¬nitive surgical management of thyroid disease in adults Ann Surg e21e93 X Chen P Wan Y Yu M Li Y Xu P Huang Z Huang Typesand timing of therapy for vocal fold paresisparalysis after thyroidectomy a systematic review and metaanalysis J Voice 0c' | 2 |
" 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 patients 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 dhebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall dhebron 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 | 0 |
"The known molecular characteristics of each cell line are shown in . The 25 NSCLC cell lines consisted of 8 EGFR-mutant 6 KRAS-mutant 1 HER4-mutant 1 NRAS-mutant 1 PIK3CA-mutant 1 EML4-ALK fusion 1 HER2-amplified and 6 cell lines without gene alterations listed. Nine of the 17 EGFR-wild type cell lines were sensitive to Ad-REIC. HCC827 and its resistant subline HCC827-GR-high2 showed a similar degree of sensitivity to Ad-REIC. No trend in molecular genotype was seen between the sensitive and non-sensitive cell lines. These results suggested that the effect of Ad-REIC does not depend on a known molecular genotype. .0087900.g001 Sensitivity and predictive factors of sensitivity for Ad-REIC treatment in 25 NSCLC cell lines. The inhibition rates of 25 NSCLC cell lines transfected with Ad-REIC compared to Ad-LacZ are shown as black bar in 20 MOI and white bar in 200 MOI. Thirteen cell lines with over 40% inhibition rate in 20 MOI are defined as highly sensitive and 12 cell lines with lower inhibition rate in 20 MOI are defined as resistant. All the resistant cell lines shows over 40% inhibition rate in 200 MOI. The cell lines are classified into 3 categories based on the GRP and CAR protein expression level as follows; category A (low GRP/high CAR) category B (low GRP/low CAR or high GRP/high CAR) category C (high GRP/low CAR). All 8 highly sensitive cell lines were included in category A and all 5 resistant cell lines were included in category C. Sq; squamous cell carcinoma AD; adenocarcinoma LC; large cell carcinoma ADSQ; adenosquamous cell carcinoma MM; malignant mesothelioma NHF; normal human fibroblast. Hoechst 33342 staining was performed in A549 cells to examine the induction of apoptosis. Apoptotic cells were observed in Ad-REIC-treated A549 cells (a). The mean rate of apoptosis was 22% and it was significantly (p<0.001 by Cochran-Mantel-Haenszel test) increased in comparison with the control Ad-LacZ treatment. .0087900.g002 Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. (a) Induction of apoptosis after in vitro Ad-REIC treatment as examined in A549 cells using Hoechst 33342 staining. The upper panel indicates the appearance of apoptotic cells after Ad-REIC treatment. The lower panel shows the apoptotic rate of A549 cells after the indicated treatment. A total of 5 different fields were examined under a microscope to determine the apoptotic rate. A significant difference was observed (*p<0.001) between the Ad-LacZ and the Ad-REIC treatment. (bar: 100 µm) (b) Western blot analysis for proteins involved in signal transduction triggered by Ad-REIC. Cells were harvested at 48 h after transfection with Ad-LacZ or Ad-REIC at 20 MOI. (c) H460 cells which are resistant to adenovirus transduction were harvested at 48 h after transfection with Ad-LacZ or Ad-REIC at 20100 and 200 MOI. The effect of recombinant REIC/Dkk-3 protein on NSCLC cell lines was examined in 7 randomly selected cell lines (NCI-H522 NCI-H611 NCI-H1299 NCI-H1819 NCI-H2009 PC-9 and A549). The MTS assay showed that REIC/Dkk-3 protein did not affect cell viability in the examined cell lines when administered at a concentration ranging from 1 to 200 µg/mL (data not shown). Expression of GRP78 and CAR in response to Ad-REIC therapy As predictive factors of Ad-REIC sensitivity in NSCLC we examined the expressions of GRP78 and CAR; these expression statuses were correlated with the inhibition of cell viability by Ad-REIC in 13 cell lines. A previous study reported that the overexpression of GRP78 inhibited ER-stress which may be oppositely correlated with the effect of Ad-REIC. CAR expression is tightly associated with the efficacy of adenovirus infection which may be positively correlated with the effect of Ad-REIC. Western blotting was performed and the expression level was quantified as shown in and . The median (range) of GRP78 and CAR expressions were 0.24 (0.0750.98) and 0.60 (0.0802.1) respectively. Based on these data cells with a GRP78 expression level more than 0.25 were defined as High CRP78 expression while those with a GRP78 less than 0.24 were defined as Low GRP78 expression. Regarding the CAR 15 cell lines significantly high level of CAR expression (over 0.50) were defined as High CAR expression while 10 cell lines those with significantly low level of CAR expression (under 0.20) were defined as Low CAR expression. GRP78 expression was low in 8 of the 13 Ad-REIC-sensitive cells (62%) and in 4 of the 12 Ad-REIC-resistant cells (33%). CAR expression was high in 12 of the 13 Ad-REIC-sensitive cells (92%) and in 3 of the 12 Ad-REIC-resistant cells (25%). Next we classified the cell lines into three categories based on the GRP78 and CAR expression statuses; cells with a Low GRP78/High CAR expression were classified as Category A those with Low GRP78/Low CAR or High GRP78/High CAR expression were classified as Category B and those with High GRP78/Low CAR expression were classified as Category C. The high sensitive cell rates were 100% in Category A (8 out of 8 95% confidence interval [CI]: 63100) 42% in Category B (5 out of 12 95% CI: 1572) and 0% in Category C (0 out of 5 95% CI: 052) (). " | 1 |
Genomic characterization of malignant progressionin neoplastic pancreatic cystsMicha«l No«et alIntraductal papillary mucinous neoplasms IPMNs and mucinous cystic neoplasms MCNsare noninvasive neoplasms that are often observed in association with invasive pancreaticcancers but their origins and evolutionary relationships are poorly understood In this studywe analyze samples from IPMNs MCNs and small associated invasive carcinomas from patients using whole exome or targeted sequencing Using evolutionary analyses weestablish that both IPMNs and MCNs are direct precursors to pancreatic cancer Mutations inSMAD4 and TGFBR2 are frequently restricted to invasive carcinoma while RNF43 alterationsare largely in noninvasive lesions Genomic analyses suggest an average window of overthree years between the development of highgrade dysplasia and pancreatic cancer Takentogether these data establish noninvasive IPMNs and MCNs as origins of invasive pancreatic canceridentifying potential drivers of invasion highlighting the complex clonaldynamics prior to malignant transformation and providing opportunities for early detectionand interventionA list of authors and their afï¬liations appears at the end of the paperNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178Pancreatic cancer is a deadly disease with a dismal prognosisthat is predicted to soon be the second leading cause ofcancer death in the United States1 However like otherepithelial malignancies pancreatic cancer arises from noninvasiveprecancerous lesions that are curable if detected and treated earlyenough Although the majority of pancreatic cancers are believedto originate in microscopic precancerous lesions pancreaticintraepithelial neoplasia or PanIN a significant minority arise inassociation with larger cystic neoplasms that can be detectedusing currently available imaging technologies2 These neoplasmswhich includeintraductal papillary mucinous neoplasmsIPMNs and mucinous cystic neoplasms MCNs are frequentlydiagnosed incidentally on abdominalidentifying acohort of atrisk patients with an important opportunity forprevention of invasive pancreatic cancer2 However preventionmust be balanced with potential overtreatment of lowrisk lesionsas pancreatic resection carries significant morbidity and evenoccasional mortality3 There is a critical need to understand themolecular alterations that are associated with the development ofinvasive cancer as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thusrequiring clinical interventionimagingAlthough genomic analyses have been performed on hundredsof invasive pancreatic cancers relatively few noninvasive neoplasms have been analyzed comprehensively Whole exome andtargeted sequencing of small cohorts of IPMNs and MCNs haverevealed driver genes characteristic of each type of cystic neoplasm4 while targeted analyses in larger cohorts have conï¬rmed the prevalence of speciï¬c driver gene mutations thatcorrelate with grade of dysplasia or histological subtype7 Thesestudies have conï¬rmed that hotspot mutations in the oncogenesKRAS and GNAS occur in lowgrade lesions while mutations inother driver genesincluding CDKN2A TP53 RNF43 andSMAD4 occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma8 Targeted nextgeneration sequencing has been used to analyze pancreatic drivergenes in different regions of IPMNs revealing a surprising degreeof intratumoral genetic heterogeneity even with respect to wellcharacterized driver gene mutations9 However the aboveanalyses were based on studies of either single regions from eachneoplasm or a limited number of genes from multiple regionsand did not provide an analysis of the evolutionary relationshipbetween different regions of pancreatic cysts and associatedcancers These limitations highlight the need for comprehensivegenomic analysis of these cysts and associated invasive cancers tounderstand the molecular alterations that underlie the transitionto invasive carcinomaIn this study we perform whole exome sequencing of IPMNsand MCNs and their associated invasive carcinomas Importantlywe focus our study on small invasive carcinomas cm inorder to more precisely analyze the genetic alterations that occurat malignanttransformation in pancreatic tumorigenesis Inaddition in a subset of our samples we perform deep targetednext generation sequencing on a larger set of additional tissuesamples in order to assess mutated loci through entire neoplasmsincluding areas of lowgrade dysplasia highgrade dysplasia andinvasive carcinoma These analyses reveal important features ofpancreatic tumorigenesisincluding evolutionary relationshipsbetween different regions within cystic neoplasms as well asmolecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancerResultsOverall approach In order to dissect the molecular relationshipsbetween noninvasive dysplastic lesions and invasive pancreaticcancers we performed whole exome sequencing of neoplastictissue samples from patients with small invasive carcinomas cm associated with neoplastic pancreatic cysts including patients with IPMNs and patients with MCNs Supplementary Data Whole exome sequencing was performed onone sample from the noninvasive component with highgradedysplasia and one sample from the invasive cancer in each caseand for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencingMatched normalsamples were analyzed by whole exomesequencing in each case to exclude germline variants and toidentify somatic mutations Whole exome sequencing was performed with an average total coverage of distinct coverageof generating TB of sequencing data SupplementaryData In addition to whole exome analyses we performed targetednext generation sequencing of microdissected tissue samplesfrom seven of the above cases six IPMNs and one MCN Forthese targeted analyses we performed laser capture microdissection to isolate neoplastic cells from every available tissue block ofthe noninvasive cyst and cancer specimens Separate sampleswere microdissected based on grade of dysplasia cell morphollocation This resulted in ogy architecture and spatialadditional samples per case The targeted panel analyses includedall mutated loci identiï¬ed in the whole exome sequencing of theseseven cases as well as the entire coding regions of wellcharacterized pancreatic driver genes Supplementary Data The targeted sequencing had an average coverage of distinct coverage of Supplementary Data We developed an integrated mutation calling pipeline torigorously assess mutations in all sample types in our analyses inorder to conï¬dently identify even subclonal alterations in sampleswith low neoplastic purity see Methods Fig Supplementary Data In addition we utilized both on target and off targetreads to examine focal copy number changes as well as loss ofheterozygosity throughout the genome Fig SupplementaryData and From our whole exome sequencing analyses weidentiï¬ed an average of somatic mutations in samples fromnoninvasive components range and an average of somatic mutations in invasive carcinoma samples rangeFig 1a Supplementary Data An average of somatic mutations were shared between the noninvasive andinvasive components while somatic mutations were unique tosamples from noninvasive components and somatic mutationswere unique to samples from invasive cancer Fig 1a We alsoidentiï¬ed an average of ï¬ve shared copy number alterationsbetween noninvasive and invasive components as well as anaverage of one copy number alteration unique to samples frominvasive cancer A similar mean proportion of somatic mutationsand copy number alterations were unique to invasive samples for somatic mutations for copy number alterationsAnalysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCNtumorigenesis In every analyzed case there were multiple sharedmutations between the noninvasive and invasive componentsThese included both driver and passenger mutations indicatingthat they shared a common phylogenetic ancestor Fig 1a b Inaddition accumulation of unique mutations in both noninvasiveand invasive components demonstrated independent evolutionafter the divergence of the subclone that gave rise to the invasiveFig Supplementary Figs S1S18 Analysis ofcanceradditional adjacent lowgrade or highgrade samples from thesame lesions revealed a subset of shared mutations suggestingthat these dysplastic lesions preceded the development of theinvasive carcinoma Fig Supplementary Figs S1 S2 S3 S5and S16 Evolutionary analyses showed a branched phylogeny inNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178asnoitatuMCancerization onlyDuctal cancer onlyMucinous cancer onlySharedIPMN onlyMCN onlyPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMbKRASTP53CDKN2ASMAD4RNF43GLI3GNASMUC16PTPRTTGFBR2ATMCNTN5PIK3CAALKAPCRYR2STK11CTNNB1ERBB4EGFRSF3B1NOTCH1KEAP1ERBB2MYCRETTSC2PrecursorSharedCancerFig Somatic mutations identiï¬ed in matched noninvasive and invasive cancer samples a In each patient sample multiple mutations were sharedbetween the noninvasive and invasive cancer samples gray In addition some mutations were limited to the noninvasive bluegreen while others werelimited to the cancer redpink Darker colors indicate alterations that were likely restricted to one component but where sequencing coverage in thesecond component was limited The proportions of shared and distinct mutations varied between different lesions b Somatic mutations in the mostfrequently mutated genes are categorized as shared between noninvasive and cancer gray limited to noninvasive blue or limited to cancer redMutations in some genes such as KRAS were always shared while others were enriched in samples from noninvasive RNF43 or cancer SMAD4MutationsMTP1MTP2MTP3MTP4MTP5MTP6MTP7MTP8MTP9MTP11MTP13MTP14MTP18MTP19MTP23MTP24MTP26MTP30chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12chr13chr14chr15chr16chr17chr18chr19chr20chr21chr22Copy number log2SamplesLG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationFig Copy number alterations identiï¬ed in matched noninvasive and invasive cancer samples Chromosomal gains red and losses blue are shownfor each chromosome in each patient with noninvasive samples on the left and cancer samples on the rightNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP19HEbefore LCMafter LCMMTP8KRASG12DKRASG12RKRASQ61H1225380275_TGKRASQ61H1225380275_TAKRASG12VTMEM56RWDD3c5662TC [SP]KCNA5A451AC17orf98T142MDUSP27T652TADAMTS12P429LCOL12A1R933HSCAF8G961VTMEM246A372AMCM10E613KPNLIPRP1T465TC13orf35L33LSMAD6L179LGRIN2AS397SAP1G1T798TTP53E171FCHO1c22471GC [SP]LRFN1S632SLTBP4P243LLOHchr17001ABRF645SALPK1R873RLOHchrX275DELchr92182377CDKN2ABSND861NSUPT6HI104TPLEKHF1D258DLOHchr221716LTBRR425GACOT11R306LOR6N1Y120YENPP5Y341YITGB3C562BTKP116TSLC1A7G465DFGF3R104QZBED4P1100LSIM1R192CPHC1S627CMS4A1T41TLOHchr712725SH2D5R199WRNPEPL1G413VGAL3ST2P85PJADE2N352SHHLA1A97VTMEM141Q61QBPTFR296HKLHL22R603RGALEQ261LANKS4BQ368RKRI1S326SOR8I2C240CLRTM2G319VLOHchr97123LAMB3R887LSPTAN1I1484FTP53R175HTissue sourceLG IPMNHG IPMNMUCINOUS CANCERDUCTAL CANCERCANCERIZATIONSequencingTargeted SeqWhole Exome SeqDriver MutationHotspotInactivating_OtherDELLOH µm µmMPT19 LG IPMN µm µm µmMPT19 HG IPMN µm_________ µm µm µmMPT19 ductal cancer µm µm µmMPT19 cancerization T2 T1 µm µmMPT8 LG IPMN µm µm µmMPT8 HG IPMN µm__ µm µm µmMPT8 mucinous cancer T1 T2 TTC39AN83ILRRC8DI741MOBSCNR2837QRGS7A195AOR14A2A156ACCDC13S534SEGFLAMG553VITGA1V27LBDP1N622NKIAA0319V824FRIMS1R211QPRDM13A303VIFNGR1M1PHACTR2A348PGPR85F208FGNED83HHTR7T240MGRM5T946KKRASG12DUNC13CN497SBNIP2D58DCCDC33D354NCLDN9P187PNOTUMR308CPIEZO2R2407QC19orf24G52AMUC16P9201QCCNE1R85WGNASR201HPHF21BA116TTNS1R894QNDUFAF6L320ISLC15A1E26KCLUHR433PRR23AA151TBRD3R313HUSP34R3136HRHOAK27ESIM1A654TNRG1A540VVRK3S129NCCDC166A84TST3GAL4V165MTSPAN17P63PMAD1L1S327SC17orf47E142GCHERPL47ITRPC5E418DZFP37R18WGNASD464NMED12E1497KBAG2T93TRNF43R371CACNG2V255MMAGEB18A63VBRINP3A748APDE1BE401EQRICH2R160RRNF43P370fsINTS1G527VMYOM3G152SPRRT3P407STGFBR2R553HPDZD2I810IFAM120BR646CEPHB4V682MNOBOXR302CCEP164R897QMAB21L1T171MZCCHC14T80MCD68L295LGAS2L2S728LRUNDC3AE49EPHBI122VDNMT1N109NZNF865A721ACASS4S376SKCNJ6R322SLC5A4L390PLRRC8CR355HNPR1R439HPPP1CBV263LBIRC6E4424VIL1Q468QMOGAT3c2359GAUSP20A675TMEX3BS256YMUC13T30AZBTB10R456QSLC26A3V54IC9orf84E8DPPP1R36R303CPLEKHA6Q171SSH3P639PDSCAML1G1252DSLCO1C1R639KENDOVA2VCOL9A1A680TMC2RF228LBPIFB2A427SLOHchr812546DELCDKN2ARNF43P660fsRNF43C471fsRNF43E39fsRNF43M1_G4delRNF43S321RNF43P231LFig Somatic mutations identiï¬ed in MTP19 and MTP8 in targeted and whole exome sequencing We show mutations identiï¬ed in each sampleincluding lowgrade IPMN light blue highgrade IPMN dark blue ductal cancer red mucinous cancer pink and cancerization purple The type ofsequencing analysis targeted or whole exome performed for each sample is indicated in a track on the bottom Representative images of neoplastic tissuestained by hematoxylin and eosin as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP2MTP8MTP3MTP19MTP1MTP5MTP24LG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationMutationsFig Evolutionary reconstruction of samples analyzed by whole exome and targeted sequencing In all cases noninvasive samples bluegreenprecede invasive samples redpink in the evolutionary history In MTP5 different invasive cancer samples are placed in different regions of thephylogeny highlighting multiple independent invasion events in this lesion In MTP19 a sample of cancerization purple has descended from invasivecancer sampleseach case with multiple clonally related but distinct dysplasticsamples from each neoplasm Fig Importantly removal ofdriver gene mutations from these analyses did not significantlyalter the resulting phylogenies indicating that the evolutionaryrelationships are supported by mutations in addition to driveralterations which might be shared by chance Thus the inferredevolutionary relationships between IPMNMCNs and cancersamples are robust as the probability of sharing a nonhotspotmutation due to chance alone is vanishingly small in million while the mean number of shared point mutations was the vast majority of which did not occur in hotspotsAnother potentialexplanation forshared mutationsthis sample impurityinnoninvasive and invasive samples is the presence of a smallnumber of cancer cells contamination in IPMNMCN samplesOur detailed pathological characterization and macro or microdissection minimized the risk ofInaddition variant allele frequencies VAFs of shared mutationsin IPMNMCN and cancer samples can also help to evaluate thelikelihood of such contamination In mutations shared betweenthe IPMNMCN and cancer the mean VAF in the noninvasivesamples was with only of shared mutations having aVAF below in the noninvasive samples These high VAFsindicate that the shared mutations were not the result of a smallnumber of cancer cells contaminating the noninvasive samplesOverall these data provide evolutionary evidence that IPMNs andMCNs were precursors to invasive pancreatic cancer with lowgrade regions usually preceding highgrade regions and ultimatelyresulting in invasive carcinomaDriver genes of IPMNMCN tumorigenesis Through wholeexome and targeted sequencing analyses of IPMNsMCNs andassociated invasive carcinomas we conï¬rmed the high prevalenceof mutations in previously identiï¬ed pancreatic driver genesincluding mutations of KRAS of cases GNAS CDKN2A TP53 SMAD4 and TGFBR2 Fig 1b Somatic mutations were also identiï¬ed in RNF43 which has been previously highlighted for its role as a driver inmucinproducing pancreatic cysts4 Somatic mutations wereobserved at low prevalence in key positions in the PI3K PIK3CATSC2 and WNT APC CTNNA2 CTNNB1 signaling pathwaysas well as in STK11 Fig 1b Supplementary Data Alteration ofthese genes and pathways has been previously reported in afraction of IPMNs471314 The two MCNs analyzed were similarto the IPMNs in the cohort with hotspot mutations in KRAShomozygous deletion of CDKN2A and inactivating mutations inRNF43 among others but as expected these MCNs did not haveGNAS alterations Supplementary Figs S3 S76In addition to driver genes previously reported in IPMNs ourdata provide an opportunity to discover novel drivers of IPMNtumorigenesis We identiï¬ed somatic mutations in the DNAdamage response gene ATM in of lesions including onenonsense mutation Fig 1b Supplementary Data In additionwe identiï¬ed alterations in Hedgehog pathway member GLI3 in of cases Fig 1b Supplementary Data We alsoidentiï¬ed somatic mutations in a previously described hotspot inSF3B1 which encodes a protein critical for RNA splicing Fig 1bSupplementary Data Ampliï¬cations of the wellcharacterizeddriver genes ERBB2 and MYC were each observed in a single caseand have not been previously reported in IPMNs SupplementaryData Other altered genes with a previously unknown role inIPMN tumorigenesis include MUC16 four cases PTPRT fourcases and CNTN5 three cases Fig 1b Intriguingly in onecase an STK11 mutation was found in combination with bialleliclosstheATM lossand cancerspeciï¬c biallelic KEAP1NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178combination ofreported in lung cancers Supplementary Fig S315these three mutations has previously beenAlthough KRAS mutations occur in the majority of IPMNs andare thought to initiate tumorigenesis in these lesions two IPMNslacked mutations in this gene One case contained a hotspotmutation in codon of GNAS another potential initiator ofIPMN tumorigenesis as well as alterations in TP53 and RNF43Supplementary Fig S15 The other case lacked mutations in anyof the frequently altered pancreatic driver genes but containedhotspot mutations in both CTNNB1 S45P and SF3B1 H662QSupplementary Fig S10 These cases highlight alternativepathways of initiation and progression in IPMNs lacking KRASmutationsOrder of genetic alterations in IPMNMCN tumorigenesis Ourmultiregion sequencing approach of IPMNsMCNs and associated invasive carcinomas provided insights into the order ofspeciï¬c genetic alterations in pancreatic tumorigenesis In ofthe cases at least one somatic mutation in the initiating drivergenes KRAS and GNAS was shared between the noninvasivecomponent and associated invasive cancer with the remainingcase lacking mutations in these genes Somatic mutations in TP53and CDKN2A were also shared in the noninvasive componentand associated invasive cancer in the majority of cases In contrast SMAD4 had alterations conï¬ned to the invasive carcinomain three cases and was shared between noninvasive and invasivesamples in four cases Fig 1b The majority of SMAD4 alterations in all sample types were biallelic including in noninvasive samples and in invasive samples SupplementaryData Alteration of TGFBR2 which functions in the samesignaling pathway as SMAD4 was also restricted to the cancer inone case Fig 1b The other genes with mutations restricted tothe invasive cancer CDKN2A CNTN5 PIK3CA KEAP1 andRET only had this pattern in a single sample Fig 1bOur study also identiï¬ed driver mutations in subclones ofnoninvasive neoplasms that diverged from and were not presentin invasive cancer These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumorsuppressor geneseg PIK3CA pE545K CTNNB1 pS45FSMAD4 pE33fs and multiple inactivating RNF43 mutations inpatient MTP3 Supplementary Fig S3 Mutations in RNF43were a particularly striking ï¬nding in these cases as somenoninvasive components contained several different RNF43mutations each limited to a small number of sections and noneinvolving the invasive cancer Fig Supplementary Fig S3 Inaddition to heterogeneity in RNF43 in early lesions we alsoidentiï¬ed two cases with multiple mutationsin KRAS inprecursor lesions of which only one was present in the invasivecancer For example in MTP19 KRAS pG12V was present in themajority of IPMN samples as well as all the invasive cancersamples but there were an additional four other KRAS mutationsall occurring in hotspot positions that were present in a smallnumber of sectionsin lowgrade IPMN samples Fig Intriguingly these three lowgrade IPMN regions shared nomutations with the invasive cancertheyrepresented genetically independent clonessuggesting thatNotably while there were often many differences in thesomatic point mutations identiï¬ed in the matched noninvasiveand invasive samples the copy number proï¬les were quite similarbetween IPMNMCNs and invasive cancers Fig Supplementary Data and the proportion of copy number alterationsunique to cancer samples was similar to that observed forsomatic mutations While homozygous deletion of some genesoccurred in the invasive cancer but notthe noninvasivecomponent such as CDKN2A in MTP8 Fig analyses ofchromosomal gains and losses through assessment of allelicimbalance revealed that an average of of the genome wassimilar in copy number in matched noninvasive and invasivesamples Fig Supplementary Data Insights into pancreatic neoplasia revealed by sequencing Thesamples analyzed by targeted sequencing were characterizedmorphologically and meticulously isolated using laser capturemicrodissection Even with this process we identiï¬ed samples intwo cases that were characterized morphologically as IPMNs butthrough genomic and evolutionary analyses were determined tobe identical to or descendants of the associated invasive cancersFor example in MTP19 some of the samples originally identiï¬edmorphologically as noninvasive IPMN and T1 sharedall the mutations present in the invasive cancer sample T2 andcontained additional mutations suggesting that these samplesdescended from the cancer Figs Evolutionary analysessuggested that some of the morphologically identiï¬ed IPMNsamples in this case as well as MTP1 actually representedintraductal spread of invasive carcinoma also referred to ascancerization of the ducts In these cases after invading thestroma the carcinoma invaded back into and colonized the cystsuch that it was morphologically indistinguishable from IPMNwith highgrade dysplasiaIn one case MTP5 we also identiï¬ed an interesting pattern ofmultifocal invasion of the carcinoma In this case we analyzedï¬ve different samples from invasive cancerthree samples wereisolated from a mucinous carcinoma and two samples wereisolated from a ductal carcinoma Based on evolutionary analysesof the patterns of shared and distinct mutations in the cancersand IPMNs we conclude thatthere were multiple separateinvasion events in this lesion as represented by the mutationsshared between the invasive cancers and noninvasive componentsas well as those that were unique to the speciï¬c invasive cancersFig Supplementary Fig S5As our study represents the largest cohort of comprehensivelysequenced IPMNsMCNs we also analyzed mutational signaturesin our dataset Intriguingly our data contrast somewhat with themutational signatures previously reported in pancreatic ductaladenocarcinoma PDAC1617 Like PDAC the most prominentmutational signature was associated with age Signature 1Awhich was identiï¬ed in almost every case SupplementaryFig S19 However we also identiï¬ed signatures associated withAPOBEC enzymes four cases smoking three cases andmismatch repair deï¬ciency cases Although smoking isconsidered a risk factor for pancreatic cancer until now themutational signature associated with smoking has not beenreported in pancreatic neoplasia18Evolutionary timeline of highgrade IPMN to PDAC To estimate the time between the development of highgrade IPMN andPDAC we evaluated Bayesian hierarchical models for the numberof acquired mutations under a range of possible mutation ratesThese models estimate the time interval between a founder cell ofa PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division timesare constantseeMethods We performed this analysis on the paired WES datafrom of our cases Supplementary Fig S20 We excludedMTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such we lacked WESdata from an IPMN sample in this case In the analyzed casesthe average median time to progression from IPMN to PDAC was years but the models showed a bimodal distribution Thismedian time was nearly years for patients but nearly yearsthis period of developmentthroughoutNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178for patients with more than acquired mutations highlightingpotential variability in progression time between patients Forexample in patient MTP1 most models suggested an average of years between the development of the IPMN and the PDAC CI years In contrast for patient MTP2 with additional mutations acquired in the PDACthe transitionappears to have been slower with an average estimate of years CI years from the Bayesian models Overall theseanalyses suggest that for most patients there is a significantwindow of time between development of highgrade dysplasiaand pancreatic cancer providing an opportunity for surveillanceand interventionDiscussionThis study representsthe largest dataset of whole exomesequencing of IPMNs and MCNs to date Importantly our dataestablished that both IPMNs and MCNs are direct precursors ofinvasive pancreatic cancer Fig This conclusion has beenpreviously suggested by the morphological relationship betweenthe noninvasive neoplasms and invasive cancer on traditionalhistologic sections as well as shared driver gene mutations intargeted sequencing studies679 However the presence ofmany shared driver and passenger mutations clearly demonstrated the common origin of IPMNsMCNs and invasive pancreatic cancers in our study and evolutionary analyses revealedthat dysplastic lesions precede invasive cancers Evolutionaryanalyses suggested that highgrade noninvasive lesions occur over years before invasive carcinoma providing a window ofopportunity for early detection and interventionIn this study we identiï¬ed somatic mutations in driver genesthat had not been previously implicated in IPMNsMCNs Forexample we identiï¬ed alterations in the DNA damage responsegene ATM in of the analyzed cases Germline mutations inATM have been recently reported in patients that developedIPMNs highlighting the potential importance of this gene inIPMN risk19 In addition mucinous colloid carcinomas aresignificantly more common than typical ductal carcinomas inpatients with germline ATM mutations further highlighting thelink between mutations in this gene and IPMNs20 Although theATM gene is large potentially increasing the likelihood of passenger or artifactual mutations even larger genes such as TTNhad a lower mutation prevalence suggesting that at least some ofthe alterations identiï¬ed in ATM are likely to be bona ï¬desomatic mutations Somatic mutations in GLI3 which encodes acomponent of the Hedgehog signaling pathway were identiï¬ed in of cases Somatic mutations in GLI3 were recently reportedin a distinct morphological variant of pancreatic carcinomaundifferentiated carcinoma with osteoclastlike giant cells aswell as at a low prevalence in sporadic PDAC suggesting that theimportance of GLI3 mutations and its signaling pathway inpancreatic tumorigenesis may extend beyond IPMNsMCNs21The hotspot mutations in SF3B1 which encodes a protein criticalfor RNA splicing are also potential drivers in the IPMN pathwayHowever somatic mutations in this gene have been reported in avariety of other neoplasms including hematologic malignanciesand uveal melanoma24We highlight somatic alteration of the SMAD4 pathway as aputative driver of progression to invasive cancer as mutations inSMAD4 or TGFBR2 occurred only in invasive cancer samples in of the cases analyzed SMAD4 was the only gene with cancerspeciï¬c mutations in more than one case highlighting thepotentially unique role this gene plays in pancreatic carcinogenesis This role has been previously suggested by next generationsequencing of highgrade PanINs showing an absence of SMAD4mutations in precancerous lesions as well as cancerspeciï¬cSMAD4 mutationsreported in a paired PanINcarcinomaanalyses2728 Loss of SMAD4 expression limited to invasivecarcinomas has been reported in MCN and IPMNassociatedinvasive cancers and targeted sequencing of a small number ofIPMNs and matched cancers identiï¬ed a single case with aSMAD4 mutation occurring only in the cancer72829 In our datathere were also four cases where mutations in SMAD4 wereshared between noninvasive and invasive cancer samples and twowhere SMAD4 mutations were limited to the noninvasive component Although our wholeexome approach could not detect alltypes of SMAD4 alterations such as rearrangements or epigeneticchanges the majority of SMAD4 mutations observed in bothnoninvasive and invasive components affected both alleles Takentogether this suggests that the role of SMAD4 mutations may notbe universal and may depend on other factors including cellintrinsic such as somatic mutations in other driver genes andcell extrinsic such as stromal and immune microenvironmentmechanismsAlthough some of our cases had SMAD4 mutations limited tothe invasive cancer most of the IPMNMCNassociated cancerslacked driver gene alterations that were associated with invasivedisease suggesting that malignant progression is not universallydriven by point mutations Previous studies have speciï¬callydemonstrated the importance of copy number alterations andchromosomal rearrangements in pancreatic tumorigenesis30 Wedid not identify large differences in the copy number proï¬lesbetween noninvasive components and associated invasive cancers suggesting that global genomic instability may be importantas an early feature of tumorigenesis but is not likely to drivemalignant transformation in many casesOur study also revealed prevalent genetic heterogeneity indriver gene mutations in early lesions demonstrating morecomplex processes than previously suggested by traditional lineartumorigenesis models Similar to our recently reported polyclonalorigin of IPMNs12 we identiï¬ed multiple independent clonesinitiated by distinct KRAS mutations in two cases in the currentstudy In addition our study identiï¬ed multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones apattern previously observed by our group and not shared by othergenes in our whole exome sequencing analyses1231 In most casesRNF43 mutations were enriched in noninvasive components andabsent from the associated invasive cancers More generally weobserved multiple instances of clear driver mutations includinghotspot mutations in oncogenes as well as inactivating mutationsin tumor suppressor genes that were limited to the noninvasivecomponents and not present in the associated invasive cancerThus these mutations occur and clonally expand in the IPMN orMCN but are not present in the subclone that subsequentlyinvades Such independent evolution of premalignant lesions hasbeen observed in other an sites and does not diminish theconclusion of our evolutionary analyses that IPMNsMCNs aretheseprecursors ofobservations suggest unique selective processes at different timepoints in tumorigenesis such that mutations selected in theprecancerous lesion are not selected for or are even selectedagainst in the invasive cancerinvasive pancreatic cancer32 RatherIn addition to these observations about clonal evolution innoninvasive lesions our data also provide genetic evidence formultiple underappreciated processes in pancreatic neoplasiaFirst we provide genetic evidence for intraductal spread ofinvasive carci | 2 |
high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with Åukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation 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 give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in omics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a Åukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a symptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that symptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie Åukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see membership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in fuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of theÅukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see fuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see dendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 [ ] and lettingd be a dissimilarity relation which is obtained from r bydx y rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie Åukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute s if sielse ci sti si ª si ¦ sicid8 si replace si with si si and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ¤ i ¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let a be the identity matrixb for each ci calculate e a a ª a ¦ cic if a is not changed a a and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the Ï2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the Ï2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have Ï2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logranks test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logranks test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ] to determine the number of clusters for a method we used the elbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ] 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrankstest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean log10 of logranks testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg high medium low in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of log10 of logranks testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes ac results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliparthenope tecniche di machine learning e soft computing per lelaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli parthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason ciaramella a cocozza sand clustering of genomic data neural netw iorio f miele g napolitano f pinelli m raiconi g tagliafer | 0 |
This study was performed assess the clinical outcomes of elderly patients withosteoporotic femoral neck fractures FNFs AOOTA 31BC treated by initial uncementedtotal hip arthroplasty UTA or cemented total hip arthroplasty CTAMethods This study involved consecutive elderly patients with osteoporotic FNFs AOOTA31BC treated by initial UTA or CTA in our medical centre from to The primaryoutcomes were the Harris hip score HHS and the rates of revision loosening periprostheticfracture and dislocationResults In total patients were included in the final analysis UTA n¼ CTA n¼ The mean followup duration was months range months The mean HHS was1Department of Microsurgery Trauma and Hand SurgeryThe First Affiliated Hospital Sun Yatsen UniversityGuangzhou China2Department of Pediatrics The First Affiliated HospitalSun Yatsen University Guangzhou China3Department of Orthopaedics The First AffiliatedHospital Sun Yatsen University Guangzhou China4Department of Orthopaedics The Third PeoplesHospital of Wuxi Jiangsu Province The Affiliated Hospitalof Jiangnan University Wuxi China5Department of Urology The First Affiliated Hospital SunYatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsJunxing Ye Department of Orthopeadics The ThirdPeoples Hospital of Wuxi Jiangsu Province The AffiliatedHospital of Jiangnan University No Xingyuan NorthRoad Liangxi District Wuxi Jiangsu ChinaEmail yejunxing0514163comJintao Zhuang Department of Urology The First AffiliatedHospital Sun Yatsen University No Zhongshan 2ndRoad Yuexiu District Guangzhou ChinaEmail brianzg86163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical Research 06 for UTA and 06 for CTA Significant dissimilarities were detected in therates of revision loosening and periprosthetic fracture between UTA and CTA vs vs and vs respectively A significant difference was also detected inthe probability of revision between the two groupsConclusion Elderly patients with osteoporotic FNFs AOOTA 31BC treated with CTA showgreater improvements in functional outcomes and key orthopaedic complications than thosetreated with UTAKeywordsFemoral neck fracture arthroplasty outcome complication retrospective osteoporosisDate received December accepted July Introduction31BCAOOTAfemoral neck fracturesManagement ofin elderlyFNFspatients is still undergoing considerableresearch12 Uncemented total hip arthroplasty UTA or cemented total hip arthroplasty CTA for displaced FNFs tendsto be a recognised surgical strategy3Comparisons between UTA and CTA forelderly individuals with an FNF generallyfavour CTA this is primarily attributed tothe exceptional clinical outcomes of CTA interms of relieving pain and improving dailyactivities as well as the higher rate of majororthopaedic complications ie revisionloosening periprosthetic fracture and dislocation associated with UTA4 Howeverrecent studies of UTA in elderly individualshave demonstrated encouraging shorttermclinical outcomes67 Moreover cementedprosthesis syndrome tends to occur morefrequently in CTA than UTA8 Cementedprosthesis syndrome theoretically poses asignificantlifealthough the speciï¬c probability of thisthreat has not been calculated89 Hencewhether to utilise CTA for elderly individuals may present the clinician with a dilemma9 The lack of consensus regarding whichtechnique UTA or CTA is preferable fortreating FNFs AOOTA 31BC in elderlyindividuals is related to the remarkableto the patientsthreatdistinction in clinical outcomes betweenthe two types of implants610Most previousconcentrated medicalstudies have involvedhighlycentresand several brands of prostheses356Additionally shortterm followup is commonplace in these studies To overcomethese drawbacks of previous studies and tocompare the midterm results of the twoprostheses we assessed the clinical outcomesof elderly patients with osteoporotic FNFsAOOTA 31BC treated with initial UTAor CTA with a mean followup of yearsMaterials and methodsStudy populationtheandrequirementEthical approval was obtained from theFirst Afï¬liated Hospital of Sun YatsenUniversityforinformed consent was waived by theInvestigational Review Board Consecutiveelderly individuals with the principle diagnosis of an FNF AOOTA 31BC whounderwentinitial UTA or CTA from March to March and forwhom detailed information was availablethroughoutidentiï¬edfrom the orthopaedics department of theFirst Afï¬liated Hospital Sun YatsenUniversity The manufacturer details offollowup were 0cMao et alTable Manufacturer details of stems and cupsemployed in the arthroplasty proceduresProcedureStemCupCORAIL1Exeter3Exeter3REFLECTIONUncemented2UTA n¼ CTA n¼ 1DePuy Synthes Warsaw IN USA2Smith Nephew London UK3Stryker Corporation Kalamazoo MI USAUTA uncemented total hip arthroplasty CTA cementedtotal hip arthroplastyclosed FNFsthe stems and cups employed in the arthroplasty are shown in Table The surgicalprocedure and postoperative rehabilitationprotocol were described in our previouslypublished study11 The inclusion criteriawereAOOTA 31BCactive and cognitively intact patients ageof 15 years independently mobile priorto the injury and a bone mineral densityTscore of at the femoral neck Themajor exclusion criteria were multiple fractures or contralateral limb fractures pathological FNFs lower limb dyskinesia priorto surgery cancer planned surgery polytrauma severe comorbidities eg thyroiddisorder with calcium and phosphorusmetabolism disorderwithcomplications drug abuse affecting bonehealing or bone metabolism early interruption of followup months and cognitive impairment Clinical and radiographicassessments were performed at and months after surgery and every monthsthereafter The primary outcomes were theHarris hip score HHS and the rates ofrevision loosening periprosthetic fractureand dislocationdiabetesStatistical analysisRevision was deï¬ned as partial or completereplacement of the prosthesis12 Looseningof the acetabulum andor stem componentstomographycompared usingas well as dislocation were deï¬ned based ona previous description13 Periprostheticconï¬rmed by Xray orfracture wascomputedexaminationContinuous data ie age bone mineraldensity body mass index followup timeand HHS wereanindependentsamples t test and categoricalvariables ie sex side [leftright] fracturetype comorbidities mechanism of injuryAmerican Society of Anesthesiologists classiï¬cation and major orthopaedic complications were compared using the chisquaretest or the MannWhitney test A KaplanMeier survival curve was used to assess theprobability of revision Hazard ratios werecalculated using a Cox proportional hazards model The signiï¬cance threshold wasset at p The statistical analysis wasperformed using SPSS IBM CorpArmonk NY USAResultsIn total consecutive patientsarthroplasties with an FNF AOOTA31BC who underwentinitial UTA orCTA met our inclusion criteria and wereincluded for analysis Figure The meanfollowup duration was months range months The patients mean age was 06 years for UTA and 06 for CTA The mean body mass indexfor UTA andwas 06 kgm2 for CTA The patientsbaselinesimilarbetween the two groups Table 06 kgm2characteristics werePrimary outcomesImproved functional outcomes were notedin both groups as indicated by the HHSUTA 06 prior to surgery vs 06 at ï¬nal analysis p CTA 06 prior to surgery vs 06 at ï¬nal analysis p At the end of followup the HHS was 0cJournal of International Medical ResearchFigure Flow diagram exhibiting methods for identifying patients with FNFs AOOTA 31BC whounderwent an initial UTA or CTAFNFs femoral neck fractures UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysignificantly different between the twoUTA 06 vs CTAgroups 06 p¼ and patients whounderwent CTA had higherfunctionalscores than those who underwent UTANo distinct betweengroup differenceswere observed at any time point before months postoperatively Table No early year postoperative complications were detectedincluding revisionloosening periprosthetic fracture or dislocation The rate of key orthopaedic complications wasfor UTAand for CTA p Table In the UTA group patients underwent revision UTA developed prosthesis loosening developed periprosthetic fracturesand developed prosthesis dislocation In the CTA group patientsunderwent revision UTA developed prosthesis loosening developed periprosthetic fractures and developed prosthesis dislocation The average time interval from the initial surgery torevision UTA was months range months for UTA and months rangefor CTA p¼ monthsSigniï¬cant differences in revision looseningand periprosthetic fracture were observedbetween the UTA and CTA groups revision vs p¼ loosening 0cMao et alTable Patient demographics and outcomesVariableSex femalemaleAge yearsBMI kgm2BMDSide leftrightFNFs AOOTA type31B31CComorbiditiesDiabetes mellitusHypertensionCerebrovascular diseaseMechanism of FNFsTraffic accidentFallingTamping accidentASA classificationUTA n¼ 06 06 06 06 HHS prior to surgeryData are presented as n n or mean 06 standard deviationpvalueCTA n¼ 06 06 06 06 UTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty HHS Harris hip score ASA AmericanSociety of Anesthesiologists BMI body mass index BMD bone mineral density FNFs femoral neck fracturesTable Comparison of hip functional scoresMonths postoperativelyFinal followupData are presented as mean 06 standard deviationUTA n¼ 06 06 06 06 06 06 06 06 CTA n¼ 06 06 06 06 06 06 06 06 pvalueStatistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty vs p¼ and periprosthetic fracture vs p¼ respectively A significant difference in theprobability of revision was also detectedbetween the groups hazard ratio interval conï¬dencep¼ Figure No significant difference was found in the rate of prosthesis dislocation between the UTA and CTA groups vs respectively 0cJournal of International Medical ResearchTable Rates of key orthopaedic complicationsComplicationsProsthesis revisionProsthesis looseningPeriprosthetic fractureDislocationUTA n¼ CTA n¼ pvalueData are presented as n Statistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplastyFigure KaplanMeier curves showing probability of revision after primary surgery HR was calculatedper the Cox proportional hazards model with age sex American Society of Anesthesiologists classificationbody mass index bone mineral density and femoral neck fracture type as covariates and surgery as the timedependent factorHR hazard ratio CI confidence interval UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysurgeryProbableDiscussionThis review characterised the outcomes of asolitary brand of a total hip arthroplastyimplant during a mean followup of years in elderly patients with osteoporoticFNFs AOOTA 31BC The data demonstrated that patients treated with CTAshowed better improvements in functionaloutcomes and key orthopaedic complications than those treated with UTAThe current ï¬ndings are consistent withprevious studies361415 Although the betterfunctional outcomes and lower rates ofrevision loosening periprosthetic fractureand dislocation are apt to favour CTA nosignificant betweengroup differences in theHHS were detected during the initial yearsafterexplanationsinclude the timedependent clinical efï¬cacyof the implants and the properties of theprostheses34616 Whether UTA or CTA ispreferable in elderly patients with a discontroversial6917placed FNF remainsA recent retrospective study involving patients with an FNF AOOTA 31B whounderwent primary unilateral UTA or CTAshowed that the mean HHS was 06 for CTA and 06 for UTAp¼ A singleblinded randomisedcontrolled trial CHANCEtrial involving individuals treated with an uncementedor cemented tapered hydroxyapatitecoatedfemoral stem and a cemented cup demonstratedtaperedhydroxyapatitecoated femoral stem andcementedthatthe 0cMao et alcemented cup provided better functionalresultsthan the uncemented taperedhydroxyapatitecoated femoral stem andcemented cup16ratesrevisionthe bone microstructureIn the present study the KaplanMeiersurvival curve demonstrated that at the2year analysis neither group showed evidence of a target event and no significantbetweengroup differences were found inofthelooseningfracture or dislocationperiprostheticNeverthelessit would be interesting toexplore whether the prosthesis materialinï¬uencesthepeak effect and the duration of the effectand if so what mechanisms affect the bonemicrostructure and whether there is a wayto change the outcome by blocking thiseffect during a 2year followup We currently have one option for prevention oravoidance of adverse events and thesechanges in treatment strategies may play akey role in improving the clinical resultsif the effects of the prosthesis materialitself cannot be blocked18 There is still alack of consensus on standards for prosthesis revision in this context19When assessing the impact of CTA onthe target events we did not observe anincreased incidence of severe orthopaediccomplications other than the complicationsmentioned in this study In one systematicreview the authors presumed that CTA wassuperior to UTA with respect to functionalscores and tolerable orthopaedic complications20 We obtained analogous results interms of hiprelated complications andfunctional scores Multicentre hip arthroplasty data indicate that UTA remains ahighrisk factor for late revision looseningand periprosthetic fractures810 The notabledissimilarities in the results of these variousstudies on hiprelated complications may belargely attributed to the design of the prosthesis prosthesis size and material selection and the surgeons experience46affected theThis study has several limitations It hada small sample and its retrospective designis association with some inherent disadvantages We did not stratify the patientsaccording to fracture type or sex In addition the potential comorbidities betweengroups were not well exposed and compared The statistical power used to addressdifferences between the groups was insufï¬cient Differences in the patients baselinedata may haveresultsFurthermore our analysis did not determine whether the deaths were instigatedby bone cement The risk of hiprelatedcomplications was not analysed The survivalcurve of other prosthesisrelatedcomplications was estimated using theKaplanMeier method and competitiverisks ie death could have affected thesurvival of the prosthesis Patients whodied lostrevisionHence the revision rate might have beenunderestimated during this long followupwith a fairly high mortality ratethe opportunity forevidenceIn conclusion the ï¬ndings described inthe current review uphold an increasingbody ofthat CTA provideshigher functional scores and lower rates ofhiprelated complications than does UTAin elderly patients with osteoporotic displaced FNFs AOOTA 31BC In thiscontext we recommend CTA for the treatment of such FNFs Our ï¬ndings may beconducive to alleviating continuing debateregarding which prosthesis UTA or CTAis more suitable for the elderly populationA future prospective study may be essentialto conï¬rm whether our conclusion continues to be acceptable as the followup timeincreasesDeclaration of conflicting interestThe authors declare that there is no conï¬ict ofinterest 0cFundingThis research received no speciï¬c grant from anyfunding agency in the public commercial ornotforproï¬t sectorsORCID iDsWeiguang YuGuowei HanReferencesorcid00000001orcid00000003 Chammout G Muren O Laurencikas Eet al More complications with uncementedthan cemented femoral stems in total hipreplacement for displaced femoral neck fractures in the elderly a singleblinded randomized controlled trial with patientsActa Orthop Gjertsen JE Lie SA Fevang JM et al Totalhip replacement after femoral neck fracturesin elderly patients results of fracturesreported to the Norwegian ArthroplastyRegister Acta Orthop Hailer NP Garellick G and Karrholm JUncemented and cemented primary totalhip arthroplastyin the Swedish HipArthroplasty Registerof operations Acta Orthop evaluation Makela KT Eskelinen A Paavolainen Pet al Cementless total hip arthroplasty forprimary osteoarthritis in patients aged years and older results ofthe mostcommon cementless designs compared tocemented reference implants in the FinnishArthroplasty Register Acta Orthop YliKyyny T Sund R Heinanen M et alCemented or uncemented hemiarthroplastyfor the treatment of femoral neck fracturesActa Orthop Yang C Han XL Wang J et al Cementedversus uncemented femoral component totalhip arthroplasty in elderly patients with primary osteoporosisretrospective analysiswith 5year followup J Int Med Res Journal of International Medical Research Solarino G Zagra L Piazzolla A et alceramiconResults of consecutiveceramic cementless hip arthroplastiesinpatients up to years of age a yearsof followup study J Arthroplasty S232S237 Hanly RJ Whitehouse SL Lorimer MFet al The outcome of cemented acetabularcomponents in total hip arthroplasty forosteoarthritis deï¬nes a proï¬ciency thresholdresults of cases from the AustralianOrthopaedic Association NationalJointReplacement Registry J Arthroplasty Imam MA Shehata MSA Elsehili A et alContemporary cemented versus uncementedhemiarthroplasty for the treatment of displaced intracapsular hip fractures a metaanalysis offortytwo thousand fortysixhips Int Orthop Jameson SS Baker PN Mason J et al Thedesign of the acetabular component and sizeof the femoral head inï¬uence the risk of revision following singlebrand cementedhip replacements a retrospective cohortstudy of mediumterm data from a nationaljoint registry J Bone Joint Surg Br 94B Zeng XS Zhan K Zhang LL et alConversion to total hip arthroplasty afterfailed proximal femoral nail antirotationsor dynamic hip screw ï¬xations for stableintertrochanteric femur fractures a retrospective study with a minimum followupof years BMC Musculoskelet Disord Johnson RL Abdel MP Frank RD et alImpact of frailty on outcomes after primaryandarthroplastyJ Arthroplasty 64e5revisiontotalhip Chen KH Tsai SW Wu PK et al Partialcomponentretained twostage reconstruction for chronic infection after uncementedtotal hip arthroplasty results of sixteen casesafter ï¬ve years of followup Int Orthop DeAngelis JP Ademi A Staff I et alCemented versus uncemented hemiarthroplasty for displaced femoral neck fracturesa prospective randomized trial with early 0cMao et alfollowup J Orthop Trauma Rolfson O Donahue GS Hallsten M et alPatientreported outcomes in cemented anduncemented total hip replacements Hip Int Chammout G Muren O Boden H et alCemented compared to uncemented femoralstems in total hip replacement for displacedfemoral neck fractures in the elderly studyprotocol for a singleblinded randomizedCHANCEtrial BMCcontrolled trialMusculoskelet Disord Liu T Hua X Yu W et al Longtermfollowup outcomes for patients undergoingprimary total hip arthroplasty with uncemented versus cemented femoral components a retrospective observational studywith5year minimum followupJ Orthop Surg Res a Engesaeter LB Espehaug B Lie SA et alDoes cement increase the risk of infection inprimary total hip arthroplasty Revisionrates in cemented and uncementedprimary THAs followed for years inthe Norwegian Arthroplasty Register ActaOrthop Schmale GA Lachiewicz PF and Kelley SSEarly failure of revision total hip arthroplasty with cemented precoated femoralcomponents Comparison with uncementedcomponents at to years J Arthroplasty Azegami S Gurusamy KS and Parker MJCemented versus uncemented hemiarthroplasty for hip fractures a systematic reviewof randomised controlled trials Hip Int 0c' | 2 |
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu | 0 |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | 2 |
test the hypothesis that levobupivacaine has antitumour effects on breast cancer cellsResults Colony formation and transwell assay were used to determine breast cancer cells proliferation Flow Cytometry annexin V and PI staining was used to investigate breast cancer cells apoptosis The effects of levobupivacaine on cellular signalling and molecular response were studied with Quantitative Polymerase Chain Reaction and western blot Induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates The results of the western blot and Quantitative Polymerase Chain Reaction indicated activation of active caspase and inhibition of FOXO1 The results of the flow Cytometry confirmed that levobupivacaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells Quantitative Polymerase Chain Reaction and Western blot analysis showed increased p21 and decreased cyclin D Quantitative Polymerase Chain Reaction and western blot analysis showed that levobupivacaine significantly increased Bax expression accompanied by a significant decreased Bcl expression and inhibition of PI3KAktmTOR signalling pathway These findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitroKeywords Levobupivacaine Proliferation Invasion Apoptosis Breast cancerIntroductionBreast cancer is one of the most recorded cancer illness among women [] In the United States it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments []Correspondence yanqiu63126com wqp89163com Department of Anaesthesiology Dalian Medical University Dalian China Department of Biochemistry and Molecular Biology Dalian Medical University Dalian ChinaFull list of author information is available at the end of the Molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment [] The mechanisms of the PI3KAktmTOR signalling pathway have present some promising targets for cancer treatments This signalling pathway hinders the functions of several tumour suppressor genes such as Bad GSK3 FOXO transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [] Suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deathThe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited [] 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 0cKwakye a0et a0al BMC Res Notes Page of At the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor EGFR which is a potential target for antiproliferation in cancer cells [] Evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [] To the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined The present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsMain textMaterials and a0methodsEthics statementThe ethical committee of the Dalian Medical University First Affiliated Hospital approved for this study to be carried outCell cultureWe purchased MCF7 and MDAMB231 breast cancer cells from the ATCC Beijing Zhongyuan limited China We maintained the MCF7 and MDAMB231 cells with highglucose DMEM or DMEMF12 Gibco USA medium The medium was supplemented with fetal bovine serum FBS Gibco USA penicillin a0unitsml and streptomycin a0µgml TransGen Biotech China to maintain the cells The MCF7 and MDAMB231 cells were then maintained in an incubator at a0ºC humidified air with CO2 atmospheric condition The cells were routinely subcultured subsequentlyAntibodies and a0reagentsEPR17671 Akt monoclonal Antibody Abcam China Y391 mTOR Polyclonal Antibody Abcam China A2845 Bcl2 Polyclonal Antibody ABclonal Technology A11550 Bax Polyclonal Antibody ABclonal Technology A0265 PIK3CA Polyclonal Antibody ABclonal Technology A2934 FOXO1 Polyclonal Antibody ABclonal Technology EPR21032 Active caspase monoclonal Antibody Abcam China AFO931 Cyclin D1 Polyclonal Antibody Affbiotech China AF6290 p21 Polyclonal Antibody Affbiotech China AntimTOR phospho S2448 Antibody Abcam China PA517387 PhosphoPI3K p85p55 Tyr458 Tyr199 Polyclonal Antibody ThemoFisher Scientific PosphopanAKT123 Ser473 Antibody Affbiotech China Peroxidaseconjugated goat antirabbit IgG Proteintech China PRAP antibodies Proteintech China and GAPDH antibodies Proteintech ChinaCell viability assay and a0IC50We determined the MCF7 and MDAMB cells viability using CCK8 assay Levobupivacaine at a concentration of or a0mM was used to treat MCF7 and MDAMB cells plated in 96well plates a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °C with CO2 The rest of the procedures used for the CCK8 assay were the same as described elsewhere []Flow cytometryAnnexin V and propidium iodide PI staining assay were used to investigate the apoptosis of MCF7 and MDAMB cells following levobupivacaine treatment After treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min The MCF7 and MDAMB treated cells were again suspended with Binding Buffer and then a0 µl of fluorochromeconjugated annexin V SigmaAldrich Saint Louis USA was added into a0µl of the cell suspension to stain intracellular phosphatidylserine PS The cells were then incubation in a dark under room temperature The cells were again suspended and a0 µl propidium iodide staining solution SigmaAldrich Saint Louis USA added into a0µl of the cell suspension We detected the percentage of the apoptotic cells via FlowJo software Treestar Ashland USA and Flow cytometry FACS Calibur Becton Dickinson and Sunnyvale CA USAQuantitative polymerase chain reaction qPCRThe procedures used for the qPCR were the same as previously described [] The primers sequences were BAX 5TGG CAG CTG ACA TGT TTT CTG3 F 5TCC CGG AGG AAG TCC AAT G3 BCL2 5ACG GTG GTG GAG GAG CTC TT3 F 5GCC GGT TCA GGT ACT CAG TCAT3 R p21 5GCG ACT GTG ATG CGC TAA TG3 F 5GAA GGT AGA GCT TGG GCA GG3 R GAPDH ²CAT GTT CGT CAT GGG TGT GAA² F ²GGC ATG GAC TGT GGT CAT GAG3² RR Western blotAt the log phase of treated MCF7 and MDAMB cells growth we harvested the cells and then washed twice with icecold PBS The rest of the procedures used for the western blot were the same as described elsewhere []Colony formation assayThe procedures used for the colony formation assay were the same as previously described [] 0cKwakye a0et a0al BMC Res Notes Page of Transwell assayThe MCF7 and MDAMBA231 cells that were pretreated with different dose of Levobupivacaine a0mM for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores Merck KGaA P18P01250 China The procedures used for the Transwell assay were the same as previously describe []Data analysisValues were expressed as the mean ± SD Statistical analysis was performed with GraphPad Prism version 501GraphPad Software La Jolla CA US Oneway ANOVA was used to measure significance p Dunnetts post hoc tests were used to test the difference between groupsResultsLevobupivacaine decreases breast cancer cell invasionTranswell assay analysis showed significantly decreased in the invasion ability of MCF7 and MDAMB231 cells in a dosedependent manner compared with the untreated cells Additional file a0 Fig S1a b Levobupivacaine inhibits proliferation in a0breast cancer cellsThe MCF7 and MDAMBA231 cell viability decreased as the concentrations of levobupivacaine or a0mM increased The MCF7 cells showed a cytotoxic effect while the MDAMB231 cells showed a similar cytotoxic effect of Fig a01a Under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates Additional file a0 Fig S2a b The viability of breast cancer cells decreased in a dosedependent manner The results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells Fig a01b c The data showed that the mRNA level of p21 significantly increased following levobupivacaine treatment Fig a0 1d e Western blot analysis showed a similar increased in p21 and decreased in FOXO1 and cyclin D1 expressions in a dosedependent manner compared with the untreated cells Fig a01f g Additional file a03f gLevobupivacaine promote apoptosis in a0breast cancer cellsLevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells Fig a0 2a b The qPCR data showed a decreased in Bcl2 and increased in Bax expressions in MCF7 and MDAMB231 cells compared with the untreated cells Fig a0 2c d Western blot analysis also showed a similar decreased in Bcl2 and increased expressions of active caspase and Bax compared with the untreated cells Fig a02e f Additional file a03e fLevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0PI3KAktmTOR signalling pathwayWestern blot analysis showed a significant decreased in the expression of the nuclear localization of pPI3K pAkt and pmTOR compared with the untreated cells Fig a03a b Additional file a03a bDiscussionBreast cancer remains a common cause of mortality among women worldwide Though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited These therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed Several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [] Recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition [] A study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells HCC by increasing the Caspase activity whereas ropivacaine inhibits the growth of HCC cells by stopping the cell cycle in G2 phase [] Lee et a0al demonstrated that local anaesthetics potentiate TNFα mediated apoptosis in HK2 cells [] The cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local See figure on next pageFig Levobupivacaine inhibits proliferation in breast cancer cells MCF and MDAMB cells were treated with different concentrations of levobupivacaine a Cell viability was measured by CCK assay IC50 results of levobupivacaine on MCF and MDAMB cells b c Colony formation of MCF and MDAMB cells treated with various concentrations of Levobupivacaine and stained with crystal violet d e The mRNA expression levels of p21 and GAPDH were analysed by qPCR f g Protein expression assessment of MCF and MDAMB cells by western blot against antibodies FOXO1 p21 Cyclin D1 and GAPDH used as control The data was statistically significant at indicates P indicates P indicates P compared with untreated cells This data corresponds to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of 0cKwakye a0et a0al BMC Res Notes Page of Fig Effects of levobupivacaine on apoptosis of breast cancer cells a b MCF and MDAMB cells were treated with different concentrations of levobupivacaine for h The cells were then stained with fluoresceinconjugated annexin V and PI and analysed by flow cytometry Error bars represent standard error of the mean P versus the control c d Relative gene expression of Bax and Bcl following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qPCR e f MCF and MDAMB cells were treated with different concentrations of levobupivacaine for h and the activities of Bax Bcl and Active caspase were examined by Western blot analysis using specific antibodies GAPDH was used as internal controls The data was statistically significant at indicates P indicates P compared with control The data correspond to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of Fig MCF and MDAMB cells were treated with different concentrations of levobupivacaine for h a b The cells were lysed and subjected to SDSPAGE and analysed by western blotting and probed with specific antibodies pPI3K pAkt and pmTOR The results showed a decrease in the expressions of pPI3K pAkt and pmTOR proteins GAPDH was used as internal controls The data represent the mean ± SD of three independent experimentsanaesthetic [] In this study we employed MCF and MDAMB231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro The antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerPI3KAktmTOR signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response Breast cancer cell resistance to therapies can result from the activation of PI3KAktmTOR signalling pathway [] This has made the PI3KAktmTOR signalling pathway an important object of study for understanding the development and progression of breast cancer In patients with breast cancer PI3KAktmTOR signalling pathway can be a target for diagnostic prognostic and treatment purposes [] Akt plays a role in the activation and inactivation of many transcription factors Activation of Akt correlated with the activation of mTOR Phosphorylation of the FOXO proteins by Akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes Cyclin D1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers [] Reports show that overexpression of cyclin D1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin D1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages [] Datta et a0al reported that Akt can phosphorylate the proapoptotic Bcl2 family member Bad causing its isolation from the mitochondrial membrane by other proteins [] Local anaesthetics modify the protein levels of key members of the Bcl2 family in a manner that presents an increase in the ratio of BaxBcl2 which may contribute to the response of cancer cells to apoptosis In the present study the role of levobupivacaine on the expression of PI3K Akt and mTOR was investigated to illustrate the potential molecular mechanism We observed a significantly decreased expression of pAkt pPI3K pmTOR and subsequent decreased expression of FOXO Cyclin D1 and Bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis These emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing PI3KAktmTOR signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyConclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the PI3KAktmTOR signalling pathway These findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0cKwakye a0et a0al BMC Res Notes Page of LimitationsNumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [] However our work is not without limitations In a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededBiology Dalian Medical University Dalian China Department of Anaesthesia and Critical Care School of Medicine University of Health and Allied Sciences Ho Ghana Department of Biochemistry and Molecular Medicine School of Medicine and Health Sciences University for Development Studies Tamale Ghana Departments of Anaesthesia and Critical Care Ridge Hospital Accra Ghana Department of Medicine Princefied University Ho Ghana Received June Accepted July Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 Additional file a0 Figure S1 Levobupivacaine decreases breast cancer cell invasionAdditional file a0 Figure S2 Effect of levobupivacaine on the morphology of MCF and MDAMB cellsAdditional file a0 Original gelsblots scan used in Fig 1f g Fig 2e f and Fig 3a b for MCF and MDAMB cellsAbbreviationsEGFR Epidermal growth factor receptor HCC Hepatocellular carcinoma cells NC Nitrocellulose PI Propidium iodide PS Phosphatidylserine qPCR quantitativepolymerase chain reactionAcknowledgementsWe thank the First Affiliated Hospital and The Department of Biochemistry of Dalian Medical University for making available all the necessary materials needed for this work We also thank the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO of China for supporting this work Our thanks also go to the China Scholarship Council and the Government of the Republic of Ghana for giving financial aid to some of the authors to study at Dalian Medical UniversityAuthors contributionsAKK SK QY and QPW conceived and designed this study QPW and QY were responsible for the supervision and coordination of this study AKK SK JL MNR QY and QPW conducted the data collections SK led the data analysis with inputs from AKK QY and QPW AKK and SK wrote the first draft of the manuscript and JL MNR SAR AAF JA and EAN contributed to revising and reviewing the manuscript All authors read and approved the final manuscriptFundingThis study was supported by the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO Availability of data and materialsThe data used andor analysed in this study are available from the corresponding author upon reasonable requestEthics approval and consent to participateThe ethical committee of the First Affiliated Hospital of Dalian Medical University approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyConsent for publicationsNot applicableCompeting interestsAuthors declare that they have no competing interestsAuthor details Department of Anaesthesiology Dalian Medical University Dalian China Department of Anaesthesiology First Affiliated Hospital of Dalian Medical University Dalian China Department of Biochemistry and Molecular References American Cancer Society Breast Cancer Facts and Figures Atlanta American Cancer Society American Cancer Society Cancer Facts and Figures Atlanta Ameri can Cancer Society Siegel R Naishadham D Jemal A Cancer statistics CA Cancer J Clin Chang YC Hsu YC Liu CL Huang SY Hu MC Cheng SP Local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogenactivated protein kinase pathway PLoS ONE 20149e89563 GomezGutierrez JG Souza V Hao HY de Montes OcaLuna R Dong YB Zhou HS McMasters KM Adenovirusmediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells Cancer Biol Ther Sunters A de Fern¡ndez Mattos S Stahl M Brosens JJ 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factoralpha TNFα serum levels in rats subjected to faecal peritonitis and treated with intraperitoneal ropivacaine Acta Cirurgica Brasileira Piegeler T Schl¤pfer M Dull RO Schwartz DE Beat A Minshall RD BeckSchimmer B Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFαinduced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase Br J Anaesth Shankar S Chen Q Srivastava RK Inhibition of PI3KAKT and MEKERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factor J Mol Signal Qian J Zou Y Rahman JSM Lu B Massion PP Synergy between phosphatidylinositol kinaseAkt pathway and BclxL in the control of apoptosis in adenocarcinoma cells of the lung Mol Ortega MA FraileMartınez O AsUnsolo A Bujan J GarcıaHonduvilla N Coca S Signal transduction pathways in breast cancer the important role of PI3KAktmTOR J Oncol Royds J Khan AH Buggy DJ Update on existing 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1i32i3838 Li K Yang J Han X Lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the upregulation of RARβ2 and RASSF1A demethylation Int J Mol Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c' | 2 |
"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 speciï¬city 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 ï¬ndings 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 speciï¬city [ ] In addition the results of TCT interpretation byï¬lmreading 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 theeï¬cacy 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 ï¬lamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1which 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 ï¬nding 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 signiï¬cance 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 HuaianMaternal and Child Health Care Hospital Huaian JiangsuChina were recruited The diagnoses of all patients were veriï¬ed 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 ofHuaian 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 classiï¬ed 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 deï¬ned 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 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°SD 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 Youdens 0cDisease Markerslymph 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 ï¬rst 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 ï¬lament ï¬exibility 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 eï¬cacy 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 andspeciï¬city 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 speciï¬city 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 P pgmL as the cutoï¬ value and the sensitivity and speciï¬city 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 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 speciï¬cityof 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 signiï¬cance it still seems to beable to inï¬uence 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 signiï¬cance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the speciï¬c 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 PMultivariate 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 conï¬rm 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 conï¬icts of interestAuthors ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as coï¬rst authors[] M H Forouzanfar K J Foreman A M Delossantos et alBreast 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 Cervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp [] Y Zhou W Wang R Wei Serum 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 The role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya Eï¬cacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp [] N Tanaka T Yamashita S Yamamoto Histologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp [] Y Watabe T Mori S Yoshimoto Copy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp [] HT An S Yoo and J Ko αActinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp [] F Niu T Wang J Li The 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 Serum 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 αActinin4 is essential for maintaining the spreading motility andcontractility of ï¬broblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida Actinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp [] D G Thomas and D N Robinson The ï¬fth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp screening on the diagnostic signiï¬cance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp [] KH Wang C J Lin C J Liu Global 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 Diagnostic 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 Actinin4 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 Thestructure and regulation of human muscle αactinin Cellvol no pp [] D Wang X W Li X Wang Alphaactinin4 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 Possible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda The biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim αActinin 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 DynamicRegulation 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 Actinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp [] T Watanabe H Ueno Y Watabe ACTN4 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 ACTN4 gene ampliï¬cation 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 Alphaactinin 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 The alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp 0c" | 2 |
" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri °p rri ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in childrens lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the possibleincidence considering countable possibility ofaht cases at most and another one is the presumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the possible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ Ï pafi 03original dalyi°i different child abuse related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review 2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of japans plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancetnumber of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse childhood experiences on healtha systematic review and metaanalysis lancet public health 201728e356fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai11901000koyoukintoujidoukateikyokusoumuka002_1pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile06seisakujouhou11900000koyoukintoujidoukateikyoku0000180499pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai12601000seisakutoukatsukansanjikanshitsu_shakaihoshoutantou0000058633pdf] accessed july the economics of child abuse a study of san francisco [httpssafeandsoundwpcontentuploads201709economicsofabuse_report_sfcapc1pdf] accessed july fang x brown ds florence cs mercy ja the economic burden of childmaltreatment in the united states and implications for prevention childabuse negl habetha s bleich s weidenhammer j fegert jm a prevalencebasedapproach to societal costs occurring in consequence of child abuse andneglect child adolesc psychiatry ment health mccarthy mm taylor p norman re pezzullo l tucci j goddard c thelifetime economic and social costs of child maltreatment in australia childyouth serv rev wada i igarashi a the social costs of child abuse in japan child youth servrev miller tr steinbeigle r wicks a lawrence ba barr m barr rgjp disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e1545fang x fry da brown ds mercy ja dunne mp butchart ar corso psmaynzyuk k dzhygyr y chen y the burden of child maltreatment inthe east asia and pacific region child abuse negl corso ps fertig ar the economic impact of child maltreatment in theunited states are the estimates credible child abuse negl macroeconomics and health investing in health for economicdevelopment [httpwhqlibdocwhointpublications2001924154550xpdf]accessed july segel je costofillness studiesa primer rtiunc center of excellence inhealth promotion economics jo c costofillness studies concepts scopes and methods clin molhepatol metrics population attributable fraction paf [httpwwwwhointhealthinfoglobal_burden_diseasemetrics_pafen] accessed july norman re byambaa m de r butchart a scott j vos t the longtermhealth consequences of child physical abuse emotional abuse and neglecta systematic review and metaanalysis plos med 2012911e1001349kalmakis ka chandler ge health consequences of adverse childhoodexperiences a systematic review j am assoc nurse pract unicef child maltreatment prevalence incidence and consequences inthe east asia and pacific region new york unicef rothman kj epidemiology an introduction oxford university press joyce t huecker mr pediatric abusive head trauma shaken babysyndrome [updated aug ] in statpearls [internet] treasure islandfl statpearls publishing available from httpswwwncbinlmnihgovbooksnbk499836 yamaoka y fujiwara t fujino y matsuda s fushimi k incidence and agedistribution of hospitalized presumptive and possible abusive head traumaof children under months old in japan j epidemiol httpsdoi102188jeaje20180094japanese population projection [httpwwwstatgojpdatajinsui2016np] accessed july summary of patient survey [httpswwwmhlwgojpenglishdatabasedbhsssps_2014html] accessed july kirigia jm mburugu gn huka gs the indirect cost of disability adjusted lifeyears lost among the elderly in kenya int arch med httpsdoi1038232483 mortality and global health estimates [httpwwwwhointghomortality_burden_diseaseen] accessed july japan gdp gross domestic product [httpscountryeconomycomgdpjapanyear2016] accessed july the results of verification of death cases caused by child abuse threport [httpswwwmhlwgojpstfseisakunitsuitebunya0000173329_00001html] accessed july miller tr steinbeigle r wicks a lawrence ba barr m barr rg disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e154550 httpsdoi101542peds20141385shiroiwa t fukuda t ikeda s takura t moriwaki k development of anofficial guideline for the economic evaluation of drugsmedical devices injapan value health moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1d2 were conï¬rmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelialmesenchymal transition thisstudy for the ï¬rst time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the ï¬rst line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difï¬cult3 it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelialmesenchymal transition emt7 it is a wellorchestrated process involved in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40ofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10 are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14 it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21 recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the ï¬rst report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within months of their procurementresuscitation achn cells were cultured in mem mediaofï¬cial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® ¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidiï¬ed atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientiï¬c and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientiï¬c according to the manufacturers protocol toverify transfection efï¬ciency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydeï¬xedparafï¬nembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically conï¬rmed for clear cellrcc ccrcc and slides were reviewed by a boardcertiï¬edpathologist for the identiï¬cation of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturers instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturers protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was ï¬rst conï¬rmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconï¬rm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modiï¬cation was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturers protocol bisulï¬tetreated dna wasanalyzed by methylationspeciï¬c quantitative polymerasechain reaction msqpcr with primer pairs speciï¬c formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturersinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbrieï¬y h posttransfection cells were counted andplaced on inserts at à cellsml for migration and à for invasion in serumfree medium and wereallowed to migrateinvade for h at °c cellsmigrated or invaded through the pores were ï¬xed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantiï¬ed at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturerscoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofï¬cial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing fireï¬y luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was conï¬rmed by rip assay an imprint rip kitwas used following the manufacturers protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunoï¬uorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantiï¬cationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunoï¬uorescencetransfected achn andcaki1 cells were ï¬xed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice weekold n charles river lab were subcutaneously injected with à caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups ï¬ve in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantiï¬ed data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mannwhitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the students ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and conï¬dence interval kaplanmeier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identiï¬ed putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofï¬cial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andï¬bronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results conï¬rmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3be compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3ce patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identiï¬ed a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mannwhitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade grade and stages normal stage iii and stage iiiiv c overall survival in tcgakirc cohort as performedby kaplanmeier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mannwhitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers ï¬bronectinand vimentin at both protein fig 5e and mrnaofï¬cial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32 we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also conï¬rmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical signiï¬cance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade grade and stages normal stage iii and stage iiiiv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade andin different stages normal stage iii stage iiiiv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplanmeier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6fhs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g ij s8cf inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic ï¬bronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofï¬cial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36 human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39 or tumorsuppressive roles42 in speciï¬c types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by ï¬owcytometric analysis of annexinvfitc 7aadstained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspeciï¬c genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofï¬cial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be conï¬rmedin a larger independent sample cohortinterestingly we found tha | 0 |
"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e | 2 |
" the ampactivated protein kinase ampk is an evolutionarily conserved regulator of cellular energyhomeostasis as a nexus for transducing metabolic signals ampk cooperates with other energysensing pathwaysto modulate cellular responses to metabolic stressors with metabolic reprogramming being a hallmark of cancerthe utility of agents targeting ampk has received continued scrutiny and results have demonstrated conflictingeffects of ampk activation in tumorigenesis harnessing multiomics datasets from human tumors we seek toevaluate the seemingly pleiotropic tissuespecific dependencies of ampk signaling dysregulationmethods we interrogated copy number variation and differential transcript expression of ampk pathway genesacross diverse cancers involving over patients cox proportional hazards regression and receiver operatingcharacteristic analyses were used to evaluate the prognostic significance of ampk dysregulation on patientoutcomesresults a total of and seven ampk pathway genes were identified as having loss or gainoffunction featuresthese genes exhibited tissuetype dependencies where survival outcomes in glioma patients were most influencedby ampk inactivation cox regression and logrank tests revealed that the 24ampkgene set could successfullystratify patients into high and lowrisk groups in glioma sarcoma breast and stomach cancers the 24ampkgeneset could not only discriminate tumor from nontumor samples as confirmed by multidimensional scaling analysesbut is also independent of tumor node and metastasis staging ampk inactivation is accompanied by the activationof multiple oncogenic pathways associated with cell adhesion calcium signaling and extracellular matrixanization anomalous ampk signaling converged on similar groups of transcriptional targets where a commonset of transcription factors were identified to regulate these targets we also demonstrated crosstalk between procatabolic ampk signaling and two proanabolic pathways mammalian target of rapamycin and peroxisomeproliferatoractivated receptors where they act synergistically to influence tumor progression significantlycontinued on next page correspondence alvinalaiuclacukinstitute of health informatics university college london euston roadlondon nw1 2da uk 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 0cchang and lai bmc cancer page of continued from previous page genetic and transcriptional aberrations in ampk signaling have tissuedependent pro or antitumorimpacts pancancer investigations on molecular changes of this pathway could uncover novel therapeutic targetsand support risk stratification of patients in prospective trialskeywords ampk glioma lossoffunction tumor metabolism pancancer the ampactivated protein kinase ampk is an evolutionary conserved key player responsible for energy sensing and homeostasis orthologous copies of ampkprevail universally as heterotrimeric complexes wherethe human genome encodes two genes for the α catalyticsubunit two regulatory subunit genes and three Πsubunit genes historically ampk was discovered as a crucial regulator of lipid metabolism since then ampkis implicated in a wide variety of fundamental metabolicprocesses as well as in metabolic diseases such as cancerand diabetes the first link between ampk and cancer was identified through the tumorsuppressive function of lkb1 which is upstream of the mtor pathway theroles of ampk werepharmacologically demonstrated by the application ofmetabolic inhibitors such as the antidiabetic metforminand the mimetic of amp aicar [] numerousstudies have since compellingly established the promiscuous nature of these pharmacological agents wherebythe inhibition of cancer cell proliferation occurs throughnonspecific ampkindependent avenues [ ]tumorsuppressivestressorssuchasin contrastof metabolicto the tumorsuppressive results frompharmacological studies genetic experiments on cancercells have credibly demonstrated that ampk activationis crucial for tumor progression and survival [] amyriadoxygendeprivation nutrient starvation and oxidative stress exists within the tumor microenvironment metabolic reprogramming during carcinogenesis would thus triggerampk activation to enable cells to survive under conditions of stress typically found in the tumor microenvironment hence conferring an overall tumorpromotingeffect ampk is also shown to support cancer growthand migration through crosstalk with other prooncogenicpathways for instance overexpression of oncogenes mycand src or the loss of the tumor suppressor folliculincould lead to ampk activation []genetic and pharmacological studies have paved theway for our understanding of the function of ampk incancer however anti and proneoplastic features ofampk remain controversial potentially due to the oversimplification of ampkmodulated processes in in vitroand nonhuman invivo models the genetic and clinicallandscape of ampk signaling has not been systematically investigated thus our study aims to address anunmet need to rigorously investigate the role of ampkin diverse cellular context using multiomics data fromactual tumors where we examined somatic copy numberalterations transcriptional and clinical profiles of tumorsfrom cancer types our analyses of clinical samples atscale would complement evidence from pharmacologicaland genetic studies to better elucidate the multifacetedand cellspecific nature of ampk signaling on tumorprogressionmethodsampk pathway genes and cancer cohortsninetytwo ampk pathway genes were retrieved fromthe kyoto encyclopedia of genes and genomes keggdatabase additional file clinical genomic and transcriptomic datasets of cancers involving patients were downloaded from the cancer genome atlastcga copy number variation differential expressionmultidimensional scaling and survival analysesdetailed methods of the above analyses were previouslypublished and thus will not be repeated here as per the guidelines [] to summarize discrete amplification and deletion indicators for copy number variation analyses were obtained from gistic geneleveltables gistic values of and were annotatedas shallow amplification and shallow deletion heterozygous events respectively gistic values of and were annotated as deep amplification and deep homozygous deletion events respectively multidimensionalscaling analyses and permutational multivariate analysisof variance permanova were performed using the rvegan package survival analyses were performed usingcox proportional hazards regression and the logranktest sensitivity and specificity of the 24ampkgene setwere assessed using receiver operating characteristicanalyses differential expression analyses were performed on patients stratified into high 4th quartileand low 1st quartile expressing groups using the genesetto determine the transcriptional effects ofanomalous ampk signalingpathway and transcription factor analysesgenes that were differentially expressed degs betweenthe 4th and 1st quartile patient groups were mapped to 0cchang and lai bmc cancer page of kegg gene ontology and reactome databases using gprofiler to ascertain biological processes and signaling pathways that were enriched the enrichr tool was used to map degs to the chea and encodetranscription factor tf databases to identify tfs thatwere significantly enriched as regulators of the degsirs2pik3r1sirt1 tbc1d1calculating the 24ampkgene score peroxisomeproliferatoractivated receptors ppar score andmammalian target of rapamycin mtor scoreampk scores were calculated from the mean expressionthe following genes slc2a4 foxo3 ppp2cbofpik3cd cab39l ccna1 fbp1 fbp2 foxo1 hmgcrppargc1appp2r2c mlycd pfkfb3 ppp2r2b prkaa2 leprcab39 irs1 and pfkfb1 ppar scores for each patientwere calculated by taking the mean expression of pparsignature genes plin5 pparg acadm gk cpt2scp2 acaa1 apoa1 ppara acox2 angptl4fabp3 plin2 aqp7 acsl1 fabp5 acadl andpck2 mtorpi3kakt scores for each patientwere calculated using the following equation mtorpi3kakt score akt mtor gsk3 s6k s6 pten all figures were generated using r version andadobe illustrator version cs6resultspancancer genomic and transcriptional alterations ofampk pathway genesfocusing on the genomic and transcriptomic landscapeof genes associated with ampk signaling retrievedfrom kegg across cancer types involving patients additional file we interrogated somatic copynumber alterations scna and mrna expression seeadditional file for a flowchart illustrating the study design to determine the effects of genomic alterations inampk pathway genes we classified genes as havinghighlevel amplifications gains lowlevel amplificationsdeep homozygous deletions and shallow heterozygousdeletions to evaluate pancancer patterns of scnaswe considered genes that were gained or lost in at least of samples within a cancer type and in at least onethird of cancer types ie at least seven cancer types atotal of genes were recurrently amplified while genes were recurrently lost fig additional file ampk is the central regulator of cellular energy levelswhich controls a number of downstream targets an example being the nuclear receptor hnf4a remarkablyhnf4a was found to be the most amplified gene identified as being recurrently amplified in of samplesin all cancers fig additionalfile this isfollowed by cftr cancer types and four other genesthat were amplified in cancer types adipor2 lepfile fig additionalprkag2 and rhebincontrast ppp2r2a was the most deleted gene found in of samples across cancers followed by the deletion of slc2a4 in cancers and five additional genesfoxo3 ppp2cb ppp2r2d ppp2r5c and ppp2r5ein cancer types fig additional file among allcancer types the highest number of amplified ampkpathway genes was observed in esophageal carcinomaesca genes followed by bladder cancer blca genes and lung cancer genes in both lung squamouscell carcinoma [lusc] and adenocarcinoma [luad]fig glioma tumors gbmlggin contrast hadonly five genes that were recurrently amplified fig in terms of somatic deletions lusc and esca both had genes deleted while no recurrent deletions were observed in papillary renal cell carcinoma kirp fig lossoffunctionevents differentialwe reasoned that scnas associated with transcriptional alterations could be considered as putative gainorexpressionanalyses between tumor and nontumor samples in eachcancer revealed that and genes were significantlyupregulated and downregulated in at least seven cancertypes respectively additional file of these differentially expressed genes seven and genes were alsorecurrently amplified and deleted respectively venn diagram in fig both gene sets were mutually exclusiveie the genes either had gainorfunction or lossoffunction features but not bothmolecular underpinnings of patient survival involvingputative lossoffunction ampk pathway componentswe next investigated the impact of transcriptional dysregulation ofthe putative gain and lossoffunctiongenes identified previously on patient survival outcomesacross all cancer types employing cox proportional hazards regression we observed that all genes sevengainoffunction and lossoffunction genes wereprognostic in at least one cancer type fig 2a thehighest number of prognostic genes was observed inglioma gbmlgg tumors genes while none ofthe genes were significantly associated with overallsurvival outcomes in esca and cholangiocarcinomachol fig 2a intriguingly although esca had thehighest number of scnas fig none of the genesharbored prognostic information suggesting that alterations in ampk signaling components have minimalroles in driving tumor progression and patient outcomes fbp1 was significantly associated with overallsurvival outcomes in cancers while ppp2r2c andppp2r2b in cancers fig 2a fbp2 is the least prognostic gene in only one cancer type cervical squamouscell carcinoma and endocervical adenocarcinoma cescfig 2a 0cchang and lai bmc cancer page of fig the landscape of somatic copy number alterations of ampk pathway genes heatmaps depict a fraction of samples within each cancertype that harbor somatic deletions and b somatic amplifications fortynine genes are recurrently deleted in at least of tumors within eachcancer and in at least seven cancer types fortysix genes are recurrently amplified in at least of tumors within each cancer and in at leastseven cancer types stacked bar charts on the yaxes illustrate the fraction of samples that possess copy number variation of a gene underconsideration grouped by shallow and deep deletions or amplifications stacked bar charts on the xaxes illustrate the fraction of samples withineach cancer type that contain shallow and deep deletions or amplifications the bar charts on the right of each heatmap depict the number ofcancer types with at least of samples affected by gene deletions and amplifications the venn diagrams demonstrate the identification of putative loss and seven gainoffunction genes from gene sets that are somatically altered and differentially expressed cancer cohorts analyzedwith corresponding tcga abbreviations are listed in parentheses bladder urothelial carcinoma blca breast invasive carcinoma brca cervicalsquamous cell carcinoma and endocervical adenocarcinoma cesc cholangiocarcinoma chol colon adenocarcinoma coad esophagealcarcinoma esca glioblastoma multiforme gbm glioma gbmlgg head and neck squamous cell carcinoma hnsc kidney chromophobekich pankidney cohort kipan kidney renal clear cell carcinoma kirc kidney renal papillary cell carcinoma kirp liver hepatocellularcarcinoma lihc lung adenocarcinoma luad lung squamous cell carcinoma lusc pancreatic adenocarcinoma paad sarcoma sarcstomach adenocarcinoma stad stomach and esophageal carcinoma stes and uterine corpus endometrial carcinoma ucec number ofsamples for each cancer type are indicated in parentheses blca brca cesc chol coad esca gbm gbmlgg hnsc kich kipan kirc kirp lihc luad lusc paad sarc stad stes and ucec given the prevalence of lossoffunction phenotypes indetermining clinical outcomes fig 2a we proceeded toexamine the combined impact of all lossoffunctiongenes on patient survival and oncogenic dysregulationto determine the extent of ampk pathway variationacross the cancers we calculated pathway scores foreach of the tumor samples by taking the meantranscript expression values of the genes slc2a4foxo3 ppp2cb pik3cd cab39l ccna1 fbp1fbp2 foxo1 hmgcr irs2 pik3r1 sirt1 tbc1d1ppargc1a ppp2r2c mlycd pfkfb3 ppp2r2bprkaa2 lepr cab39 irs1 and pfkfb1 we observedinteresting patterns when cancers were ranked from lowto high based on their median pathway scores fig 2bgbmlgg had the highest median pathway score whileblca and cesc were found at the lower end of thespectrum fig 2b as expected kaplanmeier analysisrevealed a significant difference in overall survival between glioma patients p stratified by low andhigh 24gene pathway scores fig 2c interestingly thecontribution of ampk signaling in cancer prognostication is cancertype dependent as in gliomalogranktests revealed that patients with high 24gene scores hadsignificantly improved survival outcomes in breast cancer p and sarcoma p fig 2c incontrast high expression of the genes was associated 0cchang and lai bmc cancer page of fig prognostic significance of ampk loss and gainoffunction genes a heatmap illustrates significant hazard ratio values from coxproportional hazards regression analyses on the lossoffunction and seven gainoffunction genes across all cancers b the distributions of ampkgene scores in each cancer are illustrated in the boxplot cancers are sorted from low to high median scores refer to fig legend forcancer abbreviations c kaplanmeier analyses and logrank tests revealed the prognostic significance of the 24ampkgene set in four cancertypes patients are stratified into q1 1st quartile and q4 4th quartile groups based on their 24gene scores for logrank tests dmultidimensional scaling analyses of the 24gene set depicted in 2dimensional space significance differences in the distribution between tumorand nontumor samples are confirmed by permanovawith increased mortality rates in stomach adenocarcinoma p fig 2c these results were in agreement when independently validated using the coxregression approach breast hazard ratio [hr] p glioma hr p sarcomahr p and stomach hr p cancers additionalfile since the 24genescore could be used to stratify patients into high andlowrisk groups we predict that when considered together gene expression values could discriminate tumorfrom nontumor samples although analysis could notbe performed on sarcoma this dataset only had twonontumor samples multidimensional scaling analysesand permanova tests of breast p gliomap and stomach p cancers revealed significantseparation between tumor and nontumor 0cchang and lai bmc cancer page of samples in twodimensional space fig 2d overall thissuggests that the 24gene set could be harnessed as adiagnostic biomarker for early cancer detectionconsistent with our previous observation that highpathway scores were associated with good prognosis insarcoma fig 2cto determine the independence of the 24gene setfrom other clinicopathologicalfeatures we employedmultivariate cox regression and observed that the gene set is independent of tumor node and metastasistnm staging where available in breast hr p and stomach cancers hr p additional file similarly kaplanmeier analyses andlogrank tests confirmed that the 24gene set allowedfurther risk stratification of patients with tumors of thesame tnm stage breast p and stomach p fig 3a furthermore we observed that within ahistological subtype of sarcomaleiomyosarcoma patients with elevated ampk signaling had significantlybetter3asurvival outcomesp figexploredthepredictivewe nextperformancesensitivity versus specificity of the 24gene set in allfour cancer types using receiver operating characteristicanalysis the area under the roc curve auc is an indication of how well the gene set could predict patientsurvival which ranges from to we found that thecombined model uniting both 24gene set and tnm staging outperformed the 24gene set when considered onits own in breast cancer patients auc vs fig 3b for stomach cancer the 24gene set onlycontributed to a marginally higher auc when used incombination with tnm staging when compared to the24gene set alone auc vs fig 3baucs of the 24gene set in glioma and sarcoma werefig the 24ampkgene set is independent of tumor stage and histological subtype a kaplanmeier analyses of patients grouped by tumornode and metastasis tnm stage breast and stomach cancers or by the histological subtype of leiomyosarcoma and the 24gene score forleiomyosarcoma the logrank test reveals a significant difference in survival rates between 1st and 4th quartile patients b receiveroperatingcharacteristic roc analyses on the 5year predictive performance of the 24gene set roc curves generated by the 24gene set are compared tocurves generated from both 24gene set and tnm staging where available or histological subtype auc area under the curve 0cchang and lai bmc cancer page of and respectively fig 3b within the leiomyosarcoma histological subtype auc was even higherat fig 3boncogenic transcriptional alterations associated withampk pathway inactivationampk pathway inactivation was associated with alteredsurvival outcomes in patients figs and we predictthat this could be due to broad transcriptional dysregulation arising from abnormal ampk signaling to investigate this phenomenon we performed differentialexpression analyses between patients stratified by the24gene set into high 4th quartile and low 1st quartileexpression groups and found that an outstanding number of common genes that were significantly differentially expressed in all four cancer types fig 4a thehighest number of differentially expressed genes degswas observed in stomach cancer genes followedby sarcoma genes glioma genes and breastcancer genes fig 4a additionalfile thedegs were mapped to kegg gene ontology andreactome databases to determine whether they were associated with any functionally enriched pathways intriguingly all four cancer types share similar patterns offunctional enrichments fig 4b and c for instance biological processes associated with cell communicationsignal transduction cell differentiation cell signalingcell adhesion and cell morphogenesis were enriched inall four cancers fig 4c in terms of specific signalingpathways calcium signaling camp signaling and processes associated with extracellular matrix anizationwere among the most enriched fig 4cto further identify potential transcriptional regulatorsof the degs we mapped the degs to encode andchea transcription factor tf binding databases remarkably we identified common tfs shared across allfour cancers that were implicated as direct binding partners of the degs fig 4c five tfs suz12 smad4rest ezh2 and nfe2l2 were found to be enriched inall four cancers suggesting that transcriptional dysregulation of tumors with aberrant ampk signaling involveddirect physical associations of these tfs with targetdegs fig 4c curiously foxm1 and e2f4 wereenriched only in glioma tumors which deserves furtherexploration in the next section overall our analysesdemonstrated that impaired ampk signaling resulted incommon patterns of oncogenesis which affect the severity of cancer and consequently mortality ratesinpatientsdownstream targets of ezh2 nfe2l2 rest smad4 andsuz12 were associated with survival outcomespathways modulating energy homeostatic may transducesignals to influence other cognate signaling modulesezh2 nfe2l2 rest smad4 and suz12 were all implicated as common transcriptional regulators of degsin glioma sarcoma breast and stomach cancers suggesting that altered ampk signaling converged on similargroups of transcriptional targets of all the target degsof the aforementioned tfs and geneswere found to be common targets of ezh2 nfe2l2rest smad4 and suz12 respectively in all four cancers fig 5a concatenating all five gene sets yielded unique genesie genes that were binding targets ofmore than one tf were considered only once to gainfurther insights into how ampk inactivation influencestumor progression we performed cox regression analyses to determine the association between each of the genes and survival outcomes the highest number ofprognostic genes was observed in glioma genes good prognoses and five adverse prognoses fig 5b incontrast out of genes were associated with adverseprognosis in stomach cancer fig 5b these observations were consistent with the 24ampkgene set beingpositive and negative prognostic factors in glioma andstomach cancer respectively fig which mirrored thebehavior of degs identified as a result of aberrantampk signaling fig 4c of the genes only andten were significantly associated with survival outcomesin sarcoma and breast cancer respectively fig 5b collectively our results suggest that the ampk pathwayand its interaction with other signaling modules are keydeterminants of patient outcomes in multiple cancertypesprognostic significance of joint ampk pathway activityand transcriptional levels of five oncogenic tfs inpatients with gliomahaving discovered the importance of the 24ampk geneset we sought to explore the crosstalk between ampksignaling and tf activity in glioma as previously mentioned glioma had the highest 24ampkgene scorefig 2b with a vast majority of the genes conferringprognostic information fig 2a moreover of the transcriptional targets of the five common tfs identifiedin patients with altered ampk signaling were significantly associated with survival outcomes in glioma fig5b additionally tfs foxm1 and e2f4 were identifiedto be enriched only in glioma tumors fig 4c thus wepredict that a joint model uniting ampk and tf expression profiles would allow further delineation of patientsinto additional risk groups and if so allowing combinedtargeting of ampk and candidate tfs for therapeuticaction as done previously we calculated ampk scoresfor each patient based on the mean expression of the genes interestingly we found that ampk scores weresignificantly negatively correlated with tf expressionlevels in glioma e2f4 rho p ezh2 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig ampk inactivation drives oncogenic transcriptional alterations in diverse biological processes and signaling modules a venn diagramillustrates the number of differentially expressed genes degs between 1st and 4th quartile patients as stratified using the 24ampkgene set infour cancer types a total of degs were common in all four cancers b dot plots depict the number of significantly enriched pathways andbiological processes upon the mapping of degs to kegg gene ontology and reactome databases each dot represents an enriched event contologies that exhibit similar patterns of enrichment across four cancers are shown degs are also mapped to encode and chea transcriptionfactor tf databases to determine enriched tf binding associated with degsrho p foxm1 rho p smad4 rho p and suz12rho p fig 6a we subsequentlycategorized patients into four groups using the mediancutoff of the ampk scores and tf expression values lowlow highhigh low ampk score and high tfexpression and high ampk score and low tf expression logrank tests revealed that patients stratified intothe four groups had survival rates that were significantlydifferent e2f4 p ezh2 p foxm1p smad4 p and suz12 p fig 6b for e2f4 ezh2 foxm1 and suz12patients with low ampk scores and high tf expressionperformed the worst e2f4 hr p ezh2 hr p foxm1 hr p and suz12 hr p fig6c for smad4 patients within the lowlow categoryhad the highest mortality rates hr p fig 6ccrosstalk between ampk and other anabolicrelatedpathways ppar and mtorampks antianabolic and procatabolic activities maywork in concert with other metabolic pathways toinvestigate the synergistic effects of ampk and two proanabolic pathways peroxisome proliferatoractivated receptors ppar and mammalian target of rapamycinmtor signaling in tumor progression we calculatedppar and mtor pathway scores detailed in themethods section for each glioma tumor low ampkscores were associated with poor outcomes in gliomafig to evaluate ampk and ppar or mtor ascombined models patients were separated into fourgroups using the median cutoff as mentioned previously interestingly when ampk and ppar scores werecollectively used for patient stratification we found thatpatients with low ampk and high ppar scores had thehighest death rates hr p confirmingthat ppar hyperactivation is associated with poor outcomes in glioma tumors with low ampk activity fig in contrast when considering mtor activitypatients with low ampk and low mtor scores performed the worst hr p fig theresults overall suggest that the ampk pathway could actsynergistically with ppar and mtor signaling to influence cancer progression significantlydiscussionwhile the role of ampk in energysensing is wellunderstood its full potential in metabolic diseases suchas cancer remains an open topic of debate despite extensive efforts spent on elucidating the role of ampksignaling [ ] there remains no consensus onwhether ampk promotes or suppresses tumor progression exploiting a rich reservoir of pancancer datasetsafforded to us by tcga we performed a thoroughexamination of genomic and transcriptomic profiles of ampk pathway genes in diverse cancer types ourcurrent understanding of ampk signaling is fueled bygenetic studies in cell lines and animal models although useful in determining causal relationships resultsfrom in vitro cell lines and animal models may have limited translational relevance as they do not accurately reflect human pathology animal models may offeradditional mechanistic insights but limitations in ethicsand costs remain moreover the complexity of humancancers is not accurately modeled in animals less than of results from animal models are translated to clinical trials despite analyses on tumor genetic datasets providing mostly correlative outcomes they remainvaluable in understanding diseasespecific molecularpathology when interrogated at scale on large patientgroups [] and when results are considered in relation to those obtained from celllines and animalmodelsemploying pancancer population data our studyidentified conserved and unique patterns of ampk signaling across diverse cancer types analyses at two molecular levels genetic and transcriptional yielded amore comprehensive depiction of ampk signalingwhere we identified genes that were both somatically altered and differentially expressed these putative lossor gainoffunction genes are more likely to impacttumor progression as they are altered at both macromolecular levels as reported in other studies we confirmedthat ampk signaling could either be oncogenic ortumor suppressive depending on the cellular context intuitively since ampk is antianabolic its function maynot be fitting for tumor growth and proliferation this isconsistent with reports demonstrating ampks tumorsuppressive activity [ ] a study on lymphoma demonstratesthewarburg effect and hypoxia signaling in mice that ampk downregulation induces 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig prognostic significance of degs targeted by enriched tfs a venn diagrams illustrate the extent of overlap between degs targeted byezh2 nfe2l2 rest smad4 and suz12 across four cancers b forest plots depict degs that are significantly associated with overall survivaloutcomes hazard ratios are denoted as purple squares while pink bars represent the confidence intervals significant wald test p values areindicated in blueits loss ofampk is proposed to act as a metabolic gatekeeper tolimit cancer cell division hencefunctionwould contribute to tumor aggression because of theloss in metabolic checkpoints [ ] ampk regulatesthe tumorsuppressive function of the serinethreoninekinase lkb1 ablation of lkb1 results in enhanced riskof developing gastrointestinal lung and skin squamouscell cancers [ ] moreover ampk is shown to inhibit pi3kaktmtor signaling which is activated inmany cancers [ ] also metabolic inhibitors such asmetformin which indirectly activates ampk could suppress tumor growth via autophagy induction and mtorinhibition [ ] metformin is shown to inhibit theproliferation of estrogen receptor α erα negative andpositive breast cancer cell lines through ampk stimulation however when tested in mice models metformin contributes to enhanced tumor progression andincreased angiogenesis providing us with a glimpse ofpotential proneoplastic effects of ampk activation in our study we observed that high levels of ampkpathway activity were associated with better outcomes inglioma breast cancer and sarcoma fig corroboratingprevious results on the tumorsuppressive function ofthe opposite is true in stomachampk converselyfig prognostic relevance of candidate tfs and the 24ampkgene set in glioma a scatter plots illustrate significant negative correlationsbetween ampk scores and tf expression levels in glioma patients are separated and colorcoded into four categories based on median ampkand tf scores density plots appended to the y and xaxes demonstrate the distribution of ampk and tf scores b logrank tests are performedon the four patient grou | 0 |
distribution and reproduction in any medium provided the original work isproperly citedLacrimal gland neoplasms comprise up to of all orbital masses clinically and histologically Much of our current coreknowledge regarding lacrimal gland tumors stems from prior study of their more common counterparts the salivary glandsThe prognosis for each lacrimal gland tumor is contingent upon proper clinical evaluation and ultimately the histopathologicdiagnosis We describe a case of an invasive carcinoma expleomorphic adenoma CaexPA with a cystadenocarcinomacomponent arising from the lacrimal gland in the absence of any previously diagnosed pleomorphic adenoma benign mixedtumor or prior incisional surgery This case illustrates the importance ofincludingimmunohistochemistry and genetic testing to narrow a diï¬erential diagnosis and potentially aid or guide therapy in the futureOur ï¬nding suggests that carcinoma of the lacrimal gland may be derived from previously undiagnosed and perhaps evensubclinical pleomorphic adenomathe histopathologic assessment IntroductionMalignant mixed tumor of the lacrimal gland also knownas carcinoma expleomorphic adenoma CaexPA is thethird most frequent epithelial lacrimal gland tumor afterpleomorphic adenoma PA and adenoid cystic carcinomaACA [] Clinically aï¬ected patients tend to be to years older than those with PA and often present with aninsidiously progressive mass of a lacrimal fossa that suddenly becomes symptomatic Other reported clinical settings are patients without any previously known lacrimalgland tumor who develop a rapidly growing mass associated with pain and patients with one or more recurrencesof PA who undergo malignant transformation [] Diagnostic imaging is critical for the clinical diagnosis of a lacrimalgland neoplasm Computed tomography CT and magnetic resonance imaging MRI are ideal but MRI remainsthe preferred method to visualize surrounding tissue anddetect radiographic features concerning aggressive malignancy [] The incidence of a malignant transformation ofPA increases with the duration of the tumor Prior studiesreport that approximately less than of PA undergoesmalignant change within years after the ï¬rst treatmentand by the end of years [] CaexPA can arisein patients without a known preexisting lacrimal glandtumor The malignant component is most often adenocarcinoma not otherwise speciï¬ed however other histologicsubtypes have been described such as adenoid cystic carcinoma ductal carcinoma mucoepidermoid carcinoma andsquamous cell carcinoma 0cCase Reports in PathologyHerein we report a case of an invasive CaexPA with anepithelial component consistent with a cystadenocarcinomain the absence of any previously diagnosed PA and supportedby immunohistochemical and molecular studies Case PresentationA 64yearold male patient presented to the emergencydepartment due to left lateral canthal pain tearing and ipsilateral hearing loss over four months The initial examinationrevealed a visual acuity of bilaterally The pupils werereactive without an aï¬erent pupillary defect Intraocularpressures were and mmHg respectively Confrontational visual ï¬elds and color plates were unremarkable Therewas a complete abduction restriction of the left eye Theexternal examination revealed edematous upper and lowereyelids predominantly overlying the lateral orbital rim associated with temporal sloping and a nontender palpable andï¬xed mass of the temporal fossa a There wasptosis of the left upper eyelid with a reduced marginal reï¬exdistance of two and a half millimeters mm Exophthalmometry measured mm and mm with a base measurement of mm The anterior segment was otherwisenormal The fundus exam revealed symmetrically sharp andpink disc margins without pallor or edemaMaxillofacial CT scan with contrast showed a lytic lesion ofthe left orbital wall with associated heterogeneous soft tissuemass measuring cm medially displacing the left lateralrectus muscle b Magnetic resonance imaging of thebrain and orbits revealed an enhancing ltrating mass of theleft lateral orbital wall extending into the left supra zygomaticmasticator space c A core guided needle biopsywas performed and the hematoxylineosin HE stainedslide revealed a moderately diï¬erentiated adenocarcinomainvolving ï¬brous connective tissue and demonstrating a cribriform architectural pattern with moderate cytologic atypia andindividual cell necrosis Figures 2a and 2bPositron emission tomography and CT of the chest abdomen and pelvis did not reveal any underlying malignancy orevidence of metastases Subsequently the patient underwentleft orbital exenteration with eyelid sparing Grossly the specimen included orbital contents frontal bone portions of thefrontal sinus and zygomatic bone Serial sectioning revealeda cm multilocular cystic mass involving the lacrimalgland fossa abutting the globe superotemporallyHistopathologically the HEstained sections disclosedpredominantly neoplastic cystic structures in the proximityof the lacrimal gland acini measuring to mm in diameterltrating ï¬brous connective tissue and bone cA small focus of pleomorphic adenoma was identiï¬ed associated with a lowgrade ductal carcinoma in situ The invasivecystic component revealed intraluminal papillary architecture and cribriform arch formations of the lining epitheliumd The neoplastic epithelium was composed ofmedium to largesized cuboidal cells with intercalated ductcell appearance eosinophilic cytoplasm and apocrine features Small foci of invasive solid components were observeddemonstrating cribriform architecture moderate to severenuclear pleomorphism and up to mitotic ï¬gures per highpower ï¬eld Columnar cells with pseudostratiï¬ed nucleiwere also present with moderate nuclear atypia Foci of ruptured cysts with hemorrhage granulation tissue lymphocyticltrate and dystrophic calciï¬cation were also seen No lymphovascular or perineural invasion was identiï¬edImmunohistochemical studies using monoclonal antibodies were performed with appropriate positive controls encompassing Gata3 mouse monoclonal antibody predilute Cell Marque Rocklin CA gross cystic disease ï¬uidprotein GCDFP15 mouse monoclonal antibody dilute Thermo Fisher Scientiï¬c Fremont CA androgen receptor AR rabbit monoclonal antibody dilute Cell Marque Rocklin CA SOX10 rabbit polyclonalantibody dilute Cell Marque Rocklin CAprostatic speciï¬c antigen PSA mouse monoclonal antibody predilute Cell Marque Rocklin CAthyroid transcription factor TTF1 mouse monoclonalantibody predilute at micrograms Ventana Medical Services Tucson AZ p63 mouse monoclonal antibody predilute Biocare Medical Concord CA p53mouse monoclonal antibody predilute at microgramsVentana Medical Services Tucson AZ high molecularweight cytokeratin HMWK903 mouse monoclonalantibody predilute Cell Marque RocklinCA CAM mouse monoclonal antibody predilute Ventana Medical Services Tucson AZ estrogen receptor ERmouse monoclonal antibody diluted VectorLaboratories Burlingame CA progesterone receptor PRmouse monoclonal antibody prediluted Leica BiosystemsNewcastle UK Her2neu rabbit monoclonal antibody predilute Ventana Medical Services Tucson AZ and Ki67rabbit monoclonal antibody predilute Ventana MedicalServices Tucson AZ Immunohistochemical studies wereperformed on an automated stainer Leica BiosystemsInc BOND III System Buï¬alo Grove IL All antibodiesand testing were performed in a CLIAcertiï¬ed laboratoryThe invasive component of the tumor was positive fata3 AR HMWK903 and CAM52 and focally positivefor GCDFP15 while negative for p63 ER PR SOX10 PSAand TTF1 stains Figures 2e2h p53 was positive in lessthan of the tumor cells The Ki67 proliferative index ofthe tumor cells was low p63 and SOX10 stainshighlighted the PA and the in situ component of the tumorDetection of HER2 status by immunohistochemistry wasequivocal and reï¬ex testing using ï¬uorescence in situhybridization FISH was negative for HER2neu geneampliï¬cation based on the updated guidelines of theAmerican Society of Clinical OncologyCollege of AmericanPathologists criteria for HER2neu testing in breast cancerMolecular proï¬ling using a nextgeneration sequencingNGS based assay Foundation One was performed onthe lacrimal gland tumor paraï¬nembedded tissue to identify genomic alterations within cancerrelated genes Thefollowing genomic alterations were detected HRAS G13RPIK3CA E542K and BCORL1 H215fs Microsatellitestatus MDstable and tumor mutation burden TMB low1MutsMbThe combined histopathologic immunohistochemicaland molecular ï¬ndings supported the diagnosis of invasive 0cCase Reports in PathologyaabcbcFigure a Clinical photo of the patient at time of presentation direct view b Computed tomography maxillofacial axial cut Illustrating a cm mass of the lateral orbit eroding the lateral orbital wall with a soft tissue component extending into the orbit causing proptosis andnonaxial displacement of the globe c MRI brain and orbit T1 fatsuppressed image with gadolinium showing a lobulated mass withmixed cystic and solid components and inhomogeneous enhancement involving the left lateral orbital wall and suprazygomatic leftmasticator space Additionally the mass demonstrates edema and enhancement suggesting ltration by the massCaexPA disclosing a carcinomatous component with apredominant cystic growth pattern and focal solid areasreminiscent of a cystadenocarcinoma The surgical marginswere negative In addition the microscopic examination ofthe left eye globe revealed pseudoexfoliation syndrome andFuchs endothelial dystrophyThe patient underwent postoperative adjuvant chemoradiation followed by excision of the eyelids with no residualtumor Followup examination showed no evidence of recurrence or metastatic disease nine months after completingadjuvant therapy DiscussionMuch remains unknown about both the natural histologicprogression and malignant transformation of CaexPAWe know that lacrimal gland neoplasms comprise oforbital masses clinically [ ] and of orbital masseshistologically [ ] and nearly half of epithelial tumorsare malignant [] Although uncommon malignant transformation of PA can occur with incomplete excision InvasiveCaexPA can represent malignant transformation of PAmany of which develop recurrence or distant metastasis tothe lung bone abdomen and CNS [] This casehighlights the importance of an immunohistochemical andgenetic evaluation in complex lacrimal tumorsThe malignant epithelial component of CaexPA hasmorphological varieties including adenocarcinoma adenoidcystic carcinoma squamous cell carcinoma mucoepidermoid carcinoma and ductal carcinoma It could be likelya mixture of subtypes [] Rarely the only evidence ofpleomorphic adenoma is the presence of large areas of hyalinized stroma composed of myoepithelial cells and a fewductal structuresIn the current case the carcinomatous component of thetumor discloses predominantly an ltrative cystic growthpattern reminiscent of a cystadenocarcinoma previouslydescribed in neoplasms of the salivary and lacrimal glandsThe term cystadenocarcinoma of the salivary gland hasevolved It encompasses a variety of tumors depicting a cysticpattern of growth to a subset of papillary and cystic malignant neoplasms that have indolent behavior This is alsoobserved in other lowgrade salivary gland carcinomashowever it is important to note that they can demonstrateltrative growthlocal recurrence and metastasize toregional lymph nodes at the time of diagnosisPreviously Foss [] in a review of cases ofcystadenocarcinoma of the salivary gland used the followingdiagnostic criteria occurrence within a salivary gland invasive growth a predominantly cystic pattern ofgrowth with or without a papillary component and theabsence of acinar or mucoepidermoid diï¬erentiation or evidence of origin in a PA In the same review the predominantcell type varies among tumors and includes small cuboidalintercalated ductlike cellslarge cuboidal cells and tallcolumnar cells The subgroups of the large cuboidal cells 0cCase Reports in PathologyababcHMWCK903Androgen receptordedp53ep63fcfghghFigure a Moderately diï¬erentiated adenocarcinoma involving cores of ï¬brous connective tissue HE 2x b High power view of thetumor revealed a cribriform pattern moderate nuclear pleomorphism and scattered mitoses HE 40x c Neoplastic cystic structures inclose proximity to lacrimal gland tissue star in a hyalinized stroma and lymphoid proliferation HE 2x d Tumor that arose from afocus of pleomorphic adenoma star depicting cystic structures with small papillae HE 4x Inset papillary projections lined by largecuboidal cells with moderate nuclear atypia and apocrine features HE 20x e Tumor cells were strongly positive for HMWK903 4xf Nuclear expression of androgen receptor was present in the tumor 20x g p53 positive in less than of tumor cells 4x h p63was negative within the invasive component of the tumor 4xhave central nuclei abundant eosinophilic cytoplasm largenucleoli and apocrine features which are similar to thoseobserved in this case The fourth group has a combinationof cell types Furthermore Foss suggest that tumorswith predominantly columnar cells are associated withincreased metastatic potentialThe cystic growth pattern characteristic of this tumor isoften associated with an ammatory response due toruptures of the dilated structures as noted in this case Cystformation in neoplasias of the salivary and lacrimal glandscan behave as a mimicker of an ammatory process Pakdel report a case of a spontaneous rupture of a PAmasquerading as orbital cellulitis [] Histologically aruptured cystic space of a PA surrounded by a monocyticltrate and foreign bodytype granulomas is described Inthis paper the authors consider the spontaneous rupture ofthe cystic space as an underlying mechanism for the acutepresentation of this tumorCystadenocarcinoma can have a broad diï¬erentialdiagnosis The principal considerations include papillary cystic acinic cell adenocarcinoma secretory carcinoma mucoepidermoid carcinoma MEC and ductal carcinoma Aciniccell carcinoma and secretory carcinoma are typically indolent monotypic tumors that can disclose a papillary cysticarchitecture Histologicallysecretory carcinoma sharesnearly identical growth patterns to acinic cell carcinoma 0cCase Reports in Pathologybut instead shows a multivacuolated eosinophilic cytoplasmoften with luminal and intracytoplasmic mucin and no truezymogen granules Immunohistochemically secretory carcinoma is S100 and mammaglobinpositive and typicallynegative for DOG1 while acinic cell carcinoma shows theopposite staining proï¬le Additionally secretory carcinomaharbors t1215p13q25 resulting in an ETV6NTRK3 genefusion [] MEC is usually composed of a mixture ofpredominantly epidermoid squamoid cells abundant intermediate cells ranging from small basal cells with basophiliccytoplasm to larger cells with eosinophilic cytoplasm andmucous cells Welldiï¬erentiated MEC is a circumscribedtumor that can disclose glandular and cystic structures linedby a single layer of mucussecreting cells however the intermediate and highgrade tumors show solid nests or sheets ofcells composed of primarily epidermoid cells with a scant cystic component and obvious invasion severe pleomorphismnecrosis and increased mitosesOn the other hand primary ductal adenocarcinoma ofthe salivary gland SDC originates from the excretoryportion of the salivary duct is a rare aggressive malignantepithelial tumor and accounts for only of epithelial lacrimal gland tumors Histologically the tumor is highly ltrative and usually solid with occasional cystic areas disclosingbreastlike ductal carcinoma features with central necrosisOccasionally cystadenocarcinoma with large cuboidal cellseosinophilic cytoplasm and highgrade nuclear atypia bearssome similaritiesto ductal adenocarcinoma Howeverpapillarycystic invasive growth is not usually seen in ductaladenocarcinoma [] The invasive component features trabeculae ducts and sheets of neoplastic cells in a desmoplasticstroma with perineural and vascular invasion The latter isnot commonly observed in cystadenocarcinoma Immunohistochemically the tumor cells are positive for a low molecular weight cytokeratin CAM52 CK7 CEA EMA andGCDFP15 Other than epithelial markersthis tumorexpresses AR in up to of invasive cases HER2 positivityin of cases and p53 overexpression in all reportedcases The Ki67 proliferative index is over The tumoris ER and PR negativeFurthermore in this case the tumor also demonstratescytomorphologicalfeatures similar to that of a low tointermediategrade ductal carcinoma in situ of the breast Thelatter ï¬ndings might correlate with the previously describedlowgrade cribriform cystadenocarcinoma of the salivary glandalso known as lowgrade salivary ductal carcinoma and salivaryductal carcinoma in situ currently categorized as intraductalcarcinoma low grade and high grade respectively These typesof tumors show a variety of growth patterns both solid and cystic ranging from cribriform to solid to micropapillary and arereminiscent of lowgrade ductal carcinoma in situ of the breastFocal ltration may be noted []The exact pathogenesis of CaexPA remains controversial Prior studies have demonstrated that PA and CaexPA of the salivary and lacrimal glands share similar genomicproï¬les and frequently overexpress the PLAG1 oncoprotein[ ] Harrison [] in their review indicate that thedevelopment of CaexPA follows a multistep model of carcinogenesis with the progressive loss of heterozygosity at 8qthen 12q and ï¬nally 17p Alterations including ampliï¬cationgene fusion and translocations in 12q genes such as HMGICHMGA2 and MDM2 may play a role in the malignant transformation In the same study the authors also mention thatloss of 17p is usually common in CaexPA indicating tumorsuppressor gene p53 loss as this tumor evolves Additionallyit appears that CaexPA and other malignant epithelialtumors other than ACC do not harbor MYB gene rearrangements or fusions It is important to note that there does notappear to be a correlation between rearrangement statusand clinical outcome []Additional mutational analysis of the lacrimal gland carcinomas has been also evaluated [] demonstrating that RASKRAS NRAS PIK3CA and MET mutations are frequent indiverse epithelial neoplasms of the lacrimal gland with thehighest proportion of mutations found in adenoid cystic carcinoma PIK3CA and MET mutations can coexist with RASmutationsPIK3CA and HRAS mutations are detected in this casewhich correlates with alterations already described in lacrimal gland carcinomas however alterations in MDM2HMGA2 NTRK3 p53 PLAG1 and ETV6 among others werenot observedIn summary this case demonstrates an invasive CaexPA with a malignant epithelial component that resemblesthe cystadenocarcinoma mixed cell type described by Foss [] Immunohistochemically the tumor was ARpositive while negative for ER PR and Her2 with expression of p53 in less than of the tumor cells Theprevalence of AR varies between the diï¬erent subtypes ofsalivary gland carcinomas SGC In recent medical literature data AR expression has been detected in as many as of SDC [] Dalin [] in the same study alsoindicate that adenocarcinoma and acinic cell carcinoma ofthe salivary gland express AR in and of the casesrespectivelyOur ï¬ndings emphasize the importance ofThe current classiï¬cation of the salivary gland tumors[] and the combined histopathologic and immunohistochemical features suggest that this tumor could representthe results of the natural course of a lowgrade cystadenocarcinoma with focal transformation into an intermediatehighgrade invasive ductal carcinoma expleomorphic adenomafurtherexploration of CaexPA pathogenesis especially the extentof disease and the histologic subtype In additiontheimmunohistochemical and genetic testing provides important support for the diagnosis as well as potentially guidesfuture therapy and prognostic evaluation Correctly identifying the type of malignant epithelial component is a signiï¬cant factor in the survival of aï¬ected patients To ourknowledge this patient represents the ï¬rst case in whichCaexPA of the lacrimal gland reveals an indolent epithelial malignancy with a low proliferative index arising froman undiagnosed PA after many likely consecutive molecular alterationsLastly due to the rarity of these tumors arising from thelacrimal gland further studies are necessary to evaluate theirbiologic behavior and determine any correlation betweenCaexPA and pseudoexfoliation syndrome 0cCase Reports in PathologyEnd Results International Journal of Ophthalmology vol no pp [] F A Jakobiec J R Bily and R L Font Orbit in OphthalmicPathology An Atlas and Textbook W H Spender Edpp WB Saunders Co Philadelphia 4th edition[] M H Devoto and J O Croxatto Primary cystadenocarcinoma of the lacrimal gland Ophthalmology vol no pp [] R H Simpson Salivary duct carcinoma new developmentsmorphological variants including pure in situ high gradelesions proposed molecular classiï¬cation Head and NeckPathology vol Supplement pp [] R D Foss G L Ellis and P L Auclair Salivary gland cystadenocarcinomas a clinicopathologic study of cases TheAmerican Journal of Surgical Pathology vol no pp [] L Barnes J W Eveson P Reichart and D Sidransky Pathology and Genetics of Head and Neck Tumours IARC [] J Antony V Gopalan R A Smith and A K Y Lam Carcinoma ex pleomorphic adenoma a comprehensive review ofclinical pathological and molecular data Head and NeckPathology vol no pp [] F Pakdel N Pirmarzdashti S Soltani Z Nozarian F AAmoli and A Kassaee Spontaneous rupture of lacrimalgland pleomorphic adenoma Ophthalmic Plastic and Reconstructive Surgery vol no pp e41e43 [] R R Seethala Head and neck pathology Surgical PathologyClinics vol no [] S L von Holstein A Fehr M Persson Lacrimal GlandPleomorphic Adenoma and Carcinoma ex PleomorphicAdenoma Genomic Proï¬les Gene Fusions and Clinical Characteristics Ophthalmology vol no pp [] T Y Chen M G Keeney A V Chintakuntlawar et alAdenoid cystic carcinoma of the lacrimal gland is frequentlycharacterized by MYB rearrangement Eye vol no pp [] M Dalin P Watson A Ho and L Morris Androgen receptor signaling in salivary gland cancer Cancers vol no p Conflicts of InterestNone of the authors declare any competing interests Currently Dr Del Valle Estopinal is the Director of OphthalmicPathology and Assistant Clinical Professor of the Departments of Ophthalmology and Pathology at University ofCalifornia Irvine The City Dr S Orange CA Authors ContributionsDr Maria Del Valle Estopinal was the ocular pathologistthat performed the histopathologic immunohistochemicaland genetic evaluation Dr Bryant Carruth wastheoculoplasticstrained ophthalmologist who performed thecore biopsy The other authors participated in the clinicalcare and preparation of this manuscript All authors readand approved the ï¬nal paperAcknowledgmentsThe authors acknowledge the cooperation of the patient Theyacknowledge the help of the SUNY Upstate Departmentof Pathology and Eye Plastic and Reconstructive Surgeonsof Central New York in the contribution to evaluationand treatment of this patientReferences[] C L Shields J A Shields R C Eagle and J P Rathmelllacrimal glandClinicopathologic review of cases oflesions Ophthalmology vol no pp [] R L Font J O Croxatto and N A Rao Tumors of the Eye andOcular Adnexa American Registry of Pathology WashingtonDC [] W Harrison P Pittman and T Cummings Pleomorphicadenoma of the lacrimal gland a review with updates ontransformation and molecular genetics SaudimalignantJournal of Ophthalmology vol no pp [] SO Baek YJ Lee SH Moon YJ Kim and YJ JunPrimary adenocarcinoma of the lacrimal gland Archives ofPlastic Surgery vol no pp [] D Bell M C Sniegowski K Wani V Prieto and B EsmaeliMutationallacrimal gland carcinomas andimplications for treatment Head Neck vol Supplement pp E724E729 landscape of[] J A Shields C L Shields J A Epstein R Scartozzi and R CEagle Jr Primary epithelial malignancies of the lacrimalgland the Ramon L Font lecture Ophthalmic Plastic Reconstructive Surgery vol no pp [] Lacrimal Gland Tumor Study Group An epidemiologicalsurvey of lacrimal fossa lesions in Japan number of patientsand their sex ratio by pathological diagnosis Japanese Journalof Ophthalmology vol no pp [] S L von Holstein M H TherkildsenJ U PrauseG Stenman V D Siersma and S Heegaard Lacrimal glandlesions in Denmark between and Acta Ophthalmologica vol no pp [] W M Hassan M S Bakry H M Hassan and A S AlfaarIncidence of orbital conjunctival and lacrimal gland malignant tumors in USA from Surveillance Epidemiology and 0c' | 2 |
Ofï¬cial Case Reports Journal of the Asian Paciï¬c Society of RespirologyRespirology Case ReportsEndobronchial metastases from a primary embryonalcarcinomaChiKang Teng1ChihYen Tu121Division of Pulmonary and Critical Care Medicine Department of Internal Medicine China Medical University Hospital Taichung Taiwan2School of Medicine China Medical University Taichung Taiwan3Division of Pathology China Medical University Hospital Taichung Taiwan WenChien Cheng12 ChiehLung Chen1 TingHan Chen1 YunShan Lin3 AbstractWe report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung Bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus Cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma The patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin BEP chemotherapy regimenKeywordsCryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourCorrespondenceWenChien Cheng Division of Pulmonary and Critical Care Medicine Department of Internal MedicineChina Medical University Hospital No YudeRoad North Dis Taichung City TaiwanEmail wcchengdrgmailcomReceived July Revised July Accepted July Associate Editor James HoRespirology Case Reports e00644 101002rcr2644IntroductionLung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases EBMs from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] Primary lung cancer is the most common cause of endobronchialtumours Extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] Although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma EBMs from embryonal carcinomas are extremely rare [] In this report we present acase of EBM from a primary embryonal carcinomaCase ReportA 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days Upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days He reported no fever chills coldsweats weight loss or decreased appetite A chest radiograph at admission revealed complete collapse of the leftlung Fig 1A Computed tomography CT was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumBronchoscopytumourobstructing theFig 1B Theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung Immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 TTF1negative sallike protein SALL4positive and cluster of differentiation CD30positive These ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomaFig We checked the levels of tumour markers in thepatient including those of alphafetoprotein AFP betahuman chorionic gonadotropin BhCG and lactate dehydrogenase LDH Each tumour marker was found to be The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of RespirologyThis is an access under the terms of the Creative Commons AttributionNonCommercialNoDerivs License which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made Vol Iss e00644Page 0cEBM from embryonal carcinomaCK Teng et alFigure Chestradiograph and bronchoscopic view of the endobronchial metastasesEBM A Complete collapse of the left lungon chest radiograph B Bronchoscopic viewof the endobronchial tumour within the leftmain bronchusFigure Tumour pathology of metastatic embryonal carcinoma A Embryonal carcinoma with a complex glandular growth pattern The characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation B Immunohistochemical staining with antithyroid transcription factor1 TTF1 highlighting cells in the alveolar space original magniï¬cation C Immunohistochemical staining with antisallikeprotein SALL4 revealed diffuse nuclear staining original magniï¬cation D Immunohistochemical staining with anticluster of differentiation CD30 highlighting diffuse membranous staining original magniï¬cation presentin high levels AFP ngmL BhCG mIUmL and LDH IUL The patientunderwent a right orchiectomy followed by a BEPbleomycin etoposide cisplatin chemotherapy regimenDiscussionWe report here the case of a young man with an EBMfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumoursLikewise manykindsLung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverEBMs from extrapulmonary malignancies are rare [] Primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith EBM primarily breast colon and renal carcinomas[] EBMs from testicular seminomas are also extremelyrare The majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0cCK Teng et alEBM from embryonal carcinomaTable Reports of previous cases of EBMsLocationDiagnostic methodPathologyzt¼rk []MoreiraMeyer []Case Case The oriï¬ce of right upper lobeRight main bronchusLeft main bronchus main carina andright main bronchus Fibreoptic bronchoscopy Mixed GCTFibreoptic bronchoscopy Mixed GCTVideobronchoscopyEmbryonal carcinomazsu []The oriï¬ce of the right upper lobeFibreoptic bronchoscopyTesticular seminomaTuran []Varkey []Our caseand right intermediary lobeRight intermediate bronchusLeft main bronchusLeft main bronchusEBM endobronchial metastases GCT germ cell tumourembryonic carcinomas or seminomas There are only a few published reports of primary testicularembryonic carcinomas resulting in EBMs []The mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently Howeversome patients are asymptomatic [] In our patient symptoms on presentation included haemoptysis and shortnessof breathResults from chest radiography in patients with EBMcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation [] Our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyHowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone Toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue The ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy The former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure [] The patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomaWe had evaluated the presence of AFP BhCG andLDH tumour markers Elevated AFP levels can be secretedby germ cell tumours GCTs including embryonal carcinoma yolk sac tumour or teratoma In GCTs detectableRigid bronchoscopyBronchoscopyFibreoptic bronchoscopyand cryosurgerySomatictype GCTEmbryonal carcinomaEmbryonal carcinomaBhCG elevation is observed in both seminomas and nonseminomas The serum level of LDH was directly correlated with tumour burden in nonseminomatous GCTswhich is also useful for the surveillance of patients withadvanced seminoma [] The tumour markers in ourpatient showed elevated levels of AFP BhCG and LDHThis was compatible with the diagnosis of embryonal carcinoma MoreiraMeyer also evaluated the patienttumour markers and found elevated levels of AFP ngmL and BhCG mIUmL The elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma [] zsu onlyevaluated the patients BhCG level which was found to beelevated mIUmL and the ï¬nal diagnosis wasmetastatic testicular seminoma []On comparison with previous case reports Table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery Other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation APC to control bleedingCryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining Cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree samplesHaemorrhage was observed to be similar during both procedures [] Further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesIn conclusion EBMs from primary GCTs notably thoseassociated with total or partial collapse are extremely rareWe have presented this case to emphasize the importanceof distinguishing EBM from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0cEBM from embryonal carcinomaDisclosure StatementAppropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisReferences zt¼rk A Aktas¸ Z and Yılmaz A Endobronchialmetastasis of mixed germ cell tumors two cases TuberkToraks Lee SH Jung JY Kim DH Endobronchialmetastases from extrathoracic malignancy Yonsei Med J Ikemura K Lin DM Martyn CP Endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing Lung MoreiraMeyer A BautistaHerrera D Hern¡ndezembryonal EndobronchialGonz¡lez MCK Teng et alcarcinomaJ BronchologyInterv Pulmonol zsu S Erol MM Oztuna F Endobronchial metastasis from testicular seminoma Med Princ Pract tumoraltesticular germ cell Turan D Akif zg¼l M Kirkil GEndobronchial metastasis ofEurasian J Pulmonol et Varkey B and Heckman MG Diagnosis of a case ofembryonal carcinoma by bronchial biopsy Chest Paradis TJ Dixon J and Tieu BH The role of bronchoscopy in the diagnosis of airway disease J Thorac Dis Aktas Z Gunay E Hoca NT Endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisAnn Thorac Med Barlow LJ Badalato GM and McKiernan JM Serumtumor markers in the evaluation of male germ cell tumorsNat Rev Urol The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0c' | 2 |
"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 [1315] including lung cancer [16]. Moreover recent studies have suggested a link between expression of some miRNAs and radiotherapy particularly in lung cancer [1719]. microRNA-21 (miR-21) is a miRNA which has been reported to be overexpressed in many human malignancies including NSCLC [2022]. 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." | 1 |
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o | 0 |
"Despite recent interest in the use of ketogenic diets KDs for cancer evidence of beneficial effects islacking This study examined the impact of a randomly assigned KD on quality of life physical activity andbiomarkers in patients with breast cancerMethod A total of patients with locally advanced or metastatic breast cancer and without a history of renaldisease or diabetes were randomly assigned to either a KD or a control group for this 12week trial Concurrentwith the first third and fifth chemotherapy sessions quality of life physical activity and biomarkers thyroidfunction tests electrolytes albumin ammonia ALP lactate and serum ketones were assessed Dietary intake wasalso recorded on admission and the end of the treatmentResults No significant differences were seen in quality of life or physical activity scores between the twogroups after weeks however the KD group showed higher global quality of life and physical activityscores compared to the control group at weeks P P Also serum lactate and ALP levelsdecreased significantly in the KD group compared to the control group at the end of the intervention ± vs ± ± vs ± P and P respectively A significant inverse associationwas observed between total carbohydrate intake and serum betahydroxybutyrate at weeks r P No significant differences between groups were observed in thyroid hormones electrolytes albuminLDH or ammonia Compliance among KD subjects ranged from to as assessed by dietary intakeand serum ketones levels of Continued on next page Correspondence khodabakhshiadelehyahoocom6Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranFull 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 0cKhodabakhshi Nutrition Journal Page of Continued from previous pageConclusion According to our results besides a higher global quality of life and physical activity scores compared tothe control group at weeks KD diet combined to chemotherapy in patients with breast cancer does not bringadditional benefit about quality of life and physical activity at weeks However decreases seen in levels of lactateand ALP in the KD group suggest that a KD may benefit patients with breast cancerTrial registration This trial has been registered on Iranian Registry of Clinical Trials IRCT under the identification codeIRCT20171105037259N2 wwwirctirtrial30755Keywords Ketogenic diet Breast cancer quality of life Physical activity Lactate Alkaline phosphatase chemotherapythere are stillIntroductionKetogenic diets KDs are high in fat and very low incarbohydrate They have been used as a dietary treatment in epilepsy for nearly a century [] RecentlyKDs have gained the attention of cancer researchersdue to their potential impact on cancer cell metabolism [] Despite the growing evidence of possibleantitumor benefitssome concernsabout potential adverse effects of KDs in cancer patientsincluding micronutrient deficiencies appetitereduction nausea constipation [] fatigue [] hyperlipidemia and especially unintended weight loss [ ]KDs are perceived as restrictive in nature which mayadd to the burden of cancer patients who already suffer from considerable physical emotional and financialstress all of which are known to negativelyimpact quality of life QoL In addition alterations inphysical and cognitive function during cancer treatment are pervasive It is estimated that of patients undergoingfromcancerrelated fatigue [] Prior studies have foundthat KD may improve physical and mental wellbeing[] Less fatigue has been reported in healthy overweight and obese adults following lowglycemic compared to highglycemic diets [] Results ofthreestudies using the validated European anization forResearch and Treatment core QoL questionnaire tofindings [] Aassess fatigue lacked consistentin advanced cancer patients showed imsmall trialprovementin sleep and emotionalfunction after athreemonth KD intervention [] Other studies havesuggested enhanced cognitive function [ ]cancertreatmentsufferTo date only four studies have assessed QoL in adultpatients with cancer [ ] Hunger is a reported sideeffect of restricted KDs however previous research hasfound that perceived hunger is reduced in low carbohydrate diets compared to low fat diets [] A recent systematic review has highlighted the need for additionallarger investigations on the impact of ketogenic diets onQoL [] The goal of this present trial was to assesswhether a KD had beneficial effects on QoL dietary intake physical activity and specific biomarkersinindividuals with breast cancer while also evaluating compliance to KD guidelines in these patientsThe protocol used in this trial [] and part of the resultsfrom this trial have been previously published [ ]MethodsThe study protocol was approved by the National Nutrition and Food Technology Research InstituteNNFTRI Shahid Beheshti University of MedicalSciencesIRSBMUNNFTRIREC1396187 All participants provided writteninformed consent prior to participating in the studySBMU TehranIranThis trial was a randomized controlled labelclinical trial to breast cancer patients with locally advanced or metastatic disease who were receiving chemotherapy for atleast weeks The studythe medical oncology clinic atwas conducted atShohadaeTajrish hospital Cancer Research CenterTehran Iran from July to October of Participation was to patients to years of ageExclusion criteria screened forsignificant cardiacrenal or neurologic comorbidities symptoms of malnutrition diabetes pregnancy and Karnofsky indexless than Using a block balanced randomizationmethod patients were assigned to the interventionn or controln groups Randomizationwas computergenerated by a statistician who was nota member of the medical team Blinding the participants or study personnel was not deemed feasible inthis dietintervention The project coordinator enrolled the participants and assigned them to their interventions Both the KD and the control diet werecalculated to be eucaloric using the MifflinSt Jeorformula The KD consisted of of calories fromCHO from protein from mediumchain triglyceride MCT oil and from fat A dietitianprovided specific nutritional counseling to each participantfacetoface meetings Patientsengaged in ongoing weekly counseling sessions viaphone WhatsApp or Telegram and were assessed forcompliance and possible adverse effects To furtherenhance compliance dietary recommendations werein individual 0cKhodabakhshi Nutrition Journal Page of individualized and appropriate recipes were providedto patients in the KD group were asked to refrainfrom eating any grains grain products starchy vegetables fruit or sugar Dietary carbohydrates were limited to nonstarchy vegetables and dietary proteinswere obtained primarily from egg meat poultry andfish Small amounts of lower carbohydrate berries andnuts were allowed as long as they did not exceed thecarbohydrate limit in the diet prescription Subjectswere encouraged to increase their fat intake and toselect from a variety of sourcesincluding olive oilbutter and cream cheese Patients were asked tochoose only the foods specified in the diet plan provided to them Patients were also encouraged to usemediumchain triglyceride MCT oil MCT oil anodorless and tasteless saturated fat does not requirebile or pancreatic enzymes for digestion It is easilyconverted to ketones in the liver thereby enhancingketosis Every weeks ml of MCT oilfromNutricia Erlangen Germany was provided to eachsubject in the KD group For better tolerance initialdosage of MCT was kept low and increased daily overa 6day period until maximum tolerable dosage wasachieved Dosage was reduced in a similar steppedprocessThe patients in the control group were instructed tofollow a standard diet consisting of CHO protein and fat Dietary compliance was checked byassessing blood betahydroxybutyrate levels every weeks and dietary intake at baseline and end of thestudyQoL assessmentQoL was assessed using the EORTC QLQC30 version and IORTC QLQBR23 questionnaires developed bythe European anization for Research and Treatmentof Cancer The validity and reliability of the questionnaires has previously been evaluated in Iran [ ]The questionnaires were completed at enrollment at weeks and at the end of the interventionDietary intake assessmentHospital dietitians used a 24h dietary recall 24HR toobtain a total of days intake one weekend day andtwo workdays through telephone and facetoface interviews both at the beginning and end of the study Theamount of each food consumed was estimated usingcommon household containers bowls cups and glassesand standard measuring cups and spoons as referencesThe mean quantity of total energy carbohydrate proteinand fat were estimated from the 24HRDietary intake wasanalyzed by Nutritionist IV software Version USPhysical activity assessmentPhysical activity was measured using the IPAC International Physical Activity questionnaire at baseline at weeks and at the end of the studyBiomarker assessmentFasting blood sampling for serum Na K Ca P lactate Mg LDH albumin ammonia and ALP were performed at baseline midway through the intervention weeks and at weeks T3 T4 and TSH were measuredat baseline and the end of the interventionStatistical analysisConsidering the power and α the sample sizewas calculated as individuals per group Assuming a dropout during the weeks of the study the finalnumber of participants was calculated as patients ineach groupStatistical analysis was carried out according to theintentiontotreat protocol Continuous variables weretested for normal distribution by the KolmogorovSmirnov test and then reported as mean ± standard deviation or median as appropriate Student ttest or MannWhitney U test was used to compare the continuousvariables between the two groups Paired sample ttestor Wilcoxon was used to compare the continuous variables within the two groups The ANCOVA test wasused to eliminate the effect of confounding factorsPearson correlation analyses were used to estimate associations between total carbohydrate intake and serumbetahydroxybutyrateData were analyzed using the SPSS version software Chicago IL USA and Stata version P was considered as statistically significantResultsDetailed patient demographics and a flow diagram werereported previously [] A total of women withbreast cancer were enrolled and randomly assigned to either the intervention n or control n groupsThree patients in the control group withdrew before beginning their assigned diet while10 patients in the KDgroup and patients in the control group withdrewfrom the study after beginning their assigned diet Ultimately patients in each group completed the studyand were included in the analysis No significant differences were seen between the two groups with regard toage cancer type metastasis and marriage or educationstatus P The intervention group included patients with locally advanced disease and patients withmetastatic disease liver bone lung liver andbone while the control group consisted of patientswith locally advanced disease and patients with 0cKhodabakhshi Nutrition Journal Page of metastatic disease bone liver lung liver andbone at other sites P Table Data related to quality of life are shown in Tables and No significant differences were seen in QoL betweenthe two groups after weeks however the KD groupshowed better global QoL compared to the controlgroup at week P Also at week diarrhea increased in the control groupcompared to the intervention P Data on week not shown Using the QoL questionnaire there was awithingroup decrease in reported hunger from baselineto weeks in the KD group P A withingroupdecrease was seen in physical performance measuresfrom baseline to weeks in both groups which was significant only in the KD group P In addition rolefunctioning and socialfunctioning scores significantlydecreased in the control group compared to the baselinebut not in the KD group P P Table Mean dietary intake is shown in Table and Fig The mean caloric and carbohydrate intake decreasedsignificantly at the end of the study compared to control P and P respectively while fatintake increased significantly in the KD group compared to the control group P After adjustingfor total energy intake this difference remained significant When data from both groups was combineda significant inverse association was observed betweentotalserum betaandr P hydroxybutyratealthough this effect was not seen when the KD groupwas analyzed separatelyintakeat weekscarbohydrateWithingroup analysis showed significant decreasesin energy carbohydrate and protein intake in bothgroups compared to the baseline Fat intake increasedsignificantly compared to the baseline in the KDgroup P and decreased significantly in thecontrol group P During the intervention of the subjects in the KDarm limited carbohydrates to g and of subjects consumed of calories from carbohydratesAt weeks of patients in the KD group hadserum ketones mmolL at 6weeks had ketone levels of mmolL As previously reportedserum ketone concentrations increased significantly inthe KD group ± to ± mmollP []At weeks physical activity improved in the KD groupcompared to the control group adjusted for cancer typeand baseline value P but after weeks physicalactivity did not show any significant differences in a between or withingroup analysis Fig No significant difference was observed in a betweenor withingroup analysis of thyroid hormones electrolytes albumin Ammonia and LDH Howeverlactateand ALP decreased significantly after intervention in theKD group compared to the control group P andP respectively ALP is adjusted for baseline valueand cancer type Table Data on thyroid hormones notshownDiscussionThe effect of KD on QoL physical activity dietary intake and biomarkers in patients with locally advancedand metastatic breast cancers was evaluated in thisstudy Based on our findings in the KD group globalQoL was higher at weeks perhaps in part because diarrhea was more frequent in the control group than theKD group No significant differences were seen in theQoL physical activity and biomarkers between the twogroups after the week intervention Lactate and ALPwere lower in the KD group compared to the controlEffect of diet on QoLIn our study in the KD group global QoL was higher at weeks No adverse effects were observed in thoseTable Baseline characteristics in breast cancer patients before interventionScale categoriesCancer TypeLocally AdvancedIntervention Ketogenic dietn Control Ordinaryn ERPRHER2Metastaticpositivenegativepositivenegativepositivenegative ER Estrogen receptor PR Progesterone receptorHER2 Human epidermal growth factor receptor aCalculated by chi square testbCategorical data shown as No p value008a057a043a079a 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before andafter intervention in KD group and control group as measuredby the EORTC QLQC30aFunctioningaPhysical functioningMD CIControlKDpvalueTable Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQC30SymptomsaFatiguepvalueControlKD Week Week pvalue33aNausea and vomitingWeek Week pvalueRole functioningWeek Week pvalue ± 11a ± ± ± Cognitive functioningWeek Week pvalue ± ± Emotional functioningWeek Week pvalueSocial functioningWeek Week pvalue ± ± ± ± Global quality of lifeWeek Week pvalue ± ± ± ± ± ± ± ± ± ± ± ± ± ± After adjusting for baseline value and chemotherapy status no significantdifferences were observedStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the two groupsData shown as mean and SDaThe higher values indicate higher level of functioning and quality of lifeparticipants assigned to the KD compared to the controlgroup after weeks Withingroup analysis showed decreased hunger and physical function in the KD groupcompared to the baseline In the control group role andsocial functioning decreased significantly compared tobaselineResults of a systematic review and metaanalysis haveshown that KDs suppress appetite [] Decrease in hunger or appetite in our study may be due to the high fatcontent of the KD as it decreases the ghrelin releasewhich in turn may reduce appetite High fat intake alsoslows digestion which could also impact the perceptionof hunger Previously we have shown that the KD resultsin weight loss [] As a clinical benefit KDinduced Week Week pvaluePainWeek Week pvalueReduction in appetiteWeek Week pvalueSleep difficultiesWeek Week pvalueDyspneaWeek Week pvalueConstipationWeek Week pvalueDiarrheaWeek Week pvalueFinancial concernsWeek Week pvalue MannWhitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate a higher grade of symptoms Data shown asmedian and quartile 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQBR23aKDControlpvalue aFunctioningFuture perspectiveWeek Week pvaluebSymptomsArmWeek Week pvalueBreastWeek Week pvalue Week Systemic therapy side effects Week pvalueConcerns over hair lossWeek Week pvalue Table Comparison of mean ± SD macronutrient intake atbaseline and 12weeksVariableMD CIpvalueKDMean ± SDControlMean ± SDEnergy KcaldayBefore ± ± ± AfterpvalueCarbohydrate grBefore ± ± ± ± AfterpvalueProtein grBeforeAfterpvalueFat gr ± ± ± ± ± 0001a 041aBefore ± ± ± ± AfterpvalueStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the groupsMD Mean differenceCI Confidence intervalaAncova Adjusted for baseline value and energy0001aMannWhitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate higher level of functioning and quality of lifebThe higher values indicate a higher grade of symptomsData shown as median and quartile decreases in appetite weight and body fat may result infavorable changes in breast cancer patients notably inoverweight or obese women [ ]In contrast with our findings Cohen found that aKD significantly enhanced physical function scores inwomen with ovarian or endometrial cancer comparedto the control group but appetite did not change atthe end of the study compared to the baseline []Part ofstudy andCohens trial may be explained by the design of thestudy While only of the participants in the Cohen study were undergoing chemotherapy all of ourpatients were receiving treatmentthe inconsistency between ourAlso timing of the administration of the questionnaires and whether the participants were in positive ornegative energy balance may have influenced ourfindingsNo significant difference was reported in QoL at theend of study compared to the baseline by TanShalaby [] However a slight decrease in physical androle functioning as well as temporary constipation andfatigue were reported in the KD group in one study []In our study constipation was noted by participants inthe KD arm during the early days which was managedby dietary changesAlso after weeks in the KD group physical activityscores was higher compared to the control group but at weeks differencessignificantbetween the two groupsin scores were notDietary intake and adherenceOur study data showed a significant decrease in carbohydrate intake and a significant increase in fat intake inthe KD group compared to the control Protein intakewas not significantly different between the two groupsbut decreased overall in both groups when compared tobaseline Total daily carbohydrate intake was similar toresults in the Cohen study [] We also assessed serumbetahydroxybutyrateIn the KD group ofpatients at weeks and at 6weekshad serum ketones and patients at weeks and weeks 0cKhodabakhshi Nutrition Journal Page of Fig Mean caloric intake and distribution of macronutrients as percentage of total kilocalories before and after week intervention in breastcancer patients in two groupsFig Comparison of trend changes in physical activity in breast cancer patients in two groups 0cKhodabakhshi Nutrition Journal Page of LBsvskwleuavpitnopdmisvskwleuavpLBsviitnopdmeuavlpinorrefnoBnodesabldetauclacerewseuavlpllaerusaemdetaepeRepytlsisyanAlavretnIecnedifnoCICecnereffiDnaeMDMpuwolloftsalroskeewskeewkeewropuwolloftsitnopdMiepytrecnacdnaeuavlenilesabrofdetsudAjavocnAsnosirapmoclepitlumrofnoitcerroc±±±±skeew±±itnopdMi±±enilesaB±±±±±±smArlairTDKICDMlortnoClortnoCDKlebairaVetatcaLluHDLstneitaprecnactsaerbdetaertDKdnalortnocnislevelrekramoBielbaTICDM±±±±lortnoCDKldgcmianommAICDMICDM±±±±±±lortnoCDK±±±±±±lortnoCDKICDMICDM±±±±±±±±±±±±ICDMlortnoCDKlortnoCDK±±±±±±±±±±lortnoCDKICDM±±ICDMlortnoCDKICDM±±±±±±lortnoCDKLdgmgMldginmubAllqemKlDgmPPLAldgmaClqemaNenilesaBLB 0cKhodabakhshi Nutrition Journal Page of had serum ketones mmoll Cohen reported that of patients had betahydroxybutyrate concentrations mmollA recent systematic study of KDs in adult cancerpatients reported a range of to with a adherence rate overall reported by [] According toour data the level of adherence to the KD interventionsuggests that the diet is a feasible option for women withbreast cancer who are receiving chemotherapyDespite the lack of any restriction in calorie intakein the study design and consistent with findings ofCohen [] the KD group showed a significant reduction in calorie intake compared to the control groupThe decrease in calorie intake may be due to reductions in appetite associated with ketosis as the subjects in the KD arm did not consume all of the fatcalculated for their diet This may also be due in partto customary practices surrounding meal preparationA decrease in appetite and subsequentinadvertentcalorie restriction most often results in weight loss inthe absence of malnutrition or cachexiathis mayhave antiinflammatory and proapoptotic propertieswhich in turn may exert a positive effect on thesehallmarks of cancer Ketosis may also enhance theeffectiveness of chemotherapy while reducing the sideeffects of treatment [ ]Effect of diet on biomarkersConsistent with the outcomes of the previous studiesour results revealed that the KD had no adverse effecton thyroid hormones electrolytes LDH urea and albumin Significant decreases were seen in serum levels oflactate KDs reduce glycolytic activity which in turn mayslow metastases by reducing the acidity of the tumormicroenvironment and lowering the availability of lactate as a substrate for biomass synthesis [] Decreaseswere also seen in ALP High levels of ALP in breast cancer patients is a negative prognostic marker often indicating progression of metastatic disease [] Moreresearch is needed to assess whether lower ALP and lactate as seen in this study contributes to slower rates ofdisease progressionTo our knowledge this is the first randomized controlled trial examining the effects of a KD on QoL inbreast cancer patientsThe primary limitation of this study was the heterogeneous nature of the sample in regards to cancer stageA secondary limitation was the small sample sizeConclusionAccording to our results besides a higher global QoLand physical activity scores compared to the controlgroup at weeks KD diet combined to chemotherapy inpatients with breast cancer does not bring additionalbenefit about QoL and physical activity at weeksWhile many blood biomarkers did not differ significantlybetween the two groups ketosis may still offer benefit tosome patients with breast cancer in part by decreasinglactate and ALPSupplementary informationSupplementary information accompanies this paper at doi101186s1293702000596yAdditional file figure Flow diagram of the patient treatmentprocessAdditional file figure Median confidence interval tyroidhormones in baseline and 12week by two trial arms in breast cancerpatientsAcknowledgmentsWe would like to thank all the patients at our clinic who took part in thisstudyAuthors contributionsKhodabakhshi carried out the conception Methodology performed theexperiments design of the diet and wrote the Davoodi and Seyfriedcollaborated in the design of the study Davoodi supervise on the thesisKalamian and Beheshti collaborated in the design of the diet Kalamian gavecritical review of the manuscript All authors have read and approved thefinal manuscriptFundingNot applicableAvailability of data and materialsData described in the manuscript code book and analytic code will bemade available upon request pendingEthics approval and consent to participateAll participants provided written informed consent prior to participating inthe studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nutrition School of Public Health Kerman University ofMedical Sciences Kerman Iran 2Physiology Research Center KermanUniversity of Medical Sciences Kerman Iran 3Biology Department BostonCollege Chestnut Hill MA USA 4Dietary Therapies LLC Hamilton MT USA5Department of Nutrition and Dietetics Mofid childrens hospital ShahidBeheshti University of Medical Sciences Tehran Iran 6Cancer ResearchCenter Shahid Beheshti University of Medical Sciences Tehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranReceived March Accepted July ReferencesNeal EG Chaffe H Schwartz RH Lawson MS Edwards N Fitzsimmons G The ketogenic diet for the treatment of childhood epilepsy arandomised controlled trial Lancet Neurol Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab 0cKhodabakhshi Nutrition Journal Page of adverse effects on blood lipids a randomized controlled trial Nutr CancerFine EJ SegalIsaacson CJ Feinman RD Herszkopf S Romano MC TomutaN Targeting insulin inhibition as a metabolic therapy in advancedcancer a pilot safety and feasibility dietary trial in patients Nutrition Epub Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab Mukherjee P Mulrooney TJ Marsh J Blair D Chiles TC Seyfried TNDifferential effects of energy stress on AMPK phosphorylation and apoptosisin experimental brain tumor and normal brain Mol Cancer Gatenby RA Gawlinski ET Gmitro AF Kaylor B Gillies RJ Acidmediatedtumor invasion a multidisciplinary study Cancer Res Singh A Pandey A Tewari M Kumar R Sharma A Singh K Advancedstage of breast cancer hoist alkaline phosphatase activity risk factor forfemales in India Biotech Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsHuebner J Marienfeld S Abbenhardt C Ulrich C Muenstedt K Micke O Counseling patients on cancer diets a review of the literature andrecommendations for clinical practice Anticancer Res Champ CE Palmer JD Volek JS WernerWasik M Andrews DW Evans JJ Targeting metabolism with a ketogenic diet during the treatment ofglioblastoma multiforme J NeuroOncol Zuccoli G Marcello N Pisanello A Servadei F Vaccaro S Mukherjee P et alMetabolic management of glioblastoma multiforme using standard therapytogether with a restricted ketogenic diet case report Nutr Metab Servaes P Verhagen C Bleijenberg G Fatigue in cancer patients during andafter treatment prevalence correlates and interventions Eur J Cancer Cohen C Fontaine K Arend R Soleymani T Gower B Favorable effects of aKetogenic diet on physical function perceived energy and food cravings inwomen with ovarian or endometrial Cancer a randomized Controlled TrialNutrients Breymeyer KL Lampe JW McGregor BA Neuhouser ML Subjectivemood and energy levels of healthy weight and overweightobesehealthy adults on highand lowglycemic load experimental dietsAppetite TanShalaby JL Carrick J Edinger K Genovese D Liman AD Passero VA Modified Atkins diet in advanced malignanciesfinal results of a safetyand feasibility trial within the veterans affairs Pittsburgh healthcare systemNutr Metab Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Klement RJ Sweeney RA Impact of a ketogenic diet intervention duringradiotherapy on body composition I Initial clinical experience with sixprospectively studied patients BMC Res Notes Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Tóth C Clemens Z Halted progression of soft palate Cancer in a patienttreated with the Paleolithic Ketogenic diet alone a 20months followupAm J Med Case Rep Gibson AA Seimon RV Lee CM Ayre J Franklin J Markovic T Doketogenic diets really suppress appetite A systematic review and metaanalysis Obes Rev Sremanakova J Sowerbutts A Burden S A systematic review of the use ofketogenic diets in adult patients with cancer J Hum Nutr Diet Khodabakhshi A Akbari ME Mirzaei HR Kazemian E Kalantari K Kalamian M Effects of ketogenic diet for breast cancer treatment A protocol forrandomized controlled clinical trial J Biochem Technol Khodabakhshi A Akbari ME Mirzaei HR MehradMajd H Kalamian MDavoodi SH Feasibility safety and beneficial effects of MCTbasedKetogenic diet for breast Cancer treatment a randomized controlled trialstudy Nutr Cancer Khodabakhshi A Akbari ME Mirzaei HR Seyfried TN Kalamian M DavoodiSH Effects of Ketogenic metabolic therapy on patients with breast Cancer arandomized controlled clinical trial Clin Nutr in press Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe European anization for Research and Treatment of Cancer quality oflife questionnaire EORTC QLQC30 translation and validation study of theIranian version Support Care Cancer Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe EORTC breast cancerspecific quality of life questionnaire EORTC QLQBR23 translation and validation study of the Iranian version Qual Life Res Champ CE Volek JS Siglin J Jin L Simone NL Weight gain metabolicsyndrome and breast cancer recurrence are dietary recommendationssupported by the data Int J Breast Ca | 2 |
"evaluate the clinicopathologic characteristics of Lymph Node metastasisbetween investing layer of Cervical fascia and deep fascia of infrahyoid strap Muscles LNCM in papillary thyroidcarcinoma PTCMethods Retrospective review of patients with PTC who underwent thyroidectomy and central compartment neckdissection CND from January to January was performed in two tertiary referral academic medicalcenters A total of consecutive patients with PTC who underwent thyroidectomy and CND were included inthe retrospective review The LNCM was resected as a separate specimen by the surgeon and the clinicopathologiccharacteristics of the patients were recorded Multivariate logistic regression analysis was performed to identify riskfactors for LNCM metastasisResults Of PTC patients patients had lymph nodes in the LNCM Among them caseswere confirmed to be positive in the LNCM In total the metastasis rate of LNCM in PTC patients was Univariate analysis revealed that the metastasis of LNCM were more likely to have a primary site in theinferior pole extrathyroidal extension ETE central cervical metastasis level III and level IV metastasis Multivariateanalysis further showed tumor location in the inferior pole ETE level III and level IV metastasis conferred asignificantly increased odds ratio for LNCM metastasisConclusion Attention should be paid to the lymph tissue in the LNCM for PTC patients especially in presence of aprimary site in the inferior pole ETE level III and level IV metastasisKeywords Thyroid carcinoma Surgery Central compartment neck dissection Recurrence Suprasternal spaceIntroductionPatients with papillary thyroid carcinoma PTC have afavorable prognosis with central neck locoregional recurrence varying from to [] The goal of a prophylacticor therapeutic central compartment neck dissection pCNDor tCND is to decrease the incidence of local recurrenceby removing all lymphatic tissue within the levels VI and Correspondence wugaosongwhueducn1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei Peoples Republic of ChinaFull list of author information is available at the end of the VII compartments which are generally the first and themost commonly involved with metastasis [] For patientswithout evidence of lymph node metastasis on preoperativeevaluation the additive value of a pCND at the time ofthyroidectomy is controversial Some authors advocatepCND considering high rate of occult metastaticnodal disease in cN0 PTC [] while other authors considerthat there is no highlevel evidence in favor of pCND []The performance of pCND is dependent on the weightgiven to the risks and benefits of pCND [] Consideringthe oncologic benefits of CND and the risks of a repeat The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of neck operation performing pCND is recommended toevery patient in China [ ]Although American Thyroid Association ATA guideline has defined the boundary of central neck compartment there is also significant variability in terms of theextent of CND In routine clinical practice CND canrange from sampling a few nodes in the paratrachealregion to a complete clearance from left carotid artery toright carotid artery and down to and including the uppermediastinum [] Owing to the variant extent of CNDsome central compartments are easily to be neglectedFor thyroid carcinoma patients with specific clinicopathologic characteristics incomplete lymph node dissectionmay result in increased recurrence reoperation andreoperationassociated complications [] Lymph Nodebetween investing layer of Cervical fascia and deep fasciainfrahyoid strap Muscles LNCM has not beenofreported The LNCM compartment is defined as followssuperiorly by the hyoid bonelaterally by the carotidarteries anteriorly by the investing layer of cervicalfascia and posteriorly by the deep fascia of infrahyoidstrap muscles LNCM space includes suprasternal spaceand intrainfrahyoid strap muscle spaceAnatomically LNCM is located anterior to the strapmuscles We consider that what is special about the concept of the LNCM is that it is belong to level VI but is aneasily overlooked anatomical area by a strap musculatureinvolving the sternohyoid and sternothyroid musclesduring selective or modified neck dissection Although themetastasis in LNCM was seldom it did occur in somePTC patients with regional recurrence As part of LCNMsuprasternal space metastasis for thyroid cancer wereinvestigated in three studies [] Thus we routinelydetected the suprasternal space and intrainfrahyoid strapmuscle space Fig This study was performed to identify the clinicopathologic characteristics and indication forlymph node metastasis dissection in the LNCMMaterials and methodsPatientsA retrospective review from the clinical and histopathologydatabase of two tertiary referral academic medical centersTongji Hospital of Huazhong University of Science andTechnology and Zhongnan Hospital of Wuhan Universityfrom January to January were conducted In theinstitutions preoperative examinations consisted of athorough physical examination neck ultrasound a clinicalevaluation of thyroid nodules and neck lymph nodes Fineneedle aspiration cytology FNAC were performed in patients who were suspected thyroid nodules or lymph nodeWith a pathological confirmation of PTC all the patientsreceived a thyroidectomy with CND Accordingly a pCNDwas performed for cN0 patients and a therapeutic CNDwas performed for cN1 patients The inclusion criteria forthe patients were as follows the clinical history completely recorded the LNCM was resected as a separatespecimen by the surgeon PTC patients who underwentthyroidectomy plus CND with or without lateral neckdissection A total of consecutive PTC patients wereenrolled The medical ethic committee of ZhongnanHospital of Wuhan University approved the procedure andinformed written consent was obtained from all patientsSurgical approachAll the operations were performed by the same seniorsurgeon Gaosong Wu with the patients under generalFig Four subdivisions Level VI 1st Level VI 2nd Level VI 3rd and Level VI 4th of central neck compartment are divided by deep fascia ofinfrahyoid muscles pretracheal visceral fascial and right recurrent laryngeal nerve 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of anesthesia Thyroidectomy was performed with a standard technique of fine capsular en bloc dissection andresection from inferior pole to superior pole []Intraoperative neuromonitoring was employed for all ofthe thyroidectomies [] Superior parathyroid glandswere identified and preserved in situ inferior parathyroidglands were protected in situ or autotransplanted in thesternocleidomastoid muscle according to three certaintypes based on their blood supply and location [ ]After the incision of the investing layer of cervicalfascia the interval between sternohyoid and sternothyroid muscles and the space anterior to the sternohyoidmuscle above the clavicle and the sternum weredetected If there was fibrofatty tissue Instead of the enbloc removal of the entire centrallymph nodes theLNCM was resected as a separate specimen if occurredThe presence or absence of lymph node metastasis wasdefined according to postoperative pathological reportsWhile dissecting paratracheal lymph nodes intraoperative neuromonitoring was employed to detect RLN fromdistally to proximally minimizing morbidity from injuryto RLN during compartment nodal dissection LNCMand other compartment lymphatic tissue were processedfor routine hematoxylin and eosine HE separatelyThe pathologic results were independently determinedby two qualified pathologists without any prior knowledge of the patients clinical dataData collection and statistics analysisTo determine the relation between LNCM metastasis andclinicopathologic factors such as age sex primary tumorsite lateral cervical lymph node metastasis level VI metastasis the chisquare test and Fishers exact test were usedas appropriate Multivariate logistic regression analysiswas performed to identify risk factors for LNCM metastasis of PTC P was considered statistically significantAll calculations were performed using SPSS for windows Postthyroidectomy hypocalcemia lasting for morethan months was considered as permanent VCP All patients were followed up every months postoperativelyResultsPatients detected with LNCMAfter reviewing patients who underwent thyroidectomy plus CND with or without lateral neck dissectionfrom January to January patients were detected with LNCM and patients wereabsent of LNCM The average tumor size of LNCM was cm and the mean number of lymph nodes sampledfrom LNCM was ranging from to Table showsthe comparison of clinicopathologic characteristics between the present LNCM group and the absent groupIn univariate analysis Hashimotos disease p multifocality leisions p the tumor located ininferior portion p extrathyroidal extensionETE p central cervical metastasis p level III and level IV metastasis p were significantly associated with high prevalence of LNCMPatients with metastatic LNCMAmong a total of patients with LNCM metastaticLNCM was found in patients Table compares the clinicopathologic characteristics between themetastatic LNCM group and the nonmetastatic LNCMgroup Three hundred eightythree patients were confirmed free of LNCM metastasis of themwith clinically negative node performed pCND and of them with clinically positive performed tCND All thepatients in the metastatic LNCM group performedtCND Lateral neck dissection was performed in cases in the metastatic LNCM group and cases in the nonmetastatic group all lateral neckdissection wastherapeutically performed Univariateanalysis was performed for the patients with and patients without metastatic LNCM Age at diagnosisgender and tumor size coexistent thyroid disease tumorfocality and level II metastasis were not correlated withLNCM metastasis Univariate analysis identified tumorlocated in the inferior pole central cervical metastasisETE level III and level IV metastasis as significant predictors of LNCM metastasis in our study population Multivariate analysis further showed that tumor location ETElevel III and level IV metastasis conferred a significantlyincreased odds ratio for LNCM metastasis Table ComplicationsThe median followup time was months range of patients had voice changes all of whomrecovered within months Temporary vocal cord paralysis was confirmed in patients by laryngoscope andthirteen permanent hypocalcemia was observed aftersurgeryDiscussionIn order to achieve the best chance of cure and effectivedisease control thoroughness of dissection has to betaken into account We prospectively performed comprehensive CND for PTC patients who underwent thyroidectomy and CND In addition data were analyzed for PTC patients to investigate the clinicopathologic characteristics for LNCM metastasis The occurrence rate ofLNCM was and of the patients harbored metastatic LNCM In total the positiverate of the LNCM was In this studymultivariate analysis revealed that a primary site in the inferior pole ETE level III and level IV metastasis were ofhigher LNCM metastasis rate which was consistent withthe findings by the previous report oflymph node 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for patients who had lymph nodes in LNCM compared tothose who did notVariablesAge mean ± SDAbsent n Present n P value¤ GenderFemale MaleTumor size cm ¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimotos diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes No LNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid Musclesmetastasis between sternocleidomastoid and sternohyoid muscle []Several studies have emphasized the importance of similar compartment in neck dissection for thyroid carcinomaSun pioneered the confirmation of the significantinvolvement of lymph node metastasis between sternocleidomastoid and sternohyoid muscle LNSS in lateralneck dissection [] which anatomically classified as part ofthe space of Burns They concluded that the positive rate ofLNSS was in clinically nodepositive cN PTCwhich was correlated with a primary site in the inferiorpole the lateral nodal metastasis level III and level IV nodalmetastasis [] Then Homma [] reported two casesof PTC patients with level III and IV lymph node metastases as well as metastasis in the suprasternal space Yu et al[] investigated the clinical significance of the suprasternalspace lymph node SSLN in pathological nodepositivepN PTC patients They concluded that metastasis rate ofSSLN was and the high SSLN metastasis of PTC wascorrelated with primary cancer site in the inferior thyroidpole strap muscle invasion level IV metastasis and LNSSmetastasis In our experience LNCM was rarely occurredin the central neck compartment and the positiveLNCM in PTC patients was infrequent as well Notably among the patients with pN PTC themetastasis rate of LNCM was which was muchlower than the metastasis incidence of SSLN described by Yu [] According to their results onefifth patients with pN PTC were performed incompleteCND and remained metastatic lymph nodes 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for positive LNCMVariablesAge mean ± SDMetastasis n Nonmetastasis n ¤ GenderFemale MaleTumor size cm ¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimotos diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes NoLNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid MusclesP value The total number of lymph nodes in the central neckcan range from to [] There is no consensus on thenumber of nodes removed or examined that would constitute an adequate dissection Aimed to allow surgeons tomore accurately report the extent of lymphadenectomyTable Multivariate analysis of predictors for LNCM positivityVariablesTumor locationp valueCentral cervical metastasisExtrathyroidal extensionLevel III metastasisLevel IV metastasisOR CI LNCM Lymph Node between superficial layer of deep Cervical fascia and deepfascia of infrahyoid Musclesvisceralwe divide the central neck compartment into four subdivisions by deep fascia of infrahyoid strap muscles pretrachealfascial and right RLN Fig Theproposed LNCM compartment is bounded superiorly bythe hyoid bone laterally by the carotid arteries anteriorlyby the investing layer of the cervical fascia and posteriorlyby the deep fascia of infrahyoid strap muscles which is defined as Level VI 1st In the current study suprasternalspace composed part of the LNCM Fig Compared toSSLN reported by Yu [] LNCM encompasseslymph nodes in the suprasternal space and lymph nodesbetween sternohyoid and sternothyroid muscles Figs and LNCM can fall under the normal subdivisions ofthe central compartment Subdivisions can actually recordthe extent of the CND which is able to provide detailedinformation for the possible second operation 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Fig LNCM coverage area in vivo Lymph nodes between sternohyoid and sternothyroid muscles a and lymph nodes in the suprasternal space bIncluding LNCM as an anatomical part of the centralneck allows for removal of previously unrecognized micrometastatic disease in of PTC patients with the inferiorportion lesions ETElevel III and level IV metastasisDissection of the LNCM space is less invasive and easy toachieve and is not timeconsuming It is at the entrance ofcentral neck compartment which is easy to expose and haslow risk of damaging RLN or parathyroid With the application ofintraoperative neuromonitoring and in situpreservation or autotransplantation of parathyroid theoccurrence of vocal cord paralysis and permanenthypoparathyroidism in the current study were lowerin this study [ ] Therefore in cases where LNCMspace metastasis is suspected or preoperative ultrasoundand CT suggests LNCM metastasis greater attentionshould be paid to the nodal tissue in the LNCM space inthyroid carcinoma patients These patients might benefitfrom a reduced risk of regional recurrence central neckreoperative morbidity and improved decision making inrelation to the use of radioiodine ablationThere are severallimitations in the present studyThe retrospective design is a limitation of the studyAnd this was two tertiary referral centers retrospective review and routine prophylactic nodal surgerywas offered in China however it is not standard elsewhere in the world which is a major limitation Aprospective randomized trial with a long time followup period may help to further evaluate the clinicalsignificance of LNCM in PTC patientsConclusionsIn summary additional dissection of nodes in theLNCM were accessible and might not increase morbidity Therefore attention should be paid to the lymphtissue between investing layer of cervical fascia and deepfascia ofinfrahyoid strap muscles for PTC patientsespecially in presence of inferior portion lesions ETElevel III and level IV metastasisAcknowledgementsThe authors thank the studied patients for their willingness to cooperatewith our studyAuthors contributionsGaosong Wu Study concepts and design Qianqian Yuan Study designmanuscript preparation and editing Jinxuan Hou Data analysis andmanuscript editing Yiqin Liao Data acquisition Lewei Zheng Manuscriptpreparation Fang Lu Data acquisition Kun Wang Quality control of dataand algorithms The authors read and approved the final manuscriptFundingThe authors have no support or funding to reportAvailability of data and materialsNot applicableEthics approval and consent to participateThis research was comprised of human participants and was approved byMedical Ethics Committee of Wuhan University Zhongnan Hospital Informedconsent was obtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei Peoples Republic of China 2Department of Thyroid and Breast Surgery Tongji Hospital of TongjiMedical College of Huazhong University of Science and Technology Jiefang Avenue Wuhan Hubei Peoples Republic of China Received April Accepted August ReferencesLang BH Ng SH Lau LL Cowling BJ Wong KP Wan KY A systematic reviewand metaanalysis of prophylactic central neck dissection on shorttermlocoregional recurrence in papillary thyroid carcinoma after totalthyroidectomy Thyroid So YK Seo MY Son Y Prophylactic central lymph node dissection forclinically nodenegative papillary thyroid microcarcinoma influence onserum thyroglobulin level recurrence rate and postoperative complicationsSurgery Hughes DT Rosen JE Evans DB Grubbs E Wang TS Solórzano CCProphylactic central compartment neck dissection in papillary thyroid 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Cancer and effect on Locoregional recurrence Ann Surg Oncol McHenry CR Is prophylactic central compartment neck dissection indicatedfor clinically nodenegative papillary thyroid Cancer the answer isdependent on how the data are interpreted and the weight given to therisks and benefits Ann Surg Oncol Selberherr A Riss P Scheuba C Niederle B Prophylactic firststep centralneck dissection level does not increase morbidity after Totalthyroidectomy Ann Surg Oncol Hartl DM Leboulleux S Al Ghuzlan A Baudin E Chami L Schlumberger M Optimization of staging of the neck with prophylactic central andlateral neck dissection for papillary thyroid carcinoma Ann Surg McAlister ED Goldstein DP Rotstein LE Redefining classification ofcentral neck dissection in differentiated thyroid cancer Head Neck Miller JE AlAttar NC Brown OH Shaughness GG Rosculet NP Avram AM Location and causation of residual lymph node metastasis aftersurgical treatment of regionally advanced differentiated thyroid CancerThyroid Sun G Wang Y Zhu Y Wang Y Xu K Wei W Lymph node metastasisbetween sternocleidomastoid and sternohyoid muscle in clinically nodepositive papillary thyroid carcinoma Head Neck Homma A Hatakeyama H Mizumachi T Furusawa J Kano S Sakashita T Lymph node metastasis in the suprasternal space from thyroidpapillary cancer Int Cancer Conf J Yu S Ge J Sun B Wei Z Lei S Lymph node metastasis in suprasternal spacein pathological nodepositive papillary thyroid carcinoma Eur J Surg Oncol Wu G Kong D Thyroidectomy with Wu Gaosong's ProcedureVideoEndocrinologr httpsdoi101089ve20150050 Wu G Wang K Intraoperative Neuromonitoring and Protection of theSuperior Laryngeal Nerve with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160070 Wu G Cui Q Wang K Carbon Nanops for Identifying Lymph Nodesand Protecting Parathyroid Glands in Thyroid Lobectomy with IpsilateralCentral Compartment Lymph Nodes Dissection VideoEndocrinology httpsdoi101089ve20160064Kong D Cui Q Gaosong W A Novel Classification of Parathyroid Glands andTheir Preservation in Thyroidectomy VideoEndocrinology httpsdoi101089ve20170093 Wang K Wu G Intraoperative Neuromonitoring in Selective Neck Dissectionfor Thyroid Cancer SND IIa Vb with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160082 Yuan Q Wu G Hou J Liao X Liao Y Chiang F Correlation betweenelectrophysiological changes and outcomes of vocal cord function in recurrent laryngeal nerves with visual integrity during thyroidectomyThyroid Cui Q Li Z Kong D Wang K Wu G A prospective cohort study of novelfunctional types of parathyroid glands in thyroidectomy Medicine 9552e5810Tavares MR Cruz JA Waisberg DR Toledo SP Takeda FR Cernea CR et alLymph node distribution in the central compartment of the neck ananatomic study Head Neck Wang K Cai H Kong D Cui Q Zhang D Wu G The identificationpreservation and classification of the external branch of the superiorlaryngeal nerve in thyroidectomy World J Surg Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
"SARSCoV2 infection associated respiratory disease COVID19 has evolved into a pandemic but being anew form of virus pathogenesis of disease causation is not fully understood and drugs and vaccinesagainst this virus are still being tested so that no effective drugs or vaccines have been advised byregulatory authority In this context the Ministry of AYUSH Government of India has recommendedAyush Kwath to improve the immunity and combat the infection Our objective of this literature reviewis to review the role of immunity in pathogenesis of COVID19 and role of Ayush Kwath against the virusand regulation of immunity Current review was conducted using a search of available literature onCOVID19 and immunity Vyadhikshamatwa Ayurveda and COVID19 Rasayana Coronavirus SARSCoV immunomodulatory effects of medicinal plants TulsiHoly BasilOcimum sanctum DalchiniCinnamonCinnamomum zeylanicum SunthiGingerZingiber ofï¬cinale and MarichBlack PepperPiper nigrum Ayurveda being an ancient science have both medicinal and cultural values and had stimulated our kitchenand uenced what we ate in different seasons and the remedies we used for common ailments Herbssuch as Tulsi Marich Sunthi Dalchini are the most commonly used and easily available drugs in homeThus Ayush Kwath due to its immunemodulatory antiviral antioxidant antiammatory antiplatelet antiatherosclerotic hepatoprotective renoprotective properties seems to be effective inimmunoregulation for controlling viral infections like COVID19 Further preclinical and clinical trialsneed to be done for the evaluation of safety and efï¬cacy of this polyherbal formulation The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation This is an access under the CC BYNCNDlicense httpcreativecommonslicensesbyncnd40 IntroductionCOVID19 also known as severe acute respiratory syndromecorona virus SARSCoV2 is an infectious disease believed to beoriginated from bats and transmitted to human beings [] Being anew form of virus pathogenesis of disease causation is not fullyunderstood and drugs and vaccines against this virus are still beingtested so that no effective drugs or vaccines have been advised byregulatory authority Not only for Coronavirus have many otherviruses also lack preventive vaccines and effective antiviral medications Studies have explored that these viruses can form drugresistant mutants which decrease the existing drugs efï¬cacy Sothese viruses can be a threat to the mankind for long time [] Corresponding authorEmail shankargautammohpgovnpPeer review under responsibility of Transdisciplinary University BangaloreHigh mortality among immunecompromised and those withsome underlying pathology implies that the factors that improveimmunity can prevent serious manifestations due to COVID19infection [] Many herbal products are found to have immunemodulatory and antiviral property so their discovery can be amilestone in the prevention and control of COVID19 [] In thiscontext the Government of India has recommended to take AyushKwath in order to boost the immunity As this is a new formulationthis needs to be validated scientiï¬cally We have made an attemptto review the immunepathogenesis of COVID19 and the role ofeach herb over it Immunopathogenesis of COVID19The S protein of coronavirus can bind to host cells through theACE2 receptor found in the oral and nasal mucosa [] Other siteswhere ACE2 receptors are found are lungs stomach intestinebladder heart and kidney [] Variable presentation of disease in101016jjaim202008003 The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda Foundation This isan access under the CC BYNCND license httpcreativecommonslicensesbyncnd40Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxdifferent age groups serious manifestations that are seen morecommonly in immunecompromised old aged and in those withunderlying pathology many asymptomatic cases in pediatric agegroup and presence of lymph ia in the majority of the casesthese factors implies that immunity has a vital role in the pathogenesis of COVID19 [16e8] It is assumed that our immune systemhas lack of memory against such a virus that gave it an edge overhumans []Viruses cause cell destruction mainly in two ways direct cytopathic effects ofthe virus and immune response mediateddestruction [] COVID19 cannot lyse the cells directly as the majorpathway of cell destruction is due to immunemediated destruction[] It has been mentioned that unlike adults less vigorous cellmediated immune response in alveoli of children results in beingasymptomatic in the majority of cases []The pathogenesis can be split into two stages Nonsevere andSevere [] Nonsevere stageThe virus fuses with the host cell membrane and enters insidethe host cell through airway epithelium [] The virus propagates and multiplies inside the host cell and can reach lower airwayand alveoli In adults with good innate cellular and humoral immunity propagation of virus can be limited and viral load reachingalveoli can be reduced thus recovery can take place within 2e3weeks with mild symptoms []Humoral immunity prevents the viruses to enter new cells whilecellmediated immunity targets on eradicating virusinfected cells[] In this stage a strong immune system can be helpful inpreventing the propagation of the virus thus reducing the severityof the disease [] Severe stageOnce the immune system is breached the virus propagates andreaches the lower respiratory tract and alveoli Then the virus canpenetrate alveoli and reaches systemic circulation causing viremia[] The virus binds to multiple ans having ACE2 receptor protein During this stage cellmediated immunity becomes robustand starts releasing various proammatory cytokines IFNaIFNg IL1B IL6 IL12 IL18 IL33 TNFa etc and chemokinesCCL2 CCL3 CCL5 CXCL8 CXCL9 CXCL10 etc causing damage tomultiple ans known as Cytokine storm [] We may need tosuppress the ammation for improvement during this severestage []Tocilizumab and antiammatory interleukin IL10 are proposed to have a therapeutic role in the reduction of severity and mortality of COVID19[] As increased risk of thromboembolic phenomena is alsofound to be associated with COVID19 prophylactic antithromboticmedications are advised during this stage []IL6 receptor antagonist Ayurveda purview Disease conceptIt seems that most early cases had a history of contact with theoriginal market for seafood but the disease has now advanced to betransmitted through human to human contact [] Thus this disease can be considered as Communicableboth contagious and infectious diseases In Ayurveda epidemics are discussed under theterm of Janapadodhwamsa [ CSVi ] by Charaka and Marakaby Sushruta [ SSSoo ] The symptoms like fever coughbreathing difï¬culty headache and vomiting resemble with clinicalfeatures of SARS [ SSSoo ] Dalhana in his commentary hasmentioned that symptoms like anosmia cough catarrh will occurafter the entry of contaminated air through the nasal ingwhich is similar to typical clinical features of COVID19 [ SSSoo] Furthermore this disease can be classiï¬ed as Adidaivika BalaPravritta Vyadhi ABPV Sansargaja Upsargaja and Aupasargic RogaABPV are those diseases arising due to causes that cannot becontrolled by human intelligence Upasargaja Vyadhi are thosefeverlike diseases that manifest due to close contact with diseasedpersons [ SSSoo ] whereas Sansargaja Vyadhi resides withpeople who are cursed by almighty god ie due to uence ofinvisible forcesforces behind human control [ SSSoo ]Aupasargic Vyadhi is deï¬ned in two different ways by Sushruta oneas a disease which spreads from one person to another person [SSNi ] and another as ¦Upadravasangyah ie complications or associated diseases that manifest after primary disease [SSSoo ] Susruta mentions the diseases like Jwara Kusthaskin diseases Shosha tuberculosis Netrabhisyandi conjunctivitis and other Aupasargika roga alike communicable diseasescan be spread through Prasanga intimate relationship GatraSansparsha direct contact Nishwasa breathing or airborneSahabhojana eating together Sahashayana sleeping togethersharing and using of others clothes ornaments ointments etc [SSNi ]Agantuja Vyadhi Ì´ diseases of exogenous origin occurs due tophysicalexternal factors like Bhuta Visha Vayu Agni and Praharatrauma etc without any involvement of Vataadi Dosha initiallyhowever in later stage dosha are involved in the disease process[ CSSoo1145] Cakrapaá¹idatta clariï¬es that Bhuta meansVisaká¹imi or a virulent anism [ CSSa1121] Krimi may beSahaja natural or Vaikarika pathogenic anisms that may bevisible macroscopic or invisible to the naked eye microscopic[ CSVi ]Thus it is difï¬cult to correlate this disease with speciï¬c Ayurveda terminology but while interpreting the disease on the basis ofSamprapti by considering the causative agent and the clinical features like fever Jwara cough Kasa anorexia Aruchi fatigueTandra generalized body ache or myalgia Angamarda andTiredness it can be contemplated as an Agantuka Vyadhi whichlater on due to the involvement of dosha develops to Nija Vyadhi asKaphaVatolvana Hina Pitta Sannipataja Jwara Severe Vata andKapha with mild Pitta [ CSSoo CSChi ] Whiletalking about the pathogenesis of fever in Ayurveda Charakamentioned that when Vataadi dosha either singly or in Sansristatwo dosha or in Sannipataja all three dosha got aggravated thenit enters Amashaya and mixed with Rasa Dhatu causing obstructionof Rasavaha and Swedavaha Srotas resulting in the destruction ofAgni Agni then spreads out from its Sthana to whole over the bodycausing the febrile condition [ CSNi120 CSChi3129]Immunity concept in AyurvedaStrength health lifespan and vital breath are dependent on thecondition of Agni [ CSSoo ] Charaka has mentioned theterm Vyadhikshamatwa and states that during certain conditions ordue to certain factors even unwholesome unhealthy food doesnot produce disease immediately all unwholesome diet are notequally harmful all dosha are not equally powerful all persons arenot capable of resisting diseases [ CSSoo ] This suggeststhat the bodys immune system plays a crucial role in diseasedevelopment The equilibrium state of Dhatu is called Swasthya [CSSoo ] The person who is desirous to be healthy should adopthealthy practices related to diet conduct and activities [ CSSooImmunity can be considered in Ayurveda as] ThusPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxVyadhikshamatwa and Oja which depends on the condition of AgniDosha and DhatuThere are three factors Aahara Swapna and Brahmacharya dietsound sleep and celibacy that support the life with which the bodywill be endowed with strength complexion and development tilllife span [ CSSoo ] Bala Ì´ StrengthImmunity is of threetypescongenital time affected and acquired Congenital is thatwhich is developed naturally in the body and mind time affected isdue to seasonal variation and age factor and acquired one is produced by the proper application of diet and exercise [ CSSoo ] Thus not only diet but also performing yoga or exercises withproper methods by giving rest in between exercises as Rasayanatherapy will increase acquired strength [ CSSoo1136] Oja isalso called Bala is the essence of all Sapta Dhatu being located inHridaya combines with Rasa and circulates through the Dhamaniand performs Tarpana or Prinanam of the whole body [ SSSoo CS1774] The equilibrium state of Kapha promotesstrength thats why normal Kapha is called Oja [ CSSoo ]Normal pure blood promotes strength complexion health andlifespan [ CSSoo ] While dealing with Sannipataja JwaraSusruta in Uttarsthana mentioned Abhinyasa Jwara also called asHataujasa Jwara indicating the loss or deranged condition of Oja[ SSUtt ]The word Rasayana Rasa þ ayana refers to nutrition and itstransportation in the body for attaining excellent Rasadi Dhatuswhich leads to gain longevity freedom from disorders optimumstrength of physique and sense ans [ CSChi ]Rasayana promotes nutrition by explicitly enriching the nutritionalvalue of Rasa by enhancing Agni ie digestion metabolism andabsorption by Srotashodhana Consequently any medication thatimproves Rasas consistency would enhance the health of all bodytissues Role of Ayurveda and traditional medicineEvery society has its own medical system which is deeplyrooted in its culture and guided by its philosophy of life Beingculturally and linguistically diverse countries there developedseveral types of traditional medicines TM based on practices skilltraditional knowledge based on beliefs theories and experiencesindigenous to different cultures Ayurveda Traditional Chinesemedicine TCM Ancient Egyptian medicine Sowa Rigpa etc system of medicine remain the most ancient yet living traditions inSouth East Asia Western Paciï¬c Eastern Mediterranean Africaregion Up to percent of the population in some Asian and African countries depend on TM for primary health care PHC needs[] Still there is a high trend of using many herbs in religious andcultural works therapeutically for common ailments and as spicesfor foods according to occasion speciï¬c and seasonal regimes Ayurveda and TM have made a signiï¬cant contribution to the prevention and alleviation of various communicable and noncommunicable diseases for thousands of years A long history ofusing many herbal remedies and experiences passed from generation to generation has resulted in people relying on herbal remedies and some simple home remedies for common diseases can beused even by illiterate citizens The selfcare an integral part ofPHC with home remedies using various herbs is the most commontreatment for India Nepal Bhutan and China for different ï¬ucommon cold fever GI disorders etc Prevention of smallpox inChina has been an epochmaking effort in the period of mankindspreventive care One observational study found that the prevalencebetween the total number of COVID19 cases per million populationand the grams of spice supply per capita per day is clearly interrelated Most nations with lower spice intake per capita reportedmore COVID19 cases per million population and vice versa []Nevertheless with the invention of drugs many herbal remediesused historically have become modern medicines Few notableexamples include morphine digoxin artemisinin and colchicine Asmany herbs are found to have immunomodulatory role and possessantiviral activity many people are being optimistic over the traditional system of Medicine Ayurveda and TCM have descriptions ofimmunomodulation along with antiviral treatments even targeted to the coronavirus family []The key factor for COVID19 to occur and evolve is the interaction between the virus and an individuals immune system [] Asmedicinal plants enhance NK cell activity inhibit activated transcription factor ATF2 downregulate Th17related cytokinesincluding transcription factor RORc IL17A and Th2related cytokines including IL5 IL13 and IL6 inhibit GATA3 IL4 IL6 IL1bRt IL17A TNFa expression and increase the secretions of ILit shows that natural products have potentimmunemodulatory and immuneboosting effects that may behelpful during the infection course by increasing innate immuneresponse to infections []INFg etc Ayush KwathConsidering the importance of immunity boosting measures inthe wake of COVID19 outbreak the Ministry of AYUSH Government of India with the interest of health promotion of the massesrecommends Ayush Kwath or Ayush Kudineer or Ayush Joshandawhich comprises of four medicinal herbs Table [] Theherbs like holy basil cinnamon ginger black pepper are highlyavailable accessible and widely used in the kitchen and areconvenient to educate and train about its use to community healthworkers community and even to all public that they can have costeffective treatment with herbal home remedies This will help topromote immunity and to lower the gatherings at hospitals andpharmacies in this pandemic This type of public health measurewould eventually promote health for all with the theme ourhealth in our own hands making responsible to each and everypeople by active involvement in their own health instead of relyingon mass distribution of some medicine As people leave theirhomes to earn a living this herbal decoction will ensure broadaccess to health care The WHO SEARO adopted a resolution torevitalize PHC through health systems strengthening to achievehealth for all with the emphasis on health promotion and diseaseprevention [] This Kwath is not just a mechanical mixtureinvented for the COVID19 pandemic but it is a revival of healthtraditionMethod of preparation and useTake all the ingredients in dry form as per standards laid downin Ayurvedic Pharmacopoeia and make coarse powder Make sachets or tea bags each of g of powder or mg tablet of aqueousextract to be consumed like tea or hot drink by dissolving in mlof boiled water once or twice daily Gud JaggeryDraksha Resinsandor Lemon Juice can be added while consuming the formulation TulsiMany invitro animal and human experimental scientiï¬cstudies showed that due to presence of eugenol phenolic compoundslinoleic acid etc compounds Tulsi has antimicrobialincluding antibacterial antiviral antimalarial antidiarrhealantioxidantcardioprotectiveimmunemodulatory properties and is thus recommended as a treatmentfor a range of diseases including features like cough fever asthmaanxiety diarrhea gastric cardiac and genitourinary disorders[32e36] Due to its antiammatory and antioxidant properties itantiammatoryhepatoprotectiverenoprotectiveanalgesicantipyreticPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxTable Contents and properties of Ayush KwathSN NameScientiï¬c name Parts used Main chemicalRasaVirya AyurvedicSansthanika KarmaProportion RemarksTulsiOcimumsanctum LinnLeavesconstituentsVolatile oil PhenolAldehyde EugenolAscorbic acid Linoleicacid CaroteneDosha karmaKatu Tikta Ushna KaphavatashamakaPittabardhakaDalchini CinnamomumStem Bark CinnamaldehydezeylanicumBreyncuminaldehydeEugenolKatu TiktaMadhuraUshna KaphavatashamakaPitta vardhakaSunthiZingiberofï¬cinale RoscRhizomeZingiberene Zingiberol KatuUshna KaphavatashamakaVedanahara DeepanaPachana AnulomanaKrimighna HridhyaRaktashodhakaKasahara SwasaharaKshayanashakaMutrala VishaghnaJwaraghna esp useful inVatashlaishmikaVishama and Jirna JwaraDeepana PachanaVajikaranaVataanulomanaYakridutejaka GrahiHriyottejakaOjovardhakaRaktashodhakaShelshmaharaYakshmanashakaMutrajananaDeepana PachanaVrishyaShoolaprashamanaRaktashodhakaHridhyottejakaShothahara KaphaghnaSwasahara JwaraghnaAampachana partsPrabhava Specialaction Krimighna parts partsPrabhava KrimighnaContraindicationsPandu KushthaMutrakriccharaktapitta Grishma andSharada Ritu MarichPiper nigrumLinnFruitPiperine PiperidinePiperettine andChavicineKatuUshna Kaphashamaka Deepana Pachana partYakriduttejakaVatanulomanaKrimighnaHriddhyottejakaKaphaghnaKaphamissarakaJwaraghna espVishamjwarapratibandhakaprotects against toxic chemicalinduced injury enhance the antioxidant enzymes and protect cellular anelles and membranes byclearing damaged free radicals []The compounds such as ursolic acid carnosol rosmarinic acidcirsilineol apigenin eugenol and cirsimaritin present in O sanctumincrease haemoglobin concentration enhance SRBC agglutinin titers decrease cyclooxygenase CoX2 and lipoxygenase LOX5enzymes activity suppress NFkB classical pathway up regulationof IL2 IFNg and TNFa down regulation of IL1b and produce ofSRBC antigenspeciï¬c antibodies which represent a major defensemechanism to assess Tcelldependent antibody responses ie Tulsiby enhancing immune response boost the defense mechanismagainst the infection [38e40] Several studies have shown that Tulsiaqueous and methanol extract of leaf and seed oil besidesimproving vital capacity also is an immunemodulator and regulator as it enhances immune response by increasing Thelper andNK cells phagocytic activity and index with the rise in lymphocytecount neutrophil count and antibody titer []In an acute toxicity study it did not produce any hazardoussymptoms or CNS and ANS toxicities or death and did not show anychange in water and food consumption body weight and hematological and biochemical proï¬les [] DalchiniIt is a potent immune system booster and is used in variousailments like ï¬u indigestion edema cough etc [] Cinnamonbark contains cinnamaldehyde benzaldehyde cuminaldehyde andterpenes [] In one study cinnamon at high dose mgkgshowed immunestimulant activity as it signiï¬cantly increased thephagocytic index serum immunoglobulin levels and antibody titerand decreased the percentage reductions in neutrophil countCinnamon low dose mgkg increased serum immunoglobulinlevels only This showed that high dose increases both cell mediated and humoral immunity whereas low dose showed effect onlyon humoral immunity [] The studies also suggest that cinnamaldehyde can act as a strong regulator of monocytemacrophagemediated immune responses by inhibition of PI3K PDK1 and NFkBactivation of signaling components In addition to this by theactivation of CD29 and CD43 it blocked cell migration cellecelladhesion induced but not cellï¬bronectin adhesion and it wasable to suppress both the production of nitric oxide NO and upregulation of surface levels of costimulatory molecules CD69 andCD80 and pattern recognition receptors TLR2 and CR3 []Cinnamon bark decrease systemic levels ofIFNg withoutaltering the levels of IL4 or IL2 inhibit antiCD3 AbstimulatedIFNg and IL4 at the mRNA and secreted protein levels enhance IL protein secretion at the cellular level which helps to decrease celldeath inhibit IL2mRNA expression inhibit antiCD3induced p38JNK ERK12 and STAT4 activation but not IkBa degradation orSTAT6 and ultimately alter the ammatory responses in T cellsThis shows the immunemodulatory effect of cinnamon on cytokine secretion and the involvement of intracellular signaling molecules in activated T cells It also causes a reduction in the subG1Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxphase accompanied by an increased ratio of apoptotic cells tonecrotic cells [] The constituents like cinnamaldehyde cinnamophilin etc are found to be a thromboxane A2 receptor antagonistanticoagulative antiatherosclerotic and thus prevents unnecessary clumping of platelets and atherosclerotic CVD []In a systematic review of its adverse events relatively few selflimiting adverse effects were reported like allergic reactions andgastrointestinal disorders on clinical trials case reports and caseseries The evidence available show that cinnamon is safe for use asspice in daily diets or as a medication [] However its use fortherapeutic reasons in high doses or for prolonged periods cancause some adverse effects and should be observed clinically SunthiAn alcohol extract increases the immunological status of micewith increased phagocytosis by macrophages whereas crudeextract was also shown to increase humoral and cellmediatedimmune responses [] The bioactive compounds of ginger suchas nevirapine bsitosterol 6gingediol germacrene methyl6shogaol 6gingerol alinalool 6shogaol gingerdion zingibereneetc are known to inhibit viral replication among these the mostpotent inhibitors of reverse transcriptase RT enzyme is bsitosterol which is predicted to be used as nonnucleoside reversetranscriptase NNRTIs HIV1 inhibitors [] It is reported thatGinger contains TNFa which is also known as an antiuenzacytokine [] The rhizome of Ginger and its main componentslike gingerols shogaols etc inhibit prostaglandin and leukotrienebiosynthesis inhibit cyclooxygenase and lipoxygenase activitiesinhibits the synthesis of proammatory cytokines such as IL1TNFa and IL8 without any signiï¬cant effect in IL6 levelsinhibit the excessive production of NO PGE TNFa and IL1beta reduce the elevated expression of NFkB and TNFa downregulate ammatory iNOS and COX2 gene expression inhibitthromboxane synthetase raise levels of prostacyclin without aconcomitant rise in PGE or PGE alpha inhibit platelet aggregation decrease agerelated oxidative stress markers and enhanceï¬brinolysis [53e58]The concentration of IgM and eosinophil count in nonsmokerswas signiï¬cantly increased in a comparative study of the effect ofginger extract among male smokers and nonsmokers whereas theconcentration of hemoglobin and lymphocyte count in smokerswas strongly increased This indicates that in nonsmokers gingerresults in a stronger antibody response or humoral immunity thanin smokers []According to Ayurveda it is contraindicated to be used in a fewdiseases Kushtha Pandu Mutrakriccha Raktapitta and in Grishmasummer and Sharada autumn Ritu There are few minor adverseeffects recorded that did not need care such as mild gastrointestinal symptoms sleepiness mild diarrhea during prior few days oftreatment It is also explained that ginger has the ability to induceheartburn and as a gastric irritant with doses above g [] Duringpregnancy ginger did not pose a major risk for side effects oradverse events [] MarichIt has been also found to increase bioavailability thus enhancethe therapeutic efï¬cacy of many drugs vaccines and nutrients andhave immunemodulatory antioxidant antiplatelets antihypertensive antiasthmatic antipyretic analgesic anticarcinogenicantiammatory antidiarrheal antispasmodic anxiolytic antidepressants hepatoprotective antiulcer antithyroids antiapoptotic antimetastatic antimutagenic antibacterial antifungal[62e65] The extract and itsand antiamoebic propertiesconstituents like piperine regulate the balance of the cytokinesproduction of Th1 Th2 Th17 and Treg cells reduce the accumulation of ammatory cells inhibit the expressions of GATA3 IL4IL6 IL1b Rt IL17A and TNFa increase INFg and IL10 secretions in BALF Bronchoalveolar lavage ï¬uid and increasemacrophage activation and T and B cell proliferation []Beside this Marich possess cytotoxic activity suppresses thelevels of total IgE antiOVA IgE antiOVA IgG1 and histaminerelease in serum ameliorates ï¬brosis and ltration of ammatory cells inhibits the allergic responses inhibitsTh2Th17 responses and mast cells activation inhibits NFkB cFos cAMPresponse elementbinding CREB and activated transcription factor ATF2 suppresses PMAinduced MMP9 expression inhibitsPKCaextracellular signalregulated kinase ERK and reducesNFkBAP1 activation In addition piperine also inhibits the Pglycoprotein Pgp and CYP3A4 functions [67e69] Piper nigrum isfound to have dose dependent antifertility effects on mice [] DiscussionAccording to Ayurveda therapeutics is of two types Swasthasyorjaskarawhich promotes strength immunity in the healthyand Roganutwhich alleviates disorders Both of these groupsperform both of these functions but Rasayana and Vajikarana aremostly used for promotive treatment CSChi [] AyushKwath has both immune promoting and disease alleviating properties which can be achieved by various treatment modalities likeRasayana Satwawajaya Yuktivyapashraya Vyadhi Viparitarthakarichikitsa etcThe Katu and Tikta Rasa Usna Virya and Deepana PachanaYakriduttejaka properties of Ayush Kwath help to improve Agni andSrotosodhana improves microcirculation and tissue perforationthus promotes proper digestion metabolism and absorption andacts as Rasayana for the development of preceding Dhatu andï¬nally form Oja Oja itself acts as immunity to prevent diseaseImmunity is dependent on the condition of Agni Ayush Kwath withits Agni promoting and Kaphashamaka properties balance Kaphaand with Raktashodhaka Hridhya Krimighna properties purify theblood It is already mentioned that natural Kapha and pure bloodpromote Oja and Bala respectively Krimighna is the Prabhavaspecial action of Tulsi and Sunthi which directly acts againstpathogens The properties like Jwaraghna esp VatashlaishmikaVishama Kasahara Swasahara Kshayanashaka ShoolaprashamanaSwothahara Kaphaghna Hridayaottejaka Yakridutejaka have directrole to alleviate various clinical signs symptoms and complicationsAs this disease is considered as KaphaVatolvana Hina PittaSannipataja Jwara the Kapha Vata Shamaka properties of AyushKwath can play a signiï¬cant role in balancing the vitiated doshasAfter six days of Jwara Charaka suggests the decoction of Pachanadrugs in the case of Amdosha and Shamaniya drugs in Niramadosha[ CSChi ] This shows that Yuktivyapashraya and Vyadhiviparita chikitsa can be done even after the involvement of Dosha inlater stages Ayush Kwath has potential psychoneuroimmunemechanisms via evidence of a reduction in depression anxietyand stress in controlled trials and shows meaning response as it is aspeciï¬c remedy for cough and respiratory problems this shows therole of Satvawajaya Chikitsa in its management []Immunity plays a key role in the pathogenesis of COVID19 bothduring the early nonsevere stage and during the severe stage ofthe disease The earlystage strong immune response may preventthe propagation and spread of viruses inside the body thusreducing the severity of cases and early termination of infectionWhile during later stage strong cellmediated immunity of thebody against the virus itself can be a factor responsible for graveconsequences due to cytokine storm The target during the earlyPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxlikesteroidsand IL6 receptorstage should be to reduce viral propagation while at a later stageshould be to reduce the ammatory response of the immunesystem Medicinal herbs with immune booster property can be anoption during the early nonsevere stage while herbs with antiammatory and antithrombotic properties can be an optionduring a later or severe stage Cytokine storm that is believed as amajor factor responsible for complications and death of COVID19patients has been found to be reduced with antiammatorydrugsantagonists Antiammatory interleukins IL10 in modern medicine [] Therole of medicinal herbs with antiammatory property on thecytokine storm is still lacking in research Like antiammatoryinterleukins and IL6 receptor antagonist Tocilizumab IL thatare proposed in modern medicine to have a therapeutic role in thereduction of severity and mortality of COVID19 Cinnamon barkthat is found to decrease INFg and IL4 Its antiatheroscleroticanticoagulative and antiplate | 2 |
" lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11284f219 in lung cell lines and tissues was measured using reverse transcriptionquantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc nonsmall cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinomaassociated 0cli rp11284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfrtyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11284f219mir3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11284f219mir3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flashfrozen in liquid nitrogen and transferred to Ëc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no atcc and 1x penicillinstreptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc clonetics corporation according to the manufacturer's instructions ncih460 and ncih1299 cells were cultured in rpmi medium cat no atcc and a549 cells in f12k medium cat no atcc supplemented with fbs cat no atcc and 1x penicillinstreptomycin thermo fisher scientific inc all cells were culture at Ëc with co2rna extraction and reverse transcriptionquantitative pcr rtqpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the firststrand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rtqpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward 'aac gac ccc ttc att gac c' and reverse 'tcc acg aca tac tca gca cc' rp11284f219 forward 'agg att ggc act cac ttc gg' and reverse 'tct ctc acc acg tct ggt ct' and ccar1 forward 'ctg atg gct agc cct agt atg ga' and reverse 'tgc ctt tca tgc cca cta aaa ' the temperature protocol used to perform rt was Ëc for h followed by Ëc for min thermal conditions of pcr reactions were initial denaturation at Ëc for min followed by cycles for sec at Ëc and sec at Ëc the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir3p mimics and mir3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck assay and edu labeling of proliferating cells a cck was used for cell proliferation assay the cells were seeded into well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at Ëc and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckmancoulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at Ëc for h a total of 1x105 cells in µl serumfree medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides sinc sirp11284f219 mirnc mir3p mimics mir3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence ''uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at Ëc in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut 'untranslated region 'utr and ccar1 wt or mut 'utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were cotransfected with mirnc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigmaaldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sdspage transferred to a pvdf membrane and blocked with nonfat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at Ëc followed by incubation with goat antimouse or goat antirabbit igghorseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of Ëc with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogenfree conditions temperature Ëc humidity and fed with sterile food and water free access to evaluate the effect of rp11284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated ncih1299 cells in µl serumfree medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant bodyweight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ¥ independent experiments a twotailed unpaired student's ttest or oneway anova with tukey's posthoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure rp11284f219 expression is upregulated in lc tissues and cell lines a expression of rp11284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas2b n3 lc lung carcinoma rtqpcr reverse transcriptionquantitative pcrresultsexpression of rp11284f219 is upregulated in lung carcinoma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11284f219 exerts antioncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck and edu assays fig 2bd the results suggested that knocking down rp11284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of sirp11284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rtqpcr assay was performed to detect the expression levels of invasionrelated genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11284f219 directly interacts with mir3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathenainnovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir3p was partially complementary with the 'utr of rp11284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 'utr with or without mir3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11284f219wt transfected cells but not the rp11284f219mut transfected cells indicating a direct interaction between the two noncoding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11284f219 leading to a significant increase in mir3p expression fig 3b thus these findings suggested an inhibitory effect of rp11284f219 on the expression of mir3p in lung carcinoma cellsthe expression of mir3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir3prp11284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir3p inhibitor which specifically downregulates the expression of mir3p was transfected into ncih1299 and ncih460 cells fig 4a the results from the cck and edu assays demonstrated that treatment with sirp11284f219 0concology reports figure rp11284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11284f219 knockdown was achieved via rp11284f219 sirna and the knockdown efficiency was verified using reverse transcriptionquantitative pcr n3 cell counting kit assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the sinc group n5 d an edu assay was performed to measure the proliferation of ncih1299 and ncih460 cells after transfection with sinc and sirp11284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11284f219 knockdown on apoptotic rates in ncih1299 and ncih460 cells n3 f transwell assay was performed to determine the effects of rp11284f219 knockdown on ncih1299 and ncih460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mirnc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure rp11284f219 directly interacts with mir3p a binding site between rp11284f219 and mir3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mirnc n3 p005 vs mirnc b expression of mir3p in ncih1299 and ncih460 cells transfected with sirp11284f219 was analyzed using rtqpcr p001 vs sinc n3 mir3p expression in c lc tissues and d ncih460 ncih1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rtqpcr n3 p005 p001 vs adjacent tissue or beas2b cells nc negative control sirna small interfering rna wt wildtype mut mutant mir microrna lc lung carcinoma mir3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by sirp11284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mirnc group fig s3 in addition transfection with mir3p inhibitor also diminished the proapoptosis effect of sirp11284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir3prp11284f219 regulates ccar1 via targeting mir3p to further evaluate how rp11284f219 exerts an oncogenic role via mir3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir3p and the ccar1 'utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1wt treated cells but not the ccar1mut treated cells suggesting a direct binding of mir3p to the 'utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir3p in lung carcinoma cells and tissuesrp11284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir3p a expression of mir3p in ncih1299 and ncih460 cells transfected with mirnc or mir3p inhibitor was detected using rtqpcr analysis n3 p005 vs mirnc cell counting kit assay was performed in b ncih1299 and c ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor n5 d edu assay was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor magnification x200 e flow cytometry analysis was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor n3 f transwell assay was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor magnification x200 n3 p005 vs sinc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure mir3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 'untranslated region and mir3p b luciferase reporter assay in ccar1wt or ccar1mut treated cells in the presence of mirnc or mir3p mimic n3 p005 vs mirnc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rtqpcr n13 p001 vs adjacent tissue expression of ccar1 in ncih1299 and ncih460 cells transfected with mirnc or mir3p mimics was detected using d rtqpcr and e western blotting n3 p005 vs mirnc mir microrna nc negative control wt wildtype mut mutant rtqpcr reverse transcriptionquantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the sirp11284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the sirp11284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11284f219mir3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir3p directly interacts with rp11284f219 by binding to its 'utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir in vitamin denhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzymemediated intratumoral drug metabolism mir is also reported to be a potential noninvasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the highmobility group box protein to inhibit its expression thus improving transforming growth factorβ1induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11284f219 on the expression of mir3p in addition it was identified that the mir3p inhibitor can neutralize the antitumor effects of rp11284f219 knockdown indicating that rp11284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir3p this antitumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with ncih1299 cells transfected with sinc or sirp11284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of ncih1299 cells transfected with sinc or sirp11284f219 n5 expression levels of d mir3p and e ccar1 in the tumor tissues from nude mice injected with ncih1299 cells transfected with sinc or sirp11284f219 for weeks were detected using reverse transcriptionquantitative pcr n5 p005 p001 p0001 vs sinc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11284f219 furthermore the regulatory axis of rp11284f219mir3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11284f219 and mir3p and the interaction between mir3p and ccar1 were demonstrated by the dualluciferase assay although this method has been used to validate rnarna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rnaprotein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicininduced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir3p the expr | 0 |
bladder cancer is the tenth most common cancer globally but existing biomarkers and prognostic models are limitedmethod in this study we used four bladder cancer cohorts from the cancer genome atlas and gene expression omnibus databases to perform univariate cox regression analysis to identify common prognostic genes we used the least absolute shrinkage and selection operator regression to construct a prognostic cox model kaplanmeier analysis receiver operating characteristic curve and univariatemultivariate cox analysis were used to evaluate the prognostic model finally a coexpression network cibersort and estimate algorithm were used to explore the mechanism related to the modelresults a total of genes were identified from the four cohorts to construct the prognostic model including eight risk genes serpine2 prr11 dsel dnm1 comp elovl4 rtkn and mapk12 and three protective genes fabp6 c16orf74 and tnk1 the 11genes model could stratify the risk of patients in all five cohorts and the prognosis was worse in the group with a highrisk score the area under the curve values of the five cohorts in the first year are all greater than furthermore this models predictive ability is stronger than that of age gender grade and t stage through the weighted coexpression network analysis the gene module related to the model was found and the key genes in this module were mainly enriched in the tumor microenvironment b cell memory showed low infiltration in highrisk patients furthermore in the case of low b cell memory infiltration and highrisk score the prognosis of the patients was the worst the proposed 11genes model is a promising biomarker for estimating overall survival in bladder cancer this model can be used to stratify the risk of bladder cancer patients which is beneficial to the realization of individualized treatmentkeywords bladder cancer cox regression prognostic model overall survivalcorrespondence yyzhucmueducn yumengcmueducn jiaxing lin and jieping yang contributed equally to this work department of urology the first hospital of china medical university shenyang liaoning china department of reproductive biology and transgenic animal china medical university shenyang liaoning chinafull list of author information is available at the end of the bladder cancer is the tenth most common cancer in the world it is more common in men than in women and the morbidity and mortality rate in men is four times higher than that in women a significant risk factor for bladder cancer is smoking with half of all cases are linked to smoking [ ] about of patients with nonmuscular invasive bladder cancer are treated by radical tumor resection followed by intravesical instillation of 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 0clin a0et a0al cancer cell int page of bacille calmettegurin vaccine approximately of patients have muscular invasive or metastatic bladder cancer and are treated with radical cystectomy and neoadjuvant chemotherapy bladder cancer is a complex disease although many clinical factors and molecular markers have been identified that can predict prognosis these have low accuracy and it does not have universal applicabilitywith the continued development of gene sequencing technology and expansion of public databases it is possible to take advantage of biological information to mine sequencing data and identify biomarkers this method can utilize large sample sizes with less investment making it an important new direction to screen disease biomarkers of available databases the cancer genome atlas tcga cance rgeno menihgov database is an authoritative oncology database and the gene expression omnibus geo httpwwwncbinlmnihgovgeo database stores curated gene expression datasets many studies have constructed a multiqueue verification model based on these two databases such as nonsmall cell lung cancer [ ] and ovarian cancer prognostic models provide effective guidance for doctors and patients to make optimal treatment decisions however in the study of the bladder cancer model many models can only be verified in two or three cohorts [ ] and do not have clinical extensibilityin this study gene expression and clinical data related to bladder cancer were obtained from tcga and geo databases and common prognostic genes were screened by univariate cox proportional hazard regression this prognostic model of bladder cancer was constructed by least absolute shrinkage and selection operator lasso regression and then verified using five cohorts this robust model can help patients with bladder cancer to achieve individualized treatmentmaterials and a0methodsdata obtaining and a0processingto reduce the error of the data we searched the tcga and geo databases for bladder cancer cohorts with a sample size of more than and these cohorts need to include survival status and survival time we found a total of five cohorts the raw rna sequencing and clinical data of bladder urothelial carcinoma blca n were obtained from tcga database and the raw rna sequencing and clinical data of gse13507 n gse32548 n gse32894 n and gse48075 n from the geo database these five cohorts were analyzed on the illumina sequencing platform in r programming language software the r package edger was used to standardize the raw rna expression matrix and obtain the corresponding log valuesconstruction of a0prognostic modelthe cox proportional hazard regression model was applied to perform univariate cox proportional hazard analysis of all genes in tcgablca gse13507 gse32548 and gse32894 cohorts the hazard ratio hr from univariate cox regression analysis was used to select the genes that were positively or negatively related to prognosis a gene with hr was considered a risk gene and a gene with hr was considered a protective gene statistical significance was defined as p the genes with hr and p were selected from the four cohorts and then risk genes were obtained by overlapping four groups of genes similarly genes with hr and p were selected for the four cohorts and combined to obtain the set of protective genes a venn diagram was constructed using the online tool bioinformatics and evolutionary genomics httpbioin forma ticspsbugent bewebto olsvenn the identified risk and protective genes make up a set of prognostic genesthe data from tcgablca as a training set was used to construct a prognostic model to simplify the model the genes were selected by univariate cox regression analysis with a p value less than the r package glmnet and survival were used to do lasso regression to further screen genes and construct a cox module first the function glmnet was randomly simulated times to construct the model and establish the relationship between lambda punishment coefficient and regression coefficients coef a higher value of lambda corresponds to greater punishment with the increase of lambda some gene coef become zero indicating that the expression of the gene will not affect the model so this gene can be removed from the model then the function cvglmnet was randomly simulated times for crossvalidation cv cv is usually divided into holdout kfold and leaveoneout cv the function used kfold cv and k took the default parameter in tenfold cross validation the data set is divided into equal parts and then nine part are tested as training sets and one is used as the validation set the deviance of the tests were used to estimate the accuracy of the model when the deviance is minimum the model is the best and the coef of the model can then be obtained by using the corresponding lambda value finally we obtained the genes and the corresponding coef to build the model the prognostic model was defined as risk score ni expi · coefi where n is the number of genes expi is the expression of the ith gene and coefi is the regression coefficient of the ith gene the algorithm can prevent overfitting of the model remove highly coexpressed 0clin a0et a0al cancer cell int page of genes and finally construct a simplified model using the obtained model we calculated the risk score of each patient in the four cohortsbetween genes modulemembership and the correlation between genes and clinical traits genesignificance kaplanmeier analysisr packages survival and survminer were used for kaplanmeier analysis and the function rescat was used to find the best cutoff value of factors the cutoff was used to divide the sample into a highrisk group and a lowrisk group to construct the kaplanmeier curve with the smallest p value the risk score distribution gene expression and patient survival status data were plotted using the r package pheatmapreceiver operating characteristic curvereceiver operating characteristic roc curves of a0 years were plotted and the area under the curve auc values were calculated using the r package survivalrocunivariate and a0multivariate cox regression analysisthe risk scores and clinicopathological factors were analyzed by univariate and multivariate cox regression analysis using the r package survival the multivariate cox analysis included age sex primary tumor range t stage grade and risk score tcgablca and gse13507 also include stage lymph node and metastasisexploration of a0gene methylationcbioportal for cancer genomics is an openaccess opensource resource wwwcbiop ortal for interactive exploration of multiple cancer genomics data sets [ ] use this tool to query the relationship between gene expression and dna methylation in the bladder cancer tcga cell dataset the tool can also download gene methylation data which can be combined with clinical data for kaplanmeier analysisweighted coexpression network analysisthe set of mrna genes in the tcgablca cohort with univariate cox analysis values less than were selected to construct a bladder cancer coexpression network by weighted gene coexpression network analysis wgcna the r package wgcna was used to construct the coexpression network this method takes advantage of similarities of gene expression and groups the genes with similar expression patterns into the same module with the idea that genes in the same module may share physiological function we then explored the relationship between the clinicalfactorriskscore and module and applied pearson correlation to determine the module that was most related to the risk score the key genes were selected by the calculated correlation pathway and a0process enrichment analysisidentified genes were entered into the metascape database httpmetas cape for pathway and process enrichment analysis the enrichment analysis included kegg pathway go biological processes reactome gene sets canonical pathways and corum to evaluate the potential biological functions and pathways of the selected genescibersort and a0estimate algorithmcibersort celltype identification by estimating relative subsets of rna transcripts is a bioinformatics algorithm to calculate cell composition from gene expression profiles of complex tissues the combination of cibersort and lm22 leukocyte signature matrix can be used to calculate the content of kinds of human leukocyte subsets we used the r package cibersort to calculate the number of immune cells in each sample of the tcgablca cohort estimate estimation of sttromal and immune cells in malignant tumours using expression data is a tool that uses gene expression trends to infer the fraction of stromal and immune cells in tumor samples the immune score of each patient in tcgablca was calculated by the r package estimate immune score represents the content of immune cells and the higher the score the higher the cell contentstatistical analysisall the statistical analyses were carried out by using r programming language software rx64 all r packages were obtained from cran cranrproje ct or bioconductor httpwwwbioco nduct or the two groups were compared by the wilcoxon test and comparison between multiple groups was performed by kruskalwallis test statistical significance was defined as p difference scatter plots were constructed using the r package beeswarm we used the r package vioplot to draw violin pictures and the r package corrplot to draw correlation heat mapresultsdata processing and a0research processwe obtained the raw rna sequencing and clinical data of tcgablca n gse13507 n gse32548 n and gse32894 n we utilized data only from patients associated with rna sequencing data survival time survival status and primary tumor for further analysis the basic clinical information of the remaining patients is summarized in table a0 the sample 0clin a0et a0al cancer cell int page of table basic clinical information for a0the a0four cohortsclinical factorstcga_blcan age ¦ gender male femalet stage t2 § t2grade who2004 low highgrade who1999 g1 g2 g3vital status alive deadfollowup mean ± sd year ± gse13507n gse32548n gse32894n ± ± ± sd standard deviationsizes of these four cohorts are all greater than the grade of bladder cancer is closely related to recurrence and invasive behavior two grading methods were used in these four cohorts tcgablca and gse13507 used the who grading standard of which was divided into punlmp papillary urothelial neoplasms of low malignant potential low grade and high grade gse32548 and gse32894 used the who grading standard of which was divided into grade g1 grade g2 and grade g3 the research process is shown in fig a0construction of a0prognostic modelunivariate cox proportional hazard analysis was carried out in tcgablca gse13507 gse32548 and gse32894 cohorts there were genes in tcgablca genes in gse13507 genes in gse32548 and genes in gse32894 that met the criteria hr and p there were genes in tcgablca genes in gse13507 genes in gse32548 and genes in gse32894 that met the criteria hr and p combining the four datasets allowed identification of risk genes and protective genes fig a0 because of the large sample size of tcgablca we used this cohort to build the prognostic model first genes with univariate cox pvalues less than in tcgablca were selected then the genes were analyzed by lasso regression analysis fig a0 2a when the number of genes in the model was the deviance was the smallest fig a02b according to the lambda value the corresponding coef of the selected genes could be determined the prognostic model could then be constructed by using the corresponding coef of the genes to see more intuitively whether these genes are collinear we analyze the coexpression of these genes as shown in the fig a02c the coexpression index of none of these two genes is greater than finally we successfully constructed a prognostic module risk score serpine2 prr11 fab p6 \u200b c 16orf74 \u200b ds el d nm1 \u200b comp t nk1 \u200b elov l4 rtkn mapk12 the basic information and coef values of the genes are listed in additional file a0 table a0 s1 the average expression values transcripts per million of genes in the four cohorts are greater than which is of practical significance for detection additional file a0 table a0s2 the results of univariate regression analysis of these genes in cohorts are shown in additional file a0 table a0s3 0clin a0et a0al cancer cell int page of fig flow chart of analysiskaplanmeier analysis of a0 geneseleven genes were taken kaplanmeier analysis in cohorts using the heat map to show the results of the study fig a0 2d except for dsel in gse32894 and c16orf74 in gse32548 the other analyses were statistically significant p serpine2 rtkn prr11 mapk12 elovl4 dsel dnm1 and comp showed that the prognosis of patients with high expression was worse and the analysis of elovl4 in tcgablca was taken as an example p fig a0 2e tnk1 fabp6 and c16orf74 showed that the prognosis of the low expression group was worse and the analysis of fabp6 in tcgablca was taken as an example p fig a02fthe degree of a0dna methylation of a0tnk1 and a0c16orf74 was a0negatively correlated with a0gene expressiondna methylation can regulate gene expression we explored the relationship between expression and methylation of these genes additional file a0 figure s1ak the results showed that there was a negative correlation between tnk1 gene methylation and gene expression spearmen cor p 144e so did as c16orf74 spearmen cor p 897e then we took tnk1 and c16orf74 methylation data combined with clinical data for kaplanmeier analysis we found that the degree of methylation of these two genes can predict the prognosis of bladder cancer p additional file a0 figure s1i m and the prognosis is worse in the case of hypermethylation the expression of tnk1 and c16orf74 is inhibited by hypermethylation which leads to a worse prognosis of bladder cancerverification of a0the a0prognostic modelthe prognostic model was used to calculate the risk scores of each patient in the training set tcgablca and three test sets gse13507 gse32548 and gse32894 we identified the best cutoff value with a risk score of for tcgablca using this method the cutoff values of gse13507gse32548gse32894 were the kaplanmeier curves showed that the prognosis of patients with highrisk was significantly worse than that of patients with lowrisk in the four cohorts p fig a03ad the receiver operating characteristic roc curves of the four cohorts were drawn the a0year area under the curve auc values for the tcgablca cohort were and respectively fig a0 3e those for the gse13507 cohort were and respectively fig a03f those for the gse32548 cohort were and respectively fig a03g and those for the gse32894 group were and fig a03h additional file a0 figure s2 shows the risk score distribution gene expression values and survival status of patients in both the highrisk group and the lowrisk groupthe clinical factors and risk scores of the four cohorts were analyzed by univariate cox and multivariate cox regression analysis table a0 the results of univariate analysis showed that t stage was more effective in predicting prognosis among the clinical factors 0clin a0et a0al cancer cell int page of fig lasso cox and kaplanmeier analysis a lines of different colors represent different genes with the increase of lambda value the coef of some genes become zero indicating that they do not affect the model b the deviance of the cross validation when the partial likelihood deviance is minimum the corresponding model is the best c the coexpression heat map of genes red indicates a positive correlation blue indicates a negative correlation and the cross indicates no statistical significance d the heatmap of kaplanmeier analysis results of genes red means high expression of the gene lead to worse prognosis blue means low expression of the gene lead to worse prognosis grey means there are no significance of the analysis p means it is statistically significant the darker of the color shows the smaller of the pvalue e kaplanmeier analysis of elovl4 in tcgablca f kaplanmeier analysis of fabp6 in tcgablcaand three cohorts had statistical significance the risk scores were statistically significant in all four cohorts and the p value of three cohorts was lower than that of the t stage in multivariate cox analysis risk scores were statistically significant in three cohorts indicating that the three cohorts were independent of other clinical factors in predicting prognosis in this analysis only two cohorts of t stage had statistical significance so it is obvious that t stage is not as strong as risk score to predict the prognosis finally we compared the risk scores for different grades and t stage in the four cohorts and found that the risk scores increased 0clin a0et a0al cancer cell int page of fig the kaplanmeier analysis and roc curves of the risk score kaplanmeier curves of tcgablca a gse13507 b gse32548 c and gse32894 d red indicates highrisk group and blue indicates lowrisk group p means it is statistically significant ci confidence interval roc curves of tcgablca e gse13507 f gse32548 g and gse32894 h in years and their corresponding auc valueswith the increase of grade and t stage p additional file a0 figure s3a b in the gse32548 cohort we compared the risk scores of fgfr3 and tp53 or with the mdm2 alteration for wild type and mutant type additional file a0 figure s3c shows that a lower risk score of mutant type than that of wild type for the fgfr3 groups p in tp53 or with the mdm2 alteration the score of mutant type was higher than that of wild type p additional file a0 figure s3cseven genes and a0model were successfully verified in a0gse48705we evaluated the prognostic ability of genes and models in gse48075 n the results showed that serpine2 rtkn prr11 mapk12 elovl4 dsel and comp were statistically significant p additional file a0 figure s4 and the prognosis was worse in the high expression group which was consistent with the analysis result of the previous four cohorts the risk score of patients was calculated according to the model 0clin a0et a0al cancer cell int page of table univariate and a0 multivariate cox regression analysis of a0 clinicalfactorsriskscore with a0 overall survival rate in a0patientsvariablesunivariate analysismultivariate analysishr ciphr ciptcgablca age gender grade stage t stage node metastasis risk scoregse13507 age gender grade stage t stage node metastasis risk scoregse32548 age gender grade t stage risk scoregse32894 age gender grade t stage risk score0inf 120e 556e 991e 262e 657e 806e 544e 246e0inf 269e 214e 996e 763e 223e 164e 824e 367e 453e 129e 246e 400e 506e 388e 364e 114e 934e 613e 326e 742e 637e 871e 181e 997e 870e 183e 271e 221e 737e 200e 427e 183e 341e446e 179e 445e 445e 942e 154e 163e 561e 408e 986e 136eitalic font means statistically significanthr hazard ratio ci confidence interval inf infinityand the risk score was analyzed by kaplanmeier analysis the prognosis of the high riskscore group was worse and the difference was statistically significant p fig a04a we drew the roc curves of the risk score and the auc value of a0 year was fig a0 4b figure a0 4c showed the risk score distribution gene expression values and survival status of patients between high and lowrisk groupsweighted coexpression network analysis and a0enrichment analysisthe coexpression network was constructed with coding genes and samples in tcgablca cohort first the expression matrix was transformed into a topological overlap matrix according to β then the genes were divided into different modules fig a05a using the dynamic pruning tree method next the association analysis of clinical traits and modules fig a05b showed a high correlation between the turquoise module and risk score cor p 2e there was also a high correlation between the turquoise module and survival status cor p 9e07grade cor p 1e09stage cor p 1e we selected key genes fig a0 5c in the turquoise module according to the standard to explore the potential function of these key genes pathway and process enrichment analysis of these key genes were performed as shown in fig a05d the three most highly significantly enriched terms were extracellular matrix anization collagen fibril anization and ecm proteoglycans all related to the tumor microenvironment tmeimmune cells can be combined with a0risk scores for a0prognostic analysiswe used cibersort to calculate the infiltration ratio of immune cells in tcgablca samples and used a bar chart to show the infiltration of high and lowrisk groups fig a06a then the wilcoxon test was used to compare the difference between high and lowrisk groups the results showed that b cells naive macrophages m0 and macrophages m1 showed high infiltration in the highrisk group b cells memory dendritic cells resting and dendritic cells activated showed high infiltration in the lowrisk group p fig a06b furthermore we took the risk score and the infiltration degree of these six kinds of immune cells for joint prognostic analysis the samples were divided into four clusters for kaplanmeier analysis according to the median value of the risk score and immune cell infiltration degree the results showed that these groups could also be used for prognostic analysis p fig a06ch among them the prognostic ability of b cells memory is the best when the degree of b cells memory infiltration is low and the risk score is high the prognosis of this cluster is significantly worse than that of other clusters we used estimate to calculate the tcgablca cohorts immune score and then combined with the risk score for kaplanmeier analysis the results showed that the cluster with low immunescore and high riskscore had the worst prognosis additional file a0 figure s5 0clin a0et a0al cancer cell int page of fig validation of the model in gse48075 a kaplanmeier analysis of the riskscore b roc curves of riskscore in years c the risk score analysis from top to bottom patients risk distribution gene expression profile and survival status mapdiscussionbladder cancer is a heterogeneous disease with a high incidence and recurrence rate but there is no robust predictive tool to guide clinical treatment some recent studies have also constructed a new model for bladder cancer such as dna methylationdriven genes related model and immune genes related model these models prefer to take a kind of gene set to build the model rather than the whole genome into the screening in this study prognostic genes were screened from four cohorts with the whole transcriptome and the common prognostic genes were selected to construct the model the model successfully predicted the overall survival of five cohorts about bladder cancer patients and it is the research with the largest cohort size in the same type of researcha variety of regional source cohorts are used to jointly develop the model which makes the model have higher credibility and broader applicability in our study all genes in all cohorts were then analyzed by univariate cox proportional hazard analysis to screen common prognostic genes in four cohorts after further screening a prognostic model was constructed using the data from the tcgablca cohort instead of using the genes obtained by analysis of a single cohort to construct a prognostic model the prognostic genes common to multiple cohorts were used to make the model more stable and reliable the patients in the tcgablca cohort were from north america gse13507 was from asia and gse32548 and gse32894 were from europe it is concluded that this model has a wide range of applicabilitythe main finding of this study is that the 11gene model we developed has a robust prognostic ability and successfully predicted the prognosis of five cohorts kaplanmeier analysis showed that the prognosis of the highrisk group was worse in all the four cohorts p the 1year auc values of the tcgablca gse13507 gse32548 and gse32894 cohorts were and respectively indicating that the risk score has the ability to predict prognosis univariate and multivariate cox analysis of clinical factors and risk scores showed that the ability of risk scores to 0clin a0et a0al cancer cell int page of fig weighted coexpression network and enrichment analysis a genes were divided into different modules according to the dynamic cutting tree method and different colors represent different modules b the heatmap of the correlation between the gene module and clinical traits p indicates statistical significance c gene significance and module membership scatter diagrams of the turquoise module the dots in the red box are represented as key genes d the left side of the picture shows the interaction network map of enriched proteins and the same color indicates the same enrichment the right side of picture shows the enriched terms decreasing from top to bottom by the significance of enrichmentpredict prognosis was better than age gender grade and t stage we also analyzed the relationship between risk score and different clinical status and found increased risk score with the increase of bladder cancer t stage and grade p there are also significant differences in risk scores between wild type and mutant types of different genes we analyzed the gse32548 mutation data and found lower risk score in the group of fgfr3 mutation in contrast in the presence of a tp53 mutation or with mdm2 alteration the risk score was higher according to previous reports mutations in fgfr3 is associated with better prognosis but tp53 mutation or with mdm2 alteration is associated with worse prognosis [ ] these s indirectly verify the prognostic ability of the risk model finally the 11gene model was successfully verified in independent cohort gse48075 the model is verified by four internal cohorts and one external cohort which shows that the model has the potential to be used in the cliniceleven genes are potential prognostic markers and therapeutic targets for bladder cancer these genes have a stable prognostic ability in tcgablca gse13507 gse32548 and gse32894 cohorts and kaplanmeier analysis showed that serpine2 rtkn prr11 mapk12 elovl4 dsel and comp was successfully verified in gse48075 besides the methylation level of tnk1 and c16orf74 can also predict the prognosis of bladder cancer among them only c16orf74 and 0clin a0et a0al cancer cell int page of fig combined analysis of risk score and immune infiltrating cells a the bar chart of the infiltration of kinds of immune cells in the samples of high and lowrisk groups b the difference of kinds of immune cells between the lowrisk group and the highrisk group was analyzed an | 0 |
"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu HawaiiParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conï¬ict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR conï¬dence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conï¬ict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulï¬lling ï¬lial obligations in order to ensure equitable access to ACP AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals values goals and preferences regardingendoflife EOL care12 Contemplation of individuals EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conï¬icts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610 AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the beneï¬ts ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proï¬ciencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships uence individuals ACP engagement To ï¬ll thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawaiiwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentiï¬ed asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was afï¬liated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andï¬nancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conï¬ict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conï¬ict was measured using the 5item Family CulturalConï¬ict scale which assesses cultural and intergenerational conï¬ictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation ï¬nancial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conï¬ict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with conï¬dence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconï¬ict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not signiï¬cantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proï¬ciency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conï¬ictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and dont want tonever but want toreluctant to yes never and dont want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conï¬icte10 most cultural conï¬ict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents life which maysometimes be in opposition to their parents own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this populations lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The ï¬nding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conï¬ict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The ï¬nding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential beneï¬t and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved onesdeath and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the signiï¬cantrelationship between family conï¬ict and ACP engagement could bethat higher levels of family conï¬ict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These ï¬ndings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizeï¬ndings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conï¬ict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conï¬ictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education ï¬nancial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulï¬lling ï¬lial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Deï¬nition and recommendations foradvance care planning An international 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ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneï¬ciaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conï¬ict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difï¬cult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawaii Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspeciï¬c psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratiï¬cation of older adults preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c" | 2 |
we report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma the patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin bep chemotherapy regimenkeywordscryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourcorrespondencewenchien cheng division of pulmonary and critical care medicine department of internal medicinechina medical university hospital no yuderoad north dis taichung city taiwanemail wcchengdrgmailcomreceived july revised july accepted july associate editor james horespirology case reports e00644 101002rcr2644introductionlung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases ebms from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] primary lung cancer is the most common cause of endobronchialtumours extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma ebms from embryonal carcinomas are extremely rare in this report we present acase of ebm from a primary embryonal carcinomacase reporta 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days he reported no fever chills coldsweats weight loss or decreased appetite a chest radiograph at admission revealed complete collapse of the leftlung fig 1a computed tomography ct was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumbronchoscopytumourobstructing thefig 1b theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 ttf1negative sallike protein sall4positive and cluster of differentiation cd30positive these ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomafig we checked the levels of tumour markers in thepatient including those of alphafetoprotein afp betahuman chorionic gonadotropin bhcg and lactate dehydrogenase ldh each tumour marker was found to be the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirologythis is an open access under the terms of the creative commons attributionnoncommercialnoderivs license which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made vol iss e00644page 0cebm from embryonal carcinomack teng figure chestradiograph and bronchoscopic view of the endobronchial metastasesebm a complete collapse of the left lungon chest radiograph b bronchoscopic viewof the endobronchial tumour within the leftmain bronchusfigure tumour pathology of metastatic embryonal carcinoma a embryonal carcinoma with a complex glandular growth pattern the characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation b immunohistochemical staining with antithyroid transcription factor1 ttf1 highlighting cells in the alveolar space original magniï¬cation c immunohistochemical staining with antisallikeprotein sall4 revealed diffuse nuclear staining original magniï¬cation d immunohistochemical staining with anticluster of differentiation cd30 highlighting diffuse membranous staining original magniï¬cation presentin high levels afp ngml bhcg miuml and ldh iul the patientunderwent a right orchiectomy followed by a bepbleomycin etoposide cisplatin chemotherapy regimendiscussionwe report here the case of a young man with an ebmfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumourslikewise manykindslung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverebms from extrapulmonary malignancies are rare [] primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith ebm primarily breast colon and renal carcinomas[] ebms from testicular seminomas are also extremelyrare the majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cck teng ebm from embryonal carcinomatable reports of previous cases of ebmslocationdiagnostic methodpathology¶zt¼rk moreirameyer case case the oriï¬ce of right upper loberight main bronchusleft main bronchus main carina andright main bronchus fibreoptic bronchoscopy mixed gctfibreoptic bronchoscopy mixed gctvideobronchoscopyembryonal carcinoma¶zsu the oriï¬ce of the right upper lobefibreoptic bronchoscopytesticular seminomaturan varkey our caseand right intermediary loberight intermediate bronchusleft main bronchusleft main bronchusebm endobronchial metastases gct germ cell tumourembryonic carcinomas or seminomas there are only a few published reports of primary testicularembryonic carcinomas resulting in ebms []the mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently howeversome patients are asymptomatic in our patient symptoms on presentation included haemoptysis and shortnessof breathresults from chest radiography in patients with ebmcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyhowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue the ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy the former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure the patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomawe had evaluated the presence of afp bhcg andldh tumour markers elevated afp levels can be secretedby germ cell tumours gcts including embryonal carcinoma yolk sac tumour or teratoma in gcts detectablerigid bronchoscopybronchoscopyfibreoptic bronchoscopyand cryosurgerysomatictype gctembryonal carcinomaembryonal carcinomabhcg elevation is observed in both seminomas and nonseminomas the serum level of ldh was directly correlated with tumour burden in nonseminomatous gctswhich is also useful for the surveillance of patients withadvanced seminoma the tumour markers in ourpatient showed elevated levels of afp bhcg and ldhthis was compatible with the diagnosis of embryonal carcinoma moreirameyer also evaluated the patienttumour markers and found elevated levels of afp ngml and bhcg miuml the elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma ¶zsu onlyevaluated the patients bhcg level which was found to beelevated miuml and the ï¬nal diagnosis wasmetastatic testicular seminoma on comparison with previous case reports table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation apc to control bleedingcryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree sampleshaemorrhage was observed to be similar during both procedures further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesin conclusion ebms from primary gcts notably thoseassociated with total or partial collapse are extremely rarewe have presented this case to emphasize the importanceof distinguishing ebm from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0cebm from embryonal carcinomadisclosure statementappropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisreferences ¶zt¼rk a aktas¸ z and yılmaz a endobronchialmetastasis of mixed germ cell tumors two cases tuberktoraks lee sh jung jy kim dh endobronchialmetastases from extrathoracic malignancy yonsei med j ikemura k lin dm martyn cp endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing lung moreirameyer a bautistaherrera d hern¡ndezembryonal endobronchialgonz¡lez mck teng carcinomaj bronchologyinterv pulmonol ¶zsu s erol mm oztuna f endobronchial metastasis from testicular seminoma med princ pract tumoraltesticular germ cell turan d akif ¶zg¼l m kirkil gendobronchial metastasis ofeurasian j pulmonol et varkey b and heckman mg diagnosis of a case ofembryonal carcinoma by bronchial biopsy chest paradis tj dixon j and tieu bh the role of bronchoscopy in the diagnosis of airway disease j thorac dis aktas z gunay e hoca nt endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisann thorac med barlow lj badalato gm and mckiernan jm serumtumor markers in the evaluation of male germ cell tumorsnat rev urol the authors respirology case reports published by john wiley sons australia ltdon behalf of the asian paciï¬c society of respirology 0c' | 0 |
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 Tianjins 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 ï¬rst 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 identiï¬esand cuts speciï¬c sequences on the bottom DNA strand atthe consensus site 3cid48AATTTT5cid48 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 identiï¬es and cuts speciï¬csequences on the bottom DNA strand at the consensus site3cid48AATTTT5cid48 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 speciï¬c 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 ï¬rst 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 copyandpaste 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 ampliï¬cation In breast cancer Morse andcolleagues ï¬rst proposed that hypomethylation activates LINE which can utilize the target primed reverse transcriptionpathway to insert into the oncogene MYC causing tumorspeciï¬crearrangement and ampliï¬cation Morse LINE was found to induce the ampliï¬cation of CCND1 oncogenein esophageal tumor by inducing the breakagefusionbridgeFrontiers 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 histologicalspeciï¬city 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 speciï¬c 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 speciï¬c Briggs But recentlytumor proteome proï¬ling studies based on mass spectrometryhave shown that ORF2p was diï¬cult to be detected and afteraï¬nity 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 ATMdeï¬ciency LINE1 retrotransposition increased and ORF2 copynumber increased in AT neurons thus verifying the correlationbetween LINE1 retrotransposition and ATM deï¬ciency 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 Alzheimers diseaseis still being studiedLINE1 and Genetic DisordersLINE1 is reported to be related to chromosome disordersThe ï¬rst 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 Neuroï¬bromatosis 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 deï¬ciencyand AicardiGoutieres syndrome AGS of threeprime repairexonuclease mutations LINE1 expression was upregulated andproammatory cytokines were produced through the cGASSTING 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 Sjögrens 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 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 speciï¬cmechanism 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 fulï¬llthe 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 antiammatory M2 macrophages and inhibits the production ofIFNγ in tumorltrating 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 isoï¬avone 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 isoï¬avone supplement mgday orally for weeks before surgery a positivecorrelation was found between LINE1 methylation level anddaily isoï¬avone 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 modiï¬cations Histone demethylaseKDM4B may enhance the LINE1 retrotransposition eï¬cacywhereas 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 Speciï¬creverse 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 deï¬ciency suppressesLINE1 mobilization by inhibiting nuclear import of LINERNP 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 identiï¬ed 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 ï¬ndings suggest that targetingAHR with small molecule inhibitors may be a novel therapeuticapproach ORF1P phosphorylation by protein kinase A is alsorequired for LINE1 Kinase inhibitors speciï¬cally 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 bodysmetabolism 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 CDK6ampliï¬cation 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 | 2 |
"days for untreated control and 59 days (p?=?0.03 Log rank test). B. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous RS4 leukemia subcutaneous xenografts treated with thiaminase 850 units SC BIW or buffer control. The median time to endpoint was 16.5 days for the control group and not reached after 60 days of observation in the treated group (p<0.001 Log rank test). C. Primary ALL and AML specimens were thawed and plated in triplicate in two concentrations of thiaminase 0.4 units/ml and 4 units/ml and assessed for viability at 48 hours relative to untreated cells. The ALL specimen with the asterisk was used for the in vivo study shown in D. D. Primary ALL cells were injected IV on Day 1; three thiaminase treatments of 2000 units/kg SC were administered on days 17 20 and 24. The animals were sacrificed on Day 33 and bone marrow was examined by flow cytometry for human ALL cells (percent human CD45); Untreated n?=?4; vehicle treated n?=?10 native thiaminase n?=?8 (* p<0.01 Mann-Whitney test). The studies in along with previous reports demonstrate that the enzyme thiaminase has activity against breast and lymphoid leukemia cell lines. To first determine whether the activity of thiaminase was related to thiamine depletion we explored the ability of small molecule thiamine antagonists to potentiate the cytotoxicity of thiaminase. We screened thiamine analogs pyrithiamine oxythiamine and N3 pyridylthiamine (N3PT) [13] [14] for activity as inhibitors of thiaminase and substrates of thiaminase. We found that oxythiamine and N3PT were thiaminase inhibitors and that pyrithiamine was a thiaminase substrate (data not shown). Therefore we hypothesized that oxythiamine and N3PT would reverse thiaminase toxicity if given simultaneous with thiaminase (because they would inhibit enzyme activity) but would act synergistically if given in sequence (because cells deprived of thiamine would become sensitive to small molecule thiamine antagonists due to TDE apoenzyme formation during thiamine starvation). In A simultaneous incubation of thiaminase and thiamine antagonists inhibits thiaminase activity while in B sequential administration of thiaminase followed by N3PT demonstrates synergistic cytotoxicity. Importantly the effect of pre-incubation in thiaminase is similar to the effect of pre-incubation in thiamine-free medium indicating that TDE apoenzyme formation is required for the cytotoxicity of small molecule TDE inhibitors and also demonstrating that extracellular thiamine starvation is sufficient to make small molecule TDE inhibitors cytotoxic. In order to further examine the interplay of N3PT and thiaminase in an in vivo model we administered PEGylated thiaminase in sequence with N3PT choosing PEGylated enzyme over the native enzyme shown in B to be able to separate extracellular thiamine deprivation caused by PEGylated thiaminase from the intracellular effect of N3PT [6]. In C the RS4 xenograft experiment shows that sequential administration of PEGylated thiaminase followed by N3PT produced an increased median TTE (55 days) compared to the untreated control (16.5 days) PEG-thiaminase (25 days) or N3PT (23 days) alone (p<0.01) confirming the in vitro data. .0085702.g002 Effect of thiamine antagonists on thiaminase activity. A. Growth inhibition of Reh and RS4 leukemia cells in the presence or absence of thiaminase and either N3PT or oxythiamine. B. Growth inhibition of RS4 and Reh leukemia cells incubated in normal medium thiamine-free medium or medium containing thiaminase prior to exposure to different concentrations of N3PT. C. RS4 subcutaneous xenografts showing untreated control N3PT alone 1k-PEGylated thiaminase and 1k-PEGylated thiaminase followed by N3PT. The time-to-endpoint was 16.5 days for control 23 days for N3PT 25 days for 1k-PEG thiaminase and 55 days for 1k-PEG thiaminase (p<0.01 Log rank test). D. Oxygen consumption rate of RS4 cells treated with thiaminase N3PT or thiaminase followed by N3PT (clear: control; thin-stripe: thiaminase; thick- stripe: N3PT; solid: thiaminase + N3PT). Since thiaminase should inhibit two key TDEs involved in Krebs cycle metabolism pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase (2-OGDH aka alpha-ketoglutarate dehydrogenase) we examined respiration (as oxygen consumption rate; OCR) and the rate of extracellular acidification (a measure of lactate production; ECAR) in leukemia and breast cancer cell lines. In D the oxygen consumption rate is decreased in an additive fashion with the sequential administration of thiaminase followed by N3PT showing further evidence of interaction of thiaminase and a small molecule thiamine antagonist. In addition since rapamycin reversed thiaminase-mediated growth inhibition in leukemia cell lines [7] but not breast cancer cell lines (data not shown) we examined the effect of rapamycin alone and in combination with thiaminase. In A thiaminase consistently decreased OCR in two lymphoid leukemia cell lines RS4 and Reh in the breast cancer cell line MCF-7 and in the non-malignant breast cell line MCF-10A. Also as expected rapamycin similarly suppressed OCR in the four cell lines. However adding rapamycin resulted in an inhibition of the thiaminase-mediated decrease in OCR rather than resulting in an additive effect as was seen with N3PT in B. .0085702.g003 OCR and ECAR of leukemia and breast cell lines incubated for 48(clear: control; thin-stripe: thiaminase; thick- stripe: rapamycin; solid: thiaminase + rapamycin). A. Quantification of OCR parameters. The ATP-linked rate is the basal rate minus the rate measured after the addition of oligomycin. The maximal capacity is the rate measured after the addition of FCCP. The reserve capacity is the basal rate minus the FCCP rate. All data are the mean ± SEM of triplicate samples and are representative of 3 independent experiments ( p<0.05 *p<0.01 two way ANOVA (Newman Kruskal Wallis test). B. Quantification of ECAR parameters. Glycolysis is the rate determine from subtracting the basal rate from the rate after the addition of glucose. Glycolytic reserve is the rate determined by subtracting the rate following the addition of oligomycin from the rate following the addition of glucose. All data are the mean ± SEM of triplicate samples and are representative of 3 ( p<0.05 *p<0.01 two way ANOVA (Newman Kruskal Wallis test). Similarly thiaminase increased ECAR in the two breast cell lines: the MCF-7 cell line and the MCF-10A cell line (B). Although rapamycin alone did not have a significant effect on ECAR the addition of rapamycin to thiaminase once again blunted the effect of thiaminase in all four cell lines. The consistent inhibition of thiaminase effect on OCR and ECAR by rapamycin in all four cell lines demonstrates a previously unidentified activity of mTOR inhibition in relation to TDEs. However the observation that the rapamycin has similar effects on the leukemia cell lines where it reverses growth inhibition [7] and MCF-7 and MCF-10A cell lines where it has no effect on thiaminase cytotoxicity (data not shown) suggests that the effect of thiaminase on OCR and ECAR may not be the determinant of thiaminase anticancer activity. To rule out the possibility that rapamycin could directly inhibit thiaminase enzymatic activity which could explain the reversal of the effects of thiaminase by rapamycin we directly examined the ability of rapamycin to inhibit thiaminase enzyme activity and found no evidence that rapamycin was a thiaminase enzyme inhibitor (data not shown). To further understand how thiaminase alters cellular metabolism and how rapamycin reverses thiaminase effects we undertook a metabolomic analysis of RS4 leukemia cells and MCF-7 breast cancer cells exposed to thiaminase rapamycin or both. A shows internal validation of the effects of thiaminase in RS4 cells. As expected the levels of thiamine and thiamine diphosphate were decreased in the thiaminase treated conditions and thiazole (a catabolic product of thiaminase cleavage of thiamine) is increased in the thiaminase treated cells confirming the finding that rapamycin does not interfere with the enzymatic activity of thiaminase. In fact thiamine was below the limit of detection in thiaminase treated cells in the absence of rapamycin. Also rapamycin was detected only in the rapamycin-treated cells. Evidence for inhibition of the TDE transketolase was observed with significant increases in ribose and ribulose surrogates for transketolase substrates ribose 5-phosphate and ribulose 5-phosphate respectively in both RS4 and MCF-7 cells (B). These results demonstrate a common signature of transketolase inhibition in the two cell lines. Furthermore the accumulation is partly to completely reversed by rapamycin demonstrating an antagonistic effect of rapamycin on thiamine-induced transketolase inhibition in addition to its antagonistic effects on OCR and ECAR. .0085702.g004 Validation of thiaminase action in of RS4 leukemia cells and MCF-7 breast cancer cells by metabolomic analysis. Both cell lines were analyzed under six conditions: control for 24 hours (C-24); incubation in thiaminase for 24 hours (T-24); control for 48 hours (C-48); thiaminase for 48 hours (T-48); rapamycin for 48 hours (R-48); and both rapamycin and thiaminase for 48 hours (R+T-48). The median is indicated by the bar in the center of the rectangle the rectangle dimensions reflect the range of the two mid-quartile values and the outer bars represent the ranges of all of the values. The data represent four independent experiments. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). A. Internal validation in RS4 cells showing the expected decrease in thiamine and thiamine diphosphate in thiaminase treated cells an increase in thiazole the product of thiaminase cleavage of thiamine in the thiaminase-treated cells and the appearance of rapamycin only in the rapamycin treated cells. B. Validation of thiaminase-mediated inhibition of transketolase by demonstration of the accumulation of substrate surrogates ribose and ribulose in both RS4 and MCF-7 cell lines at 48 hours. C. Immunoblot analysis of thiamine pyrophosphate kinase (TPK1) and thiamine triphosphatase (THTPA) in RS4 and MCF-7 cells treated with thiaminase rapamycin or both. D. Immunoblot analysis of pyruvate kinase isozymes M1 and M2 and carnitine palmitoyl transferase (CPT1) in RS4 and MCF-7 cells treated with thiaminase rapamycin or both. Although there was no evidence that rapamycin altered the level of thiaminase-induced thiamine depletion we sought to confirm this observation by examining the expression of enzymes that regulate thiamine phosphorylation. In C the expression of both thiamine pyrophosphokinase TPK1 an enzyme that phosphorylates thiamine and thiamine monophosphate into its active diphosphate form and thiamine triphosphatase (THTPA) an enzyme that dephosphorylates intracellular thiamine triphosphate into the active diphosphate form are shown. A modest increase in THTPA expression is seen in RS4 cells treated with both thiaminase and rapamycin which may be a result of feedback from intracellular thiamine diphosphate depletion but no other changes are seen in either RS4 or MCF-7 cells. In D the expression of enzymes involved in energy metabolism pyruvate kinase isoforms M1 and M2 and carnitine palmitoyl transferase 1 (CPT1A) were also examined by immunoblot. PK-M1 expression was decreased in MCF-7 cells relative to RS4 cells. In MCF-7 cells both PK-M2 and CPT1A expression levels were modestly reduced by rapamycin and this suppression was reversed by thiaminase in the case of CPT1A but not PK-M2. However similar findings were not observed in RS4 cell lines adding more evidence to the overall observation that different cell lines will respond differently to acute thiamine depletion. Figure 5 shows effects of thiaminase on the TDE branched-chain keto acid dehydrogenase (BCKDH) and demonstrates different metabolomic signatures between RS4 leukemia cells and MCF-7 breast cancer cells. In Figure 5A the metabolic pathways for the catabolism of branched chain amino acids is illustrated. The metabolomic profile for these metabolites is shown in Figure 5B and Figure S1. In RS4 cells thiaminase increases the levels of BCKDH substrates alpha-hydroxyisocaproate 2-hydroxy-3-methylvalerate and alpha-hydroxyisovalerate all metabolites that are increased in maple syrup urine disease the congenital form of BCKDH deficiency. In contrast a decrease in the products of BCKDH are observed in MCF-7 cells with decreased isobutyrylcarnitine 2-methybutyrylcarnintine and isovalerylcarnitine. Once again the effects of thiaminase were offset by rapamycin. These findings suggest that the substrate load for BCKDH is much higher in RS4 leukemia cells. Therefore we investigated the expression of the cytosolic form and the mitochondrial form of the branched chain aminotransferases cBCAT and mBCAT - the enzymes immediately upstream of BCKDH in both RS4 and MCF-7 cells. As shown in Figure 5C MCF-7 cells do not appear to express cBCAT providing a potential explanation for the lack of BCKDH substrate accumulation in this cell line after thiaminase treatment. Figure 5C also appears to show an apparent increase in phosphorylated BCKD-E1 after thiaminase treatment in MCF-7 cells which is reversed by co-treatment with rapamycin. Phosphorylation of BCKDH inactivates the enzyme and may contribute to the decrease of BCKDH products seen in the MCF-7 cells and suggests that there may be an interaction between thiaminase rapamycin and the enzymes that regulate BCKDH phosphorylation. To determine whether the accumulation of BCKDH substrates contributed to thiaminase-induced growth inhibition in RS4 cells we examined thiaminase growth inhibition in the presence of gabapentin a structural analog of leucine and an inhibitor of cBCAT [15]. As shown in Figure 5D gabapentin partially protected RS4 cells from thiaminase growth inhibition while having no effect on MCF-7 cells. .0085702.g005 Figure 5 Differential effects of thiaminase on branched chain amino acid catabolism. A. Schematic diagram showing branched chain amino acid catabolism. B. Different metabolomic signatures indicating inhibition of BCKDH by thiaminase. The top three panels show accumulation of BCKDH substrates after 48-7 cells after thiaminase treatment- most notably isovalerylcarnitine which is also reversed by rapamycin. C. An immunoblot of cytosolic and mitochondrial branched chain amino acid transferase (cBCAT and mBCAT) the enzymes that catalyzes the reactions that produce BCKDH substrates and total and phosphorylated BCKDH subunit E1 (BCKD-E1 and pBCKD-E1 respectively) in RS4 leukemia cells treated with thiaminase rapamycin or both. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). D. Cytotoxicity assay of RS4 and MCF-7 cells treated with increasing concentrations of thiaminase (in milliunits mu) under control conditions or in medium that contains gabapentin. Thiaminase also affected the catabolism of aromatic amino acids in RS4 leukemia cells. As shown in Figure 6A the breakdown products of both phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (4-hydroxyphenylpyruvate (HPP) and 3-(4-hydroxyphenyl)lactate (HPLA)) are increased in the thiaminase treated cells. Furthermore as shown in Figure 6B tryptophan catabolites are increased through one pathway which produces indolelactate but not through the kynurenine pathway. As in the approach we took with the alteration of branched chain amino acids we examined the possibility that the accumulation of these aromatic amino acid catabolites mediated the growth inhibitory effects of thiaminase. The drug nitisinone which inhibits 4-hydroxyphenylpyruvate dioxygenase an enzyme that catalyzes an alternative catabolic pathway for phenylalanine [16] and would be expected to increase the accumulation of aromatic amino acid catabolities did not however alter the dose-response curves of thiaminase in either cell line (data not shown). .0085702.g006 Figure 6 Effects of thiaminase on aromatic amino acid catabolism. A. Accumulation of the products of phenylalanine (phenylpyruvate and phenyllactate) and tyrosine (hydroxyphenylpyruvate and hydroxyphenyllactate) catabolism in RS4 cells after treatment with thiaminase for 48 hours. B. Accumulation of tryptophan catabolites after thiaminase treatment showing accumulation of indolelactate but not kynurenine. For C-48 vs T-48 and C-48 vs T+R-48 comparisons ** indicates p<0.05 and * indicates 0.05<p<0.1). Discussion Any systematic difference between cancer cells and normal cells provides a potentially exploitable therapeutic opportunity. The altered energy metabolism in cancer cells known as the Warburg effect represents such an opportunity. The vitamin thiamine is a key cofactor in two critical enzymes in energy metabolism: PDH and 2-OGDH. It is also a cofactor for two other critical enzymes; TK which is required for the formation of the biomass of a cell through the pentose phosphate shunt; and BCKDH which regulates the catalysis of branched chain amino acids. Thiamine requires active transport for cellular uptake. We have previously shown down-regulation of thiamine transporters in human tumors [2] [3] suggesting that tumors may have a nutritional vulnerability that could be exploited clinically analogous to the down-regulation of asparagine synthase in acute lymphoblastic leukemia which is exploited therapeutically by the bacterial enzyme asparaginase. The enzyme thiaminase catalyzes the cleavage of thiamine into two molecules: one is a pyrimidine (conjugated to a nucleophile for type I thiaminases) and the other is a thiazole that may be phosphorylated depending on the thiamine substrate that enters the reaction [5]. Several forms of thiaminase exist in nature including plant animal and bacterial forms of the enzyme thus serving as an example of convergent evolution even though the physiologic role of an enzyme that destroys an essential nutrient is not clear. We have developed methods to produce purify and modify Bacillus thiaminolyticus thiaminase I enzyme and have shown in preclinical xenograft models that therapy directed at thiamine dependent enzymes (TDEs) a completely novel concept and strategy has the potential to treat leukemia [7]and breast cancer [8] and despite its anti-metabolite role thiaminase can cause tumor responses at a systemically tolerable dose. To determine which TDE is responsible for the cytotoxic effects of thiaminase we examined the effect of the enzyme on cellular respiration and metabolism with the goal of identifying the critical TDEs responsible for cytotoxicity in the setting of acute thiamine deprivation. Since thiaminase is a bacterial enzyme it is less-than-ideal as a pharmaceutical product and the identification of the specific pathway disrupted by thiamine deprivation that leads to cytotoxicity could reveal an alternative pharmacologic approach. The metabolomic changes in the global biochemical profiles of RS4 leukemia cells and MCF-7 breast cancer" | 1 |
"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t 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 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyers patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials []f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2 mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1nod2 mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ ] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti | 0 |
CASE REPORTpublished August 103389fbioe202000929SecondGeneration RTQuIC Assayfor the Diagnosis ofCreutzfeldtJakob Disease Patientsin BrazilBreno Jos Alencar Pires Barbosa1 Bruno Batitucci Castrillo1 Ricardo Pires Alvim1Marcelo Houat de Brito1 Helio R Gomes1 S´nia M D Brucki1 Jerusa Smid1Ricardo Nitrini1 Michele C Landemberger2 Vilma R Martins2 Jerson L Silva3 andTuane C R G Vieira3 Department of Neurology Hospital das Clnicas University of S£o Paulo Medical School S£o Paulo Brazil Tumor Biologyand Biomarkers Group International Research Center AC Camargo Cancer Center S£o Paulo Brazil National Centerof Nuclear Magnetic Resonance Jiri Jonas Institute of Medical Biochemistry Leopoldo de Meis National Institute of Scienceand Technology for Structural Biology and Bioimaging Federal University of Rio de JaneiroUFRJ Rio de Janeiro BrazilThe recent development of IQCSF the second generation of realtime quakinginducedconversion RTQuIC using cerebrospinal ï¬uid CSF for the diagnosis of CreutzfeldtJakob Disease CJD represents a major diagnostic advance in the ï¬eld Highly accurateresults have been reported with encouraging reproducibility among different centersHowever availability is still insufï¬cient and only a few research centers have access tothe method in developing countries In Brazil we have had suspected cases of CJDsince when surveillance started Of these were undiagnosed This lack ofdiagnosis is due among other factors to the lack of a reference center for the diagnosisof these diseases in Brazil resulting in some of these samples being sent abroad foranalysis The aim of this research study is to report the pilot use of IQCSF in a smallcohort of Brazilian patients with possible or probable CJD implementing a referencecenter in the country We stored CSF samples from patients with possible probable enetic CJD one case during the time frame of December through June AllCSF samples were processed according to standardized protocols without access tothe clinical data Eight patients presented to our team with rapidly progressive dementiaand typical neurological signs of CJD We used CSF samples from seven patientswith other neurological conditions as negative controls Five out of seven suspectedcases had positive tests two cases showed inconclusive results Among controls therewas one falsepositive a CSF sample from a 5yearold child with leukemia undertreatment The occurrence of a false positive in one of the negative control samplesraises the possibility of the presence of interfering components in the CSF sample frompatients with nonneurodegenerative pathologies Our pilot results illustrate the feasibilityof having CJD CSF samples tested in Brazilian centers and highlight the importance ofinterinstitutional collaboration to pursue a higher diagnostic accuracy in CJD in Braziland Latin AmericaKeywords CreutzfeldtJakob disease prionconversion biomarkersrapidly progressive dementiarealtime quakinginducedEdited byMaria Dos Anjos PiresUniversity of Tr¡sosMontes and AltoDouro PortugalReviewed byAssia AngelovaGerman Cancer Research CenterDKFZ GermanySumit GhoshThe Research Institute at NationwideChildrens Hospital United StatesCorrespondenceBreno Jos Alencar Pires BarbosabrenojbgmailcomTuane C R G VieiratuanebioqmedufrjbrSpecialty sectionThis was submitted toBiosafety and Biosecuritya section of the journalFrontiers in Bioengineering andBiotechnologyReceived May Accepted July Published August CitationBarbosa BJAP Castrillo BBAlvim RP de Brito MH Gomes HRBrucki SMD Smid J Nitrini RLandemberger MC Martins VRSilva JL and Vieira TCRG SecondGeneration RTQuIC Assayfor the Diagnosis of CreutzfeldtJakobDisease Patients in BrazilFront Bioeng Biotechnol 103389fbioe202000929Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alINTRODUCTIONtogroupTSEsconditionsencompasstransmissibleasecondaryspongiformPrion diseases also known asofrareencephalopathiesneurodegenerativeabnormalconversion of a constitutively expressed cellular glycoproteinthe prion protein PrPC into an abnormally folded isoformPrPsc Geschwind Zanusso SporadicCreutzfeldtJakob disease sCJD is the most common priondisease in humans and usually presents as rapidly progressivedementia in combination with variable degrees of multisystemneurologicalimpairment Zerr and Hermann Sinceits clinical and molecular manifestations are heterogeneousand nonspeciï¬c early diagnosis of prion diseases remainschallenging in clinical practice Geschwind and Murray Brain histopathological evaluation andor detection of PrPScare still the standard criteria for establishing a deï¬nitive diagnosisfor CJD CDC However invasiveness when performingthis type of analysis antemortem brings very little beneï¬t to thepatient since these diseases are still incurable Clinical signs andparaclinical tests are the most commonly used approaches duringthe course of the disease and can be used to classify it as a possibleor probable prion disease Brown CDC Among the paraclinical tests brain diï¬usion weightedMRIDWMRI and cerebrospinal ï¬uid CSF analysis have increaseddiagnostic accuracy Eisenmenger Staï¬aroni Bizzi The presence of protein Tauprotein neuronspeciï¬c enolase NSE the astroglial proteinS100B and PrPSc itself are used as biomarkers in CFS for TSEdiagnosis Schmitz Connor Overallthe only protein that is a speciï¬c biomarker for TSE is PrPScPrPSc can be detected in CSF based on its selfpropagating abilityconverting and seeding the aggregation of the nonpathogenicPrPC into PrPScThe realtime quakinginduced conversion RTQuIC assayits experimentalwas developed in and since thenconditions have been improved and tested on a large number ofsamples in several laboratories worldwide Wilham Green It ultrasensitively detects limited amounts of PrPScin CSF and other tissue samples Wilham The recentdevelopment of the secondgeneration IQCSF RTQuIC assayusing CSF for the diagnosis of CJD represents a major diagnosticadvance in the ï¬eld Wilham Orrº In humans RTQuIC analysis showed a sensitivity of and speciï¬city of with encouraging reproducibility amongdiï¬erent centers Franceschini RTQuIC started to be clinically used in and becamea criterion of the Centers for Disease Control and PreventionCDC to diagnose CJD as probable CDC Its highsensitivity and speciï¬city make it an important clinical laboratorytestits global availability is stillinsuï¬cient only a few research centers have access to the methodespecially in developing countriesfor widespread use butSurveillance of TSE cases has been compulsory in Brazil since Martins and suspected CJD cases werereported up to June Of these were undiagnosed deOliveira Cardoso Ministrio da Saºde RTQuIC for the Diagnosis of Brazillian PatientsinimagingperformingexaminationsandDiï¬cultiesneuropathological analyses are found in several medicalunits in the country making diagnosis problematic Somediagnosed patients had samples sent to centers outside thecountry for biomarker analysis allowing for greater coverageof the diagnostic criteria The implementation of a speciï¬ctest in Brazil such as RTQuIC which can provide diagnosisfor diï¬erent TSEs with ease and conï¬dence is urgent to havenotiï¬cation and diï¬erential diagnosis for these diseases Thisis also important to guide medical decisions Here we reportthe pilot use ofthe secondgeneration IQCSF RTQuICassay in a small cohort of Brazilian patients with possibleor probable CJD to implement a reference center for thisanalysis in the countryMATERIALS AND METHODSClinical InvestigationPatients with suspected CJD were admitted for investigationunder a protocol for rapidly progressive dementia RPD aspreviously reported Studart Neto Following a fullneurological examination all patients underwent complete bloodcell count serum electrolytes blood glucose acute Creactiveprotein liver renal and thyroid function tests antithyroid andantinuclear antibody testing as well as treponemal and HIVserology Patients also underwent brain magnetic resonanceimaging [T1 T2 ï¬uidattenuated inversion recovery FLAIRgradient echo and diï¬usion weighted imaging sequences] EEGand CSF cell count biochemistry and g globulin levels CSF protein was assessed in cases of suspected prion diseasesas required by Brazilian norms Selected patients underwentchest and abdomen computed tomography mammography forwomen testicular ultrasound for men and thyroid ultrasoundto rule out paraneoplastic RPD In addition we obtainedonconeural andor neuronal surface antigen antibody testingwhen paraneoplastic or autoimmune encephalitis was suspectedA ï¬owchart for patient inclusion is provided in Figure Three cases had access to molecular analysis of the PRNP genefor polymorphisms in the codon Samples were analyzedusing denaturing highperformance liquid chromatographyTechnical details about this procedure as well as ampliï¬cationreactions and DNA extraction have been previously describedCastro Smid CSF SamplesWe analyzed eight CSF samples from seven patients withpossible probable or genetic CJD referred to the Departmentof Neurology at University of S£o Paulo from December to June One patient named ABT had her CSF testedtwice at diï¬erent times We used CSF samples from sevenpatients with other neurological conditions as negative controlsCSF samples mL were collected by lumbar puncture LPfollowing a standard procedure Two milliliters of the CSFsample were then centrifuged at g for min andstored in polypropylene tubes at ¦C until blind analysis byresearcher TCRGV at UFRJFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsof CJD The patient named ABT had her CSF tested twice atdiï¬erent times ABT1 and ABT2 samples therefore renderinga total of eight CSF samples ABT patient results will be betterreported in Case descriptionWe randomly used CSF samples from seven patients withother neurological conditions as negative controls Five out ofeight suspected samples had positive RTQuIC results in ourhands Figure 2A Clinical diagnosis CSF analysis and RTQuICresults are summarized in Table Samples from patients LRC and DAS were used as positivecontrols given that they were previously analyzed using RTQuICin a reference center outside the country The NationalPrion Disease Pathology Surveillance Center Cleveland OHUnited States Our RTQuIC analyses were also positivecorroborating this result Figure 2A Patient ASM was a geneticCJD positive for the E200K mutation and in our analysis thispatient was also positive presenting a very short lag phase and ahigh ï¬uorescence signal Figure 2AAmong the negative controls there was one false positivesample CT2 Figure 2A The falsepositive case was a5yearold child with leukemia who was receiving intrathecalchemotherapy GBTLI protocol with methotrexate aracitincitrabin dexamethasone during the time frame of the studyIn the following two cases are presented in which the resultsobtained with RTQuIC show interesting aspects related toRTQuIC sensitivity and disease progressionCase ABT was a 72yearold woman with years of formal educationShe had an atypical presentation characterized by a 4monthhistory of rapidly progressive cognitive impairment associatedwith visual hallucinations and gait disturbances with repeatedfalls She had no relevant past medical history or family history ofany neurological conditions On neurological examination shescored on the MiniMental State Examination MMSEand physical tests revealed only a prominent axial syndrome asshe could not sit or stand up without bilateral support There wereno pyramidal signs parkinsonism or visuospatial impairmentA comprehensive investigation with metabolic inï¬ammatoryparaneoplastic and infectious tests was unremarkable Brainmagnetic resonance imaging MRI revealed symmetric diï¬usionweighted image DWI hyperintensities in the basal gangliaFigure a ï¬nding that raised the suspicion of CDJ TheCSF analysis was unremarkable with elevated tau protein levelsand negative The RTQuIC results were inconclusiveonce from wells only two crossed the threshold ABT1sample in Figure 2B Electroencephalogram EEG revealeddisanized electrical brain activity with no periodic wavesThree months later she developed a signiï¬cant worsening withthe need for aid for most activities of daily living severe cognitiveimpairment she could not be tested with the MMSE myoclonusand the presence of prominent frontal reï¬exes on neurologicalexamination At this point a prolonged EEG eventually showedbilateral periodic sharp waves and another lumbar puncturewas performed with a second sample sent for RTQuIC analysiswith a positive result with a high ï¬uorescence ABT2 samplein Figure 2B She eventually died of clinical complicationsFIGURE Flowchart of samples included for RTQuIC analysisRTQuICThe RTQuIC assay was performed using the improvedQuICCSF IQCSF conditions as published Orrº Brieï¬y µL of CSF was added to µL of reaction mixturein each well of a black well plate with a clear bottomNunc The ï¬nal solution contained mM phosphate buï¬er atpH mM ethylenediaminetetraacetic acid tetrasodium saltdihydrate EDTA at pH mM NaCl µM thioï¬avinTThT sodium dodecyl sulfate SDS and mgmLrecombinant Syrian hamster truncated form of prion proteinHa rPrP Samples were tested in quadruplicate threeor four times independently generating a total of or wellsevaluated for each sample The plates were sealed and incubatedin a FLUOstar OMEGA plate reader BMG Labtech Germanyat ¦C with intermittent cycles of shaking s doubleorbital rpm and rest s ThT ï¬uorescence was collected every min using ± nm excitation and ± nmemission wavelengths The threshold was deï¬ned as the averageï¬uorescence for all samples within the ï¬rst h of incubationplus standard deviations SD A sample was consideredpositive when at least two of four replicate wells crossed thisthreshold All IQCSF RTQuIC analyses were performed at theFederal University of Rio de JaneiroUnseeded reaction not shown and random CSF samplesfrom patients with other neurological conditions Table wereselected as negative controls Given the descriptive nature of thisstudy no statistical analyses were performedRESULTSIn the referred time frame seven patients presented to our teamwith rapidly progressive dementia and typical neurological signsFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsTABLE Results of diagnostic investigations in the tested patient cohortSample ageDiagnosisaCSF analysisRTQuIC statusWBCµ LProt mgdLGlu mgdLTaub pgmLCT1 CT2 CT3 HCS HBV RMC ABS LRC DAS ABT1 ABT2 ASM NAJJN GMT TN Control chronic meningitisControl leukemiaControl cranial nerves syndromeControl multiple sclerosisControl HIVControl SAHventriculitisControl chronic meningitisRPD probable CJDRDP probable CJDRPD possible CJDRPD possible CJDRPD genetic CJDRPD probable CJDRPD possible CJDRPD probable CJD1cNANANANegativePositiveNegativeNANANANegativeNegativePositiveNegativeNegativeNegativeNegativeNegativePositivedPositivedNegativePositivePositivePositiveNegativePositiveWBC white blood cells Prot protein levels Glu glucose levels RPD rapidly progressive dementia SAH subarachnoid hemorrhage NA not available aControls andtheir suspected diagnosisinvestigation bReference value for total tau levels was pgmL cThis sample had red blood cells dThese patients also had their CSFsamples sent abroad with positive results The National Prion Disease Pathology Surveillance Center Cleveland OH United States months after the ï¬rst symptoms appeared PRNP gene analysisrevealed codon heterozygous MVin hisCase JJN was a 65yearold male who presented to our clinicwith an 8month history of behavioral changes He had years of education and a medical history of hypertensionand gouty arthritis There was no family history of anyneurological conditions His wife described the ï¬rst symptomsas prominent changesfood preferences with anunusual demand for rice and chicken Two months laterhe developed visual hallucinations mostly described as thepresence of spiders in the ceiling In the ï¬rst evaluationhe was independent of instrumental activities of daily livingNeurological examination revealed an MMSE of withan unremarkable physical examination A laboratory workupincluding metabolicinï¬ammatory and serology studies wasnegative At this point we were surprised by the ï¬nding ofbrain MRI diï¬usion weighted imaging revealing marked bilateralhypersignal in the frontotemporoparietooccipital cortex basalganglia thalamus and less markedly in the hippocampusraising the suspicion of sporadic CreutzfeldtJakob DiseasesCJD Figures 4AB The patient was hospitalized for furtherinvestigation The EEG study revealed bilateral and synchronousslow waves A brain 18FDG PET showed hypometabolism in thetemporal and frontal lobes caudate nuclei and temporoparietalcortex Figure 4C The CSF study was initially unremarkableexcept for the high total tau protein levels whereas ptau and betaamyloid values were within the normal rangeA CSF sample was sentfor autoantibodies and RTQuICtesting The patient was empirically treated with intravenousmethylprednisolone for days and was discharged for outpatientfollowup Atthis point we received CSF results negativefor autoantibodies with an inconclusive RTQuIC responsefrom wells only two crossed the threshold Figure 2CIn the following months he eventually developed cognitivedeterioration and parkinsonism with the presence of morecomplex visual hallucinations He eventually died of clinicalcomplications approximately months after initial symptomsPRNP gene analysis revealed codon heterozygous MVDISCUSSIONsyndromes ofencephalopathiesThe present study reports the results of pilot secondgenerationreferred forRTQuIC testing in a small patient cohortrapidly progressive neurologicalsuspectedprion nature Our center previously reported cases ofrapidly progressive dementia among patients in a 3yearintervalrepresentedthe majority of the samples followed by CJD Studart Neto All cases from that study werediagnosed with CJD according to the University of CaliforniaSan Francisco Modiï¬ed Criteria Vitali which doesnot include CSF testingImmunemediatedAs we described here IQCSF RTQuIC proved to be anextremely important tool in the diagnosis of ABT and JJN casesCases and Both patients presented with rapidly progressiveneurological syndromes and could not initially be classiï¬ed withpossible or probable CJD according to the most recent criteriaCDC Both ABT and JJN showed slightly elevated levelsof total Tau protein but although this biomarker has sensitivity it is only speciï¬c for CJD Connor protein was not elevated in both cases despitetheir genetic subtype MV which is often associated with positive protein levels Manix Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsFIGURE Blind RTQuIC analysis of CSF samples A RTQuIC responses from reactions seeded with µl of CSF from suspected CJD cases LRC DAS ASMJJN GMT TN The RTQuIC responses from reactions seeded with CSF samples from patients with other neurological disorders CT1 CT2 CT3 HCS HBV RMCABS were used as a negative control Each curve represents the mean of four replicate readings of three or four repetitions B Singlewell analysis of CSF samplesfrom patient ABT ABT1 and ABT2 refer to the ï¬rst and second collections respectively C Singlewell analysis of CSF samples from patient JJNIn the case of ABT patient the clinical presentation ofrapid cognitive impairment with visual hallucinations and gaitimpairment was considered atypical In this case the brain MRIwas an important diagnostic clue with DWI hyperintensitiesin the basal ganglia leading to a diï¬erential diagnosis withmetabolic encephalopathies hypoxicischemic lesions or toxicFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsdisease progression To our knowledge this approach performedwith samples of a patient collected at diï¬erent times has not yetbeen tested and this is the ï¬rst report suggesting that RTQuICresults change according to disease evolution A more feasiblehypothesis would be that the ï¬rst sample had some interferencesuch as blood that could mask the positive result Cramm despite a only modest presence of red blood cells in thisparticular sample cellsµL see Table Atypical features for sCJD in JJN Case included a presentation with mild behavioral changes the initialsparing of motor systems with evidence of motor ï¬ndingsin the neurological examination only almost months afterï¬rst symptoms an unremarkable EEG with months ofevolution and the presence of a bilateral T2 hypersignalin the hippocampus The RTQuIC was inconclusive buttogether with MRIit could suggest early diagnosis ofprobable CJD Perhaps with disease progression JJNs CSFsample would obtain a positive RTQuIC result as observedfor ABT patientThe occurrence of one falsepositive case in our RTQuICtest weakens our diagnostic accuracy and underscores the needfor improvements in the protocol Despite having an extremelyhigh speciï¬city IQCSF RTQuIC falsepositive results have beenreported in the literature Hayashi reported thecase of a 61yearold man who presented with rapid cognitiveimpairment myoclonus and recurrent seizures A brain MRIrevealed cortical hyperintensities and the CSF analysis showedelevated and tau levels with a positive RTQuIC Despiteaggressive treatment with corticotherapy the patient died andpostmortem assessment revealed only pathological changes afterconvulsion with no signs of prion disease The authors concludethat convulsion may cause falsepositive RTQUIC results andthat a postmortem evaluation remains the gold standard fordiagnosing similar cases The shaking eï¬ect was analyzed in vitroby Orrº and they showed that long shaking periodsreduced scrapieseeded reaction times but continuous shakingpromoted falsepositive reactionsFIGURE Diffusionweighted images DWI from ABT case revealingenlarged ventricles and symmetric hyperintensities in the basal ganglia Ayellow arrows B shows corresponding apparent diffusion coefï¬cient ADChypointensities in the same territorylesions ie carbon monoxide intoxication Finelli and DiMario A careful clinical assessment made all possibilities lesslikely and CJD became our main hypothesis Despite aninconclusive RTQuIC result for the ï¬rst collected sample thepatient eventually evolved with a more typicalimpairmentand the second CSF sample with a 3month interval yieldedpositive RTQuICThe diï¬erent results obtained with the ABT patient sampleswere very intriguing We hypothesize that this diï¬erence could beattributed to higher loads of PrPsc following disease progressionAlthough disease duration does not seem to be related toRTQuIC results McGuire it is not yet clear whetherthere is a change in the presence of seeds in CSF according toFIGURE A MRI diffusionweighted images from JJN case revealing hyperintensities on frontal temporoparietal and posterior cingulate cortical areas Ayellow arrow B MRI apparent diffusion coefï¬cient maps with corresponding hypointensities in the same regions above B yellow arrow C 18FDG PETCTshows hypometabolism on the bilateral temporoparietal cortex posterior cingulate precuneus and caudate nucleusFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsRegarding our falsepositive case we did not ï¬nd any similarcases reporting the use of intrathecal medications as a possiblereason for a positive RTQuIC result A positive case in a5yearold child without any neurological symptoms wouldnever be expected an 18yearold woman was the youngestperson diagnosed with probable sCJD using RTQuIC Yao Therefore this sample was selected as a negative controlalthough infant CSF was never RTQuIC analyzed Despiteoptimized protocols sample processing issues can always be apossibility for such unexpected resultsThe present study is limited by its pilot nature with a modestsample size Improvements in the establishment of the protocolare necessary requiring a greater number of analyses The useof nasal brushes to collect patient samples for RTQuIC analysisis also valid to improve protocol sensitivity and speciï¬cityHowever this is the ï¬rst study to our knowledge to report speciï¬cbiomarkerbased feasible results performed in a developingcountry for prion diagnosis in addition to pointing out newpossible interferences in the protocol and the need to understandhow the diï¬erent current diagnostic approaches can revealdisease progressionCONCLUSIONThe cases reported here illustrate the importance of usingRTQuIC for patients with neurological syndromes enabling thediagnosis of probable CJD while no other method was suï¬cientto support this diagnosis even with atypical clinical presentationIn addition the identiï¬cation of a false positive in a sample froma leukemic pediatric patient undergoing intrathecal treatmentwith chemotherapy suggests new possible interferences in themethod This will require future investigation of the eï¬ect of thesechemotherapeutic agents for inclusion or not as a limitation forcarrying out the assayCSF samples are often evaluated only once due to theinvasiveness of CSF collection and the absence of curativetreatment for TSEs in addition to rapid disease progressionOne of the cases we report points out the importance ofcarrying out studies that evaluate the progression of the diseaseand that RTQuIC is a useful approach for such studies Inthis case the use of nasal brushes might be prioritized overCSF analysis since this provides greater sensitivity and enablesmore frequent sample collection given the less invasive natureof the procedureFinally our study illustrates the feasibility of having CJDCSF samples tested in Brazilian centers and highlights theimportance of interinstitutional collaboration in order to pursuea greater diagnostic accuracy for CJD in developing countriesREFERENCESBizzi A Pascuzzo R Blevinsetal Evaluation ofM E Mdetecting prion disease with diï¬usion magneticJAMA Neurol101001jamaneurol20201319print]J Grisoli M Lodi R Moscatellia new criterion forimagingofresonance[EpubaheadIt also demonstrates that RTQuIC can be clinically availablefor testing patients from Brazil and other Latin Americancountries and points to the need and feasibility of establishinga national reference center for the diagnosis of these rare butdevastating diseasesDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialETHICS STATEMENTEthical review and approval was not required for the studyon human participants in accordance with the local legislationand institutional requirements Written informed consent toparticipate in this study was provided by the participants legalguardiannext of kin Written informed consent was obtainedfrom the individuals and minors legal guardiannext of kinfor the publication of any potentially identiï¬able images or dataincluded in this AUTHOR CONTRIBUTIONSBB study design patient data collection and manuscript writingBB BC RA MB HG and SB patient data collection andinterpretation JS and RN study design data interpretation andmanuscript critical revision ML and VM genetic analysis JLSand TV RTQuIC facility implementation TV study designRTQuIC evaluations RTQuIC data analysis and manuscriptwriting All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by research grants from the CarlosChagas Filho Foundation for Research Support in the State of Riode Janeiro FAPERJ and the National Council of Technologicaland Scientiï¬c Development CNPq to JLS and TVACKNOWLEDGMENTSWe would like to acknowledge all the patients and their familiesfor their contributions to this studyBrown P Brunk C Budka H Cervenakova L Collie D Green A WHO Manual for Surveillance of Human Transmissible SpongiformEncephalopathies Including Variant CreutzfeldtJakob Disease Geneva WorldHealth anizationCastro R M R P S Landemberger M C Walz R Carlotti C GHuang N Cunha D R High capacity and low costdetection of prion protein gene variant alleles by denaturing HPLCFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsJ Neurosci Methods101016jjneumeth2004CDC CDCs Diagnostic Criteria for CreutzfeldtJakob Disease CJD[Internet] Atlanta Centers for Disease Control and PreventionConnor A Wang H Appleby B S and Rhoads D D Clinical laboratorytests used to aid in diagnosis of human prion disease J Clin Microbiol57e0076919Cramm M Schmitz M Karch A Mitrova E Kuhn F Schroeder B Stability and reproducibility underscore utility of RTQuICfor diagnosis of CreutzfeldtJakob disease Mol Neurobiol 101007s1203501591332de Oliveira Cardoso C A de Albuquerque Navarro M B M Soares B E Cand Oliveira Cardoso T A Avalia§£o epidemiol³gica dos ³bitos pordoen§as pri´nicas no Brasil sob o enfoque da biosseguran§a Cadernos SaºdeColetiva 1015901414462x201500010002Eisenmenger L Porter MC Carswell C J Thompson A Mead S RudgeP Evolution of diï¬usionweighted magnetic resonance imagingsignal abnormality in sporadic creutzfeldtjakob disease with histopathologicalcorrelation JAMA Neurol Finelli P F and DiMario F J Diagnostic approach in patients withsymmetric imaging lesions of the deep gray nuclei Neurologist 10109701nrl0000087718555976aFranceschini A Baiardi S Hughson A G McKenzie N Moda F Rossi M High diagnostic value of second generation CSF RTQuIC acrossthe wide spectrum of CJD prions Sci Rep Geschwind M D Rapidly progressive dementia Continuum 101212con0000000000000319Geschwind M D and Murray K Diï¬erential diagnosis with other rapidprogressive dementias in human prion diseases Handb Clin Neurol 101016b9780444639455000209Green A J E RTQuIC a new test for sporadic CJD Pract Neurol 101136practneurol2018001935Hayashi Y Iwasaki Y Yoshikura N Asano T Mimuro M Kimura A An autopsyveriï¬ed case of steroidresponsive encephalopathywith convulsion and a falsepositive result from the realtime quakinginduced conversion assay Prion Manix M Kalakoti P Henry M Thakur J Menger R Guthikonda B CreutzfeldtJakob disease updated diagnostic criteria treatmentalgorithm and the utility of brain biopsy Neurosurg Focus 39E2Martins V R Gomes H R Chimelli L Rosemberg S and Landemberger M C Prion diseases are under compulsory notiï¬cation in Brazil surveillanceof cases evaluated by biochemical andor genetic markers from to Dement Neuropsychol 101590s198057642008dn10400004McGuire L I Peden A H Orrº C D Wilham J M Appleford N EMallinson G Real time quakinginduced conversion analysisof cerebrospinal ï¬uid in sporadic CreutzfeldtJakob disease Ann Neurol 101002ana23589Ministrio da Saºde Protocolfor Notiï¬cation and Investigation ofCreutzfeldtJakob Disease with a Focus on the Identiï¬cation of the New Variant[Internet] Brazil Ministrio da SaºdeOrrº C D Bongianni M Tonoli G Ferrari S Hughson A GGroveman B R A test for CreutzfeldtJakob disease usingnasal brushings N Engl J Med 101056nejmoa131Orrº C D Groveman B R Hughson A G Zanusso G Coulthart M Band Caughey B Rapid and sensitive RTQuIC detection of humancreutzfeldtjakob disease using cerebrospinal ï¬uid mBio 6e0245114Orrº C D Hughson A G Groveman B R Campbell K J Anson K J MancaM Factors that improve RTQuIC detection of prion seedingactivity Viruses 103390v8050140Schmitz M Ebert E Stoeck K Karch A Collins S Calero M Validation of protein as a marker in sporadic creutzfeldtjakob diseasediagnostic Mol Neurobiol Smid J Landemberger M C Bahia V S Martins V R Nitrini R SmidJ Codon polymorphism of prion protein gene in is nota risk factor for Alzheimers disease Arquivos Neuro Psiquiatr 1015900004282x20130055Staï¬aroni A M Kramer A O Casey M Kang H Rojas J C Orrº C D Association of blood and cerebrospinal ï¬uid tau level and otherbiomarkers with survival time in sporadic creutzfeldtjakob disease JAMANeurol Studart Neto A Soares Neto H R Simabukuro M M Solla D J F Gon§ | 2 |
Pathwayspecific model estimation for improved pathway annotation by network crosstalkMiguel CastresanaAguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new networkbased method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gn gncid30 S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P S Notice that S Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iQcid31jPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP Q P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 pcid30 respectively This means that µ ¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n k logBα βk cid28 logŴα k logŴβ n klogŴα β n logŴα logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactomes specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the BenjaminiHochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A Betaalanine metabolism B Prostate cancer and C Alzheimers disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the Betaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway Prostate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the Prostate cancer pathway with a dispersion in the second quartile and Fig a03C shows the Alzheimers disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the Prostate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the Prostate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for Prostate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the Alzheimers disease and Parkinsons disease pathways share of their genes The Alzheimers disease the Parkinsons disease and the Huntingtons disease pathways have of the genes in common from the union between them Further the Oxidative phosphorylation the Nonalcoholic fatty liver disease the Alzheimers disease the Parkinsons disease and the Huntingtons disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each methods ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stabletheir variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU | 2 |
" tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1 irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6 a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27 jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30 thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with à gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with à gy or à gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturers instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated à gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with à cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at °c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar à objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturers instructions à caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of Ã106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoys 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of Ãà cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in à ripa buffer mm trishcl ph mm nacl np40 sodium deoxycholate sds containing à halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with à ripa buffer containing à halt protease inhibitor cocktail to obtain à concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grids front side and blotted for s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired students t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired students t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated à gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56 nk cells cd56cd3 and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified à e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified à f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired students t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3 or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa | 0 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annotated fluorescence image dataset for training nuclear segmentation methods \u2009 Eva Bozsaky19 Fikret Rifatbegovic Florian Kromp Magdalena ambros1 Maria Berneder1210 Tamara Weiss1 Daria Lazic1 Wolfgang D¶rr410 Allan Hanbury Sabine taschnerMandl Lukas Fischer Klaus Beiske7 Peter F ambros Inge M ambros12 \u2009Fullyautomated nuclear image segmentation is the prerequisite to ensure statistically significant quantitative analyses of tissue preparationsapplied in digital pathology or quantitative microscopy The design of segmentation methods that work independently of the tissue type or preparation is complex due to variations in nuclear morphology staining intensity cell density and nuclei aggregations Machine learningbased segmentation methods can overcome these challenges however high quality expertannotated images are required for training Currently the limited number of annotated fluorescence image datasets publicly available do not cover a broad range of tissues and preparations We present a comprehensive annotated dataset including tightly aggregated nuclei of multiple tissues for the training of machine learningbased nuclear segmentation algorithms The proposed dataset covers sample preparation methods frequently used in quantitative immunofluorescence microscopy We demonstrate the heterogeneity of the dataset with respect to multiple parameters such as magnification modality signaltonoise ratio and diagnosis Based on a suggested split into training and test sets and additional singlenuclei expert annotations machine learningbased image segmentation methods can be trained and evaluatedBackground SummaryBioimage analysis is of increasing importance in multiple domains including digital pathology computational pathology systems pathology or quantitative microscopy1 The field is currently rapidly expanding mainly facilitated by technological advances of imaging modalities in terms of resolution throughput or automated sample handling Moreover publicly available databases and platforms allow the access to image datasets and annotations enabling the research community to develop sophisticated algorithms for complex bioimage analysisDigital pathology relies on tissue sections as a basis for diagnosing disease type and grade or stage78 Moreover the accurate quantification of subcellular information including nuclear features at the single cell level is critical for the characterization of tumor heterogeneity plasticity response to therapeutic intervention910 and others Several approaches to visualise subcellular compartments such as nuclear morphology andor biological markers in tissues or cell preparations are well established Hematoxylin and eosin HE histological stainings immunohistochemical IHC and immunofluorescence IF stainings Whereas HE and IHC stainings and visualisation by bright field microscopy are standard procedures in routine diagnostic laboratories and pathology departments IF is more often employed in research settings than in routine diagnostics Prominent examples recently proved 1Tumor biology group Childrens Cancer Research Institute Zimmermannplatz Vienna Austria 2Labdia Labordiagnostik GmbH Zimmermannplatz Vienna Austria 3Software Competence Center Hagenberg GmbH SCCH Softwarepark Hagenberg Austria 4ATRABApplied and Translational Radiobiology Department of Radiation Oncology Medical University of Vienna Whringer G¼rtel Vienna Austria 5Institute of Information Systems Engineering TU Wien Favoritenstrasse Vienna Austria 6Complexity Science Hub Josefstdter Strae Vienna Austria 7Department of Pathology Oslo University Hospital Ullernchaussen N0379 Oslo Norway 8Department of Pediatrics Medical University of Vienna Whringer G¼rtel Vienna Austria 9These authors contributed equally Florian Kromp Eva Bozsaky 10Deceased Maria Berneder Wolfgang D¶rr email floriankrompccriat sabinetaschnerccriatScientific Data 101038s4159702000608wwwwnaturecomscientificdata 0cthe feasibility and power of IFbased quantitative analysis for eg detection of blood circulating or bone marrow disseminated tumor cells for minimal residual disease MRD diagnostics or the detection of genetic alterations by fluorescence in situ hybridization FISH11 Although the digitization of fluorescence stainings is more challenging and time consuming when compared to the digitization of HE or IHC stainings up to or more cellular or subcellular markers can be visualized in multiplex assays1415 This depicts a beneficial gain of information on individual cells and cell compartmentsA prerequisite for any reliable quantitative imagebased analysis however is the accurate segmentation of nuclei in fluorescence images5616 In order to reach sufficient power for statistical analysis fully automated segmentation algorithms are preferred While tissues and cell preparations presenting with well separated nuclei can be segmented based on simple intensity thresholds densely packed tissues or cell aggregations require more sophisticated algorithms for nuclear segmentation Segmentation algorithms focusing on the separation of objects instances are called instance segmentation algorithms or instance aware segmentation algorithms Although designed to split aggregating nuclei they frequently fail to separate each nucleus in cases of tightly clustered nuclei17 Remaining aggregations of nuclei lead in the worst case to their exclusion from the downstream analysis potentially causing a biologically significant biasTo overcome these drawbacks novel deep learningbased image segmentation approaches are currently developed in many labs worldwide They promise to solve most segmentation problems as long as highquality expertannotated datasets are available to train the systems However there are only a limited number of annotated nuclear image datasets publicly available While annotated datasets outlining nuclei in HE or IHC images can be obtained a comprehensive dataset including IF images of tissue sections of diverse origin and annotated nuclei is currently not available to the best of our knowledge Apart from the tedious process of annotation this may be due to the fact that it is challenging to decide whether an aggregation consists of one or multiple nuclei This is because in epifluoresecence microscopy images nuclei often appear blurry and within cell aggregations their borders are frequently not definableIn summary the time consuming and challenging process of annotation hampered the generation and publication of annotated fluorescence image datasets including tightly aggregated and overlapping nucleiWe hereby present an expertannotated comprehensive dataset18 that can be used to train machine learningbased nuclear segmentation algorithms The presence and annotation of tightly aggregating and partially overlapping nuclei enable the algorithms to learn how to solve the task of accurate instance aware nuclear segmentation The dataset consists of annotated IF images of different biological tissues and cells of pathological and nonpathological origin covering the main preparation types used in imagingbased biomedical research settings Schwann cell stromarich tissue from a ganglioneuroblastoma cryosections a Wilms tumor tissue cryosection a neuroblastoma tumor tissue cryosection bone marrow cytospin preparations infiltrated with neuroblastoma cells neuroblastoma tumor touch imprints cells of two neuroblastoma cell lines CLBMa STANB10 cytospinned on microscopy glass slides and cells of a normal human keratinocyte cell line HaCaT cytospinned or grown on microscopy glass slides The characteristics of neuroblastoma and the Schwann cell stromarich ganglioneuroblastoma tumors have been previously described by Shimada in detail19 Multiple modalities Zeiss Axioplan II Zeiss and Leica laser scanning microscopes LSM were used for image acquisition while using different magnifications 10x 20x 40x 63x objectives Nuclei in IF images were annotated by trained biologists carefully curated by an experienced disease expert and finally reviewed and curated by an external disease expert and pathologist The final annotated dataset forming the ground truth dataset was split into a training set and a test set to be used for machine learningbased image segmentation architectures The training set consists of images with similar characteristics while the test set partially consists of images with varying image characteristics To enable a detailed assessment of the generalizability of trained segmentation algorithms with respect to image parameters such as the magnification or the signaltonoise ratio the images of the test set were divided into classes based on common image characteristics In addition to the expertannotated ground truth randomly selected nuclei from images of each class were marked on the raw images and presented to two independent experts subject to annotation These annotations further called singlenuclei annotations serve to validate the quality of the annotated dataset by comparing the ground truth annotations to the singlenuclei expert annotations and to set a baseline for machine learningbased image segmentation architecturesIn conclusion the proposed expertannotated dataset presents a heterogeneous realworld dataset consisting of fluorescence images of nuclei of commonly used tissue preparations showing varying imaging conditions sampled using different magnifications and modalities The dataset can be used to train and evaluate machine learningbased nuclear image segmentation architectures thereby challenging their ability to segment each instance of partially highly agglomerated nucleiMethodsPatient samples Tumor n and bone marrow n samples of stage M neuroblastoma patients the Schwann cell stromarich part of a patient with a ganglioneuroblastoma tumor and one wilms tumor patient were obtained from the Childrens Cancer Research Institute CCRI biobank EK18532016 within the scope of ongoing research projects In addition two patientderived neuroblastoma cell lines CLBMa STANB10 were used Written informed consent has been obtained from patients or patient representatives Ethical approval for IF staining and imaging was obtained from the ethics commission of the Medical University of Vienna EK12162018 All authors confirm that we have complied with all relevant ethical regulationsPreparation and IFstaining of tumor tissue cryosections The freshfrozen tumor tissues of one ganglioneuroblastoma patient one neuroblastoma patient and one Wilms tumor patient were embedded into tissuetekOCT and µm thick cryosections were prepared Sections were mounted on Histobond glass slides Scientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cMarienfeld fixed in formaledhyde and stained with 46diamino2phenylindole DAPI a blue fluorescent dye conventionally used for staining of nuclei for cellular imaging techniques Finally slides were covered with Vectashield and coverslips were sealed on the slides with rubber cementPreparation and IFstaining of HaCaT human skin keratinocyte cell line The HaCaT cell line a spontaneously transformed human epithelial cell line from adult skin20 was cultivated either in culture flasks or on microscopy glass slides Cell cultures were irradiated and Gy harvested cytospinned airdried and IF stained Cells grown and irradiated on the glass slides were directly subjected to IF staining Cells were fixed in formaldehyde for minutes at °C and were permeabilized with sodium dodecyl sulfate SDS in PBS for minutes Slides were mounted with antifade solution Vectashield containing DAPI and coverslips were sealed on the slides with rubber cementPreparation and IFstaining of tumor touch imprints and bone marrow cytospin preparations Touch imprints were prepared from fresh primary tumors of stage M neuroblastoma patients as previously described21 Mononuclear cells were isolated from bone marrow aspirates of stage M neuroblastoma patients by density gradient centrifugation and cytospinned as described22 After fixation in formaldehyde for minutes cells were treated according to the Telomere PNA FISH Kit Cy3 protocol Dako mounted with Vectashield containing DAPI covered and sealedPreparation and IFstaining of neuroblastoma cell line cytospin preparations STANB10 and CLBMa are cell lines derived from neuroblastoma tumor tissue of patients with stage M disease Preparation and drugtreatment were conducted as described23 Briefly cells were cultured in the absence or presence of nM topotecan a chemotherapeutic drug for weeks detached and cytospinned to microscopy glass slides Preparations were airdried fixed in formaldehyde immuno and DAPI stained covered and sealedFluorescence imaging Samples were imaged using an AxioplanII microscope from Zeiss equipped with a Maerzhaeuser slide scanning stage and a Metasystems Coolcube camera using the Metafer software system V386 from Metasystems an AxioplanII microscope from Zeiss equipped with a Zeiss AxioCam Mrm using the Metasystems ISIS Software for microscopy image acquisition an LSM microscope from Zeiss equipped with an Argonlaser nm a photomultiplier tube PMT detector nm and a motorized Piezo Zstage using the Zeiss Zen software package and a SP8X from Leica equipped with a Diode Laser and a PMT detector nm For the presented dataset we digitized the DAPI staining pattern representing nuclear DNA Additional immunofluorescence or FISH stainings were in part available An automatic illumination time was set as measured by pixel saturation Metasystems Metafer and ISIS or defined manually Zeiss and Leica LSMs Objectives used were a Zeiss PlanApochromat objective Zeiss Axioplan II numerical aperture air a Zeiss PlanApochromat Zeiss LSM numerical aperture oil a Zeiss PlanApochromat Leica SP8X nuermical aperture oil a Zeiss PlanNeofluar objective Zeiss Axioplan II numerical aperture and a Zeiss PlanApochromat objective Zeiss Axioplan II Zeiss LSM and Leica SP8X numerical aperture oil Representative field of views FOVs were selected according to the following quality criteria sharpness intact nuclei and a sufficient number of nucleiGround truth annotation Nuclei image annotation was performed by students and expert biologists trained by a disease expert To accelerate the time consuming process of image annotation a machine learningbased framework MLF was utilized supporting the process of annotation by learning characteristics of annotation in multiple steps24 The MLF annotations result in a coarse annotation of nuclear contours and have to be refined to serve as ground truth annotation Therefore annotated images were exported as support vector graphic SVG files and imported into Adobe Illustrator AI CS6 AI enables the visualization of annotated nuclei as polygons overlaid on the raw nuclear image and provides tools to refine the contours of each nucleus An expert biologist and disease expert carefully curated all images by refining polygonal contours and by removing polygons or adding them if missing Finally an expert pathologist was consulted to revise all image annotations and annotations were curated according to the pathologists suggestions In cases where decision finding was difficult a majority vote including all experts suggestions was considered and annotations were corrected accordingly Images were exported and converted to Tagged Image File Format TIFF files serving as nuclear masks in the ground truth dataset A sample workflow is illustrated in Fig a0Dataset split As the dataset is intended to be used to train and evaluate machine learningbased image segmentation methods we created a dataset split into training set and test set The training set consists of multiple images of ganglioneuroblastoma tissue sections normal cells HaCaT as cytospin preparations or grown on slide and neuroblastoma tumor touch imprints and bone marrow preparations For each of these types of preparation multiple images using the same magnification 20x or 63x imaged with the same modality Zeiss Axioplan II and the Metasystems Metafer Software and showing a good signaltonoise ratio were included The test set consists of additional images of these preparation types and moreover includes images of different preparation types eg neuroblastoma cell line preparations Wilms tumor and neuroblastoma tumor tissue sections imaged with different modalities Zeiss and Leica confocal LSM Zeiss and Metafer ISIS software and different signaltonoise ratiosScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cacS1S2S3bdFig Workflow for ground truth image annotation a Raw image visualizing HaCaT cytospinned nuclei b A machine learning framework was used to annotate the raw image learning from user interaction within three consecutive steps S1 foreground extraction S2 connected component classification red nonusable objects blue nuclei aggregations green single nuclei and S3 splitting of aggregated objects into single nuclei resulting in an annotation mask c Zoomin of the SVGfile showing the nuclear image overlaid with polygons representing each annotated nucleus Polygons were modified by expert biologists to fit effective nuclear borders Challenging decisions on how to annotate nuclei mainly occurring due to aggregated or overlapped nuclei were presented to an expert pathologist and corrected to obtain the final ground truth d The curated SVGfile was transformed into a labeled nuclear maskTo enable an objective comparison of image segmentation architectures to the ground truth annotations with respect to varying imaging conditions we classified each image of the test set into one of classes according to criteria such as sample preparation diagnosis modality and signaltonoise ratio The details are presented in Table a0 The recommended dataset split into training set and test set and the test set classes can be downloaded along with the datasetSinglenuclei annotation To set a baseline for machine learningbased image segmentation methods and to validate the proposed dataset nuclei were randomly sampled from the ground truth annotations for each of the classes marked on the raw images and presented to two independent experts for image annotation Annotation was carried out by a biology expert with longstanding experience in nuclear image annotation further called annotation expert and a biologist with experience in cell morphology and microscopy further called expert biologist Nuclei were annotated using SVGfiles and Adobe illustrator The singlenuclei annotations described as singlecell annotations within the dataset can be downloaded along with the datasetAnnotation criteria The annotation of nuclei in tissue sections or tumor touch imprints is challenging and may not be unambiguous due to outoffocus light or nuclei damaged nuclei or nuclei presenting with modified morphology due to the slide preparation procedure We defined the following criteria to annotate nuclear images¢\t Only intact nuclei are annotated even if the nuclear intensity is low in comparison to all other nuclei present¢\t Nuclei have to be in focus¢\t¢\t Nuclear borders have to be annotated as exact as resolution and blurring allows forIf parts of a nucleus are out of focus only the part of the nucleus being in focus is annotatedScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cAcronymGNBIGNBIINBINBIINBIIINBIVNCINCIINCIIITSDescriptionganglioneuroblastoma tissue sectionsganglioneuroblastoma tissue sections with a low signaltonoise rationeuroblastoma bone marrow cytospin preparationsneuroblastoma cell line preparations imaged with different magnificationsneuroblastoma cell line preparations imaged with LSM modalitiesneuroblastoma tumor touch imprintsnormal cells cytospin preparationsnormal cells cytospin preparations with low signaltonoise rationormal cells grown on slideother tissue sections neuroblastoma WilmsTable Test set split into classes to evaluate the generalizability of machine learningbased image segmentation methods with respect to varying imaging conditions¢\t Nuclei are not annotated if their morphology was heavily changed due to the preparation procedure¢\t Nuclei from dividing cells are annotated as one nucleus unless clear borders can be distinguished between the resulting new nucleiData recordsThe dataset presented here is hosted in the BioStudies database under accession number SBSST265 identifiersbiostudiesSBSST26518 It consists of fluorescence images of immuno and DAPI stained samples containing nuclei in total The images are derived from one Schwann cell stromarich tissue from a ganglioneuroblastoma cryosection images2773 nuclei seven neuroblastoma NB patients images931 nuclei one Wilms patient image102 nuclei two NB cell lines CLBMa STANB10 images1785 nuclei and a human keratinocyte cell line HaCaT images2222 nuclei In addition the dataset is heterogenous in aspects such as type of preparation imaging modality magnification signaltonoise ratio and other technical aspects A summary of the dataset composition and relevant parameters eg diagnosis magnification signaltonoise ratio and modality with respect to the type of preparation is presented in Fig a0 Detailed information for each image is provided along with the dataset The signaltonoise ratio was calculated as follows We used the binarized ground truth annotation masks to calculate the meanforeground nuclear and meanbackground signal First we calculated the mean intensity of all raw image pixels covered by the masks foreground region resulting in the meanforeground signal By applying a morphological dilation on the binary foreground region using a diskshaped structuring element of size pixels and by inverting the resulting mask mean intensity values of raw image pixels covered by the inverted mask were calculated resulting in the meanbackground signal Dilation was applied to exclude pixels neighboring nuclei from the calculation as they do not represent the background signal but present increased intensity values due to blurred nucleiEach image of the dataset is provided in TIFFformat In addition we provide two types of annotations annotated labeled masks in TIFFformat and SVGfiles describing the exact position of each nucleus as a polygon The SVGfiles reference a nuclear image stored in Joint Photographic Experts Group JPEGformat and provide all annotated objects within an additional layerX Y XTechnical ValidationTo validate the proposed dataset and to set a baseline for machinelearning based image segmentation methods we compared the singlenuclei annotations to the ground truth annotation We calculated the Dice coefficient Y of each nucleus comparing the ground truth annotation X and the corresponding expert annotaDCtion Y Finally we computed the mean Dice coefficient for each of the classes by calculating the mean value of the Dice coefficients of all annotations of a class The overall Dice coefficient was computed by calculating the mean value of all annotations of all classes The results are presented in Table a0The overall Dice coefficients of achieved by the expert biologist and achieved by the annotation expert show that the concordance in annotations of nuclear contours on the pixel level is high between expert annotations and the ground truth A Dice score close to the optimal score cannot be achieved due to the nature of fluorescence images eg blurriness resulting in fuzzy nuclear borders and highly overlapping nuclei with partially invisible borders Varying Dice coefficients between classes are due to different imaging conditions For example images in the GNBII class were more blurry and present lower signaltonoise ratios than images of eg the NCI to NCIII classes thus exact nuclear contours cannot be determined Dice scores achieved by the annotation expert were higher than the scores achieved by the expert biologist in eight out of ten classes This was an expected result as the task of image annotation differs from image acquisition and visual biological interpretation Thus the annotation experts experience in image annotation was of benefit to achieve higher coefficients The presented scores and the released singlenuclei ground truth can be further used to benchmark the accuracy of machine learningbased image segmentation architectures in comparison to the baseline set by human annotatorsScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0c GNBNBDiagnosisNormal HaCaT TWTissue Cell line grown oCell line c y tp i nse ll li n e c ytospinCTU toucharrowe mnoB Signal to noise x worarBone mTU touch Cell line c y te ll li n e c ytospin sop i nCell line gro x xx Tissue Cell line grown worarBone mTU touchMaterial PreparationCell line grownTissue sectionCTU toucharrowe mnoBZeissLeicawnTissue sectionSISIModality Metafe rFig Heterogeneity of the proposed dataset with respect to the type of preparation GNB ganglioneuroblastoma NB neuroblastoma TU touch tumor touch imprint Tissue tissue sectionAnnotatorBiol expAnnot expGNBIGNBII NBINBIINBIIINBIVNCINCIINCIIITSOverallTable Mean Dice coefficient between randomly selected nuclei of the manual annotations and the ground truth annotations with respect to the human annotator Annot exp annotation expert Biol exp expert biologist GNBI ganglioneuroblastoma tissue sections GNBII ganglioneuroblastoma tissue sections with a low signaltonoise ratio NBI neuroblastoma bone marrow cytospin preparations NBII neuroblastoma cell line preparations with different magnifications NBIII neuroblastoma cell line preparations with different magnifications and imaged with LSM modalities NBIV neuroblastoma tumor touch imprints NCI normal cells HaCaT cytospin preparations NCII normal cells HaCaT with low signaltonoise ratio NCIII normal cells HaCaT grown on slide TS other tissue sections neuroblastoma Wilms tumor Bold values set the baseline for machine learningbased image segmentation methodsCode availabilityWe provide code to transform predicted annotation masks in TIFFformat to SVGfiles for curation by experts as well as the transformation from SVGfiles to TIFFfiles The contour sampling rate when transforming mask objects to SVGdescriptions can be set in accordance to the size of predicted nuclei Therefore new nuclei image annotation datasets can easily be created utilizing the proposed framework and a tool to modify SVGobjects such as Adobe Illustrator The code is written in python and is publicly available under githubcomperlfloccriNuclearSegmentationPipelineScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cReceived August Accepted July Published xx xx xxxxreferences Irshad H Veillard A Roux L Racoceanu D Methods for Nuclei Detection Segmentation and Classification in Digital Histopathology A Review Current Status and Future Potential IEEE Rev Biomed Eng Meijering E Carpenter A E Peng H Hamprecht F A OlivoMarin J C Imagining the future of bioimage analysis Nat Ching T Opportunities and obstacles for deep learning in biology and medicine J R Soc Interface Blom S Systems pathology by multiplexed immunohistochemistry and wholeslide digital image analysis Sci Rep Biotechnol Waters J C Accuracy and precision in quantitative fluorescence microscopy J Cell Biol Ronneberger O Spatial quantitative analysis of fluorescently labeled nuclearstructures Problems methods pitfalls Chromosome Res unfavorable groups Cancer Ambros I M Morphologic features of neuroblastoma Schwannian stromapoor tumors in clinically favorable and Gurcan M N Histopathological Image Analysis A Review IEEE Rev Biomed Eng Saltz J Spatial anization and Molecular Correlation of TumorInfiltrating Lymphocytes Using Deep Learning on Pathology Liu J Molecular Mapping of Tumor Heterogeneity on Clinical Tissue Specimens with Multiplexed Quantum Dots ACS Nano Images Cell Rep Ambros P F Mehes G Ambros I M Ladenstein R Disseminated tumor cells in the bone marrow Chances and consequences of microscopical detection methods Cancer Lett Narath R L¶rch T Rudas M Ambros P F Automatic Quantification of Gene Amplification in Clinical Samples by IQFISH Cytometry B Clin Cytom Mhes G Luegmayr A Ambros I M Ladenstein R Ambros P F Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow Clin Cancer Res Schubert W Analyzing proteome topology and function by automated multidimensional fluorescence microscopy Nat Ostalecki C Multiepitope tissue analysis reveals SPPL3mediated ADAM10 activation as a key step in the transformation of Jung C Kim C Impact of the accuracy of automatic segmentation of cell nuclei clusters on classification of thyroid follicular Biotechnol melanocytes Sci Signal lesions Cytometry Part A Xing F Yang L Robust NucleusCell Detection and Segmentation in Digital Pathology and Microscopy Images A Comprehensive Review IEEE Rev Biomed Eng Kromp F An annotated fluorescence image dataset for training nuclear segmentation methods BioStudies Database identifiersbiostudiesSBSST265 Shimada system Cancer Shimada H Ambros I M Dehner L P Hata J Joshi V V The International Neuroblastoma Pathology Classification the Boukamp P Normal Keratinization in a Spontaneously Immortalized J Cell Biol Brunner C Tumor touch imprints as source for whole genome analysis of neuroblastoma tumors Plos One Mhes G Detection of disseminated tumor cells in neuroblastoma Log improvement in sensitivity by automatic immunofluorescence plus FISH AIPF analysis compared with classical bone marrow cytology Am J Pathol TaschnerMandl S Metronomic topotecan impedes tumor growth of MYCNamplified neuroblastoma cells in vitro and in vivo by therapy induced senescence Oncotarget Kromp F Machine learning framework incorporating expert knowledge in tissue image annotation IEEE ICPR AcknowledgementsThis work was facilitated by an EraSME grant project TisQuant under the grant no and by a COIN grant project VISIOMICS under the grant no both grants kindly provided by the Austrian Research Promotion Agency FFG and the St Anna Kinderkrebsforschung Partial funding was further provided by BMK BMDW and the Province of Upper Austria in the frame of the COMET Programme managed by FFGAuthor contributionsF Kromp A Hanbury S TaschnerMandl and PF Ambros conceived the study M Berneder E Bozsaky T Weiss S TaschnerMandl and M Ambros prepared samples for the dataset W Doerr provided essential material F Kromp E Bozsaky IM Ambros M Ambros PF Ambros T Weiss and S TaschnerMandl acquired all images of the annotated dataset F Kromp T Weiss E Bozsaky F Rifatbegovic IM Ambros and K Beiske participated in annotation or curation of the ground truth dataset or the singlenuclei annotations F Kromp and L Fischer performed the statistical analysis of the singlenuclei annotations F Kromp S TaschnerMandl PF Ambros IM Ambros D Lazic and L Fischer interpreted the data F Kromp and E Bozsaky wrote the manuscript with input from all authors S TaschnerMandl F Rifatbegovic A Hanbury PF Ambros IM Ambros D Lazic and L Fischer revised the manuscriptCompeting interestsThe authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to FK or STMReprints and permissions information is available at wwwnaturecomreprintsScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cPublishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsOpen Access This article 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 article are included in the articles Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the articles Creative | 2 |
" 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 asksdo you think the chemo is workingkey 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 doc what would you do if he was your brotherkey 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 borderline 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 im a fighter not a quitter and with jesus anything is possible she then asks what comesnextkey question how to approach a patient who has a treatable 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 caregivers 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 dartmouths irb rely on dartmouths 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 studyteams 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 themethods 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 | 0 |
Rheumatoid arthritis RA is a systemic chronic inflammatory disease that affects synovial joints and has various extraarticular manifestations including atherosclerotic cardiovascular disease CVD Patients with RA experience a higher risk of CVD leading to increased morbidity and mortality Inflammation is a common phenomenon in RA and CVD The pathophysiological association between these diseases is still not clear and thus the risk assessment and detection of CVD in such patients is of clinical importance Recently artificial intelligence AI has gained prominence in advancing healthcare and therefore may further help to investigate the RACVD association There are three aims of this review to summarize the three pathophysiological pathways that link RA to CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators useful for RA patients and to understand the role of artificial intelligence in CVD risk assessment in RA patients Our search strategy involves extensively searches in PubMed and Web of Science databases using search terms associated with CVD risk assessment in RA patients A total of peerreviewed s were screened for this review We conclude that a two of the three pathways directly affect the atherosclerotic process leading to heart injury b carotid ultrasound imagebased calculators have shown superior performance compared with conventional calculators and c AIbased technologies in CVD risk assessment in RA patients are aggressively being adapted for routine practice of RA patientsKeywords Arthritis a0· Rheumatoid a0· Atherosclerosis a0· Cardiovascular disease a0· Inflammation a0· Carotid artery diseases a0· Carotid intimamedia thickness a0· Risk assessmentIntroductionRheumatoid arthritis RA is a chronic inflammatory disease that not affects only synovial joints but also has several extraarticular involvements including those related to the skin eyes heart lungs kidneys and other ans [ ] It affects of the global population with a higher prevalence in females when compared with males [ ] Cardiovascular disease CVD is a common manifestation in RA patients with a two to threefold higher risk of cardiovascular events and mortality compared with a normal population [] However this increased risk is not entirely jasjitsuriatheropointcom Jasjit S Suri Extended author information available on the last page of the explained by conventional risk factors [] Current statistically derived CVD risk calculators use conventional risk factors alone [] are not suitable for RA patients and they either underestimate or overestimate the risk [] This may be because of the paradoxical behavior of some of the conventional risk factors such as body mass index lowdensity lipoprotein highdensity lipoprotein and total cholesterol in RA [ ] Despite this lack of clarity the guidelines by the European League Against Rheumatism EULAR recommend aggressive control of these conventional risk factors [ ] Recent attempts were made to improve the CVD risk assessment in the RA population including the development of RAspecific risk factors in the CVD risk calculators [] However such calculators could not provide adequate improvement in risk Vol01234567891 0c Rheumatology Internationalprediction and reportedly still underestimated or overestimated CVD risk in RA patients [ ]To provide a better CVD risk assessment in RA a pathophysiological association between these diseases should be understood as this would help in refining CVD risk predictors in RA patients [] Atherosclerosis a common phenomenon in RA [ ] can be adequately monitored using imaging modalities such as magnetic resonance imaging [] computed tomography [] optical coherence tomography [] and ultrasound [] Each of these imaging modalities offers unique information about morphological variations in atherosclerotic plaque Ultrasound imaging specifically in carotid arteries is a comparatively lowcost noninvasive radiationfree and easytouse imaging modality that is widely adopted in preventive cardiovascular and clinical vascular practices [ ] The imagebased phenotypes of carotid ultrasound such as carotid intimamedia thickness cIMT and carotid plaque are considered surrogate markers of coronary artery disease and have been used for preventive CVD risk assessments in several studies [] These imagebased phenotypes indicate the morphological variations in the atherosclerotic plaque and are associated with the inflammatory markers of RA [] Patients with RA have elevated cIMT and have more plaque area PA when compared with nonRA patients [] Thus the inclusion of these imagebased phenotypes in risk prediction models may improve the CVD risk assessments of RA patients Recent studies have combined the effect of these imagebased phenotypes with conventional risk factors including proinflammatory markers like erythrocyte sedimentation rate ESR to perform CVD risk assessment [] Such integrated risk calculators have demonstrated better CVD risk stratification when compared to traditional CVD risk calculators in nonRA patients [ ]Besides these statistically derived CVD risk calculators artificial intelligence AIbased techniques are also penetrating several medical imaging and risk assessment applications [] AIbased algorithms such as machine learning ML methods provide a better CVD risk assessment when compared with statistically derived conventional risk calculators [ ] So far AI algorithms have been used for CVD risk assessment in the nonRA population and their potential still needs to be evaluated in RA cohorts However AI is well adapted for RA screening and diagnosis [] This review provides an insight into how the AIbased algorithms can be used for CVD risk assessment in RA patients There are three aims of this review to summarize the pathophysiological pathways that link RA with CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators for RA patients and to provide an understanding of the role of artificial intelligence in CVD risk assessment in RA patientsSearch strategyFigure a0 shows a flow diagram indicating the search strategy for this narrative review To write a comprehensive narrative review it is essential to select at least two credible databases that provide highquality peerreviewed s [] This review is the outcome of several searches in the PubMed and Web of Science databases using keywords such as cardiovascular diseases AND risk assessment AND rheumatoid arthritis carotid atherosclerosis AND rheumatoid arthritis noninvasive imaging AND rheumatoid arthritis carotid ultrasound AND rheumatoid arthritis carotid intimamedia thickness OR carotid plaque AND inflammatory markers carotid atherosclerosis AND erythrocyte sedimentation rate OR C reactive protein machine learning AND rheumatoid arthritis and machine learning AND cardiovascular risk assessment AND rheumatoid arthritis The availability of all these keywords in the and the full text was investigated to select the relevant s Peerreviewed s published in the last a0years were then given priority Citations from the published s were also shortlisted for this review All these s were subsequently filtered by the expert coauthors to select only those that met the objectives of this review leading to sPathophysiology of a0RA leading to a0CVDThe pathophysiological association between RA and CVD can be explained in two stages the role of traditional risk factors and direct vascular damage Inflammation plays a pivotal role in both of these stages []The role of a0traditional risk factors in a0the a0pathophysiology of a0RAdriven atherosclerotic CVDThe righthand panel of Fig a0 explains the pathophysiological association between RA and CVD via four pathways [IaId] governed by traditional risk factors such as hypertension proatherogenic dyslipidemia insulin resistance and obesity Patients with RA are generally found with proinflammatory cytokines such as interleukin IL IL6 and tumor necrosis factor α TNFα [] These proinflammatory cytokines are found in the synovium which triggers a systemic inflammatory response and may result in damage to the vascular endothelial cells [] Nitric oxide NO and cyclooxygenase1 are two essential components required 0cRheumatology International Fig Flow diagram for the search strategyto maintain the healthy endothelium which is inhibited by TNFα and IL6 thereby resulting in endothelial cell damage [ ] Inhibiting endothelial NO leads to arterial stiffness [] and is further associated with an increase in peripheral vascular resistance PVR [] thus leading to hypertension in RA patients Additionally several medications used to treat RA such as diseasemodifying antirheumatic drugs DMARDs leflunomide and cyclosporine glucocorticoids nonsteroidal antiinflammatory drugs NSAIDs and cyclooxygenase II inhibitors Cox IBs may also be involved in the development of hypertension in RA patients [ ]Another pathophysiological link between RA and CVD is proatherogenic dyslipidemia [] Nearly of RA patients have proatherogenic dyslipidemia [] In nonRA patients increased CVD risk is associated with elevated levels of lowdensity cholesterol LDLc total cholesterol and reduced highdensity lipoprotein cholesterol HDLc However in RA patients low levels of total cholesterol TC low levels of LDLc and suppressed levels of HDLc have been reported This condition is known as the lipid paradox [] Highly suppressed HDL levels in RA patients are proatherogenic [] Furthermore RA patients show high atherogenic index levels despite low lipid levels The atherogenic index is calculated as a ratio of TC HDLc and it may vary according to their levels [] Apolipoprotein B Apo B is a major apolipoprotein in LDL and several studies have indicated an increase in the ratio of Apo B Apo A in RA patients [] A combination of low TC LDLc and suppressed HDLc levels with a high atherogenic index and a high ApoBApoA ratio behaves as proatherogenic dyslipidemia [ ] Longstanding proatherogenic dyslipidemia causes atherosclerosis and eventually CVDRheumatoid cachexia is another important RAspecific characteristic that increases CVD risk [] It is characterized by significantly increased adiposity and reduced muscle mass while one maintains their bodyweight [] The pathophysiology [shown in pathwayI c] behind R cachexia can be explained in two ways It is characterized by the reduction of muscle mass that is largely due to increased inflammatory cytokines particularly TNFα by activating the transcriptional nuclear factorkappa B cells NFkB pathway and promoting the ubiquitin pathway which causes catabolismproteolysis muscle protein breakdown [ ] Central obesity or abdominal obesity is present in of women and of men This causes visceral adiposity in RA which has an additional adverse impact on CVD [] On the other hand increased adiposity also induces the production of inflammatory cytokines in RA which further worsens this 0c Rheumatology InternationalFig Pathophysiological association between rheumatoid arthritis and cardiovascular disease IL1 interleukin IL6 interleukin TNFα tumor necrosis factor α EC endothelial cells SMC smooth muscle cells MCP1 monocyte chemoattractant protein MCSF macrophage colonystimulating factor VCAM vascular cell adhesion molecule ICAM intercellular adhesion molecule NSAIDs nonsteroidal antiinflammatory drugs CoxIBs cyclooxygenase inhibitors HTN hypertension PVR peripheral vascular resistance TC total cholesterol HDL highdensity lipoprotein LDL lowdensity lipoprotein APOB apolipoprotein B APOA apolipoprotein A NFkB nuclear factorkappa B cellsscenario [] This syndrome may be explained in the triad of increased adiposity reduced muscle mass and low body mass index BMIEpidemiological studies have suggested a strong association between insulin resistance IR metabolic syndrome and RA [ ] [shown in pathwayI d in the dark greendotted box] Inflammation plays a crucial role in these three conditions [] In patients with RA IR serves as an independent prognostic risk factor that signifies the presence of subclinical atherosclerosis it is determined by carotid intimal thickness cIMT and is measured by carotid ultrasonography [] Longstanding inflammation due to RA promotes oxidative stress endothelial dysfunction and atherosclerosis in this population []Progression of a0atherosclerosis and a0direct vessel damage in a0RAIn RA the activation of Tcells and mast cells increases the production of proinflammatory cytokines such as IL1 IL6 and TNFα These proinflammatory cytokines stimulate endothelial cells ECs and smooth muscle cells SMCs in subendothelium [] by expressing cell adhesion molecules such as vascular cell adhesion molecule VCAM and the intercellular adhesion molecule ICAM [] and by producing chemokines including monocyte chemoattractant protein MCP and macrophage colonystimulating factor MCSF The activation of endothelial cells allows the migration of LDLc into the subendothelial layer where 0cRheumatology International it becomes oxidized and triggers the inflammatory response for the recruitment of immune cells such as T lymphocytes and monocytes in the intimal layer Once they enter the intimal layer monocytes are transformed into macrophages and they then take up the oxidized LDLc to become foam cells The completion of this complex process then leads to the formation of atherosclerotic plaque Macrophages also trigger the migration of smooth muscle cells from tunica media to tunica intima and initiate their proliferation The SMCs form a thin fibrous cap to prevent the encroachment of atherosclerotic plaque towards the lumen However over time proinflammatory cytokines enzymes and free radicals cause fibrous cap erosion and make the plaque vulnerable for rupture The amplification of the inflammatory response results in the acceleration of plaque formation eventually leading to plaque rupture and thrombotic events which damage the blood vessels Pathway II of Fig a0 represents this processCurrent conventional CVD risk prediction models for a0RAOver the last decade several CVD risk assessment calculators have been developed very few of which are recommended by the cardiovascular risk management guidelines [ ] Some standard cardiovascular risk prediction algorithms are the Framingham risk score FRS [] Systematic Coronary Risk Evaluation SCORE [] American College of CardiologyAmerican Heart Association ACCAHA risk score [] World Health anization WHO risk charts [] and Reynoldss risk score RRS [] These risk calculators use traditional risk factors such as patient demographics age gender ethnicity blood biomarkers lowdensity lipoprotein cholesterol highdensity lipoprotein cholesterol and total cholesterol behavioral markers smoking and alcohol consumption and physiological markers height weight body mass index All these risk calculators were initially developed for nonRA populations therefore when used in RA cohorts CVD risk is substantially underestimated [] The use of traditional risk factors alone while not considering RAspecific inflammatory markers could be another reason for such underestimation However RRS included an RAspecific inflammatory marker called high sensitivity Creactive protein hsCRP [] for CVD risk prediction but did not report any significant improvement in the CVD risk assessment [] Rajagopalan et a0al [] also reported a small improvement in area under the curve in females and in males when C reactive protein CRP or erythrocyte sedimentation rate ESR was added to the FRSOver the past few years several efforts have been made to improve the cardiovascular risk assessment in RA patients The EULAR guidelines recommended the use of a modified SCORE mSCORE in RA patients positive with rheumatoid factor RF or anticitrullinated protein antibodies ACPA and RA duration of more than a0years [ ] Cox et a0al [ ] developed the QRISK2 and QRISK3 algorithms which use the presence of RA as a CVD risk predictor hazard ratio confidence interval Solomon et a0al [] also developed an RAspecific CVD risk calculator called expanded risk score or ERS by including RAspecific biomarkers [such as disease activity disease duration a modified health assessment questionnaire HAQ disability index and daily prednisone use] with the traditional biomarkers used in the Coxbased model The authors reported an improvement of in cindex when validating the risk score on the reserved dataset Recently Curtis et a0al [] also proposed a CVD risk prediction tool for RA patients by combining conventional and RAspecific risk factors The authors predicted the risk of composite CVD events such as MI stroke and death during the followup period of a0years The area under the curve AUC for cardiovascular risk stratification for this model was All these RAspecific CVD risk scores reported a better risk assessment on the proprietary databases Still when compared with other risk calculators in different RA cohorts these calculators have demonstrated mixed results [] Crowson et a0al [] reported an underestimation of CVD risk by FRS and RRS in RA patients The observed risk was twice the predicted risk Furthermore the authors did not report any improvement in cardiovascular risk prediction when CRP was added to their model Arts et a0al [] investigated the roles of SCORE FRS RRS and QRISK2 in RA patients Out of these four models SCORE FRS and RRS underestimated CVD risk in RA patients whereas the QRISK2 reported an overestimation The AUC ranged between and for the four risk models A similar study by Arts et a0al [] investigated the performance of the original recalibrated and improved version of SCORE calculators to predict the CVD risk in RA patients The AUC values for these scores were CI and All these three scores underestimated the CVD risk in RA patients In short even after the SCORE was redesigned using the RAbased risk factors it did not result in an adequate CVD risk assessment In another study by Crowson et a0al [] of RA patients a CVDrisk prediction model was developed that reported better performance AUC compared with conventional risk calculators such as FRS AUC ACCAHA AUC SCORE AUC and QRISK2 AUC Furthermore conventional risk calculators either overestimated or underestimated CVD risk in RA patients Wahlin et a0al [] compared the expanded risk score ACCAHA risk score and a modified version of ACCAHA with a multiplier of for a CVD risk assessment of 0c Rheumatology International RA patients The authors also reported an underestimation of CVD risk by all calculators However the discrimination ability was slightly better since AUC for ERSRA risk was compared to AUC of for two variants of ACCAHAThe overall trend of all these risk prediction algorithms developed for general and RA cohorts indicates a poor CVD risk assessment in patients with RA One possible reason for such poor performance is the paradoxical behavior of some of the risk factors such as lipids and body mass index Another potential reason for this outcome is the inclusion of risk factors that do not provide complete information about the CVD risk profile in RA patients [] Corrales et a0al [] indicated a high prevalence of carotid atherosclerosis plaque in patients with lowCVD risk This observation demonstrated the limited ability of conventional risk factorbased algorithms to improve the CVD risk assessment process which may be improved using imaging modalities Therefore there is still room to develop more accurate automated and reliable risk calculators for RA patients by exploring and including nontraditional risk factors such as genetic biomarkers inflammatory biomarkers or imagebased atherosclerotic plaque phenotypes in the risk prediction algorithmCarotid ultrasound atherosclerosis imaging for a0CVD risk assessment in a0RA patientsImaging modalities are becoming essential for the visualization of atherosclerotic plaque and CVD risk assessment in RA patients [] Noninvasive imaging modalities such as computed tomography magnetic resonance imaging ultrasound and positron emission tomography are currently used to assess carotid atherosclerosis in RA patients [] MRI is used to measure the plaque composition including calcification lipidrich necrotic core and fibrous cap thickness [] Computed tomography is generally used to determine carotid artery stenosis [] Ffludeoxyglucosepositron emission tomography FDGPET is a nuclear imaging modality that quantifies the inflammation in carotid atherosclerotic plaque [] Noninvasive carotid ultrasound is a commonly adopted imaging modality that can capture morphological variations in the atherosclerotic plaque quantified using carotid intimamedia thickness cIMT carotid intimamedia thickness variability IMTV and plaque area [] When compared with other noninvasive counterparts carotid ultrasound is less expensive and easier to use [ ] Therefore the scope of this review is restricted to the use of carotid ultrasound for CVD risk assessment in RA patients The automated cIMT and carotid PA are considered surrogate markers of coronary artery disease and widely used for CVDstroke risk assessment []Several studies have shown a high prevalence of increased cIMT and carotid plaque in RA patients [] Studies have also demonstrated the significant association between these carotid atherosclerosis biomarkers and RAspecific markers of inflammation such as ESR CRP and IL6 [] Table a0 provides some of such studies that link both carotid atherosclerosis and RA using two sets of biomarkers ie imagebased phenotypes and inflammatory biomarkers One common observation from these studies is that patients with RA show an elevated cIMT and carotid plaque area compared with nonRA cohorts row R2R4 of Table a0 [ ] This association between carotid atherosclerosis and RA also seems independent of the three carotid artery segments common carotid artery carotid bulb and internal carotid artery from where the cIMT or plaque was measured [ ] However several studies have reported more aggressive atherosclerotic plaque formation in the carotid bulb segment when compared to other arterial segments [] The higher plaque prevalence in the carotid bulb is a consequence of turbulent blood flow and reduced shear stress which leads to endothelial dysfunction [ ] This observation of higher plaque in a bulb has also been confirmed in RA patients [] Figure a0 shows carotid ultrasound scans for RA Fig a03a b and nonRA patients Fig a03c d The lefthand side panel of Fig a03a c shows the raw carotid ultrasound scans measured using a Bmode ultrasound scanner The broad usage of carotid ultrasoundbased phenotypes and their significant association with RAspecific Similarly the righthand side panels of Fig a03b d show the processed scans tracking morphological variations in the carotid atherosclerotic plaque for the quantification of cIMT and plaque area The cIMT and plaque area are both greater in RA patients than in nonRA patientsAnother important observation from Table a0 is the significant association between carotid atherosclerotic biomarkers and RAspecific inflammatory markers such as ESR CRP and IL6 [ ] ESR is a relatively inexpensive measure of inflammation in RA patientstherefore several studies have used ESR for CVD risk assessment [] Some of such studies are listed in Table a0 All these studies indicated a substantially higher CVD event rate in patients with elevated ESR levels Besides ESR studies have also suggested the use of other popular RAspecific inflammatory markers such as CRP or hsCRP and IL6 for the improvement in the CVD risk assessment [ ] Furthermore these RAspecific inflammatory markers are also associated with the annual progression of cIMT [ ] which is a prominent surrogate marker of cardiovascular events In a study with RA patients Kaseem et a0al [] demonstrated the association of ESR CRP and IL6 with carotid atherosclerosis with significant odds ratios p of and respectively 0c hti foTM wdetaicossa era PCIc PRCdna RSE ecneserp dna TM mm a0 yb tinueno sesaercniyreve rof RSEni esaercniIc sepytonehp desabegami sisorelcsorehta ditoraC ni rehgih naht stneitapARniyltnacfiingis era noitalupopARnon eht hti wdetaicossa sawAR orehta fo AC I ditorac ACChti ytireves hgihni sisorelcs wnaht blub ni rehgih stnemges saw stneitapyretra eerht nehw ACI FIB ACC slortnoc ot derapmoc ylevitagen si tnemtaert ARhti wdetalerrocnoitammaflniTMIc oslA era srekramyrotammaflnI detaicossa elcsorehta ditorac yltnacfiingis ARni TMI ditoraChtiwsisorfiingis erew smret rotcaf ksir DVCÃRSEdna RSE hti wdetaicossa yltnacnoissergorpTM Ic ti w fo ecnelaverP euqalp dna sraey a0 ehT llomm a0 ARni euqalp ega CTditorac llew ew eb dna ega gnisu nac stneitapdetciderpCTRheumatology International yrammuSCstluseRCsrotcaf ksir egaminoNCepytonehp desabegamICsraey ega naeMC PRCdna rof p p r r RSEhti w detaicossa TMIc PRCdna RSEPC ecneserp dna TMIc dna AR roF slortnoC roF sv PCdna mm a0 sv ecnelaverp ± ARnoN ± TMIc svAR blubAC ROdna mm a0I I C PC sv rof FIBTM ACTMII c 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RSE sa eht tciderp ot desuhcus eranedrub euqalp ditorac srekramyrotammaflnI ni tnatropm DACgnitciderp rofi osla era TMIc dna thgiehPC ecneserp eht PC fodnoyeBstneitapAR mm a0 ¥ thgieh mm a0 ¥TMIc PC roF hgihPRCsh nietorpLDHdoolb cilotsaid loretselohc PBDnietorpopil erusserp evitcaer ytisnedwol cLDL loretselohc latot CPRC etar noitatnemides etycorhtyre RSE sgurdnoitacrufibFIB citamuehritna CT doolb cilotsys PBS yrotsih ylimaf HF sutillem setebaidMD gniyfidomesaesidnoisnetrepyhNTH yretra ditorac lanretni ditorac mumixam xamTMIc ssenkciht aidemamitni ditorac TMIc esaesid yretra yranoroc DAC esaesid ralucsavoidrac DVCACI yretra xedni ssamydob IMB sDRAMD rotcaf diotamuehr FR nikuelretni LI nietorpditorac nommoc ACC sitirhtra diotamuehr AR euqalp ditorac PC stneitap fo rebmunN rebmun laires NS edirecylgirt GT loretselohc evrucehtrednuaera CUA ssenkciht aidemamitni evitcaer Cytivitisnesnietorpopil ytisnedhgih oitar sddoRO erusserp cRheumatology Internationalinflammatory markers has also enabled their inclusion in the CVD risk prediction calculators [ ] Recently several CVDstroke risk prediction models have been developed that have combined the effect of conventional risk factors and the automated carotid atherosclerosis biomarkers [ ] These risk prediction models reported a better performance in identifying high CVD risk patients compared with current standardofcare risk calculators However such socalled integrated risk prediction models were developed for the general population They were based on the annual progression rates of carotid atherosclerotic biomarkers and conventional risk factors [] Therefore given the progression rates of cIMT and PA due to the RAspecific inflammatory markers such models can be updated and might be useful for CVD risk assessment in RA patientsArtificial intelligence in a0CVDstroke risk assessmentArtificial intelligence AI is expeditiously changing the landscape of the global healthcare system and assisting the healthcare workforce in clinical decisionmaking [] Machine learning ML and deep learning DL are the two common branches of AI that have broad ranges of applications in almost every medical imaging sector eg classification and plaque characterization for stroke risk assessment [] thyroid cancer characterization [] liver cancer diagnosis [] prostate cancer diagnosis ovarian cancer diagnosis [] lung cancer detection [] brain tumor classification [] and heart disease prediction and disease classification [ ] During the recent global pandemic of coronavirus diseases AI is providing promising results in the diagnosis of patients with the help of several imaging techniques such as computed tomography [] and Xrays []Since this review is on CVDstroke risk assessment we have summarized several studies that have used MLbased algorithms for CVDstroke risk assessment Table a0 All of these studies follow a supervised learning approach in which the MLbased classifier is trained to identify the correct output labels using input risk factors or features and predefined gold standards or labels Figure a0 shows the generalized framework of supervised MLbased CVD risk assessment In the case of CVD risk assessment the gold standard can be the primary endpoints such as presence or absence of cardiovascular events or surrogate endpoints such as cIMT PA and CAC score or a combination of these risk factors [ ] Several types of input features can be used to train the AIbased algorithms They can be traditional risk factors imagebased phenotypes grayscale image features or statistically derived features Once the offline ML classifier is trained using these features and gold DAC rof RO roF gnikomS MD NTH PBD PBS IMB egAaimedipilrepyH thgiehPCdna TM Ic ± ][ nosetnavSR 0cRheumatology International Fig Carotid ultrasound image of the common carotid artery for control patientsstandard its coefficients are then used in the online ML system to predict the out risk labels Online ML systems do not require a gold standard to make the final risk classification All the studies provided in Table a0 used this approach for CVD risk assessment Unlike MLbased algorithms DLbased models such as convolutional neural networks do not require input features beforehand Instead such algorithms automatically learn their offline coefficients from the input image datasets [] Currently AIbased techniques are used in the diagnosis of RA [] the identification of RA disease severity [] the classification of several RA synovial tissues [] and mortality prediction due to RA [] Although MLbased algorithms are used in the RA field no efforts have been made to assess the CVD risk in RA patients using such automated intelligencebased paradigms MLbased algorithms have been used to perform CVD risk assessments in nonRA populations and reported a better performance in identifying highrisk CVD patients when compared with the current standard of care conventional risk calculators [ ] Patients with RA experience more atherosclerotic plaque in the carotid artery which might lead to cardiovascular events [] In recent years several studies have demonstrated a better stroke risk assessment using MLbased strategies [] and DLbased strategies [] Besides all these studies attempts can be made to develop more accurate CVD risk prediction tools for RA patients using AI techniques Figure a0 conceptualizes several components required for CVD risk assessment in RA patients The AIbased CVD risk assessment for RA patients can be made possible by combining several types of risk factors such as patients demographics physiological parameters behavioral risk factors imagebased phenotypes and most importantly RAspecific inflammatory markers This combined set of features can be used as inputs along with the gold standard to identify what CVD risk category RA patients belong to As such both ML and DLbased systems can be employed to performed CVD risk assessment in patients Because of the significant association between carotid atherosclerosis and RA researchers can conduct a pilot study with cIMT and plaque areas as the surrogate markers for CVD risk assessmentSummaryIn this review we provided several | 2 |
" 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 spearmans 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 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 manufacturers 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 spearmans 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 à power time à power¼¼ 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 energy time à power time à 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 aheat 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 its 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 in 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 postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans 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 energyindex 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 wascancerspecific 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 cancerspecific 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 abscopal effecton 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 differentiation 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 ahmeds 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 writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview 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 writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview 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 six 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 suppls144rognoni 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 2013146e218jin 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 2018145e490erinjeri 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 | 0 |
" among eukaryotic anisms alternative splicing is an important process that can generate multipletranscripts from one same precursor messenger rna which greatly increase transcriptome and proteome diversitythis process is carried out by a superprotein complex defined as the spliceosome specifically splicing factor branchpoint binding protein sf1bbp is a single protein that can bind to the intronic branchpoint sequence bpsconnecting the ² and ² splice site binding complexes during early spliceosome assembly the molecular functionof this protein has been extensively investigated in yeast metazoa and mammals however its counterpart inplants has been seldomly reportedresults to this end we conducted a systematic characterization of the sf1 gene family across plant lineages inthis work a total of sequences from plant species were identified phylogenetic relationships of thesesequences were constructed and subsequent bioinformatic analysis suggested that this family likely originatedfrom an ancient gene transposition duplication event most plant species were shown to maintain a single copy ofthis gene furthermore an additional rna binding motif rrm existed in most members of this gene family incomparison to their animal and yeast counterparts indicating that their potential role was preserved in the plantlineage our analysis presents general features of the gene and protein structure of this splicing factor familyand will provide fundamental information for further functional studies in plantskeywords alternative splicing expression profile phylogenetics plants promoter splicing factor correspondence fyzhunjfueducn kailu zhang zhen feng jingfang yang and feng yang contributedequally to this work1coinnovation center for sustainable forestry in southern china college ofbiology and the environment nanjing forestry university nanjing jiangsu province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang bmc plant biology page of in eukaryotes canonical splicing removes noncoding intronic sequences and assembles the coding elements intomature mrnas while alternative splicing as generatesdifferent multiple transcripts that encode proteins withdistinct structures and functions by differential usage ofexons or splice site [ ] the resulting transcripts ofas greatly contribute to posttranscriptional regulationbiological complexity and proteome diversity in eukaryotes [ ] given that on average there are approximately exons in each transcript in the humantranscriptome and the degenerative nature of corresponding splice sites premrna splicing is sophistically catalysed by the spliceosome spliceosome is amultimegadalton protein complex which consists offive u1 u2 u4 u5 and u6 small nuclear ribonucleoprotein ps snrnps and over spliceosomalproteins furthermore the early assembly of spliceosome complex e or the commitment complex is anatpindependent process and contains u1 snrnps sf1and u2 snrnp auxiliary factors u2af large and u2afsmallthe prespliceosome complex a is formed by replacing sf1 withsf3b155sap155 of u2 snrnps [ ] stepwiseassembly of the following spliceosome during the splicing reaction has been reported as well [ ] however splice site recognition is a critical step during earlyassembly of the spliceosome the current model describes the binding of u1 snrnp and u1 snrna to ashort stretch of nucleotides at the ² splice site of splicing factor sf1mammalian branch point bindingprotein mbbp at the branch point and of u2 snrnpauxiliary factors at the ² splice site these threeciselements are necessary but usually insufficient to define a specific exonintron boundary thus additionalsplicing enhancers or silencers located at exons and introns may allow the recognition of genuine splice sitesduring early spliceosome assembly [ ] subsequentlysubunitsimportantly sf1 preferentially binds to the intronbranch point sequence bps which is adjacent to thebinding site polypyrimidine tract py of u2af largesubunits mammal u2af65 and fission yeast u2af59bridging u1 and u2af to form an intermediate lariatstructure [ ] in particular sf1 is characterized bythe presence of two types of rna binding motifs at thenterminus a k homologyquaking khqua2 domain which originated from the human heterogeneousribonucleoprotein hnrnp k protein [ ] and oneor two zinc knuckle motifs cx2cx4hx4c x represents any amino acid sf1 also contains a prolinerichregion at cterminus [ ] intriguingly the yeast khdomain specifically binds to the bps of premrnas witha glyproarggly motif and the variable loop of thekh domain and is necessary for spliceosomeassembly the first but not the second zinc knuckledomain in yeast has been demonstrated to bind rnawith high affinity moreover the stability of thesf1u2af65rna complex is further affected by thephosphorylation status of several sf1 serine residuesser20 ser80 and ser82 in vitro the prolinerichregion of sf1 interacts with u1 snrnp prp40fbp11 inyeast and human [ ] in regards to its interactionpartner the u2af large subunit the nterminal of sf1interacts with its noncanonical rna recognition motifsrrm or u2af homology motif[ ]whereas the other two rrms of u2af large subunitbind to the py region uhmsaccharomycescerevisiae common fruita previous study in fission yeast schizosaccharomycespombe suggests that the initial corecognition of thebranch site and ² splice site is pivotal for correct splicing of target premrnas because of the importance of splice site recognition for gene expression andprotein diversity sf1 has been demonstrated to play essential roles in a number of eukaryotic species includinghuman homo sapiens mice mus musculus buddingyeastflydrosophila melanogaster and roundworm caenorhabditis elegans [ ] for example in humansmissense mutation of splicing factors which are responsible for splice site recognition such as sf1 has beenlinked to tumourigenesis similarly heterozygoussf1 knockdown mice are susceptible to colontumourigenesis induced by an anotrophic carcinogenazoxymethane and sf1 has been found to associatewith betacatenintcf4 complex suggesting its role incarcinogenesis in contrast knockdown of sf1 suppresses the development of germ cell tumours in mice indicating its tissue dependency in cancer researchfurthermore the molecular function of sf1 has been extensively studied in yeast for instance a sf1 mutantstrain causes frequent exon skipping in fission yeast additionally sf1 has been proposed to recognize suboptimal sequences in specific introns and lead to nuclearaccumulation of premrna with aberrant splicing however increasing evidence indicates that this proteinis a regulator of splice site recognition and does not reduce general splicing specifically during alternative splicing by targeting a subset of genes [ ] thishypothesis is supported by the fact that knockdown ofsf1 in both yeast and human extracts only slightly affects the splicing outcome rnai targeting of thisgene has been demonstrated to not affect the splicingpattern of several splicing marker genes tested in comparison to studies in human and yeast few reports have been published related to plant sf1 genessimilar functions of the arabidopsis sf1 gene were proposed in an early study in this plant sf1homologue is reportedly responsible for the splicing of a 0czhang bmc plant biology page of to maintain itsgroup of transcripts the lossoffunction mutant atsf1 of this gene leads to abnormal development earlyflowering and dwarfism and aba or heat stress sensitivity in arabidopsis [ ] subsequently the domainstructure and its functional relationships have been substantially investigated and the rrm domain is considered crucialfunction in plantsmoreover sf1 may have a different mechanism of ²splice site recognition in plant because the plant sf1 homologs contain a different rrm domain compared withfungal and metazoan counterparts [ ] on the otherhand a study found that atsf1 may be likely to play afunctional role in the cytoplasm because it was found toshuttle between the nucleus and cytoplasm however no related investigations have been conducted onthe phylogenetic analysis of plant sf1 genes and theirregulatory mechanisms although it is a highly conserved family and has conserved functions in eukaryotesplant sf1 genes may have overlapping and distinct rolescompared to the mammalian genes hence studying thephylogenetic relationship and regulatory mechanism ofplant sf1 genes may make us understand the evolutionary history characteristics an expression profile of thisgene family and predict specific functions in plants thiscan lay the foundation for further functional studies inviridiplantae to this end we systematically identified sf1 sequences from plant species ranging fromalgae to higher plants meanwhile the gene and proteinstructure potential regulation at promoter regions andexpression pattern of these genes were further investigated in this study we hypothesize that plant sf1 isstructurally different from its counterparts in animalsand yeast but it is conserved among lower and higherplants indicating its specific role in alternative splicingin branch point recognitionthalianasf1methodssequence acquisition and identification of plant sf1genesthe arabidopsisprotein sequenceat5g51300 was used to search similar sequences inall available plant species from the phytozome v121databasehttpsphytozomejgidoegovpzportalhtml by running the blastp program with an evaluecutoff 1e10 the other parameters were the default settings then the retrieved protein sequences wereexamined and filtered using the hmmer score defaultsettings which contained pf16275 splicing factorkhomology domain kh_1 and pf00076 rna recognition motif rrm_1 finally putative sf1 sequencesfrom plant species were identified detailed information including groups plant species common namesand number of sf1 homologs reported for each planthelixhairpinsf1hhdomainpf00013species for subsequent analysis are listed in table s1subcellular location prediction of identified sf1 proteinswas carried out using wolf psort httpswolfpsorthgcjp construction of molecular phylogenetic tree of plant sf1genesprotein sequences of the aforesaid plant sf1 genes wereextracted from phytozome v121 database for phylogenetic relationship analysis the sequences with the longestcoding sequences were chosen for genes with multipledifferent splicing isoforms then multiple sf1 proteinsequences were aligned with the muscle v38 softwarewith default settings the molecular phylogenetictree of plant sf1 genes was then constructed using themaximum likelihood method ml jtt g i modelvia phyml v30 program with the following parametersinitial tree bionj discrete gamma model yes numberof categories gamma shape parameter proportion of invariant subtree patterns aliasing no figtree v143 was used to visualize and edit the phylogenetic treegene structure protein domain and multiple em for motifelicitation meme analysisrequired genomic cdna and peptide sequences and allsf1 gene structures were downloaded from the phytozome v121 database corresponding intron phases weregenerated using the online program gene structure display server gsds20httpgsdscbipkueducn correlation analysis of sf1 exons were performedby using the piece2 webserver httpwwwbioinfogenomenetpiecesearchphp tdsourcetags_pctim_aiomsg sf1 protein sequences were used to search formatching pfam families using the hmmer websitehttpswwwebiacuktoolshmmer then protein domain patterns were drawn by using tbtools software according to the full pfam resultanttableconserved motifs of plant sf1 cdna sequences andprotein sequences were analysed on the meme onlineprogram httpmemesuitetoolsmeme considering a maximum of the most preserved motifs predicted for each sequence and leaving other settings onthe default parametersmotif prediction in promoter regions of plant sf1 genesthe 15kb ²flanking sequences of plant sf1 geneswere extracted from genomic data available in phytozome database prediction of plant putative ciselementswas performed with the online server plantcarehttpbioinformaticspsbugentbewebtoolsplantcarehtml motifs related to tissuespecific expressioninternal hormones and external environmental stress response were selected for further analysis and discussion 0czhang bmc plant biology page of expression analysis base on microarray datasets and geneexpression experimentsexpression data of arabidopsis s tuberosum g max slycopersicum p trichocarpa and b distachyon includingtissue specificity and stress responses were extracted fromthe efp browser series of the bioanalytic resource forplant biology httpbarutorontoca expressionvalues of selected plant sf1 genes were log transformedlg to generate visualize expression difference heatmapsby using bar heatmapper tool program httpbarutorontocantoolscgibinntools_heatmappercgigene expression experimentstotal rna of samples from different plant tissues wereextracted by rneasy mini kit qiagen usa and subsequently reversed transcribed into cdna by fastkinggdna dispelling rt supermix fastking tiangenchina according to the manufacturers instruction rtpcr amplification were programmed asfollowings °c min °c s °c s °c s cycles °c min sybr premix ex taqtm accuratebiotechnology co ltd hunan china was used forquantitative realtime rtpcr analysis which was conducted on the stepone plus realtime pcr system following optimized program °c s °c s °c s cycles the data were normalized to the expression of internal reference genes table s6 and the transcript abundance was determined by the comparativect value method analysis of proteinprotein interaction network andstructural conservationa proteinprotein interaction network was generated bythe string website httpsstringdb withrepresentative protein sequences from arabidopsis thefollowing basic settings were employed meaning of network edges evidence line colour indicates the type ofinteraction evidence and active interaction sourcesexperimentsthere are three domains in the arabidopsis sf1 protein the phosphorylation and u2af65 binding of thenterminal domain of splicing factor during ² splicesite recognition of homo sapiens pdbid 2m0g identity evalue 7e17 was similar to that of the khomology domain the structure for recognition of theintron branch site rna by splicing factor of homo sapiens pdbid 1k1g identity evalue 9e27 canbe used as the template for the splicing factor helixhairpin domain therefore homology modelling wasperformed with modeller based on two crystalstructures the amino acid conservation scores were calculated using the consurf web server based on the mlmethod input attributes were the 3d model andmultiple sequence alignment figure s4 related figureswere created based on pymol with default settings analysis gene structure evolution with orthologue groupof sf1 genesreconstruction of the evolutionary history of the structure of the plant sf1 family of orthologous genes wascarried out by searching at5g513001 in the piece severhttpwwwbioinfogenomenetpieceindexphpthis provided an exonintron display for orthologousgenes from gene structure data sets linked to the phylogenetic treeresultssequence identification and phylogenetic analysis of theplant sf1 gene familyto identify sf1 gene family members in plants we carried out a blastp search using the arabidopsis atsf1at5g51300 amino acid sequence against the phytozome database v121 after filtering the sequence without sf1 signature or truncated sequences a total of sequences from plant species were retrieved whichwere roughly classified as algae bryophyta basicangiosperm monocots and eudicots table s1specifically the only species with four copies of plantsf1s was eutrema salsugineum salt cress table s1 inparticular three copies of sf1 genes were observed infive species including panicum virgatum switchgrasstriticum aestivum common wheat daucus carotacarrot kalanchoe laxiflora milky widows thrill andsalix purpurea purple osier willow additionally plant species contained two copies and species including the model plant arabidopsis possessed only onecopy of plant sf1s respectively the relatively largernumber of sf1 genes and higher number of plant speciesin this work demonstrated the universality and complexity of the sf1 gene family the retrieved sequences of plant species provided us with more complete information to analyse the phylogenetic relationship of the sf1gene family subsequently a rooted phylogenetic treewas constructed based on the abovementioned protein sequences by using the maximum likelihoodmethod the trees bootstrap threshold was represented by a colour gradient fig in general all sf1protein sequences were clustered into four major cladesincluding alga in yellow other land plants in greenmonocots in pink and eudicots in blue and one species amborella trichopoda belonged to basic angiosperm shown in colourless the phylogenetic tree ofsf1s figs and left panel with clear topology andoverall high bootstrap values was similar to evolutionarytrend from lower plants to higher plants reported inother studies for example the genes of algae in the yellow branch were representative members of the lineage 0czhang bmc plant biology page of fig circular phylogenetic tree of the sf1 gene family available in plants the phylogenetic tree of sf1 genes in plants was constructed basedon maximumlikelihood with jtt g model by using phyml v3037 a total of protein sequences from plant species were chosen tocalculate the phylogenetic relationship for tree construction bootstrap values are labelled at each major branch the corresponding informationof each transcript such as species name common name number of identified transcripts and their transcript id nomenclature are shown intable s1 taxonomies based on apgiv system 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig gene structure comparisons and conserved motif identification among plant sf1 genes from left panel to right panel verticalphylogenetic tree genomic anization and identified cdna conserved motifs by meme analysis intron phase and are shown on thegene structure the conserved sequence of identified motifs represented by different coloured boxes are listed below some long genes werereduced to onehalf of their original length to fit this picturethat diverged before the evolution of land plants whichwas the basal part of the phylogeny in the blue branchfive sequences from kalanchoe with higher bs valuesformed a subclade showing their closer evolutionary relationships additionally cagra3782 s00261p fromcapsella grandiflora and carubv10025900m from c rubella formed a subclade with the arabidopsis sequencesbecause they all belong to brassicaceae which is consistent with the apg iv system fig and table s1 usually some homologous sf1 sequences from the samespecies were clustered in the same small branch next toeach otherthese species included cashew soybeanapple woodland strawberry quinoa carrot coloradoblue columbine maize common wheat cereal grassmoss and bog moss fig and table s1 in contrastsome other homologous sf1 members from the samespecies were clustered into the different subclades suchas purple osier willow poplar eastern cottonwood saltcress potato diploid kalanchoe milky widows thrillhalls panicgrass switchgrass green algae and volvoxfig and table s1gene structure and conserved motif analysisit is necessary to compare the exonintron anizationand conserved motifs of the plant sf1 gene family toclarify their evolutionary process and potential functionthe gene structure models of sf1 genes were attachedto the phylogenetic tree fig and the correspondingintron phase of each was also displayed fig tables2 figure middle panel shows that the gene lengthand structure of each member of the sf1 family exhibitssignificant differences for example the gene structureof members of sf1 family genes did not containintron sequences this subset accounts for of thetotal number of members fortyeight sequences of sf1genes had exon1 intron anizations accounting for of all genes in particular some genes from algaehad multiple exons including vocar0008 s02941p volvox carteri which contained the most exons exonsmoreover different gene structures were also observedat the same subbranch for instance two sequencesfrom zea mays maize zm00008a037777_p01 exonsand zm00008a007621_p01 exons were observed tohave distinctive gene structures although the dissimilation of gene structure of each member of sf1s was substantial we found that the length of cdss did notsignificantly change fig thus whether it influencesthe differentiation of their gene function needs to befurther investigated further investigation on conservedmotifs by using multiple em for motif elicitationmeme search tool demonstrated that most sf1 genes sequences exhibited similar sequence signatures andthe same order and all contained the analysed motifsexcept one sequence of micromonas pusilla hada different position fig right panel although no obvious differences in identified conserved motifs werefound among basal angiosperm monocots and eudicotssequences from the same species were found to have different motifs fig for example aqcoe5g4069001pand aqcoe7g0393001p from the eudicot aquilegiacoerulea had motifs and motifs respectively thesame situation was found in d carota dcar_006843dcar_008506 and dcar_004968 had motifs motifs and motifs respectively intriguingly the cdslength of dcar_008506 was the longest notably somesequences from algae and moss had fewer conservedmotifs for examplein bryophyta the sequences ofphyscomitrella patens pp3c7_10890v31p and pp3c11_24710v31p sphagnum fallax sphfalx0015s00771pand sphfalx0010s01971p and marchantia polymorphamapoly0009s01891p had nine motifs in algal plantsthe sequences of and from micromonashad only motifs and motifs respectively moreoveralthough the sequences of volvox carteri vocar0007s03451p and vocar0008 s02941p and chlamydomonasandcre09g386731t11 contained multiple exons they had motifs indicating their sequence variation had little influence on function classes further correlation analysisof the sf1 exon regions were carried out to elucidate thegainloss of introns correlations between transcripts ofplant sf1s are shown in fig providing additional information for phylogenetic analysis for example thereis more similarity between pgsc0003dmt400081859and migutd025312 because of multiple exact matchesbetween the exons of the two transcriptscre12g553750t11reinhardtiianalysis of protein domain and conserved motifs inpeptidesthe protein domains were analysed by using the aboveselected peptide sequences from plant species thepeptides annotations were splicing factorrelated andconserved protein motifs were predicted according tothe retrieved peptide sequences by meme analysisfig consequently all sf1s were found having sf1_hh nterminal domain on the nterminal ofthe 0czhang bmc plant biology page of fig analysis of gene structure evolution with orthologue group of sf1 genes exonintron structure and intron phase right panel are linked tothe plant species tree left panel genes with red colour represent the members of the plant sf1 genes different coloured lines mean differentexon comparison results between species 0czhang bmc plant biology page of fig comparisons of protein domains and conserved motif identification among plant sf1 genes protein domain middle panel and identifiedprotein conserved motifs right panel identified by meme analysis are shown against the vertical phylogenetic tree left panel the conservedsequence of identified motifs represented by different coloured boxes are listed below 0czhang bmc plant biology page of peptides followed by a kh domain and a cterminal domain namely an rna recognition motif rrm fig middle panel interestingly in algae peptides from mpusilla v carteri vocar0008 s02941p andc reinhardtii cre09g386731t11 had two rrm domains the amino acid lengths of sf1 proteins rangedfrom aa to aa and most of them possessed to amino acids table s3 consistently most ofthem are approximately to amino acids inlength subcellular location prediction showed that themajority of sf1 proteins were had nuclear localization table s3 moreover proteins of 147m014250 ricinus communis and migutf011911pmimulus guttatus were located in the vacuoles proteins of traes_2dl_6f03f05fa4 t aestivum and m pusilla were predicted to be cytoplasmic proteins of gsmua_achr5p25100_001 musa acuminataand cre09g386731t11 c reinhardtii were located inthe chloroplast and endoplasmic reticulum respectivelymeme analysis for sf1 peptide sequences was used topredict a total of conserved motifs which are presented as coloured boxes and cover most of the proteinfig right panel further analysis showed that peptides had all motifs accounting for approximately of all sf1 protein sequences analysed in the studyinterestingly all sequences from moss have conservedmotifs in the analysis suggesting the conservation ofsf1 proteins in bryophyta furthermore almost all eudicots had conserved motifsexcept anacardium occidentalegrandifloracagra3782 s00261p which lacked motif and malusdomesticavescamrna211921v10hybrid and brassica rapa brarac014811p which lacked motif while most monocots had eight conserved motifs in contrast algal plantsonly possess approximately half of the predicted motifs due to their peptides with integrant protein domainsimplying the least degree of conservation and divergenceof plant sf1 proteins in algae t motifs that all algaeshared were motif motif motif and motif anaoc0018 s04251pmdp0000558834fragariaand canalysis of promoter and tissuespecific expression of sf1genesto further analyse the regulation of plant sf1 genes atthe transcriptional level the 15kb upstream sequencesof plant sf1 genes were obtained from the phytozomedatabase then the ciselements of each promoter wereidentified by using the plantcare program table s4 consequently a total of motifs were predictedgenerally eight ciselements related to tissuespecificexpression among them were selected fig and tables4 including hdzip1 for differentiation of the palisademesophyll cells the ryelement which regulates seedspecific expression the aaca_motif and gcn4_motif waspresentfurther hdzipinvolved in endosperm expression and the catboxccgtccbox doct and oct for meristem expression further analysis showed that there were only promoters of sf1 genes which had tissuespecific regulatory ciselements particularly the catbox and ccgtccbox turned up at the highest frequency and greatestabundance in the promoters of sf1 genes both of themregulate meristemspecific expression and play key rolesduring development and growth of plants consistentlypurple false brome brachypodium distachyon of monocots not only had a catbox and ccgtccbox butwas also highly expressed in young leaves internode adventitious roots and roots fig and figure s2however no motifs were found to link the highexpression of two sf1s of glycine max soybean insam and roottip figure s1 additionally the aaca_motif was only detected in solanum tuberosumpgsc0003dmp400032853 of potato suggesting itsspecific role in regulating endospermspecific negativeexpressioninpodel03g1132001p of populus deltoideseasterncottonwood and spipo17g0046100 of spirodela polyrhiza greater duckweed the ryelement was detectedin the promoter of the dicot model plant arabidopsisand low expression was also reported in dry seed inarabidopsis fig suggesting that the ryelement isinvolved in seedspecific negative expression of arabidopsis moreover expression levels in the same tissuetype showed significant differences during differentgrowth stages for example the expression level in stamen of flower stage of arabidopsis was obviouslyhigher than that of the other flower development stageshowever the expression levels of different growth stagesof solanum lycopersicum were not only similar butlower and no motifs were found in the promoter in tomato figs and s1 furthermore different expressionpatterns were detected in several sf1 genes with multiple copies figs s1 and s6 for instance similar tissue expression profiles were detected in two sf1trichocarpahomologuespotri001g1264001 and potri003g1072001 and themonocotandzm00008a037777_p01 figure s1 and s5 in contrasttwo sf1 genes of s tuberosum showed differential expression patterns similar to in g max figs and s1zm00008a007621_p01dicot populuszea maysfrom theanalysis of promoter and internal and external hormonesexpression of sf1 genesin longterm evolution and development plants havegradually formed mechanisms of adaptation and resistance to adversity to maintain their life and sustaingrowth to understand the regulatory mechanisms of internal and external stimuli on plant sf1s cisacting elements involved in hormone and stress were studied with 0czhang bmc plant biology page of fig see legend on next page 0czhang bmc plant biology page of see figure on previous pagefig analysis of motifs related to tissue specificity in the plant sf1 promoter regions eight cisacting motifs are represented in different colortriangles positions of these identified motifs are labeelled along the kb ²flanking regions of each sf1 gene the line solid and dottedrepresents regions with basic pairs and regions of no sequences or annexed base n respectively symbols on above the line represent the motifsat the plus strand whereas symbols on below the line represent the motifs at the minus strand function of motifs aacamotif involved inendospermspecific negative expression catbox cisacting regulatory element related to meristem expression ccgtccbox cisactingregulatory element related to meristem specific activation doct cisacting regulatory element related to meristem specific activationgcn4_motif cisregulatory element involved in endosperm expression hdzip1 element involved in differentiation of the palisade mesophyllcells ryelement cisacting regulatory element involved in seedspecific regulation the black vertical lines represent break at that particularbranch oct cisacting regulatory element related to meristem specific activationlowtemperatureltr droughtthe plantcare database fig table s4 finally hormone and stressrelated motifs were selected from promoter sequences of plant sf1s there are hormonerelated motifs including abscisic acid abreauxin auxre auxrecore tgabox tgaelementethylene ere gibberellin garemotif pbox tatcbox meja cgtcamotif tgacgmotif and salicylic acid tcaelement and five stressrelated motifsincludingmbswound wunmotif and anoxic are gcmotif motifs almost each sf1 sequence had a great diversity ofciselements in its promoter regions except some sequences such as araha13031 s00021 and traes_2al_3d67296921 which did not contain a single motif dueto the sequences contain n or no promoter suggestingthat multiple hormonesmediated signalling pathwaysare closely related to sf1 plants resistance analysisshowed that more than half of sf1 promoters containedabre cgtcamotif tgacgmotif and are respectively moreover external hormone signals also affect theabundance of sf1 transcripts figure s3 for examplein arabidopsis at5g513001 mj methyl jasmonateinhibited its expression fig and treatment withother hormones like acc a precursor of ethylene iaaauxin | 0 |
the diagnostic platform utilizing the detection of biomarkers in various body ï¬uids called liquidbiopsy 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 eï¬cient and reliable markerswithin the body ï¬uids 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 patternspeciï¬c 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 speciï¬c and reï¬ect 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 ï¬eldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reï¬ect the pattern speciï¬c 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 theidentiï¬cation of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artiï¬cialintelligencecan 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 speciï¬c 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 modiï¬cations of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and speciï¬city in liquid biopsybasedmethods for disease detection and classiï¬cationthe patientthe5methylcytosine5mc modiï¬cationat 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 tissuespeciï¬c 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 deï¬nitive 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 epigenomeproï¬le 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 posttranslationallymodiï¬ed at amino acid residues located on their n andcterminal tails these modiï¬cations act as epigenetic marksthat can speciï¬cally distinguish diseaserelated ccfdnas in bloodsamples various types of histone modiï¬cations 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 methylspeciï¬c pcr methylationsensitivehighresolution melting and pyrosequencing garcagimnez dna methylation speciï¬c 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 modiï¬ers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identiï¬ed 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 modiï¬cations on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodiï¬cations 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 bisulï¬te sequencing rrbs meissner is an eï¬cient method for absolute quantiï¬cation 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 bisulï¬te 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 identiï¬ed 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 proï¬lingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodiï¬cations 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 quantiï¬cation of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body ï¬uids are of clinical signiï¬cance 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 bloodbrain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 speciï¬cfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patients 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 ï¬eld 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 liquid 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 speciï¬c 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 modiï¬cations 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 identiï¬ed as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic ï¬rmkneip 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 cellspeciï¬c mirnas zhang mir was found to be a signiï¬cant 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 signiï¬cantly 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 signiï¬cantly 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 isatherapyimmunotherapyidentiï¬cation ofrapidly developingsigniï¬cant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticeï¬cacy 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 mycnampliï¬ed 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 signiï¬cantlyupregulated 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 molecularproï¬ling 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 beneï¬ts recentlynovel immunological approaches produced insightful responsessnyder in addition the identiï¬cation 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 beneï¬t 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 eï¬ciently detected atthe ï¬fth 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 downs syndrome edwardssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands eï¬cient 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 modiï¬cations are speciï¬c 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 ï¬rst reported use of fetalderivedepigenetic marker in maternal body ï¬uids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspeciï¬c methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspeciï¬c 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 ï¬rst such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been conï¬rmedby 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 bisulï¬te 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 ï¬nds 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 microï¬uidics 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 identiï¬cation of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identiï¬ed 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 | 0 |
"preadipocytes diï¬erentiate into adipocytes During this process the preadipocytes ceaseto proliferate begin to accumulate lipid droplets and develop morphologic and biochemical characteristics of mature adipocytesMesenchymal stem cells MSCs are a type of adult stem cells known for their high plasticity and capacity to generate mesodermaland nonmesodermal tissues Many mature cell types can be generated from MSCs including adipocyte osteocyte and chondrocyteThe diï¬erentiation of stem cells into multiple mature phenotypes is at the basis for tissue regeneration and repair Cancer stem cellsCSCs play a very important role in tumor development and have the potential to diï¬erentiate into multiple cell lineagesAccumulating evidence has shown that cancer cells can be induced to diï¬erentiate into various benign cells such as adipocytesï¬brocytes osteoblast by a variety of small molecular compounds which may provide new strategies for cancer treatmentRecent studies have reported that tumor cells undergoing epithelialtomesenchymal transition can be induced to diï¬erentiateinto adipocytes In this review molecular mechanisms signal pathways and the roles of various biological processes in adiposediï¬erentiation are summarized Understanding the molecular mechanism of adipogenesis and adipose diï¬erentiation of cancercells may contribute to cancer treatments that involve inducing diï¬erentiation into benign cells IntroductionAdipogenesis is the process through which mesenchymalstem cells MSCs commit to the adipose lineage and diï¬erentiate into adipocytes During this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs Currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro One isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells There are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells Anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3T3L1 and 3T3F422A cells [] Cancer cells with tumorinitiation ability designated as cancer stem cells CSCshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential [] In addition to CSCs [] cancer cells undergoing epithelialmesenchymaltransformation EMT havebeen reported to be induced to diï¬erentiate into adipocytes[] Lung cancer NCIH446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro[] The adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer[] Cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes BRCA mutations can be inducedto diï¬erentiate by poly ADPribose polymerase PARP 0cStem Cells Internationalinhibitors [] The nuclear receptor peroxisome proliferatoractivated receptor Î PPARÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells[] Thyroid cancer cells expressing the PPARÎ fusion proteinPPFP can be induced to diï¬erentiate into adipocytes bypioglitazone [] Adipogenesis can be induced in welldiï¬erentiated liposarcoma WDLPS and dediï¬erentiatedliposarcoma DDLPS cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine IBMX []In this review we highlight some of the crucial transcription factors that induce adipogenesis both in MSCs and inincluding the wellstudied PPARÎ and CCAATCSCsenhancerbinding proteins CEBPs [] as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation We focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes Adipocyte DifferentiationCell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation Theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype [] Adipogenesishas diï¬erent stages Each stage has a speciï¬c gene expression pattern [] In general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases The ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes The preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types In the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins []The diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation [] After the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge After reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes Adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors Some preadipocyte cellsmouse cell lines 3T3L1 3T3F442A undergo one or tworounds of cell division prior to diï¬erentiation [] whereasother cell lines mouse C3H10T12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion []Whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial However it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] When cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets [] Regulatory Pathways inPreadipocytes CommitmentAdipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated The most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported Adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process Figure Wnt Signal Pathway in Adipogenesis Wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo [] The Wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development Wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways[] Wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of PPARÎ and CEBPα [] Constitutive Wnt10b expression inhibits adipogenesis Wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes In vivo transgenic expression of Wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development []Wnt10a and Wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] Wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation [] indicating that preadipocytes integrate inputs fromseveral competing Wnt signals The Hedgehog HH Signaling Pathway MechanismThree vertebrate HH ligands including sonic hedgehogSHHIndian hedgehog IHH and desert hedgehogDHH have been identiï¬ed and initiated a signaling cascademediated by patched Ptch1 and Ptch2 receptors [ ]HH signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as C3H10T12 KS483 calvaria MSCslines and mouse adiposederived stromal cells [] Thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after HHsignaling was inhibited [] Although it is generally agreedthat HH expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking HH signaling andadipogenesis remain poorly deï¬ned [] ERKMAPKPPAR Signal Pathway Extracellularregulated protein kinase ERK is required in the proliferativephase of diï¬erentiation ERK activity blockade in 3T3L1 0cStem Cells InternationalDEX insulin DEMXWNT 10band othersSHHPBC SMOTGFð½P SMAD3 SMAD3Testosteroneð½catentinARIRSPI3KAKTCREBPKAPCREBFOXO1A2TCFLEF GATA23CEBPð½MAPKG3K3ð½P2CEBPð½CEBPαPPARá¿«BMPsSMAD1SREBPAdipocytegenesFigure Regulation pathways in preadipocytes commitment BMP and Wnt families are mediators of MSCs commitment to producepreadipocytes Exposure of growtharrested preadipocytes to diï¬erentiation inducers IGF1 glucocorticoid and cAMP triggers DNAreplication leading to adipocyte gene expression due to a transcription factor cascade The dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis Inthe terminal diï¬erentiation phase ERK1 activity leads toPPARÎ phosphorylation which inhibits adipocyte diï¬erentiation This implies that ERK1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedThis reduction is mediated in part by mitogenactivatedprotein kinase MAPK phosphatase1 MKP1 [ ]These extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation Adipocyte DifferentiationRegulatory Proteins PPARÎ and Adipocyte Diï¬erentiation PPARÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis [] Forced expression ofPPARÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts [] Indeedthe proadipogenic CEBPs andKrüppellike factors KLFs have all been shown to induceat least one of the two PPARÎ promoters In contrast antiadipogenic transcription factor GATA functioned in part byrepressing PPARÎ expression [] PPARÎ itself has twoisomers The relative roles of PPARÎ1 and PPARÎ2 in adipogenesis remain an question PPARÎ2 is mainlyexpressed in adipose tissue while PPARÎ1 is expressed inmany other tissues Although both can promote adipocytediï¬erentiation PPARÎ2 could do so eï¬ectively at very lowligand concentration compared with PPARÎ1 [] The twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesisPPARÎ1 can also be expressed in cell types other than adipocytes Ren [] used engineered zincï¬nger proteins tothe expression ofthe endogenous PPARÎ1 andinhibitPPARÎ2 promoters in 3T3L1 cells Ectopic expression ofPPARÎ2 promotes adipogenesis whereas that of PPARÎ1does not Zhang reported that PPARÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity []There are transcriptional cascades between adipocytesgenes including PPARÎ and CEBPα which are the coreadipocyte diï¬erentiation regulators In the early stage of adipocyte diï¬erentiation the expression of CEBP and CEBPδincrease which upregulates CEBPα expressionfurtheractivate PPARÎ PPARÎ activating CEBPα in turn resultsin a positive feedback PPARÎ binding with retinoic acid Xreceptor RXR forms diï¬erent heterodimers The variousdimmers can combine with the PPARÎ response elementPPRE and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes []CEBPs participate in adipogenesis and several CEBPfamily members are expressed in adipocytesincludingCEBPα CEBP CEBPÎ CEBPδ and CEBPhomologous protein CHOP The temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of CEBP and CEBPδ leads toCEBPα expression This notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationCEBP is crucial for adipogenesis in immortalized preadipocyte lines CEBP and CEBPδ promote adipogenesis atleast in part by inducing CEBPα and PPARÎ CEBPαinduces many adipocyte genes directly and plays an important role in adipose tissue development Once CEBPα isexpressed its expression is maintained through autoactivation [] Despite the importance of CEBPs in adipogenesis 0cStem Cells Internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of PPARÎ CEBP cannot induce CEBPαexpression in the absence of PPARÎ which is required torelease histone deacetylase1 HDAC1 from the CEBPαpromoter [] Furthermore ectopic CEBPα expressioncannot induce adipogenesis in PPARÎï¬broblasts []However CEBPα also plays an important role in diï¬erentiated adipocytes Overexpression of exogenous PPARÎ inCEBPαdeï¬cient cells showed that although CEBPα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] Figure Human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation However PPARÎ exogenousligands need to be added to promote adipocyte diï¬erentiation Therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of PPARÎ [] BMP and Transforming Growth Factor TGF inAdipocyte Diï¬erentiation A variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein BMP []transforminggrowth factor TGF [] insulininsulinlike growthfactor IGF1 [] tumor necrosis factor α and interleukin [] matrix metalloproteinase [] ï¬broblast growthfactor FGF and FGF2 [] BMP and TGF have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells []The TGF superfamily members BMPs and myostatinregulate the diï¬erentiation of many cell types includingadipocytes [] TGF inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of TGF impairsadipocyte development [] Inhibition of adipogenesis couldbe obtained through blocking of endogenous TGF with adominantnegative TGF receptor or drosophila mothersagainst decapentaplegic protein SMAD inhibitionSMAD3 binds to CEBPs and inhibits their transcriptionalactivity including their ability to transactivate the PPARÎ2promoter [ ] Exposure of multipotent mesenchymalcells to BMP4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion [] Theeï¬ects of BMP2 are more complex and depend on the presence of other signaling molecules BMP2 alone has little eï¬ecton adipogenesis and it interacts with other factors such asTGF and insulin to stimulate adipogenesis of embryonicstem cells [] BMP2 stimulates adipogenesis of multipotentC3H10T12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations [] KLFs in Adipocyte Diï¬erentiation During adipocyte differentiation some KLF family members are overexpressedsuch as KLF4 KLF5 KLF9 and KLF15 while KLF16 expression is reduced [ ] KLF15 is the ï¬rst KLF family members which were identiï¬ed to be involved in adipocytediï¬erentiation Its expression increased significantly on thesixth day of 3T3L1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in MSCs andmouse embryonic ï¬broblasts Inhibition of KLF15 by siRNAor mutation led to a decrease in PPARÎ CEBPα fatty acidbinding protein FABP4 and glucose transporter GLUT4 However overexpression of KLF15 in NIH3T3cells was found to be associated with lipid accumulation aswell as increases in PPARÎ and FABP4 [] Mice with complete absence of KLF5 showed embryonal lethality and micewith singlechromosome KLF5 knockout showed a significant reduction in white fat in adulthood suggesting thatKLF5 plays an important role in adipocyte diï¬erentiationKLF5 can be activated by CEBP or CEBPδ which isinvolved in early adipocyte diï¬erentiation KLF5 can beactivated by CEBP or CEBPδ which is involved in earlyadipocyte diï¬erentiation Direct binding of KLF5 to thePPARÎ2 promoter in combination with CEBPs inducesPPARÎ2 expression [] Transfection of KLF5 dominantnegative mutants in 3T3L1 cells reduced lipid droplet accumulation and inhibited PPARÎ and CEBPα expressionwhereas overexpression of wild KLF5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation Similar to KLF5 KLF9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as PPARÎ CEBPα and FABP4 hence inhibitingadipocyte diï¬erentiation However KLF9 overexpressiondid not upregulate the expression of PPARÎ and CEBPα[] In addition KLF4 can transactivate CEBP by bindingto the region of KB upstream of the CEBP promoter and promote lipid diï¬erentiation [] KLF6 can forma complex with histone deacetylase3 HDAC3 inhibitingpreadipocyte factor1 Pref1 expression and promotinglipid diï¬erentiation [] KLF2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation Overexpressed KLF2 can bind to theCACCC region of PPARÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of PPARÎCEBPα and sterolregulated elementbinding proteinsSREBP by inhibiting the promoter activity [] RNAsequence analysisshowed that KLFl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3T3L1 cells Adipocyte diï¬erentiation was promoted byKLF16 knockdown but was inhibited by KLF16 overexpression via inhibition of PPARÎ promoter activity [] In addition KLF3 and KLF7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] Signal Transducers and Activators of TranscriptionSTATs and Adipocyte Diï¬erentiation The activated STATprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription In the adipocyte diï¬erentiation of mouse 3T3L1 cells the expression of STAT1 andSTAT5 was significantly increased while that of STAT3and STAT6 was not significantly changed [] In the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells STAT1 expression was significantly decreased[] while the expression of STAT3 and STAT5 wasincreased and STAT6 expression was unchanged [] Therole of STAT1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process Early adipocytediï¬erentiation of 3T3L1 cells was inhibited by STAT1 0cStem Cells InternationalKLF5SREBP1cKLF15KLF2CHOPCEBPá¿«KROX20LigandCEBPð½CEBPð¿GATA23PPARá¿«CEBPð¼ProadipogenicAntiadipogenicGenes of terminaladipocytedifferentiationFigure A cascade of transcription factors that regulate adipogenesis PPARÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis PPARÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors CEBPα is regulated through a series of inhibitory proteinprotein interactions Some transcription factorfamilies include several members that participate in adipogenesis such as the KLFs Black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î Loss of STAT1 in 3T3L1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α [] Other studies have found thatSTAT1 is required for adipose diï¬erentiation and STAT1overexpression in C3H10T12 cells can prevent the inhibition of lipid diï¬erentiation caused by Bcell lymphoma6knockdown [] There was no abnormal adipose tissuein STAT1 knockout mice [] STAT3 not only aï¬ectsthe proliferation of 3T3L1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein [] The FABP4 promoter was used to speciï¬callyknock out STAT3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice [] STAT5A and STAT5B have diï¬erenteï¬ects on adipocyte diï¬erentiation Abnormal adipose tissuewas found in the mice with STAT5A or STAT5B knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown [] Histone Modiï¬cation in Adipocyte Diï¬erentiation Histone deacetylase sirtuin SIRT plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation [] During the adipocyte diï¬erentiation of C3H1012 cells SIRT1 expressiondecreased [] Overexpression of SIRT1 activated theWnt signal which caused the deacetylation of cateninThe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation SIRT1 knockdown resulted inincreased acetylation of the histones H3K9 and H4K16 inthe secreted frizzledrelated protein sFRP and sFRP2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation [] Forkhead box proteinO FOXO is a member of the transcription factor FOXOfamily It can recruit cyclic AMP response elementbindingprotein CBPhistone acetyltransferase p300 to initiate anacetylation The acetylated FOXO1 can be phosphorylatedby phosphorylated protein kinase B PKBAKT The phosphorylation of FOXO1 by AKT inhibits the transcriptionalactivation of FOXO1 The acetylation of FOXO1 lost the ability of DNAbinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm [] SIRT1 and SIRT2 can deacetylateand active FOXO1 Activated FOXO1 nonphosphorylatednuclear FOXO1 in the nucleus binds to the promoters of target genes encoding p21 p27 and PPARÎ and initiates subsequent transcriptions [] SIRT2 inhibits the acetylation andphosphorylation of FOXO1 thereby induces the accumulation of activated FOXO1 in the nucleus Activated FOXO1could inhibit adipogenesis via PPARÎ [] Lysinespeciï¬c histone demethylase LSD1 expression increasedduring the adipocyte diï¬erentiation of 3T3L1 cells LSD1could reduce the dimethylation levels of histone H3K9 andH3K4 in the CEBPα promoter region thereby promotingadipocyte diï¬erentiation [] SET domaincontaining SETD8 catalyzed the monomethylation of H4K20 andpromoted PPARÎ expression The activation of PPARÎ transcriptional activity leads to the induction of monomethylatedH4K20 and modiï¬cation of PPARÎ and its targets therebypromoting adipogenesis [] Enhancer of zeste homolog EZH2 is a methyltransferase and can bind methyl groupsto histone H3K27 which is also necessary for lipid diï¬erentiation The absence of EZH2 in brown fat precursors results inreduced levels of the Wnt promoter histone H3K27me3which is also saved by the ectopic EZH2 expression or theuse of a Wntcatenin signal inhibitor [] In addition histone demethylases such as lysinespeciï¬c histone demethylase LSDKDM KDM6 and histone lysine demethylasePHF2 are also involved in adipose diï¬erentiation andKDM2B inhibits transcription factor activator protein 2αpromoter via H3K4me3 and H3K36me2 [] Role of microRNA and Long NoncodingRNA in AdipogenesismicroRNA miR can bind and cut target genes or inhibittarget gene translation Endogenous siRNA can be producedby the action of Dicer enzyme and bind to a speciï¬c proteinto change its cellular location [] Many kinds of miRsare involved in regulating adipocyte diï¬erentiation The 0cStem Cells Internationalexpression of miR143 increased during the diï¬erentiationof adipose progenitor cells Overexpression of miR143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation Inhibition of miR143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] Additionally miR8 promotes adipocyte diï¬erentiation by inhibiting Wnt signaling [] Moreover miR miR103 miR21 miR519d miR210 miR30miR204211 and miR375 also play a certain role in promoting adipocyte diï¬erentiation while miR130 miR448and let7y inhibit lipid diï¬erentiation [ ] In additionto miRs long noncoding RNA LncRNA is a type of noncoding RNA and is important during epigenetic regulationand can form a doublestranded RNA complex with mRNAcauses protein transcription Lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of PPARÎ2[] As a novel LncRNA HOXAAS3 expression increasedduring the adipose diï¬erentiation of MSCs and HOXAAS3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation [] Zhu et al[] reported that HOXAAS3 interacted with EZH2 toregulate lineage commitment of MSCs HOXA AS3 canregulate the trimethylation level of H3K27 in the Runx2promoter region by binding to EZH2 Therefore HOXAAS3 is considered to be an epigenetic switch regulating MSCslineage speciï¬city [] Adipocyte diï¬erentiationassociatedLncRNA can act as a competitive endogenous RNA of miR in the process of lipid diï¬erentiation thereby promotingthe expression of SIRT1 the target gene of miR204 and thusinhibiting lipid diï¬erentiation [] The LncRNA NEAT1can also regulate adipocyte diï¬erentiation under the uence of miRNA140 [] Other LncRNA including LncRNABlnc1 and Plnc are also involved in regulating adipocytediï¬erentiation [ ] Other Biochemical Response Involved inAdipocyte Differentiation Unfolded Protein Responses in Adipocyte Diï¬erentiationIn the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded RNAdependent proteinkinaselike ER kinase and activating transcription factorATF 6α [] Knockdown of ATF6α aï¬ects the expressionof adipocytes genes and inhibits C3H10T12 adipocyte differentiation [] The inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3T3L1 cells is also due to inducedCHOP and decorin expressions and this inhibitory eï¬ectis ameliorated by CHOP knockout [] In the adipocytediï¬erentiation process of 3T3L1 cells increases in PPARÎand CEBPα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated Eukaryotic translation initiaEIF 2α phosphorylated endoribonucleasetion factorIRE1α ATF4 CHOP and other unfolded protein responsesEndoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationAdditionally EIF2α mutation results in continuous activation or overexpression of CHOP which also inhibits adipocyte diï¬erentiation [] After the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized Exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationFigure Role of Oxidative Stress in Adipogenesis During thedirectional diï¬erentiation of MSCs mitochondrial complexI and III and NADPH oxidase NOX4 are the main sourcesof oxygen species ROS production Currently it is believedthat ROS aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the Wnt HH and FOXO signaling cascade duringMSCs diï¬erentiation [] The diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low ATPcellcontents and a high rate of oxygen consumption The lackof these characteristics indicates stem cell diï¬erentiation[] Adipocyte diï¬erentiation is a highly dependent ROSactivation factor related to mitosis and cell maturation[] Schroder found that exogenous H2O2 could stimulate adipocyte diï¬erentiation of mouse 3T3L1 cells andhuman adipocyte progenitor cells in the absence of insulinH2O2 regulates adipocyte diï¬erentiation of 3T3L1 cells ina dosedependent manner High doses of H2O2 and μM promote adipocyte diï¬erentiation [ ] Tormos found that ROS synthesis increased in humanMSCs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation Byknocking down Rieske ironsulfur protein and ubiquinonebinding protein ROS produced by mitochondrial complexIII was found to be necessary in initiating adipose diï¬erentiation [] However other studies have shown that theexpression levels of adiponectin and PPARÎ were decreasedby using H2O2 mM in 3T3L1 cells [] Free radical nitric oxide NO also promotes lipid diï¬erentiationbecause treatment with NO inducer hydroxylamine or NOsynthase NOS substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells NOSinduced adipose diï¬erentiation mainly via eNOS rather thaniNOS [] ROS can induce adipose diï¬erentiation primarily by inhibiting Wnt FOXO and HH signaling pathwaysthat inhibit lipid diï¬erentiation Autophagy in Adipocyte Diï¬erentiation The increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation[] Baerga conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene Atg agene encoding an essential protein required for autophagy[] Knockdown of Atg5 in 3T3L1 cells promotesproteasomedependent degradation of PPARÎ2therebyinhibiting adipocyte diï¬erentiation [] Zhang reportedthat autophagyrelated gene 7Atg7 is also crucial for adipose development Atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cStem Cells InternationalCEBPð½ geneEBF1 geneKLF4EGR2CEBPð½CytosolCEBPð¿ geneCEBPð¿KLF5genePPARá¿« geneKLF5NR2F2NFKB11433RELASREBF1A2RXRAPPARá¿«PPARá¿«RXRA heterodimerPPARá¿«RXRAcorepressor complexFABP4Ligands of PPARá¿«FAM120BTHRAP3EP300NCOA2NCOA3HELZ2NCOA1CREBBPEBF1ADIPOQ geneAIDRFCEBPð¼ geneZNF638ZNF467CEBPð¼NCOR1HDAC3NCOR2 SLC2A4 geneGLUT4 geneLEP geneFABP4 geneCDK4CCND3PLIN1 genePCK1 geneFABP4CD36 genePPARARXRAcoactivator complexPPARá¿«fatty acidRXRAmediatorcoactivator complexANGPTLgenePPARGC1AMediator complex consensusLPL geneNucleoplasmProteins bind to gene promotersTranscription of genes into proteinsActing on proteins compoundingTGFð½1WNT1WNT10BTNF77233ADIPOQGLUT4SLC2A4 tetramerLEPFABP4lipid dropletPLIN1PCK1PaPa Pa4xPalmCCD36PaANGPTL4LPLFigure Regulation of adipocyte diï¬erentiation A regulatory loop exists between PPARÎ and CEBP activation Transcription factor CoeEBF activates CEBPα CEBPα activates EBF1 and EBF1 activates PPARÎ CEBP and CEBPδ act directly on the PPARÎ gene bybinding its promoter and activating transcription CEBPα CEBP and CEBPδ can activate the EBF1 gene and KLF5 The EBF1 and KLF5proteins in turn bind the promoter of PPARÎ which becomes activated Other hormones such as insulin can aï¬ect the expression ofPPARÎ and other transcription factors such as SREBP1c PPARÎ can form a heterodimer with the RXRα In the absence of activatingligands the PPARÎRXRα complex recruits transcription repressors such as nuclear receptor corepressor NCoR NCoR1 andHDAC3 Upon binding with activating ligands PPARÎ causes a rearrangement of adjacent factors Corepressors such as NCoR2 are lostand coactivators such as Transcription intermediary factor TIF2 CBP and p300 are recruited which can result in the expression of CyclicAMPresponsive elementbinding protein CREB followed by PPA | 2 |
" african americans aa are at high risk for colorectal cancer crc studies report a increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc 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 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenintcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patientsdata included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln advanced adenoman crcn p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a à advanced adenoma b à and cancer c d à and à respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof n intensity which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln cnc intensity n intensity advanced adenoma n crcn c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself []our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother christodoulides c adipogenesis and wnt signalling trendsendocrinol metab world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 statuscancer res ashktorab h kupfer ss brim h carethers jm racial disparity ingastrointestinal cancer risk gastroenterology kaler p augenlicht l klampfer l activating mutations in betacatenin incolon cancer cells alter their interaction with macrophages the role of snailplos one 201279e45462enzo mv the wntbetacatenin pathway in human fibroticlikediseases and its eligibility as a therapeutic target mol cell ther stamos jl weis wi the betacatenin destruction complex cold springharb perspect biol 201351a007898 chung gg tissue microarray analysis of betacatenin in colorectalcancer shows nuclear phosphobetacatenin is associated with a betterprognosis clin cancer res miyoshi y activation of the betacatenin gene in primaryhepatocellular carcinomas by somatic alterations involving exon cancerres fukuchi t betacatenin mutation in carcinoma of the uterineendometrium cancer res brabletz t variable betacatenin expression in colorectal cancersindicates tumor progression driven by the tumor environment proc natlacad sci u s a 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut berger na obesity and cancer pathogenesis ann n y acad sci guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1t23 ross se inhibition of adipogenesis by wnt signaling science kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
" 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 didnt 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 kaplanmeier 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 didnt 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_ampxue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265pmyc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didnt 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 heà and pdl1à 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_valueÏ2test 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 didnthave 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 wasnt 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 wasnt 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 aswerdlow 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 oncollesokhin 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 publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
"The second patient with EGFR mutation achieved the longest PFS and OS (727 and 1249 days respectively). .0087629.g007 Kaplan-Meier estimates of PFS and OS. No statistically significant difference (P?=?0.007) in PFS was observed between metabolic non-progressive (mNP) patients (median PFS 292 days ; range 190727) and metabolic (mP) progressive patients (median PFS 64 days ; range: 37216). Improved PFS in non-progressive patients was associated with prolonged OS (mNP; n?=?4; median OS: 1031 days ; 296 to 1249 days versus mP; n?=?8 ; 337 5 days ; 71 to 734 days) (HR 0.34; 95% CI 0.06 to 0.84; P?=?0.03). Discussion Despite the widespread use of [18F]FDG-PET/CT in NSCLC staging a large-scale study recently failed to confirm an overall survival gain in NSCLC patients.[17] This result highlights the value of [18F]FDG-PET/CT in unmet clinical needs such as prediction of residual NSCLC after surgery[18] neoadjuvant therapy[19] or antineoplastic therapy.[20] Prediction of response to antineoplastic therapies would appear to be particularly adapted to targeted therapies that do not induce rapid tumor shrinkage. NSCLC preclinical models have validated this hypothesis with both gefitinib[21] and erlotinib.[22] This original method could compensate for the weakness of RECIST criteria and has led to the proposal of evaluation of new criteria by addition metabolic evaluation by FDG-PET to CT scan.[23] The value of PET in evaluation of response to new targeted therapies emerged in the early 2000 s with the first reports on the efficacy of imatinib mesylate in Gastro Intestinal Stromal Tumor (GIST). Subsequently many studies have confirmed that PET is able to identify very early (i.e. only 24 hours after initiation of treatment) a decrease in glucose metabolism which is correlated with overall survival and progression-free survival of patients with GIST.[24] [25] In the present exploratory study a decrease in SUVmax of at least 21.6% soon after starting therapy (9±3 days) was able to discriminate progressive from non-progressive patients and was associated with improved PFS and OS. This result confirms the results of Mileshkin et al. who showed in a series of 51 patients receiving second- or third-line treatment with erlotinib that an early (14 days) [18F]FDG-PET partial metabolic response was associated with improved PFS and OS even in the absence of subsequent RECIST response.[26] Evaluation of response by [18F]FDG-PET can be performed semi-quantitatively for instance by establishing a SUV cut-off to discriminate metabolic progressive patients from non-metabolic progressive patients. This patient classification (mP/mNP) seems to be more appropriate to assess response to cytostatic therapy that is designed to stabilize disease rather than achieve complete response. The main difficulty of this approach is the overlap of SUV changes between mP and mNP patients. Furthermore different cut-off variations can be expected depending on the types of SUV measured the types of drugs used and the types of tumors which increase the difficulty of establishing a reliable SUV cut-off. However despite the absence of consensus on the most appropriate cut-off value it is generally admitted that the rationale for metabolic response or non-progression of tumor is decreased [18F]FDG tumor uptake or at least stability of tumor uptake over time respectively. Another limitation of semi-quantitative analysis of FDG-PET is that it does not take into account the development of new lesions. However PET detection of new lesions early in the course of therapy has been reported to be a strong independent predictive factor of OS in NSCLC patients treated by EGFR inhibitor.[27] Our findings are consistent with this observation as new lesions occurred in 2/8 patients correctly classified as progressive on PET2 and in 4/5 patients correctly classified as progressive on PET3. One patient (patient #7) was reclassified as mP on PET3 due to the appearance of a new lesion despite a decrease of SUVmax to below the cut-off value. As in our study previous studies failed to demonstrate any difference between SUVmax and SUVpeak.[22] [28] However SUVmax remains the standard for semi-quantitative [18F]FDG-PET assessment probably because is a parameter that can be reliably reproduced by independent operators. It is noteworthy that in our study no significant difference in mean SUV values was observed between PET1 PET2 and PET3 which can be explained by the nature of the cytostatic therapy. 11/12 patients were correctly classified (P versus NP) by PET2 and 10/10 were correctly classified by PET3 by applying the SUV cut-off determined by ROC analysis. In 9/10 patients PET3 revealed response information concordant with PET2. The only patient with discordant [18F]FDG-PET findings was classified by SUV analysis as progressive on PET2 and non-progressive on PET3. Blood glucose injected dose or uptake time were normal and/or not significantly different between PET2 and PET3 (1.16 and 1.4 g/l; 261 and 262 MBq; 60 and 75 min respectively) excluding any to methodology-related error. A flare-up phenomenon could be proposed as described on several occasions on [18F]FDG-PET during cytotoxic treatments for squamous cell carcinoma in prostate cancer patients with bone metastases[29][33] and particularly NSCLC patients treated with erlotinib presenting an osteoblastic bone flare-up response mimicking disease progression.[34] Benz et al also described a case of flare-up on early PET in a NSCLC patient treated by erlotinib.[27] Another explanation is that the P/NP classification probably increases mismatches of response assessments related to a discordant outcome of patients with stable disease.[27] Our results suggest that therapeutic efficacy PFS and OS of erlotinib therapy can be predicted 2 weeks after starting erlotinib. These data are consistent with the data of a retrospective study recently published by Kobe et al.[26] [35] At the present time anticancer therapy is currently monitored in the context of hormone-sensitive cancers by regular assay of tumor markers (such as prostate-specific antigen in prostate cancer). The efficacy of hormonal therapy is reflected by a decrease in blood levels of the marker. When the marker remains elevated hormonal therapy is considered to be ineffective and is therefore stopped. Repeated PET imaging can be considered to be a promising approach to evaluate cancer therapy such as targeted therapies that do not induce tumor shrinkage. This new approach appears to be supported by the results of recent clinical trials. The Tarceva Versus Docetaxel or Pemetrexed for Second Line Chemotherapy of Advanced Stage NSCLC (TITAN) trial failed to demonstrate an improvement in OS with erlotinib compared to chemotherapy in unselected NSCLC patients receiving second-line treatment (HR?=?0.96; 95% CI 0.781.19; p?=?0.73).[36] In a similar group of NSCLC patients the results of the TAILOR trial indicated a highly significant increase of PFS in favor of docetaxel (HR?=?0.71; 95% CI 0.530.95; p?=?0.02) versus erlotinib.[37] We consider that evaluation of the metabolic response to erlotinib could provide useful information to rapidly identify patients in whom erlotinib therapy is ineffective especially in EGFR patients without EGFR-activating mutations or unknown status. [18F]FDG-PET could also become a theranostic tool for clinicians. By stopping ineffective therapy earlier physicians can rapidly propose other drugs to a larger proportion of patients with better performance status. This approach could increase the number of patients included in early trials and accelerate drug development. However no medico-economic study has been conducted to determine whether the additional costs induced by [18F]FDG-PET are compensated by the decreased costs of drug (erlotinib) and medical care induced by side effects. Our study highlights the need for more prospective and randomized studies to evaluate the theranostic use of [18F]FDG-PET for management of erlotinib therapy in NSCLC including medico-economic considerations. Conclusion [18F]FDG-PET performed within two weeks of starting erlotinib therapy (9±3 days) appears to be able to predict morphologic response at 2 months according to RECIST criteria. [18]FDG-PET may be clinically useful for early evaluation of targeted therapies as a theranostic tool. Nathalie BAIZE MD Université d'Angers CHU Angers Pôle des Spécialités Médicales et Chirurgicales Intégrées Département de Pneumologie Angers France References 1 FerlayJ ParkinDM Steliarova-FoucherE (2010) Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer46: 76578120116997 2 JemalA BrayF CenterMM FerlayJ WardE et al (2011) Global cancer statistics. CA Cancer J Clin61: 699021296855 3 Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials. Non-small Cell Lung Cancer Collaborative Group. BMJ311: 899909 4 SchillerJH HarringtonD BelaniCP LangerC SandlerA et al (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med346: 929811784875 5 LynchTJ BellDW SordellaR GurubhagavatulaS OkimotoRA et al (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. 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J Nucl Med52: 1871187722065872 29 BjurbergM HenrikssonE BrunE EkbladL OhlssonT et al (2009) Early changes in 2-deoxy-2-[18F]fluoro-D-glucose metabolism in squamous-cell carcinoma during chemotherapy in vivo and in vitro. Cancer Biother Radiopharm24: 32733219538055 30 MessiouC CookG ReidAH AttardG DearnaleyD et al (2011) The CT flare response of metastatic bone disease in prostate cancer. Acta Radiol52: 55756121498309 31 KrupitskayaY EslamyHK NguyenDD KumarA WakeleeHA (2009) Osteoblastic bone flare on F18-FDG PET in non-small cell lung cancer (NSCLC) patients receiving bevacizumab in addition to standard chemotherapy. J Thorac Oncol4: 42943119247091 32 BiersackHJ BenderH PalmedoH (2004) FDG-PET in monitoring therapy of breast cancer. Eur J Nucl Med Mol Imaging31 Suppl 1S11211715112111 33 MortimerJE DehdashtiF SiegelBA TrinkausK KatzenellenbogenJA et al (2001) Metabolic flare: indicator of hormone responsiveness in advanced breast cancer. J Clin Oncol19: 2797280311387350 34 LindJS PostmusPE SmitEF (2010) Osteoblastic bone lesions developing during treatment with erlotinib indicate major response in patients with non-small cell lung cancer: a brief report. J Thorac Oncol5: 55455720357621 35 KobeC SchefflerM HolsteinA ZanderT NogovaL et al (2012) Predictive value of early and late residual 18F-fluorodeoxyglucose and 18F-fluorothymidine uptake using different SUV measurements in patients with non-small-cell lung cancer treated with erlotinib. Eur J Nucl Med Mol Imaging39: 1117112722526960 36 CiuleanuT StelmakhL CicenasS MiliauskasS GrigorescuAC et al (2012) Efficacy and safety of erlotinib versus chemotherapy in second-line treatment of patients with advanced non-small-cell lung cancer with poor prognosis (TITAN): a randomised multicentre open-label phase 3 study. Lancet Oncol13: 30030822277837 37 GarassinoMC MartelliO BrogginiM FarinaG VeroneseS et al (2013) Erlotinib versus docetaxel as second-line treatment of patients with advanced non-small-cell lung cancer and wild-type EGFR tumours (TAILOR): a randomised controlled trial. Lancet Oncol14: 98198823883922 Nucleic Acids Res Nucleic Acids Res nar nar Nucleic Acids Research 0305-1048 1362-4962 Oxford University Press 24970867 4117748 10.1093/nar/gku489 15 Methods Online Integrated RNA and DNA sequencing improves mutation detection in low purity tumors Wilkerson Matthew D. 1 2 * Cabanski Christopher R. 1 3 Sun Wei 2 4 Hoadley Katherine A. 1 2 Walter Vonn 1 Mose Lisle E. 1 Troester Melissa A. 1 5 Hammerman Peter S. 6 7 Parker Joel S. 1 2 Perou Charles M. 1 2 Hayes D. Neil 1 8 * 1Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 2Department of Genetics University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 3The Genome Institute at Washington University St. Louis MO 63108 USA 4Department of Biostatistics University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 5Department of Epidemiology University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA 6Department of Medical Oncology Dana-Farber Cancer Institute Boston MA 02215 USA 7Broad Institute of Harvard and MIT Cambridge MA 02142 USA 8Department of Internal Medicine Division of Medical Oncology Multidisciplinary Thoracic Oncology Program University of North Carolina at Chapel Hill Chapel Hill NC 27599 USA *To whom correspondence should be addressed. Tel: +1 919 966 3098; Fax: +1 919 966 1587; Email: [email protected] Correspondence may also be addressed to D. Neil Hayes. Tel: +1 919 966 3786; Fax: +1 919 966 1587; Email: [email protected] 01 9 2014 26 6 2014 26 6 2014 42 13 e107 e107 15 5 2014 22 4 2014 14 10 2013 © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited. Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method called UNCeqR that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA ERBB2 and FGFR2). In summary integrating RNA-seq with DNA-WES increases mutation detection performance especially for low purity tumors. cover-date 2014 INTRODUCTION Somatically acquired sequence mutations (nucleotide substitutions insertions and deletions) fuel the initiation and progression of cancer (1). Knowledge of mutations in patient specimens informs therapeutic management (23) and in large patient cohorts provides the basis to assess recurrently altered genes that may drive molecular pathogenesis (145). DNA whole exome sequencing (DNA-WES) is currently the popular technology to sequence cancer genomes and has led to an abundance of discoveries in many cancer types (468). However detecting somatic mutations by DNA-WES with high sensitivity and specificity remains a challenge (7910) as evidenced by validation rates of 73% in repeated sequencing and by large inter-rater disagreement among different groups analyzing the same sequencing data (710). The biggest challenge is high quality mutation detection in low purity tumors " | 1 |
" the popularization of health and medical informatics yields huge amounts of data extracting clinicalevents on a temporal course is the foundation of enabling advanced applications and research it is a structure ofpresenting information in chronological order manual extraction would be extremely challenging due to thequantity and complexity of the recordsmethods we present an recurrent neural network based architecture which is able to automatically extractclinical event expressions along with each events temporal information the system is built upon the attentionbased and recursive neural networks and introduce a piecewise representation we divide the input sentences intothree pieces to better utilize the information in the sentences incorporates semantic information by utilizing wordrepresentations obtained from bioasq and wikipediaresults the system is evaluated on the thyme corpus a set of manually annotated clinical records from mayoclinic in order to further verify the effectiveness of the system the system is also evaluated on the timebank_dense corpus the experiments demonstrate that the system outperforms the current stateoftheart models thesystem also supports domain adaptation ie the system may be used in brain cancer data while its model istrained in colon cancer data our system extracts temporal expressions event expressions and link them according to actuallyoccurring sequence which may structure the key information from complicated unstructured clinical recordsfurthermore we demonstrate that combining the piecewise representation method with attention mechanism cancapture more complete features the system is flexible and can be extended to handle other document typeskeywords clinical text mining event extraction temporal extraction relation extraction piecewise representationattention mechanism precision medicine is an emerging approach for diseasetreatment and prevention it becomes the whole worldbiomedicine domain the research hot spot which needsthe support of biomedical methods eg data mining it correspondence clixjtueducn zhijing li and chen li contributed equally to this work1school of computer science and technology xian jiaotong universityxian shaanxi china2shaanxi province key laboratory of satellite and terrestrial network techrd xian jiaotong university xian shaanxi chinaassociates with key information extracted from clinicalrecords eg symptoms over a disease course the associations are often statistically concluded from the evidencecollected from the clinical records the medical bigdata mostly exists in an unstructured form eg textwhich could store useful information very well aligningbiomedical events in clinical data along the events actually occurring time is a meaningful and efficient way ofstructuring such complex data the result may assistauxiliary diagnosistreatment scheme determinationepidemic prediction and side effect discovery etc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli bmc medical informatics and decision making page of many works have been devoted in the study of application in the medical era however large data analysis ofmedicaltreatment needs to map the correspondingmedical events in the clinical records the medical eventswith temporal information are very useful in medical erathese efforts will become the foundation of understanding disease facilitating the analysis of large medical dataas well for example the clinical record in fig may bepresented in a structured manner as the occurring eventsalong with temporal information it is easier for understanding the events and corresponding time point for example using the time point april as a referencethe entity bleeding is before the time point and the entitybolus chemotherapy and nausea is after the time pointin such case the actual events and their occurring consequence becomes clear at a glancein this paper we present a novel system which is builtupon deep neural networks to automatically extractevent expressions and their related temporal expressionsfrom clinical records the system has been evaluated onthe temporal histories of your medical event thyme corpus a corpus developed by a number of professionals according to characteristics of the corpusclinical data contains very long sentences that willundoubtedlythe difficulty of processingtherefore we do not simply use neural networks wewant to make full use of the contextualinformationour proposed method anically combines piecewiserepresentation and attention mechanism by a recurrentneural network rnn and achieves the stateoftheartperformance the results show improvements in automatic extraction of clinical event expressions along witheach events temporal informationincreaserelated workextracting clinical events along with temporal information is a complicated task and the existing systems oftenaccommodate several independent components each ofwhich retrieves different parts eg events and time andassemble them together each component may use a setof hand crafted rules or be based on a pretrained mlmodel velupillai develop the blulab systeminclude the cleartk support vector machine and conditional random fields classification approach and get thefirst place in semeval2015 task clinical tempeval macavaney present the system guir includeconditional random fields and decision tree ensemblesusing lexical syntactic semantic distributional and rulebased features guir receive the best score insemeval2017 task clinical tempeval in the way oftemporal expressions extraction tourille use aneural network based approach and achieve goodperformance for both event and relation extraction insemeval2017 task clinical tempeval lin propose a recurrent neural network with multiplesemantically heterogeneous embeddings within a selftraining framework for clinical temporal relation extraction task they achieve good results for both in andcrossdomainafter event and temporal expression extractionassigning each event with the right temporal expressioninvolves more complicated process some systemsmatch event with temporal expression by a set of syntactic rules crafted by experts wang use syntacticrulebased method for automatic pathway relation information extraction from biomedicalliterature these methods are fast but not flexible enough somefig the example of the medical information extraction the texts marked by the underscores ___ are the temporal expressions the textsmarked by the dash lines _ _ _ are the event expressions 0cli bmc medical informatics and decision making page of existing methods for medical relation information extraction are based on machine learning ml models[]conditional random field crf and supper vectormachine svm are often used in the task of relation extraction lu liu propose an svm model toextract the relations between the potential named entitypairs finkel propose a crfbased informationextraction system to determine relationships deeplearning revives the popularity of neural networkswhich can learn effective relation features from the givensentences withoutengineeringsocher is the first work that employs an rnnmodel to classify relation one early work proposed byluo is based on a recurrent neural network and ableto classify relations from clinical notes compared withthe rule based methods these methods are more flexibleneural network based methods take less time by quicklyscreening out the most unlikely candidate entitypairstherefore some approaches attempt to combine bothhence we think rnn is a good choice and we adopt thernn systemcomplicated featurein recent years attention mechanism has been widelyused in various tasks of nlp based on indepth learning li propose a model that combines abidirectional long shortterm memory network with amultiattention mechanism forrelation extractionzhou propose the attentionbased bidirectional long shortterm memory networksattblstmfor relation classification and the modelresults outperforms most of the existing methods withonly word vectors on the semeval2010 relation classification task so in this paper we also introduce theattention mechanismmethodologythe system consists of three components the first component extracts temporal expressions temporal expressions enable events to be chronologically annotatedthe second component identifies the relevant medicalevents event expressions any situation relevant to thepatients clinical timeline the third component detectsthe relations between the events and the temporalexpressionsannotating clinical records are very expensive frequently only a data of disease specific type is availablebesides the regular mlbased extraction we introducedomain adaption to allow the system to be able to extract the information from one type of disease eg braincancer while it is trained on another type eg coloncancerwe show the pipeline of our system in fig thesystem is built upon an annotating pipeline adoptingthemanagementunstructuredinformationfig the dataprocessing pipeline of the system we first extracttemporal expressions and event expressions respectively frommedical records then extract the relations between themarchitecture uima framework the preprocessingincludestokenization partofspeech tagging andlemmatization which used the stanford corenlp toolkit in both time and event extractions spans oftime and event expressions are represented by the offsets in texts the automatic annotations of event expressions temporal expressions and their relations arebased on three rnn models utilizing lexical syntacticand semantic features [] at the core of deeplearning techniques for nlp lies the vector basedword representation which maps words to an ndimensional space for the choice of the word embeddings corpus we do some research work only entities time and event expressions in the clinical records can be found in the wikipedia in comparisonthe bioasq corpus which is full of biomedical information contains more than of the entities weuse word embeddings from the european projectbioasq obtained by using word2vec on pubmed abstracts [ ] and include the vectorsof distinct words each word is representedas a 200dimensional vector if a word could not befound in the bioasq corpus the embedding is generated from wikipedia by word2vec mikolov as a complement [ ]thestateinternalof rnn can demonstratedynamic timing behavior the hidden state vector can be computed by the following formulaht ¼ f wht þ uxtðþin this formula xt is the input ht is the hidden stateu and w are the weight coefficients f is the nonlinearfunction such as tanh or relu 0cli bmc medical informatics and decision making page of extraction of temporal expressions and event expressionsindependent models are trained for the extractions oftemporal expressions and event expressions figure shows the infrastructure of the system there are twoforms of temporal expressions one is numeric temporalexpressions eg etc and the other is casualtemporal expressions eg day weeks during aperiod etc firstly we generalize all the numeric temporal expressions into for example both and become which can be easily recognizedby the regular expression the regular expression is useddue to the characteristics of the data the numeric temporal expressions are not well recognized by rnn if wedo not use it secondly the casual temporal expressionsare recognized by a rnn model the casual temporalexpressions in the training set are tagged to representthe tokens position in a particular expression there are four tags in our proposed method includingb i o and e which state that the token is at thebeginning on the inside on the outside or at the end ofthe entity respectivelyin this section we propose the system arnn whichis based on a recurrent neural network combining theattention mechanism we need to predict the tokensposition tag of each word before we can train the arnnmodel to predict the type of each temporal expressionwe treat each temporal expression as an entity and eachentity is treated as a unit input we use the average valueof all the word embeddings of an entity in the nextprocess the network in the fig shows the flow chartof arnn network to predict the type of the entitythere is an example sentence from the corpus we willget a ct enterography to rule out crohns disease inthis case the given entity is enterography and thecontext words are we will get a ct to rule outcrohn disease in order to better apply the context information we employ the attention mechanism to learnthe weighted score of each context word related to thegiven entitythe higher weight the higher semantic is bound upwith the given entity 01 00αi exp vti u vhtuv is the parameter that has to be learned from fig we can see that st is the state vector that integrates theother context words information with the given entity attime t st can be computed asst ¼xihαihiwe combine st and h3 to obtain h0sent the given entity which can repre ¼ h3 þ sth0in this process the prediction of entitys type will bepredicted from the given input entity vector the methodof using regular expression are also used to match themissing temporal expressions eg similar to temporal expressions extraction the eventexpressions extraction is built on another rnn unliketime expressions event expressions are all single wordsthere is no need to sign each tokens position we usethe softmax classifier to predict the label y² of the temporal and event expressions the state vector h0 is usedas input and y² could be computed by 10 11py ¼ softmax w h0 ¼ arg maxypyyeventtime relation erer extraction is the most important task in this paperin this section we propose the novel system aprnnwhich is based on a recurrent neural network combiningthe attention mechanism and the piecewise representation the eventtime relation are regarded as a classification problem it is divided into four categories based onsome wellknown communities such as semeval orbionlp the event time relation associates the identifiedevent expressions and temporal expressions and indeedindicates the what and when of a medical event inclinical records the four types are before after beforeoverlap and overlapthe shortestsyntactic path used includespiecewise representationthe dependency parsing of each sentence has beenobtained by utilizing stanford corenlp toolkit thepreviously trained word embeddings which representeach word by a 200dimension word vector and theshortest syntactic paths are fed into the rnn modelof er extraction the word embeddings and shortestsyntactic path are used as features the informationofthewords the poss and the length we add all thesevectors as the entity feature after adding up we stillget a 200dimensional vector for each entity if theentity include severaltemporal expressions we add the vectors of each token and get theaverage vector as the entity vector the whole is divided into sentences as input units we extractall entity pairs based on the annotations given a sentence x x1 x2 ¦ xt the words are projected intoa sequence of word vectors denoted by e1 e2 etwhere t is the number of words in this part wewould like to introduce the piecewise representationtokens eg 0cli bmc medical informatics and decision making page of fig architecture of the rnn model for the extraction of temporal expressions and event expressions we propose the attentionbased rnnmodel to do the entity extraction on the left side of the figure is the details of the attention mechanism the right part of the figure is thernn modelinformation as shown in fig in other wordsthe input sentence is divided intothree parts according to the entity pair we call thisprocess piecewise representation the purpose ofpiecewise representation is to better use of the contextthe examplesentence is divided into three parts according to theentity pair will enterography the whole sentence iswe will get a ct enterography to ruleout crohnsdisease the first part is the sequence before the entity pair we the second part is the sequence between the entity pair get a ct and the third partis the sequence after the entity pair to rule¦ thereare reasons for segmenting the sentence the firstone is that in many cases some studies may choosethe sentence between the entity pair as input insteadof using the whole sentence nevertheless thiscan miss some information and some of them maybe useful only several words cannot supply enoughcontextfeatures segmented sentences can be used to extract the effectiveinformation forextractingrepresentsinformation to the greatest extent of each sequenceand avoid the absence of contextualinformationanother reason comes from the network and our corpus in the corpus the longest sentence contains words however the average length of all sentenceshas words with the rnn structure since the information of a sentence is learned word by word thefeature vector produced at the end of the sentenceactuallysentence althoughrnn has the memory in learning process but thememory time is not long accumulation by recurrentconnectionslongterm informationquickly and the feature vector atthesentence is hard to carry the information of earlysteps in model training there are many longdistancesentences more than words in the training dataso the piecewise representation can help the systembetter use the information of the sentence the syntactic analysis and pos information of the examplesentence are also shown in the fig the end ofentirethetendsto fet 0cli bmc medical informatics and decision making page of fig the flow of our proposed model aprnn on the left side of the figure is the details of the attention mechanism the right part of thefigure is the rnn model which is divided into three partsmodelthe er model contained three parts the first component the feature layer the second component thehidden layer catch the information of word sequenceand produces wordlevel features representations andthen merges wordlevel features into a sentencelevelfeature vector by selecting the most valuable featureinformation among allfeatures weshow the whole process in the fig in this part weproposeattention mechanism to obtain thethe wordleveltherepresentation of the sentence not all the words inthe context describe the er relation each word inthe context has different effects on the given entitypair therefore we introduce the attention mechanism to learn the weighted score of each context wordrelated to the entity pair for the first part the attention mechanism is used to screen the most useful information we use the bilinear operator to computethe attention weight αi for each vector h1 and h2 toreflect how the information relevant to the first entity 0cli bmc medical informatics and decision making page of in the entity pair the current state h3 the calculation method of αi and st is the same as the description in the extraction of temporal expressions andevent expressions sectionwe combine st and h3 to obtain h0 which can represent the sequence before the entity pair for the secondpart we choose the state of the last entity in the sequence ht to represent the whole sequence for thethird part the same method is used as the first part wealso use the same attention mechanism to obtain therepresentation oft the singlesentencelevel feature vector need to be obtained to represent the entire sentence for the relation classificationwe introduce the maxpooling approach as in cnnmodels to obtain the single sentencelevel feature vector the maxpooling is formulated as followsthe sequence h0htf gm ¼ maxtnext the sentencelevel feature vector m is passed tothe output layer furthermore the output layer has classes we use the softmax classifier to predict the labely² from a set of labels y from the sentence the statevector m is used as input therefore y² could be computed bypy ¼ softmax wmð ¼ arg maxypyyþin addition there are two settings in er extraction inorder to better compare our approach the first is basedon our proposed method the second is only utilize thernn network without any piecewise representation orattention mechanism as shows in fig experiment and resultsdatathe major medical data are the data of medical institutions diagnosis and treatment collection of massiveclinical data and laboratory data produced every day atall levels of hospitala golden annotated corpus marked up with temporalexpressions events and relationship between them isneeded to allow us to evaluate by our methods thethyme corpus which has been used since isone of the suitable corpora consisting of clinical andpathological notes of patients with colon cancer andbrain cancer from mayo clinic unlike other datasetsthe events in this dataset are all single words which arevery suitable for our system the notes are manually annotated by the thyme project thymehealthnlpusing an extension of isotimeml for the annotation oftemporal expressions events and temporal relations of the corpus is used for training is forfig the flow of the comparison system this is a commonrnn modeldevelopment and is for testing the developmentset is used for optimizing learning parameters thencombine it with the training set to build the system usedfor reporting results table shows the distribution ofthe thyme corpus the colon cancer data are used astraining data and are tested on both colon cancer andbrain cancer data to demonstrate its effectiveness withtable the distribution of the thyme corpus in this table weshow the different types of data in the corpusdatacolon cancertrain dev testbrain cancertrain testdocumenttemporal expressions event expressions er 0cli bmc medical informatics and decision making page of or without domain adaptation and this can reflect thatour approach is not limited to a particular field inevaluation all methods have access to the same set oftraining and testing datatable the temporal expressions extraction results on braincancer we utilize different methods to do the task the resultsare shown in part the result of previously best system isshown in part resultsthe method has been evaluated on both colon cancerdata and brain cancer data to demonstrate the effectiveness with or without domain adaptation in order to dobetter research several methods are used to de the entityextractionsix methods of temporal expression extraction arulebased method a system based on crf a system based on general rnn without any attention mechanism or context rnn a system based on rnnwith easy attention mechanism but without any contextwords rnnatt our proposed method a rnn system with attention mechanism and context words asystem combines the crf and rnn network all the results are compared part in tables and for therulebased methods firstly we find all the prepositionsaccording to our experience and experimental statisticswe extract five tokens behind their own prepositionsthrough careful observation of data we found thatmany time expressions always show up behind a preposition we then judge whether those five words are relatedto time expressions we define a time dictionary to listthe words which we think can be a part of the time exlike month week day hour maypressionsmonday morning once and so on next we contrastthe five tokens with time dictionary and findwhether it can represent a date or a precise time finallywe extract all the continuous tokens that we thoughtmay relate to the time expressions if there is a definite before those tokens extract it as well there existsome expressions do not after a preposition and onlycontain one word and most of them have the same prefix like pre post peri so we use this prefix rule tofind the remain expressions the major feature we usedfor training the crf and svm classifier is simple lexicaltable the temporal expressions extraction results on coloncancer the part shows the results of six different methodsthat we used to do the temporal expressions extraction thepart shows the result of the previously best systempart methodrulebasedcrfrnnrnnattarnnpart crfarnnblulab run prf1part methodrulebasedcrfrnnrnnattarnncrfarnnpart guirprf1features word embeddings partofspeech tag numericlower case blulab run andtype capital typeguir system are the previously best system mentionedin the section two these results are shown in tables and part in both tables and rule based methods achievethe lowest result the recalls are relatively better thanthe precisions due to the welldefined dictionary theerror analysis shows that some pre post and periare considered as time expressions while they should notbe meanwhile the rulebased method often mistakestwo independent expressions as one if they are adjacentin table the rnn systems performances are lowerthan blulab run 3a cleartk svm pipeline usingmainly simple lexical features along with informationfrom rulebased systems the rulebased information iseffective but it has limitations it can extract rules according to the characteristics of data we do not addany rules to the rnn system the observation on theerror analysis shows that without any attention mechanism and context words rnn is not very effective forsimilar combinations of numbers and letters eg h days etc because the form of the corresponding wordvectors are generated randomly and the time series contains a large number of the above type so the modelcannot learn characteristics of time series so it cannotbe correctly extracted after adding the attention mechanism and context words the arnn system achieve therelatively good results because of the good results ofcrf we combine the crf with the arnn and achievethe best result from table we can see that the rnnoutperforms the guir system which is the current bestsystem it is an ensemble of crf and decision tree withlexical syntactic semantic distributional and rulebasedfeatures the guir system can not extract the previously unseen or atypical date formats very well it is obvious that their rules are not comprehensive enoughthis problem also exists in rnn system however whenadding the attention mechanismit can extract more 0cli bmc medical informatics and decision making page of new and otherwise unknown formats the arnn andcrfarnn system achieve the best results in this partwe have two test data sets one is colon cancer anotherone is brain caner we trained all the models on thesame training data and test them on two different testdata sets except for the different test data the parameters are exactly the same the experimental results provethat our model is effective on other test data setsmeanwhilefive methods of event extraction amethod based on svm a system based on crf asystem based on general rnn without any attentionmechanism or context rnn a system based onrnn with easy attention mechanism but without anycontext words rnnatt our proposed method arnn system with attention mechanism and contextwords all the results are evaluated part in tables and for event extraction the svm and crf modelobtain the relatively good results in colon data and perform poorly in brain colon data compared to the bestsystem limsi however rnn achieves preferably results in the two sets of test data even higher than thebest system limsi as shown in both tables and when adding the attention mechanism and contextwords the results are improvedas for the er extraction which is the key point of thepaper first we compare our proposed model with thefollowing methods a general rnn system withoutany attention mechanism or piecewise representationwe use the sentence between the entity pair as the inputrnn a general rnn system without any attentionmechanism or piecewise representation we use thewhole sentence as the input rnnwhole we can seethe results of rnnwhole is better than the results ofrnn it means that the sentence length can affect theperformance of the system therefore we use the sentence between the entity pair as the input for other system a general rnn system with attention mechanismbut without piecewise representation rnnatt ageneral rnn system without attention mechanism butwith piecewise representation rnnpie our proposed system aprnn but only use the word embeddings trained from wikipedia aprnnwiki ourtable the event extraction results on colon cancer different methods are utilized by us all the results are shown inpar1 the part shows the result of the previously best systemf1methodsvmpart prcrfrnnrnnattpart arnnblulab run table the event extraction results on brain cancer we adopt methods to do the task the results can be compared in part the result of the best system is shown in part part part methodsvmcrfrnnrnnattarnnlimsiprf1proposed system aprnn but only use the word embeddings trained from bioasq aprnnbioasq our proposed system which is based on a recurrentneural network combining the attention mechanism andthe piecewise representation all these results are evaluated part in tables and except for model and the word embeddings for other models are from bothwikipedia and bioasq from the results we can seethat both attention mechanism and piecewise representation are useful they can improve the results to someextent we can directly compare the value of attentionin two groups of experiments result and result result and result the result and result result and result can directly demonstrate the performance with and without segmentation the difference between model and is that model is missing thepiecewise representation and the difference betweenmodel and is without or with the attention mechanism the result has been improved with the piecewiserepresentation the experiment and are aboutlooking at the impact of word embeddings the result and result show that different word embeddings canlead to different results after combining the two corpuswikipedia and bioasq the results increase slightlytable the er classification results on colon cancer part shows the results of the relevant methods we used the otherrelated works which achieved the very good results are shownin part part part methodrnnrnnwholernnattrnnpieaprnnwikiaprnnbioasqaprnnblulab run svmattblstmprf1 0cli bmc medical informatics and decision making page of table the er classification results on brain cancer the resultsof our proposed methods are shown in part part shows theresults of other relatedpart part methodrnnrnn wholernnattrnnpieaprnnwikiaprnnbioasqaprnnlimsisvmattblstmprf1aprnn different factors that may affect the resultsare verified from experimental results eg piecewise representation attention mechanism word embeddings allthese factors are utilized to make better use of contextual informationwe compare our work with other related work thelimsi system which achieves the best score on the ertask in semeval2017 task li rao and zhang proposed the litway which is a system that has adopteda hybrid approach that uses the libsvm classifier with arulebased method for relation extraction theyachieve the best score in the seedev task of bionlpst thus we use their approach as a benchmark forour system the bilstmattention networks proposedby zhou were chosen as another benchmarking model attblstm which outperforms most of theexisting methods they designed a bidirectional attention mechanism to extract wordlevel features from thesentence the features for the attentionbased model include word vector | 0 |
"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 speciï¬city 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 ï¬ndings 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 speciï¬city [ ] In addition the results of TCT interpretation byï¬lmreading 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 theeï¬cacy 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 ï¬lamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1which 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 ï¬nding 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 signiï¬cance 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 HuaianMaternal and Child Health Care Hospital Huaian JiangsuChina were recruited The diagnoses of all patients were veriï¬ed 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 ofHuaian 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 classiï¬ed 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 deï¬ned 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 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°SD 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 Youdens 0cDisease Markerslymph 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 ï¬rst 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 ï¬lament ï¬exibility 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 eï¬cacy 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 andspeciï¬city 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 speciï¬city 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 P pgmL as the cutoï¬ value and the sensitivity and speciï¬city 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 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 speciï¬cityof 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 signiï¬cance it still seems to beable to inï¬uence 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 signiï¬cance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the speciï¬c 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 PMultivariate 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 conï¬rm 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 conï¬icts of interestAuthors ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as coï¬rst authors[] M H Forouzanfar K J Foreman A M Delossantos et alBreast 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 Cervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp [] Y Zhou W Wang R Wei Serum 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 The role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya Eï¬cacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp [] N Tanaka T Yamashita S Yamamoto Histologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp [] Y Watabe T Mori S Yoshimoto Copy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp [] HT An S Yoo and J Ko αActinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp [] F Niu T Wang J Li The 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 Serum 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 αActinin4 is essential for maintaining the spreading motility andcontractility of ï¬broblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida Actinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp [] D G Thomas and D N Robinson The ï¬fth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp screening on the diagnostic signiï¬cance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp [] KH Wang C J Lin C J Liu Global 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 Diagnostic 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 Actinin4 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 Thestructure and regulation of human muscle αactinin Cellvol no pp [] D Wang X W Li X Wang Alphaactinin4 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 Possible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda The biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim αActinin 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 DynamicRegulation 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 Actinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp [] T Watanabe H Ueno Y Watabe ACTN4 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 ACTN4 gene ampliï¬cation 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 Alphaactinin 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 The alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp 0c" | 2 |
" levels of physical activity change throughout the year however little is known to what extentactivity levels can vary based on accelerometer determined sedentary and physicallyactive time the aim of thislongitudinal study was to examine older adults activity changes from a nonsnowfall season to a subsequentsnowfall season with consideration of the codependence of domains of time usemethods participants were older japanese adults women aged years living in a rural area ofheavy snowfall who had valid accelerometer active style pro hja750c data during nonsnowfall and snowfallseasons activity was classified as sedentary behavior sb lightintensity pa lpa and moderatetovigorous pamvpa compositional changes from the nonsnowfall to the snowfall season were analyzed using aitchisonsperturbation method the ratios of each component in the composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonal changes were calculated and were then divided by thesum of these ratiosresults in men the percentages of time spent in each activity during the nonsnowfallsnowfall seasons were for sb for lpa and for mvpa these corresponded to mean seasonal compositionalchanges δsb δlpa δmvpa of and respectively in women the percentages of time spent ineach activity during the nonsnowfallsnowfall seasons were for sb for lpa and formvpa these corresponded to mean seasonal compositional changes δsb δlpa δmvpa of and respectively the degree of seasonal change was greatest in mens in older adults activity behaviors were changed unfavorably during snowfall season particularly so formen the degree of seasonal change was greatest for sb development of strategies to keep rural older adultsactive during the snowfall season may be needed for maintaining a consistentlyactive lifestyle for their healthkeywords accelerometry aging environment exercise sedentary lifestyle correspondence inouetokyomedacjp1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo 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 0camagasa bmc public health page of global physical activity guidelines state that older adultsleast min per week of moderateshould do atintensity physical activity atleast min vigorousintensity physical activity or an equivalent combinationof both in bouts lasting min or more physical inactivity increases the risk of many adverse health outcomes including mortality cardiovascular disease type diabetes several types of cancer and cognitive decline[] in recent years there has been significant growthin research investigating the detrimental health effects ofsedentary behavior sb put simply too much sitting reducing sb and increasing physical activity are keyingredients of initiatives to reduce the global burden ofnoncommunicable diseases [ ]there is a growing body of evidence identifying the importance of natural environments the attributes of whichcan vary greatly by season as well as built environments asinfluences on physical activity [ ] one systematic review of studies from eight different countries showed thatlevels of physical activity tended to be lowest during winter to date however most of these studies have relied onselfreport physical activity assessments and have not focusedon older adults objective assessment via accelerometers can now overcome problems of recall and reportingbias and allow more accurate and finergrained assessmentsof activity behavior patterns sb lightintensity physical activityactivitymvpa there is limited evidence on how season affects devicebased activity behaviors [] compositionaldata analysis coda allows consideration ofthe codependence of time spent in all behaviors arising within aday or other fixed period [ ] to date no previous studyhas investigated seasonal changes of activity behaviors takingtime spent in each activity behavior into accountlpa and moderatetovigorous physicalwe compared times spent in accelerometermeasuredactivity behaviors during a nonsnowfall and a snowfallseason in a sample of communitydwelling older japanese adults using the coda approach we also exploredwhich activity behaviors were most affected by seasonmethodsstudy sample and data collectionwe used longitudinal data from the neuron to environmental impact across generations study neige study themethods of this study are described in previous study participants were older adults without longterm care livingin tokamachi city japan tokamachi is a rural city officiallyregistered as a heavy snowfall region during winter locatedin the southernmost region of niigata prefecture participants were from two areas matsunoyama mountain areaor tokamachi area downtown the mountain area mmaximum snow depth had more snow than the downtownarea maximum snow depth during winter theaverage temperature at tokamachi city in february was °c lowest °c highest °c brieflyin a total of residents were recruited from a resident registry using stratified randomsampling in the nonsnowfall season autumn of we conducted a questionnaire survey and health examination of older adults who agreed to enrollment inthe neige study at the same time they were asked towear an accelerometer of these participants agreedto also wear an accelerometer during the snowfall seasonwinter of accelerometermeasured activity behaviorshabitual time spent in activity behaviors were evaluatedby active style pro hja750c omron healthcarekyoto japan active style pro is a validated accelerometer [] and comparable to the devices most commonly used in studies conducted in western countries[ ] its measurement algorithm has been explainedin detail elsewhere [ ] participants were instructedto wear an accelerometer over the waist on an elasticated belt for seven consecutive days except duringsleep and waterbased activities during snowfall andnonsnowfall season respectively in the survey duringsnowfall season participants were mailed an accelerometer no acceleration signal detected for longer than consecutive minutes was defined as nonwear participants with a wear time corresponding to at least hduring waking time per day collected over four ormore valid days were included in the analysis thedata were collected in 60s epochs activity behaviorswere classified into three intensity categories based onmetabolic equivalents mets sb mets lpa mets and mvpa ¥ mets [ ]sociodemographic biological and psychological factorsparticipants reported their age gender living arrangement with others alone and selfrated health verygood good fair poor in autumn we classified participants between the ages of and years as youngoldand those between ages and years as oldold bodymass index bmi was calculated from height and weightkgm2 measured by body composition analyzer mc780a tanita corporation tokyo japanstatistical analysesall analyses were performed using r version r foundation for statistical computing vienna austria we usedr package compositions for coda approach for all analyses pvalues were considered statistically significantanalyses were applied stratified by gender since activity behavior patterns were significantly different between men andwomen 0camagasa bmc public health page of the chisquare test or t test was performed to compare participant characteristics mcnemars test wasused to compare nonsnowfall and snowfall seasonsproportions of those adhering to physical activity guidelines ie ¥ minweek of mvpa in bouts of at least min a minbout mvpa was defined as ormore consecutive minutes above the moderate intensitythreshold with the allowance of min interruptionintervals we described activity behaviors during the nonsnowfalland snowfall season using coda approach as all participantsspent some time in every behavior there was no need for amethod to deal with zeros compositional changes [δsbδlpa δmvpa] were then determined by aitchisons perturbation method [] the ratios of each component inthe composition such as [sbsnowsbnonsnow lpasnowlpanonsnow mvpasnowmvpanonsnow] for seasonalchanges were calculated and were then divided by the sumof these ratios an equal composition of these three activitiesin the nonsnowfall and snowfall seasons would result in acompositional change of[ ] compositionalchanges were plotted as ternary diagrams with some significant guide values illustratedresultsparticipant enrollment and characteristicsof the older adults who agreed to wear an accelerometer during both the nonsnowfall and snowfallseasons response rate were excluded for notmeeting wearing time criteria n bone fracturen unreturned accelerometer n and malfunction n the analytic sample was who had validaccelerometer datawhen compared to participant characteristics betweenthose who had valid accelerometer data in snowfall season and those who did not a significant difference wasfound in age groups youngold oldold chisquare test participants who participated in thesurvey in the snowfall season were significantly morephysically active approx more minutes of mvpaper day than those who did not ttesttable presents the characteristics of the participantsthe mean sd age was years womenand most of the participants were living with others kgm2 bmi and good perceived health comparedto women men were more likely to be from the mountain area be living with others and to have attained¥ minweek of mvpa there were no significant seasonal differences in proportion of those adhering to global physical activity guidelines nonsnowfall seasonoverall men women snowfall season overall men women activity behaviors in snowfall and nonsnowfall seasonmean accelerometer wear time was minday innonsnowfallseason and minday in snowfalltable participants characteristics at baseline nonsnowfall seasonoverall n mean ± sd n ± men n mean ± sd n ± women n mean ± sd n ± ± ± pvalueage yrsage categoriesyoungold yrsoldold ¥ yrsarea of residencemountain areadowntownliving arrangementliving with othersliving alonebmi kgm2bmi categoriesnormal kgm2obese ¥ kgm2perceived healthgood very good good ± poor fair poorpvalue was calculated using t test or chisquare test as appropriate 0camagasa bmc public health page of season older adults spent most of their time on sb andlpa table presents the descriptive statistics of timespent in each activity behavior in men time spent ineach activity sb lpa and mvpa was and respectively during the nonsnowfall season and and respectively during the snowfall season corresponding to mean seasonal change of forsb for lpa and for mvpa fig sincelarger distance from indicates greater change in timespent in activity the largest seasonal change was observed in sb compared to lpa and mvpa in womentime spent in each activity was and respectively during nonsnowfall season while that was and respectively during snowfall seasoncorresponding to mean seasonal change of for sb for lpa and for mvpa significant unfavorable seasonal changes in activity behaviors were observed in both men and women but the degree ofchange was larger in men in every behavior if we contrast the changes the ratios between men and womensimilar findings were observed after stratification byresidential area but the degree of change in activity behaviors was larger in mountain area than in downtownregardless of genderdiscussionwe identified seasonal differences of accelerometer determined activity behaviors in communitydwelling olderadults in a rural snowfall area using the coda approacholder adults had more unfavorable activity behavior patterns during the snowfall season compared to nonsnowfall season with the magnitude of these differencesgreater in men no significant seasonal differences werefound in the proportions adhering to global physical activity guidelinesour findings are consistent with previous studies usingselfreport [ ] and pedometer accelerometer [] data where levels of adults physical activity duringwinter was less than the other seasons a study iniceland found that both older men and women weremore active during summer than winter with less timespent in accelerometer determined sb in men in women a study in the uk found that onlytime spent in lpa was significantly higher during springthan that during winter among older adults winter ± hday spring ± hday summer ± hday and autumn ± hday we found thatthe magnitude of decline during winter seems to belarge compared to these icelandic and uk studies thismay result from winter conditions including amount ofsnowfall furthermore our study participants were relatively more active and less sedentary during autumn thenonsnowfall season more highly active participantsmay experience activity decline to a greater degree compared to low activity participants in this sample therewere no significant seasonal changes of adherence toglobal physical activity guidelines this may be due tovery little time spent in mvpa in bouts of more than mingender differences of activity patterns were in linewith our previous findings [ ] that japanese olderwomen were more physically active than older men provided that all activity behaviors were assessed in thissample men experienced greater seasonal activity declines than did women in japan women are more responsible for household chores and thus engage in acertain amount of activities throughout the year theremay be needed to develop a strategy to combat this seasonal decrease in physical activity for menin the current study activity behavior patterns wereunfavorably changed with approximately increasein sb and decrease in mvpa the degree of the changein activity behaviors may be significant for health a previous study found decline of physical activity during winter unfavorably affected the physical performance levelafter one year in communitydwelling older women particularly its effect on maximum walking speed given that the rate of muscle mass decline is higher inolder adults compared to middleaged adults maintaining physical activity may be required to keep physicaltable compositional geometric means of time spent in activity behaviors during the nonsnowfall and snowfall seasonnonsnowfall season minday wear timewear timesb lpa snowfall season minday wear timewear timesb lpa mvpa mvpa overallmenmountain areadowntownwomenmountain areadowntownabbreviation sb sedentary behavior lpa lightintensity physical activity mvpa moderatetovigorous physical activity 0camagasa bmc public health page of fig changes in sedentary behavior sb light intensity physical activity lpa and in moderatetovigorous physical activity mvpa from thenonsnowfall to snowfall season in ruraldwelling older japanese adults a stratified by gender blue men red women b stratified by genderand residential area blue mountain area green downtown abbreviation sb sedentary behavior lpa lightintensity physical activity mvpamoderatetovigorous physical activityability and prevent a negative cycle of frailty another study showed shortterm decreased physical activity with increased sb caused impairment of metabolism which increases risk of cardiovascular disease therefore promoting physical activity during wintermay be one way to tackle agerelated diseases and lossof physical functioningstrengths and limitationsstrengths of this study included objective assessment ofactivity behaviors and a novel statistical approach eventhough there has been increasing research using objective methods [ ] no study has treated with consideration to the codependence of time spent in activitybehaviors moreover we provided resultsfrom aseldomstudied elderly population in a rural snowfallarea however several limitations should be consideredto interpret the findings we may underoverestimate ofsb and lpa since active style pro cannot provide posturalinformation also some activities eg snow removal and skiing may be not captured accuratelysecond we may underestimate the decline of activitylevel since most of the days that participants wore an accelerometer were relatively good weather in spite ofheavy snowfall area it has previously been observed thatlonger day length is associated with increased devicebased physical activity in the older population [ ]third as tokamachi city is a rural area where heavysnowfall is common during winter it is not necessarilyrepresentative ofjapanese rural areas with different 0camagasa bmc public health page of climates people in areas with less snowfall may experience a smaller decline of physical activity during snowfall season more research is needed in the differentclimate zones from different geographic areas finallyselection bias may have occurred in general accelerometry respondents have been more active and healthierthan nonparticipating older adults in this studythose had valid accelerometer data in snowfall seasonwere more physically active than those who did notimplications for future research policy and practiceour findings suggest several implications in terms of bothdevelopment of interventions to protect against seasonalphysical activity decline and physical activity surveillancemonitoring further research regarding how to stay activeduring winter may be required for health promotion particularly in regions that experience long winters and withsevere weather eg heavy snow given that approximately of the land in japan has snow and a cold winter and that a quarter of the population lives in thoseareas leading an active lifestyle during winter potentially has a significant public health impactsince sb is the most affected by season it may be better tofocus on developing strategies to reduce time spent in sittingolder adults might be particularly influenced by seasonaloutside conditions due to reduced physical functioning andmobility and spend much of their time indoors it thus maybe effective to provide supplementary resources for indooractivities eg gymnastic exercise programs sharing ofhousehold chores and making educational instrumentalsupports for safe snow removal further approach includes replacing mentallypassive with mentallyactive sbmay be effective for health particularly for preventing cognitive decline [ ] and depression [ ]as for physical activity surveillance monitoring investigators should be aware of the potential for under oroverestimation of levels of activity especially when theinterest is in its betweenindividual variation includingcommunitylevel and countrylevel comparisons seasonality also should be considered when interventionstudies are performedsaccelerometer determined activity behaviors were greatlyaffected by season in communitydwelling older adults ina rural snowfall area resulting in unfavorable changesparticularly in sb time during snowfall season development of strategies to keep rural older adults active duringthe snowfall season may be needed for maintaining aconsistentlyactive lifestyle for their healthabbreviationssb sedentary behavior lpa lightintensity physical activitymvpa moderatetovigorous physical activity coda compositional dataanalysis mets metabolic equivalentsacknowledgementswe thank the city workers and the participants for their time and effort inthe neige studyauthors contributionsys hm si and tf developed the neige study ys hm si tf hk nf mmand sa collected the data sa performed the analysis and prepared themanuscript sc and no gave critical feedback and revised the manuscript allauthors advised on the data analysis and interpretation and reviewed themanuscriptfundingthis study was funded by grant from the policy research institute ministryof agriculture forestry and fisheries in japan and the pfizer healthresearch foundation and by jsps kakenhi grant number 16h03249 k19794 k10829 and 19h03910 the funding bodies were not involved inany portion of the study design data collection and analysis interpretationof the results and preparation of the manuscriptshiho amagasa is supported by jsps research fellowships for youngscientists neville owen is supported by a national health and medicalresearch council of australia nhmrc centre of research excellence grant nhmrc senior principal research fellowship and thevictorian governments operational infrastructure support programavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatethe university ethics committees niigata university and tokyo medicaluniversity granted ethical approval written informed consent was obtainedfrom all participantsconsent for publicationnacompeting intereststhe authors declare no conflict of interestauthor details1department of preventive medicine and public health tokyo medicaluniversity shinjuku shinjukuku tokyo japan 2institute ofgerontology the university of tokyo hongo bunkyoku tokyo japan 3department of global health promotion tokyo medical anddental university yushima bunkyoku tokyo japan 4school ofhealth and life science institute of applied health research glasgowcaledonian university cowcaddens road glasgow uk 5department ofsport and movement science ghent university ghent belgium6behavioral epidemiology laboratory baker heart diabetes institute level commercial road melbourne vic australia 7centre for urbantransitions swinburne university of technology po box hawthornmelbourne australia 8division of international health niigata universitygraduate school of medical and dental sciences asahimachidoriniigata city japan 9department of active ageing niigatauniversity graduate school of medical and dental sciences asahimachidori niigata city japanreceived february accepted august references world health anization global recommendations on physical activity forhealth httpappswhointirisbitstream106654439919789241599979_engpdf accessed jan lee im shiroma ej lobelo f puska p blair sn katzmarzyk pt effect ofphysical inactivity on major noncommunicable diseases worldwide ananalysis of burden of disease and life expectancy lancet kyu hh bachman vf alexander lt mumford je afshin a estep k veermanjl delwiche k iannarone ml moyer ml physical activity and risk ofbreast cancer colon cancer diabetes ischemic heart disease and ischemic 0camagasa bmc public health page of stroke events systematic review and doseresponse metaanalysis for theglobal burden of disease study bmj 2016354i3857aune d norat t leitzmann m tonstad s vatten lj physical activity andthe risk of type diabetes a systematic review and doseresponse metaanalysis eur j epidemiol arem h moore sc patel a hartge p berrington de gonzalez avisvanathan k campbell pt freedman m weiderpass e adami ho leisure time physical activity and mortality a detailed pooled analysis of thedoseresponse relationship jama intern med sofi f valecchi d bacci d abbate r gensini gf casini a macchi c physicalactivity and risk of cognitive decline a metaanalysis of prospective studiesj intern med young dr hivert mf alhassan s camhi sm ferguson jf katzmarzyk ptlewis ce owen n perry ck siddique j yong cm sedentary behavior andcardiovascular morbidity and mortality a science advisory 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" mutations in the exonuclease domain of pole a dna polymerase associated with dna replicationand repair lead to cancers with ultrahigh mutation rates most studies focus on intestinal and uterine cancers withpole mutations these cancers exhibit a significant immune cell infiltrate and favorable prognosis we questionedwhether loss of function of other dna polymerases can cooperate to pole to generate the ultramutatorphenotypemethods we used cases and data from cancer types in the cancer genome atlas to investigate mutationfrequencies of different dna polymerases we tested whether tumor mutation burden patient outcomediseasefree survival and immune cell infiltration measured by estimate can be attributed to mutations in polqand polzrev3lresults thirty six percent of colorectal stomach and endometrial cancers with pole mutations carried additionalmutations in polq eq polzrev3l ez or both dna polymerases ezq the mutation burden in thesetumors was significantly greater compared to poleonly e mutant tumors p in addition eq ez and eqz mutant tumors possessed an increased frequency of mutations in the pole exonuclease domain p colorectal stomach and endometrial eq ez and eqz mutant tumors within tcga demonstrated diseasefree survival even if the pole mutations occurred outside the exonuclease domain p however immunescores in these tumors were related to microsatellite instability msi and not pole mutation status this suggeststhat the host immune response may not be the sole mechanism for prolonged diseasefree survival ofultramutated tumors in this cohort results in this study demonstrate that mutations in polq and rev3l in pole mutant tumors shouldundergo further investigation to determine whether polq and rev3l mutations contribute to the ultramutatorphenotype and favorable outcome of patients with pole mutant tumors correspondence beatriceknudsenpathutahedu1department of biomedical sciences cedarssinai medical center losangeles ca usa2samuel oschin cancer research institute socci cedarssinai medicalcenter los angeles ca 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 0chuang bmc medical genetics page of the analysis of thousands of cancers by the cancergenome atlas tcga consortium and academic institutions revealed a small group of cancers with mutationsin pole and an ultramutator phenotype [ ] multiplestudies have found significantly improved survival in patients with pole mutated endometrial cancers [ ]while the survival benefit was not as profound in polemutated colorectal carcinoma the pole geneencodes the catalytic subunit of dna polymerase epsilon which catalyzes the leading strand synthesis duringdna replication pole possesses highfidelity dnapolymerization proofreading and ²² exonuclease activities which promote accurate dna synthesis firstidentified and reported in of colorectal carcinomas[ ] pole mutations were also noted at frequencies of amongst uterine corpus endometrialcancers [ ] and in gastric adenocarcinoma mutations can be found across the entire pole genebut those in the pole exonuclease domain are mostprevalent in cancers with ultrahigh mutation rates mutmb these cancers exhibit higher mutationrates than microsatellite instable msi tumors associated with mismatch repair abnormalities in additionpole ultramutant cancers also possess a high frequency of ctoa transversions [ ] a tumor associated inflammatory response similar to thatin msitumors has been reported to occur early on duringdevelopment of pole mutated endometrial and colorectal cancers it is thought to be caused by neoantigens that are generated as a result of the high mutation burden [ ] and render pole mutant cancersresponsive to immunotherapy polymerase theta polq is a lowfidelity dna polymerase lacking a ² to ² exonuclease function theenzyme is involved in the alternative nonhomologousendjoining pathway altnhej which is a backupmechanism of double stranded dna break repair thispathway predominates in cancer cells when other dnarepair pathways are missing or when telomere ends aredeprotected [ ] the loss of polq sensitizes cellsto ionizing radiation and polqdeficient mice exhibit increased dna instability and genomic rearrangementssuggesting a role for polq as a guardian of the genome both overexpression and loss of polq increasemutation frequencies [ ] multiple structuralmotives in polq can interact with dna rad51 andbrca1 in addition polq forms a complex withparp1 in a pathway of synthetic lethality with brca1and is thus considered a therapeutic target [ ]however which domains in polq should be targetedremains to be determined rev3l rev3 like dna directed polymerase zetapolz catalytic subunit is involved in dna synthesisthat reads through damaged dna translesion dnasynthesis tls the high efficiency of polz bypassing a broad spectrum of dna lesions led to its recognition as a master tls polymerase polzrev3l has been linked to carcinogenesis in breastlung gliomas and gastric cancers and modulatescisplatin sensitivity []because studies describe overlapping functions andsynergy among the over dna polymerases [ ] we undertook an unbiased approach to identifymutations in polymerases within the tcga tumorcompendium this analysis revealed a greater frequencyof mutations in polq and polzrev3l compared toother polymerases of similar gene length therefore wepropose that polq and polzrev3l may cooperatewith pole in generating ultrahigh mutation ratesmethodsdata acquisitionthe data used in this study are based upon the wholeexome sequence data sets generated by the tcgaresearch network httpcancergenomenihgov thelocations and frequencies of somatic mutations msistatus and clinical stage and follow up information intcga provisional datasets were obtained from cbioportal httpwwwcbioportal [ ] up to supplementary table mutation data were obtainedby first selecting the query tab from cbioportal thecancer type specific data were chosen from the tcgapancancer atlas studies we entered the genes ofinterest pole polq and rev3l to retrieve mutationdata from the genes when the result page wasdisplayed we accessed the information underneath themutations tab we downloaded the number of mutations in each sample the annotation of protein aminoacid change and the type of mutation the survivalinformation for each case months after diagnosis wasobtained through the comparisonsurvival link functional domains of the proteins were provided by pfamdatabase data visualization for mutations wasperformed with mutationmapper in cbioportalcase selection criteriawe searched all cancer types in tcga for those thatpossess or more cases with pole mutations thisyielded tumor types that we named the pancandata set in this study we then determined the frequencyof mutations within additional polymerase within pancan we selected the three adenocarcinomas uterinecorpus endometrial adenocarcinoma colorectal adenocarcinoma and stomach adenocarcinoma with the highesttriple mutated dnapolymerase status for more detailed analysisfrequency of double or 0chuang bmc medical genetics page of studies performedkaplan meier survival plots were generated using clinicalfollowup data available within the tcga database todetermine the global mutational spectrum we classified types of nucleotide transitions or transversions thefrequency of each mutation type was calculateddigital images of all eq mutant tumors and representative cases of tumors with neither pole nor polqmutations and of msi tumors were assessed by one author jr for the amount of tumor immune infiltratethe combination of tumor infiltrating lymphocytes andthe peritumorallymphoid infiltrate was graded on ascale between and tumor associated lymphocyteswere graded as none minimal rare to per highpowered field hpf to per hpf and perhpf peritumoral lymphocytes were assessed at thedeepest advancing tumor front graded at low powernone minimal mild moderate andmarked these visual semiquantitative scores werecompared with the immune scores evaluated using estimate estimation of stromal and immune cells inmalignant tumor tissues using expression data estimate score were obtained from the website ofestimate at md anderson httpsbioinformaticsmdandersonestimatediseasehtml theplatformtype selected was rnaseqv2 data for colorectalstomach and endometrial cancer types were downloaded next we separated the colorectal stomach andendometrial cancer cases into quartiles defined by thehighest intermediate and lowest of mutational counts or msi status and calculated the immunescores for each groupstatistical data analysisall statistical analyses were conducted in r v313 plotswere generated using ggplot2 package in r datavisualization methods were described previously the horizontal lines in the boxplots represent the 1st2nd and 3rd quartiles and whiskers outside the boxshow the interquartile range the significance of thedifferences of data illustrated in the boxplots wascalculated using the wilcoxon ranksum tests the chisquare test was performed to test the significance ofdifferences in frequencies of all tables the significancein the kaplanmeier survival plot was calculated usingthe log rank test statistical significance was accepted atp resultsof the cancer types in the tcga database weidentified cancer types pancan with or morecases that possessed mutations in the pole proteincoding region fig 1a and supplementary table these cancer types contained cases with polemutations anywhere in the exome of these polemutant tumors carried mutations in one or more of the other dna polymerases we observed dna polymerases polq and polzrev3l to be most frequentlymutated fig 1b these two polymerases were mutatedin of tumors with pole mutations in fact thesetwo polymerases were even more commonly mutatedthan pole in our pancan cohort supplementary figure altogether cases with pole and polq mutations eq cases with pole and polzrev3lmutations ez and cases with pole polq andpolzrev3l eqz mutations were identified in thepancan cohort fig 1c mutations in the exonucleasedomain of pole are responsible for causing the ultramutator phenotype in colorectal and uterine corpuscancers [ ] in order to determine the contribution of polq and rev3l to the ultramutator phenotype we compared the mutation frequencies of tumorswith mutations in only pole to eq ez and eqzmutant tumors mutation frequencies in the cellulargenome increased in the following order no polemutations poleonly mutations anywhere with thepole exome eq ez eqz mutations fig 1dthe median mutation count of eqz tumors was morethan 10fold higher p than that of tumors withonly pole mutations e q and e z mutant tumorsalso displayed significantly higher mutation countscompared to eonly mutant tumors suggesting a contribution of mutationally altered polq or polzrev3l tothe overall cancer mutation rates next we determinedthe number of mutations in the exonuclease and polymerase domains of pole in the cancer types withinour pancan compendium fig 1e the percentage ofpole mutations in the exonuclease domain was greaterin eq ez and eqz mutant tumors compared topoleonly mutant tumors p in contrast thepercentage of mutations in the dna polymerase domainwas similar thus our data confirm the notion thatmutations in the exonuclease domain of pole areresponsible for ultrahigh mutation rates in additionmutations in polq and rev3l may further increasethe mutation burden however why mutations in polqand rev3l preferentially increase tumor mutationfrequencies remains elusive endometrialto further investigate a potential role of these mutantdna polymerases in the ultramutator phenotype wefocused on colorectalucec andstomach stad cancers these cancer types containthe highest numbers of tumors with eq ez and eqz amongst the cancer types included in pancanfig 1a supplementary table among these cancertypes we identified cases with eq cases with ezand cases with eqz mutations fig 2a in thesecancers the mutation burden in pole eq ez and e 0chuang bmc medical genetics page of fig cancer types pancan with poleqz mutations in tcga a number of cases with pole only eq ez and eqz mutations in cancertypes cohort referred to as pancan within tcga the xaxis shows the actual number of cases with pole green eq orange ez pink and eqz blue mutations the yaxis displays the cancer types uterine corpus endometrial carcinoma ucec stomach adenocarcinoma stadcolon and rectum adenocarcinoma skin cutaneous melanoma skcm lymphoid neoplasm diffuse large bcell lymphoma dlbc lungadenocarcinoma luad breast invasive carcinoma brca sarcoma sarc cervical squamous cell carcinoma and endocervical adenocarcinomacesc pancreatic adenocarcinoma paad lung squamous cell carcinoma lusc head and neck squamous cell carcinoma hnsc bladderurothelial carcinoma blca kidney renal clear cell carcinoma kirc and liver hepatocellular carcinoma lihc b case numbers with mutationsin polymerase genes the number of cases in pancan with mutations in the following polymerases is displayed on the yaxis dntt pola1polb pold1 polg polh poli polk poll polm poln polq rev1 rev3l c venn diagram displaying the number of cases in pancan withmutations in or pol genes d mutations per mb yaxis of pancan cases without pole mutations other or with pole eq ez and eqzxaxis mutations number of cases in each group are listed in parenthesis e mutation frequencies in pole exonuclease and polymerase domainsas a percentage of total number of mutations in the pole exome other refers mutations in the entire exome outside the exonuclease orpolymerase domains the cases are grouped by their polymerase mutation status on the yaxis and the number in parenthesis represents thetotal number of pole mutations within each group 0chuang bmc medical genetics page of fig poleqz mutations in colorectal endometrial ucec and stomach stad cancers a venn diagram displaying the number of caseswith mutations in or pol genes b mutation groups of cases without polymerase mutations other or with mutations in pole only eq ez and eqz the number of cases in each group is listed in parenthesis the pvalue for comparison of pole and eq groups is not significantp c number of cases with mutations in pole exonuclease domain in various mutation groups d percentages the ratio of transitions tiand transversions tv are shown on the yaxis for core stad and ucec the xaxis shows the mutation groupsqz mutant tumors paralleled the mutation burden inthe whole pancan cohort compare fig 2b and fig1c supplementary figure 2ad eqz mutant tumors demonstrated on average an 8fold increase inmutation frequencies compared to tumors with onlypole mutations amongst pole mutant tumors tumors carried mutations outside the pole exonuclease domain while tumors carried mutations withinthe exonuclease domain the frequency of poleexonuclease mutationsin fig 2cprovides a valid explanation forthe difference inmutation rates and potential association of poleexonuclease domain mutations with mutationsinpolq and polzrev3l which are the other twomost frequently mutated dna polymerases casesoverall exonuclease domain mutations were identifiedin cases of pole only mutant tumors eqcases ez cases and of eqz cases fig 2cstratified by cancer types pole exonuclease domainmutations occurred in colorectal endometrial and stomach tumors demonstrating cancertype specific frequencies supplementary figure 3a incontrast to the pole gene that demonstrates mutationalhotspots in the exonuclease domain mutational hotspotsin the polq gene are not associated with a functionalprotein domain supplementary figure 3b c dwhile rev3l does not reveal mutational hotspotsapproximately of mutations lead to truncated protein expression supplementary figure 3e f anothercharacteristic of pole mutant tumors are c to a and g 0chuang bmc medical genetics page of to t transversions we observed the greatest increaseof nucleotide transversions in cancers with eqz mutafig 2d consistent with the loss of poletionsexonuclease activity in these tumorssince mutations in pole confer increased disease freesurvival dfs in patients with uterine cancer even inthose patients with highgrade tumors [ ] we investigated the prognostic role of polq and rev3l mutations in pole mutanttumors kaplanmeier curveswere constructed for colorectal endometrial and stomach cancer cases with followup data fig 3a using thetcga annotations of dfs in individual patients nocancer recurrences were observed in the eq ez andeqz mutant groups pole exonuclease domain mutations were observed in cases in the good survivalgroup and case in the poor survival group consistentwith the expected long dfs periods of patients withpole exonuclease domain positive tumors in additionanalysisin the pancan cohort cases with mutations in pole outside the exonuclease domain were in the good survival group of those had concurrent mutations in polq or rev3l orin both polymerases fig 3b furthermore a kaplanmeierrevealedimproved dfs associated with mutations in polq andrev3l the favorable survival outcome was observed incolorectal endometrial and stomach cancers howeverno favorable outcome was observed in diffuse bcelllymphoma p these data provide preliminaryevidence of cancertype specificfavorable survivaloutcomes in tumors with pole mutations that arelocated outside the pole exonuclease domain if concurrent mutations in polq rev3l or in both polymerasesare presentcompared to microsatellite stable tumorsmssmicrosatellite instability msiin colorectal cancerconfers a better prognosis to determine whetherfig survival and clinical characteristics of patients with polymerase mutations colorectal endometrial ucec and stomach stad cancers akaplanmeier curves of diseasefree survival dfs for groups of patients pole only n median followup months green line eqn median followup months orange line ez n median followup months pink line and eqz n median followup months blue line tumors without mutations in pole polq or rev3l exomes grey line the overall pvalue is p individual pvalues eqz vrs pole p eqz vrs none p eq or ez or eqz vrs pole p b polymerase mutation analysis ofcases in the good survival group in panel a the red bar indicates cases with pole exonuclease mutations c cancer typespecific illustration ofmutation count pole polq and rev3l mutations microsatellite instability msi and tumor stage 0chuang bmc medical genetics page of the favorable outcome of eq ez and eqz mutantcancers can be explained by msi or tmn stage we examined the relationship between msi statustumorstage and polymerase mutations in colorectal endometrial and stomach cancers fig 3c and supplementaryfigure and supplementary table despite improveddfs rates the full range of tumor stages was observedamongst eq ez and eqz tumors p supplementary table 2a comparing the poleonly and eqz mutant cancers did not reveal a significant difference in tumor stage but differed in the frequency ofmsi cases p pole mutant tumors were morefrequent in the msi group than in themss group in addition the frequency ofmsi cases in pole mutant tumors differed between the cancer types p supplementary table 2b c although msi is enriched in samples with highmutation levels supplementary table 2d as expectedp were10fold higher mutation countsobserved in cancers with eqz mutations compared tomsi without eqz mutations supplementary figure these results suggest that mutations in eq ez and eqz confer a better prognosis independent of msi statusand tmn stage in colorectal endometrial and stomachadenocarcinomaswe next examined the amount ofthe cancerassociated immune infiltrate the immune scoreobtained through estimate corresponded tothe categorical score of the immune infiltrate derivedfrom digital he images supplementary fiure therefore we used the estimate immune scoresfor further analysis of colorectal endometrial andstomach cancers as shown in fig 4a a significantdifference was observed in the median immunescores between groups with lowintermediate andhigh mutation burden grouped based on mutationburden and not on e q z mutantseemethods and as expectedthe median immunescores increased with total mutation levels surprisingly the immune scores in eqz mutant tumorsdid not differ significantly from tumors with a lowlevel of mutationsfig 4b as expected msitumors possessed higher immune scores than msstumors p immunescores of msi and eqz mutation tumors weresimilar to those in the msi group and higher thanmss and eqz mutation tumors fig 4d withinthe group of tumors with pole exonuclease domainmutations mss tumors possessed lowerimmunescores than msi tumors but the difference was notsignificanttogether results in this tcga cohort demonstratethat the immune response is driven by msi ratherthan pole exonuclease domain mutationssupplementary fig 4c finallystatusp figurediscussionan analysis of dna polymerases in tumors withmutations in the pole revealed additional mutations inspecific polymerases most commonly in polq andpolzrev3l among the cancer types colorectaluterine and stomach cancer were mostfrequentlyafflicted by these mutations cancers with mutations inpole and polq eq pole and polzrev3l ezand in all polymerases eqz were associated withthe highest mutation burden and an excellent prognosisindependent of msi status and tumor stage mutationsin the exonuclease domain were observed in of eqz mutant tumors but only in of poleonly mutant tumors or in of eq ez tumorshowever despite harboring 10fold more mutationsthan msi tumors and 8fold more mutations than themutation frequencies associated with poleonly mutanttumors eqz mutant tumors did not display significantly more inflammationthe main result from that analysis is that patients withcolorectal stomach and endometrial cancers bearing eq ez and eqz mutations have disease free survival dfs at a median follow up time of months incontrast patients with tumors bearing mutations inpole only most of which outside the pole exonucleasedomain had a dfs of at follow up of monthsthe favorable dfs in eq ez and eqz mutated tumors occurred even in tumors with mutations in polethat are located outside the pole exonuclease domainthe contribution of mutations in pole to tmb casubstitutions and cancer type associations are describedin table of raynor using a larger resourceof cases and should be used to interpret the mutationsin the current study listed in supplementary figure as a whole the current study expands the spectrum ofpole mutant tumors with an excellent prognosis thefavorable prognosis included patients with high tumorstage which echoes prior studies demonstrating a favorable outcome of uterine tumors with pole exonucleasemutations despite adverse standard clinicopathologicindicators including high grade high stage and lymphovascular invasion [ ] while the high mutationfrequencies may cause an early growth advantage as tumors evolve they may succumb to high mutationburden as new mutations can no longer be tolerated andcause tumor cell death [ ] or increased sensitivityto therapeutic agentsthe prevailing hypothesis for the favorable prognosisof cancers displaying the hypermutator phenotype is theincreased attack by the immune system evidence insupport of this theory is the observation that tumorinfiltrating and peritumorallymphocytes are increased and that cytotoxic activities in cd8 and cd4are heightened in polelymphocyte populations 0chuang bmc medical genetics page of fig estimate immune scores by mutation frequency quartiles eqz mutation groups and msi in colorectal endometrial ucec and stomachstad cancers a estimate immune scores in cancers within high intermediate and low overall mutation quartiles b immune scores of sampleswith eq ez and eqz mutations compared to the low mutation quartile from panel a c immune scores in groups of cancers separated by msistatus d immune scores in msi and mss eqz cases compared to all other msi cases for each panel the number of cases within each group isincluded in parentheses on the xaxismutated endometrial cancers [] similar to hypermutated msi tumors this observation has led tothe hypothesis that immune checkpoint inhibitors maybe efficacious in pole ultramutated tumors ourresults question a direct relationship between mutationburden tumor immune response and pdl1 expressionalso raised in a larger study across cases from cancer types in tcga while we observed a concordance between the computational and histological assessments of the immune infiltrate the immune score intumors with eqz mutations depended on msi statusthis result suggests that the immune infiltrate attributable to mutations in eqz mutant tumors may be lessor that its composition may involve immune cells otherthan lymphocytes lesser cd8 and gammainterferongene expression signatures have also been observed ingastrointestinal tumors with a large single nucleotidevariantsnv burden that was attributed largely topole exonuclease mutations perhaps eqz mutations occur at a later point in tumor evolution whenimmunosuppressive factors already dominate we alsocannot rule out the possibility of increased numbers ofcytotoxic lymphocytes intermixed with eqz mutanttumor cells because computational methods and inspection of he images are not sensitive enough to detectsmall differencesinfiltrating lymphocytestils that may have large antitumoral effectsin tumora significant limitation of the study lies in the relatively small number of tumors this limitation cautionsthe generalization of results and seemingly novel insights 0chuang bmc medical genetics page of into the hypermutator phenotype studies by othergroups attributed hypermutator phenotype to specificmutations primarily within the pole exonucleasedomain given the proofreading function of the exonuclease domain it makes sense that mutations outsidethe domain have a lesser effect on tmb in agreementwith this concept our study reveals that compared totumors harboring only a mutation in pole polesinglemutant tumors of cases pole exonucleasedomain mutations are more common in tumors withboth double ez and eq and triple eqz dnapolymerase mutations of cases and doubleand triple mutant tumors have higher mutation countsthan pole single mutant tumors while it cannot befully excluded that polq and polzrev3l mutationsare bystander events in pole mutanttumors weobserve a higher tmb in cases with mutations in allthree polymerases supplementary figure 2a and bmechanistically polq and polz are thought to function in different repair processes polq in alternativemicrohomologymediated nonhomologous dna repair pathway and polz in translesion dna synthesishow these dna repair processes cooperate with thereplicative dna polymerase pole to prevent genomeinstability remains unknown this will be an importantsubjectthe mechanismfor further understanding ofunderlying the hypermutator phenotypesif validated in additional cohorts our findings may haveimportant clinicalimplications they build upon andexpand the previously well documented good prognosticimpact of pole exonuclease mutationsin uterinecancer that have generated intense interest in part dueto the paradox of a favorable prognosis in tumors withpathologic indicators of poor prognosis while in thisstudy prolonged dfs is observed in colorectal endometrial and stomach cancers with eqz mutations thisis not the case in other noncarcinoma cancer typeswithin tcga thus we find that the positive outcomeprediction is cancer type specific altogether resultsfrom this study provide a rationale for including polqandor polzrev3l mutations in clinical outcomestudies of tumors with pole mutations however future validation is required to confirm the concept that isrevealed in the current studycolorectal core stomach stad and endometrial ucec cancers byspecific polymerase mutated groups in tcga data sets supplementaryfigure locations of mutations in pole polq and rev3l exomes inindividual colorectal core stomach stad and uterine cancers ucecsupplementary figure relationships between mutation spectrumand mutation counts pole polq and rev3l exome mutations msi andtumor stage of individual cases supplementary figure mutationrates per mb yaxis of core stad and ucec cases with msi and eq ez and eqz xaxis mutations supplementary figure relationshipbetween pathology inflammation score and estimate immune scores forcore stad and ucec supplementary figure estimate immunescores in colorectal core endometrial ucec and stomach stadcancers supplementary table number of cases with pole polq zrev3l or multiple exome mutations in the pancan cohortsupplementary table contingency tables showing number of casesof colorectal endometrial and stomach cancers in each categoryabbreviationspole polymerase epsilon polq polymerase theta polzrev3l rev3 likedna directed polymerase zeta polz catalytic subunit tcga the cancergenome atlas msi micro satellite instability mss micro satellite stabilitytmb tumor mutation burden tnm tumor lymph node metastasis estimate estimation of stromal and immune cells in malignant tumor tissuesusing expression dataacknowledgementsnot applicableauthors contributionsall authors have read and approved the content of this manuscript fhstudy design and data analysis ht data interpretation jr data analysis andmanuscript writing bsk data interpretation and manuscript writingfundingthe prostate cancer foundation funded salaries of fh and bsk forcomputational analysis steven spielberg team science award andr01ca131255 bsk funded salaries for data analysis and paper writing of fhand bsk we also acknowledge the institutional support of salaries throughthe nih g20 rr030860 to the cedarssinai biobank and translational research core for salaries of bsk and fh the content of this publication doesnot necessarily reflect the views or policies of the department of health andhuman services nor does any mention of trade names commercial products or anizations imply any endorsement by the us governmentavailability of data and materialssequencing data can be obtained from national cancer institue gdc dataportal and are published in raw genomic and clinical data can befound at the nci genomic data commons httpsportalgdccancergovlegacyarchive the mc3 mutation annotation file can be accessed at httpsgdccancergovaboutdatapublicationsmc32017 and the processed datafiles can be viewed at httpsgdccancergovaboutdatapublicationspancanatlasethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors do not declare any competing interestssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12881020010899additional file supplementary figure number of cases polemutations n and mutations in the exomes of dna polymerasegenes in pancan supplementary figure mutation counts forauthor details1department of biomedical sciences cedarssinai medical center losangeles ca usa 2samuel oschin cancer research institute soccicedarssinai medical center los angeles ca usa 3surgerycedarssinai medical center los angeles ca usa 4pathology andlaboratory medicine cedarssinai medical center los angeles ca usa 5department of pathology university of utah salt lake city ut usa 0chuang bmc medical genetics page of received january accepted july referenceskandoth c schultz n cherniack ad akbani r liu y | 0 |
RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomens Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the seed and soil theory was ï¬rst proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells seeds leave their primary site of origin and spreador metastasize the microenvironment soil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve assoil for tumor development and might also serve as a seed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on theï¬eld of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identiï¬ed receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieï¬yintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and eï¬cienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentiï¬ed as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong TDC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deï¬ciency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKLRANK interactionsand blocking bone resorption The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identiï¬edas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a speciï¬c fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The eï¬cacy of Denosumab has been conï¬rmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspeciï¬c Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeï¬cient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyers plaque structures Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeï¬cient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inï¬ammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL speciï¬cally in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinï¬ammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a signiï¬cant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespeciï¬c deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspeciï¬c rankldeï¬cient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deï¬ciency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a speciï¬c antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to speciï¬cally eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the ï¬rst to reportthat during pregnancy RANKL deï¬ciency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκBcyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and ampliï¬edproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also signiï¬cantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identiï¬ed SNPs which were signiï¬cantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in signiï¬cantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Ampliï¬cation of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also signiï¬cantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which speciï¬cally inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will beneï¬t notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was signiï¬cantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no signiï¬cant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinï¬ltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand signiï¬cantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports ï¬ndings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thismicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas ï¬broblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a vicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs ï¬broblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer prostate cancer and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANKs cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinï¬ltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are signiï¬cantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a signiï¬cantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassigniï¬cantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs Moreover the beneï¬t of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the vicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a signiï¬cantimprovement in overall survival In these patients nostatistically signiï¬cant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab signiï¬cantly improved progressionfree survivalPFS Whether this survival beneï¬t is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the ï¬rst clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a signiï¬cant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ··] p A median followup of months showeda signiï¬cant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI Cox p These data suggest that adjuvant Denosumabcan signiï¬cantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratiï¬cations eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial conï¬rmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso signiï¬cantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment signiï¬cantly reduced the time to bonemetastasis at the site of ï¬rst occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe ï¬eld of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the bodys own immunesystem to seek out speciï¬cally target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as | 2 |
colorectal cancer crc is one of the most common malignanttumors in china chen crc is one of the ï¬veleading causes of cancer death and its incidence is graduallyincreasing owing to obesity and lifestyle changes du chen postoperative treatments includingchemotherapy and radiotherapy are important for longer patientsurvival traditional chinese medicine tcm has become anoption for preventing crc metastasis and enhancing the eï¬ectsof chemotherapy shi xu xie tcm is used as an alternative or supplementary treatmentin the united states and europe and has been widely used totreat various diseases in asia especially in china wang tcm has also been widely investigated in asia for eï¬ectiveand lowtoxicity monomer compounds to develop new drugs forcancer therapy and to counteract drug resistance sui zheng xie in china patients usually choose tcm for adjuvant therapyafter curative resection xu the eï¬ectiveness oftcm has been proven in multiple cancers including breastcancer lee hepatocellular carcinoma chen pancreatic cancer kuo and crc shi xu in crc tcm significantlyimproved diseasefree survival in stage ii and iii crc in aretrospective cohort study including patients shi in a multicenter prospective cohort study including patients with stage ii and iii crc postoperative tcmtreatment was associated with better diseasefree survival andoverall survival compared to those of the untreated groupxu certain active ingredients in tcm herbsmay have stronger activity in inhibiting cell proliferation andpromoting cell apoptosis tan huang and hu for example bufalin an active component of the tcmchan su can reverse multidrug resistance by inhibiting theprotein expression and eï¬ux function of abcb1 yuan cinobufagin another cardiotonic steroid isolated fromchan su suppresses tumor neovascularization by disrupting theendothelial mtorhif1α pathway to trigger reactive oxygenspeciesmediated vascular endothelial cell apoptosis li of the frequently used tcm treatments the most eï¬ectivesingle herbs are ginseng radix ren shen hedyotis diï¬usa willdbai hua she she cao scutellaria barbata ban zhi lian andastragali radix huang qi lee wu however the underlying mechanisms of these remedies remainunknown network pharmacology can eï¬ciently and quicklyidentify the interactions between drugs and target proteinsproviding a foundation for tcm application zhang fufang yiliu yin fyy is a tcm formula that has beenused in clinical practice for cancer treatment our previousstudy found that fyy inhibited cell proliferation migration andinvasion and promoted apoptosis in hepatocellular carcinomayang fyy contains eight herbs astragali radixhuang qi ganoderma lucidum ling zhi semen armeniacaeamarum ku xing ren h diï¬usa willd bai hua she she caoaconiti lateralis radix praeparata fu zi glycyrrhiza glabralinne gan cao radix panacis quinquefolii xi yang shenfyy inhibits colorectal cancer progressionand platycodi radix jie geng of these herbs radix panacisquinquefolii ginseng radix h diï¬usa willd and astragaliradix are commonly used in anticancer formulas lee wu g lucidum and platycodi radix alsoreportedly have anticancer eï¬ects radix astragali jung g lucidum dai platycodi radix park andlee and h diï¬usa willd zhang inhibitcancer cell proliferation polysaccharides in g lucidum inhibitthe proliferation of crc cells upregulating the expression of p21protein and blocking cells at the g2m phase na in the current study we investigated the anticancer eï¬ectof fyy on crc cells in vitro and in vivo and a networkpharmacology analysis was performed to explore the potentialmolecular mechanisms the information obtained in this studywill aid in elucidating the previously unavailable mechanismsof action of fyy in crc and developing fyy as an adjuvanttherapy for crcmaterials and methodspreparation of fyy and cell culturethe components of fyy conformed to the provisions stated bythe chinese pharmacopoeia fyy was prepared at the weifanghospital of traditional chinese medicine shandong chinayang fyy mgml was stored at ¦c untiluse and was further diluted to the required concentrations insubsequent cell experiments human crc cell lines hct116and sw480 were purchased from the cell resource center of theshanghai institutes for biological sciences chinese academy ofsciences shanghai china hct116 cells were grown in rpmi medium rpmi1640 hyclone united states and sw480cells were grown in dulbeccos modiï¬ed eagles medium dmemhyclone united states containing fetal bovine serumfbs gibco brl united states and penicillinstreptomycinsigmaaldrich st louis mo united states in co2 at ¦cin a humidiï¬ed incubatorcell viability and colony formationassayscells per well were seeded into 96well plates andincubated overnight at ¦c co2 in a humidiï¬ed incubatorwhen the cells adhered to the wall hct116 and sw480 cellswere treated with or mgml of fyy or pbs asa control for and h cell viability was measured using acell counting kit8 cck8 beyotime institute of biotechnologyinc shanghai china ten microliters of the cck8 solutionwas added to each well and then samples were incubated at ¦cfor h finally the absorbance value at nm was determinedusing a multiskantm fc microplate photometer thermo fisherscientiï¬c inc united stateshct116 and sw480 cells were treated with or mgmlof fyy or pbs as a control for h the cells perwell were then cultured in sixwell plates and the medium waschanged every days for days cell colonies were ï¬xed with paraformaldehyde and then stained with giemsa beyotimeinstitute of biotechnology inc shanghai china for minfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiona colony formation assay was performed to count viable colonies cells per colonycell cycle analyseshct116 and sw480 cells were treated with or mgmlfyy or pbs as a control for h the collected cells were ï¬xed in cold ethanol and stored at ¦c overnight the nextday the cells were washed twice with cold pbs then µlrnase a µgml and µl propidium iodide µgmlsigma aldrich st louis mo united states were added to eachsample and incubated for min in the dark measurements weretaken using a ï¬ow cytometer facscan bd biosciences bedfordma united states and the data were analyzed using flowjo software tree star inc ashland or united statescell apoptosis analysescell apoptosis was detected using an apoptosishoechst staining kit beyotime biotechnology shanghai chinasamples were ï¬xed with paraformaldehyde atroomtemperature for min and stained with mgml hoechst at room temperature for min then ï¬uorescencewas detected under an olympus ix50 microscope olympuscorp tokyo japan at magniï¬cation apoptotic cellswere identiï¬ed using an alexa fluor annexin vdead cellapoptosis kit invitrogentmmolecular probes eugene orunited states after centrifugation at g for min the celldensity was counted and diluted in annexinbinding buï¬erto obtain cellsml µl per assay cells were stainedwith µl of annexin vfitc and µl propidium iodide atroom temperature for min in the dark and then µl ofbinding buï¬er was added measurements were taken using a ï¬owcytometer and the data were analyzed using flowjo softwarenetwork pharmacologyactive fyy compounds were screened using the traditionalchinese medicine systems pharmacology database tcmsp1ru with the pharmacokinetic information retrievalï¬lter based on the tcmsp platform the oral bioavailabilityand druglikeness were set to ¥ and ¥ to obtainqualiï¬ed herbal compounds the chemical structures of thecompounds were drawn using chembiooï¬ce kerwin crc targets were predicted and screened using thegenecards database2 stelzer and omim platform3amberger and hamosh venny venny wasused to screen for common targets between fyy and diseaserelated targetsdrug compounddiseasetarget networks were built usingcytoscape v software shannon and themerge function was used to analyze the core compoundsprotein interaction networks of the common fyy and crcrelated targets were built using the string database platform1httptcmspwcomtcmspphp2httpswwwgenecards3httpswwwomim4httpbioinfogpcnbcsicestoolsvennywith medium conï¬dence and rejecting the target proteinindependent of the network szklarczyk gene ontology go analysis and kyoto encyclopediaof genes and genomeskegg pathway analysis wereperformed using metascape zhou enrichedgo terms and relevant pathways with pvalues wereselected for better prediction and veriï¬cation of the biologicalprocess and mechanismwestern blot analysisthe following primary antibodies obtained from cell signalingtechnology inc danvers ma united states were used inthe immunoblotting analysis pi3k p110α akt pan pakt ser473 bcl2 bclxl bax p21 cmyc andgapdh total proteins were extracted fromcells and tissues using ripa lysis buï¬er cwbio beijing chinaequal amounts of protein from each sample were separatedby sdspage electrophoresis and then transferred onto045µm pvdf membranes biorad laboratories herculesca united states subsequently the membranes were blockedwith milk in pbs plus tween pbst for minincubated with primary antibodies overnight at ¦c and thenincubated with goat antirabbit horseradish peroxidases abcamcambridge ma united states or goat antimousehorseradish peroxidases abcam cambridge ma united states for h at room temperature finallythe bandwas detected using an enhanced chemiluminescence reagentand visualized with a fusion fx7 system vilber lourmatfrance imagej software was used to calculate the intensity grayvalue of each protein band and gapdh served as a controlfor normalizationtumor xenografts in nude miceten male balbc nude mice weeks old ± gwere purchased from beijing vital river laboratory animaltechnology co ltd beijing china the mice were housedat ± ¦c under a 12h lightdark cycle with free accessto food and water all animal experiments were completedat the speciï¬cpathogenfree medical animal laboratory of theaï¬liated hospital of qingdao university and approved bythe animal ethics committee of the aï¬liated hospital ofqingdao university ahqu20180310a hct116 cells cells per tumor were subcutaneously injected into the rightarmpit of the nude mice seven days after tumor inoculationthe tumor size was measured using a vernier caliper andthe mice were divided into two groups the fyy treatmentgroup and a control group n mice per group thefyy group was intragastrically administered ml10 g bodyweight daily in a primary concentration of mgml thecontrol group was intragastrically administered an equivalentvolume of pbs tumor sizes were measured every daysand calculated using the following formula tumor volumemm3 length width2 the nude mice werekilled by cervical dislocation on day and the tumorswere excised weighed and photographed finallytumorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontissue and liver tissue were stored in formalin or at¦c for subsequent immunohistochemistry or western blotanalyses respectivelyimmunohistochemistrytumor and liver tissues of the nude mice were ï¬xed with paraformaldehyde for h and then embedded in paraï¬nembedded paraï¬n sections were dewaxed in xylene andrehydrated in ethanol antigen retrieval was performed in m citrate buï¬er ph using a pressure cooker followed byincubation for min samples were then washed thrice with pbsand ï¬xed in ethanol for min ki67 antibody novuscolorado united states was stained with a streptavidinperoxidase detection kit zsgbbio beijing china accordingto the kit instructionsstatistical analysisdata analysis was performed using graphpad prism softwaresan diego ca united states all experimental data wereexpressed as the mean ± sd the statistical signiï¬cance of theresults was analyzed by oneway analysis of variance anovafor multiple group comparisons and students ttest for two groupcomparisons a value of p was considered statisticallysignificant all experiments were performed in triplicateresultsfyy inhibited proliferation and promotedapoptosis of crc cells in vitrofufang yiliu yin significantly inhibited the growth of hct116and sw480 cells in a dosedependent manner figure 1a thecolony formation assay showed that the number of the coloniesin the fyy group and mgml was lower than that of thecontrol group figure 1bcolony formation ability was significantly inhibited by mgml p and mgml p fyy forhct116 and for sw480 cells respectively the cell cycle analysisshowed no significant diï¬erence in the percentage of cells ins p for mgml and g2m phases p for mgml in hct116 however a significant increase in g0g1phase was found after treatment with increasing concentrationsof fyy p for mgml figure 2a in hct116similar results were obtained for sw480 cells fyy blockedcell cycle at the g0g1 phase in a concentrationdependentmanner fyy inhibited the expression of cmyc p for mgml and promoted the expression of p21 protein p for mgml figure 2b in hct116 similar results wereobserved in sw480 cells this indicated an inhibitory eï¬ect oncell proliferationcell apoptosis as shown by hoechst staining increased afterfyy treatment figure 3a flow cytometry analysis showedthat the early p for mgml and late apoptosisp for mgml of hct116 cells were significantlypromoted figure 3b by fyy treatment similar results wereobtained for sw480 cells figure 3bnetwork pharmacological analysis offyy targeting crca total of compounds from fyy were retrieved oralbioavailability ¥ and drug likeness ¥ from the tcmspdatabase supplementary table a total of genes related tothese compounds and genes related to crc were screenedout using venny figure 4a common targets wereobtained supplementary table data imported into cytoscape to construct compounddiseasetarget networks figure 4a showed that of the fyy compounds may aï¬ect disease targets the top core compounds were screened based on the topologicalproperties of degree as shown in table quercetin kaempferolluteolin betasitosterol isorhamnetin formononetin calycosinjaranol acacetin and naringenin were the top active fyyingredients against crc the other active compoundsare listed in supplementary table two networks wereconstructed for the top core compounds and the remaining active compounds figure 4a the proteinprotein interactionnetwork built using string software used to investigatethe mechanisms of fyy provided common targets aftersetting the conï¬dence level above figure 4b theprioritization of key targets was analyzed according to thedegree of the node exported from the string database andthe top ï¬ve targets were cyclind1 mapk8 egfr myc andesr1 figure 4cbiological function and pathwayenrichment of fyy on crcthe biologicalfunctions and signaling pathways from allcore targets were enriched the top biological enrichmentresults are shown in figure 4d fyy aï¬ected crc throughmultiple go biological processes including apoptotic signalingpathway response to steroid hormone and response to inanicsubstance kegg analysis results included cancer prostatecancer apoptosis and pi3kakt signaling pathwayswe further investigated how the fyy mechanism promotedapoptosis using rtpcr and western blot analysis of hct116and sw480 cells fyy inhibited the relative expression ofpi3k mrna p figure 5a fyy downregulated theexpression of pi3k pakt bcl2 and bclxl and upregulatedthe expression of bax p figures 5bc takentogether these data support the idea that fyy induces crccell apoptosis by modulating the pi3kakt pathway and bcl2family proteinsfyy inhibited tumor growth and cellproliferation in vivothe hct116 cell xenograft model used to investigate theantitumor eï¬ect of fyy showed that fyy significantlyinhibited tumor growth compared to the control figure 6athe average tumor volumesafter days oftreatmentwere ± mm3in the control group and ± mm3 in fyytreated group figure 6b whiletumor weights were ± and ± mgrespectively ki67 significantly decreased in the fyytreatedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited colorectal cancer cell proliferation a cck8 assay indicated that fyy inhibited the proliferation of hct116 and sw480cells in a dose and timedependent manner after and h of treatment pbs was used for the control treatment n per group b colony formation abilitydecreased after treatment with different concentrations of fyy for both hct116 and sw480 n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy significantly inhibited the colorectal cancer cell cycle a fyy significantly inhibited the cell cycle progress of hct116 and sw480arresting them at the g2m phase as shown by ï¬ow cytometry assay n per group b the expression of cmyc decreased and p21 increased with fyytreatment n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained usinganovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy promoted colorectal cancer cell apoptosis a hoechst staining analysis indicated that fyy promoted apoptosis includingchromatin condensation and nuclear fragmentation in hct116 and sw480 cells magniï¬cation b flow cytometry indicated that fyy promoted the earlyand late apoptosis of hct116 and sw480 cells n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure network pharmacological analysis and biological functional enrichment analysis of fufang yiliu yin fyy a venn diagram showed common targetsof fyy in colorectal cancer crc compounddiseasetarget networks of fyy against crc b proteinprotein interactions identiï¬ed by string software c thepredicted key targets of fyy treatment of crc d go and kegg pathway enrichment analysesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontable the top bioactive compounds of fufang yiliu yin are listed below according to the degree of similarity of the compounddiseasetarget networkspubchem cidmolecule namequercetinformulac15h10o7ob dlkaempferolc15h10o6luteolinc15h10o6degreestructurebetasitosterolc29h50oisorhamnetinc16h12o7formononetinc16h12o4calycosinc16h12o5jaranolc17h14o6acacetinc16h12o5naringeninc15h12o5glycyrolc21h18o67methoxy2methyl isoï¬avonec17h13no5continuedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong table continuedpubchem cidmolecule name7omethylisomucronulatolformulac18h20o5ob dllupiwighteonec20h18o5glyasperin fc20h18o6fyy inhibits colorectal cancer progressiondegreestructureob oral bioavailability dl druglikenesscrc tumor xenograft group figure 6b the expression ofpi3k pakt bcl2 and bclxl followed the same trend asthe in vitro study results figure 6cdiscussionboth retrospective and prospective studies have proven theanticancer eï¬ects of tcm on crc shi xu here we reported the anticancer eï¬ect of the fyyformula which contains eight ingredients fyy significantlyinhibited cell proliferation and promoted crc cell apoptosisin vitro fyy also inhibited xenograft tumor growth in vivousing a network pharmacology analysis we found that fyymay act on crc through active compounds targeting crcrelated genes that regulate the apoptosis and pi3kaktsignaling pathwaysto better understand the complementary eï¬ects of fyyformula ingredients we retrieved a total of compounds fromthe tcmsp database supplementary table compounddiseasetarget networks showed that of the compoundsmay aï¬ect crcrelated targets by searching pubmed wethe top compounds table exhibit antifound thatcrc eï¬ects mainly by promoting apoptosis and inhibiting cellproliferation for example quercetin was mostly related toprotective eï¬ects against crc and is found in three of the eightremedies in fyy astragali radix huang qi h diï¬usa willdbai hua she she cao and g glabra linne gan cao quercetininhibits crc progression by promoting cell apoptosis andautophagy as well as inhibiting angiogenesis and inï¬ammationdarband quercetin induces apoptosis by inhibitingdiï¬erent signaling pathways including the mapkerk pi3kaktand nfκb signaling pathways zhang xavier it also inhibits the migration and invasion of crc cells viaregulating the tolllike receptor 4nfκb signaling pathway han further kaempferol induces crc cell apoptosischoi while isorhamnetin formononetin andnaringenin show anticancer eï¬ects by inhibiting cell proliferationli abaza the similarity of theeï¬ects provided by fyy compounds may provide a mutualenhancement eï¬ect but this must be further tested using singleor mixed compoundsfufang yiliu yin induced cell cycle arrest in crc cells at theg0g1 phase and promoted apoptosis in hct116 and sw480cells to explain the mechanism by which fyy inhibits cellproliferation and promotes apoptosis we performed proteinprotein interaction network kegg and go pathway analysesproteinprotein interaction network analysis indicated the topï¬ve targets were cyclind1 mapk8 egfr cmyc and esr1biological functional analysis indicated apoptosis and cancerrelated pathways including the pi3kakt signaling pathwaythen our experimental study conï¬rmed the activation ofthe pi3kakt pathway and bcl2 family proteins as well ascmyc expressiontraditional chinese medicine formulas reportedly inhibitcancer progression by diï¬erent signaling pathways a tcmformula jianpi jiedu inhibits crc tumorigenesis and metastasisvia the mtorhif1αvegf pathway peng another tcm formula huang qin ge gen tang enhances the ï¬uorouracil anticancer eï¬ect by regulating the e2f1ts pathwayliu the zhi zhen fang formula reverses multidrugresistance mediated by the hedgehog pathway in crc sui these formulas as well as fyy all contain astragaliradix huang qi h diï¬usa willd bai hua she she caog glabra linne gan cao and radix panacis quinquefolii xiyang shen however there have been no reports regarding theanticancer eï¬ect of tcm formulas acting through the apoptosisand pi3kakt pathways in crc figure in the current studywe found that fyy decreased the transcription and protein levelof pi3k figure and further inhibited the phosphorylationof akt in both the cells and tumor tissues figures accumulating evidence indicates that the pi3kakt pathwayplays an important role in tumor development pi3k can partiallyactivate akt at the thr308 or ser473 sites by inducing thetranslocation of akt to the cell membrane via phosphoinositidedependent kinase akt inhibition is usually indicated by afrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy modulated the expression of the pi3kakt signaling pathway and bcl2 family proteins relative pi3k mrna expression wasaltered by fyy treatment in hct116 and sw480 cells a n per group expression levels of pi3k akt pakt bcl2 bclxl and bax were altered by fyytreatment in hct116 b and sw480 cells c n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited tumor growth in vivo a subcutaneous xenograft tumors after days demonstrated that fyy inhibited xenograft tumrowth n per group b tumor volume was significantly smaller after days of fyy treatment n per group ihc analysis of ki67 expression infyytreated tumor and liver tissues magniï¬cation the pvalues were obtained using anova c protein expression levels of pi3k akt pakt bcl2bclxl and bax in tumor tissues n per group values are shown as the mean ± sd p and p vs control group the pvalues were obtainedusing students ttestdecrease in the pakt ser473 level and is mostly achievedby inhibiting pi3k using pi3kspeciï¬c inhibitors ly294002or wortmannin reener and marti the regulation ofpi3kakttranscription and protein expression by a tcmtreatment has been previously reported tcm interventiondecreased pakt levels following the concentration gradientof the tcm treatment while the total overall akt level wasunchanged gu zhao calycosina component of astragali radix reportedly inhibits crcproliferation through the erβmediated regulation of the igf1rand pi3kakt signaling pathways zhao quercetinkaempferol and rutin in h diï¬usa willd also exhibit anticancereï¬ects in crc by regulating the pi3kakt signaling pathwaycai frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure schematic representation of the proposed pi3kakt signalinginduced cell cycle arrest and apoptosis triggered by fufang yiliu yin fyy by combiningthe network pharmacological analysis and our results we hypothesized that fyy activates the pi3kakt signaling pathway and modulates the expression of p21cmyc and bcl2 family proteins thereby inducing cell cycle arrest and apoptosiscellapoptosisandinhibited metastasiswe previously found that fyy inhibited cell proliferationofpromotedhepatocellular carcinoma yang fyy may havea similar eï¬ect on diï¬erent types of cancer although wedemonstrated both the anticancer eï¬ects of fyy and the actionmechanism by which it operates limitations of this study includethe following ï¬rst we did not investigate the antimetastaticeï¬ect of fyy on crc a migration and invasion assay andcrc liver metastasis model should be used to investigate thissecondfurther studies should investigate whether mutualenhancement eï¬ects exist between the applications of fyyand regular chemotherapy and also examine its eï¬ect ondrug resistancein conclusion our study ï¬ndings showed that fyy inhibitedproliferation and promoted apoptosis in crc cells by modulatingthe pi3kakt signaling pathway and bcl2 family proteins webelieve that fyy could be a promising adjuvant therapy for crcethics statementthe animal study was reviewed and approved by animalethics committee of the aï¬liated hospital of qingdaouniversity ahqu20180310a written informed consent wasobtained from the ownerstheiranimals in this studyfor the participation ofauthor contributionsbd and cz obtained funding conducted the research andprepared the manuscript zy and qj performed the experimentssz prepared and provided the fyy formula yw and hzperformed the network pharmacology analysis cs designed thestudy and interpreted the data all authors contributed to the and approved the submitted versiondata availability statementall data presented in thissupplementary materialstudy areincluded in thefundingthis work was supported by the china postdoctoral sciencefoundation grant numbers 2016m602098 and 2018m640615the taishan scholars program ofshandong provincefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiongrant number the shandong higher educationyoung science and technology support program grant number2020kjl005 the qingdao postdoctoral science foundationgrant number and the national natural sciencefoundation of china grant number supplementary materialthe supplementary material for this can be found onlineat httpswwwfrontiersins103389fcell202000704fullsupplementarymaterialreferencesabaza m s orabi k y alquattan e and alattiyah r j growthinhibitory and chemosensitization eï¬ects of naringenin a natural ï¬avanonepuriï¬ed from thymus vulgaris on human breast and colorectal cancer cancercell int doi 101186s1293501501940amberger j s and hamosh a searching online mendelian inheritance inman omim a knowledgebase of human genes and genetic phenotypes currprotoc bioinformatics doi 101002cpbi27cai q lin j wei l zhang l wang l zhan y hedyotis diï¬usawilld inhibits colorectal cancer growth in vivo via inhibition of stat3 signalingpathway int j mol sci doi 103390ijms13056117chen q shu c laurence a d chen y peng b g zhen z j eï¬ect of huaier granule on recurrence after curative resection of hcca multicentre randomised clinical trial gut doi 101016s0618chen w zheng r baade p d zhang s zeng h bray f cancerstatistics in china ca cancer j clin doi 103322caacchoi j b kim j h lee h pak j n shim b s kim s h reactive oxygen species and p53 mediated activation of p38 and caspases iscritically involved in kaempferol induced apoptosis in colorectal cancer cellsj agric food chem doi 101021acsjafc8b02656dai s liu j sun x and wang n ganoderma lucidum inhibitsproliferation of human ovarian cancer cells by suppressing vegf expressionand upregulating the expression of connexin bmc complement alternmed doi darband s g kaviani m youseï¬ b sadighparvar s pakdel f g attari j s quercetin a functional dietary ï¬avonoid with potential chemopreventive properties in colorectal cancer j cell physiol doi 101002jcp26595du z x jia x y and lin k colorectal cancer mortality characteristicsand predictions in china asian pac j cancer prev doi 107314apjcp201516177991gu z f zhang z t wang j y and xu b b icariin exerts inhibitoryeï¬ects on the growth and metastasis of kyse70 human esophageal carcinomacells via pi3kakt and stat3 pathways environ toxicol pharmacol doi 101016jetap201706004han m song y and zhang x quercetin suppresses the migrationand invasion in human colon cancer caco2 cells through regulating tolllikereceptor 4nuclear factorkappa b pathway pharmacogn mag s237s244doi huang c and hu g shikonin suppresses proliferation and inducesapoptosis in endometrioid endometrial cancer cells via modulating mir106bptenaktmtor signaling pathway biosci rep 38bsr20171546 doi101042bsr20171546jung y jerng u and lee s a systematic review of anticancer eï¬ectsof radix astragali chin j integr med doi 101007s116550152324xkerwin s m chembiooï¬ce ultra suite j am chem soc doi 101021ja1005306kuo y t liao h h chiang j h wu m y chen b c chang c m complementary chinese herbal medicine therapy improves survivalof patients with pancreatic cancer in taiwan a nationwide populationbasedcohort study integr cancer ther doi lee y w chen t l shih y r v tsai c l chang c c liang h h adjunctive traditional chinese medicine therapy improves survival inpatients with advanced breast cancer a populationbased study cancer doi 101002cncr28579li c yang x chen c cai s and hu j isorhamnetin suppresses coloncancer cell growth through the pi3kaktmtor pathway mol med rep doi 103892mmr20141886li x chen c dai y huang c han q jing l cinobufaginsuppresses colorectal cancer angiogenesis by disrupting the endothelialmammalian target of rapamycinhypoxiainducible factor 1α axis cancer sci doi 101111cas13988liu h liu h zhou z parise r a chu e and schmitz j c herbalformula huang qin ge gen tang enhances 5ï¬uorouracil antitumor activitythrough modulation of the e2f1ts pathway cell commun signal doi101186s1296401802181na k li k sang t wu k wang y and wang x anticarcinogeniceï¬ects of water extract of sporodermbroken spores of ganoderma lucidumon colorectal cancer in vitro and in vivo int j oncol doi103892ijo20173939park j c and lee y j in vivo and in vitro antitumor eï¬ects of platycodind a saponin puriï¬ed from platycodi radix on the h520 lung cancer cell evidbased complement alternat med doi peng w zhang s zhang z xu p mao d huang s jianpi jiedudecoction a traditional chinese medicine formula inhibits tumorigenesismetastasis and angiogenesis through the mtorhif1alphavegf pathwayj ethnopharmacol doi 101016jjep201805039reener m c and marti f the balancing act of akt in t cells front biol doi 101007s1151501212026ru j li p wang j zhou w li b huang c tcmsp adataba | 0 |
" trophoblast cell surface antigen trop2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion the association between trop2 expression and occurrence anddevelopment of oral squamous cell carcinoma oscc remains to be understoodmethods we investigated the role of trop2 in oscc patients using a combination of biophysical approaches atotal of oscc patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry kaplanmeier survival curve analysis and atomic force microscopy to analyzetrop2 expression morphology and mechanical properties of oscc tissuesresults trop2 was overexpressed in of poorly differentiated oscc samples high levels of trop2 wereassociated with survival rate lower than and patient age odds ratio [or] p confidence interval [ci ] tumor size or p ci [] and tnm stageor p ci [] average surface roughness of low medium and highly differentiatedoscc tissues were ± ± and ± nm respectively the pearson coefficient revealed anegative association between tumor stiffness and trop2 expression r p overexpression of trop2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics taken together our findings demonstrated that trop2 may be an indicator of osccdifferentiation leading to the altered mechanical properties of oscc tissueskeywords oral squamous cell carcinoma trop2 tissue stiffness differentiation survival oral squamous cell carcinoma oscc is a commonsubtype of head and neck and other malignant tumors[ ] the past few decades have shown increased incidence of oscc that is expected to rise further in the future thereforeimperative to determineisit correspondence zhangkllzu163com baoping zhang shuting gao and ruiping li contributed equally to thiswork1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu chinafull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of oscchuman trophoblast cell surface antigen trop2 alsocalled tumorassociated calcium signaltransduction2tacstd2 is a surface glycoprotein encoded by tacstd that has extracellular domains a single transmembrane domain and a short tail [ ] trop2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal pancreatic and laryngealcancers inhibiting trop2 expression has shownpromise in clinical applications [ ] trop2 regulates 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 bmc cancer page of tumorigenic properties including cancer cell adhesion invasion and migration tang have recentlyshown that trop2 impacts growth and metastasis byactivating pi3kakt signaling this phenomenon hasalso been observed in gallbladder cancer amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ ] this has shed light on the biological functions of trop2 and its use as a prognostic biomarker for osccvarious biochemical mechanismsatomic force microscopy afm is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesafm has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast liver and lung parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesthis study aims at investigating the correlation between trop2 expression and clinicopathological characteristics of oscc we have demonstrated the tissuemorphology and mechanics of oscc samples duringtumor development using afm we believe our findingswill help develop trop2 in accurately diagnosing osccin tumors with different grades of differentiationmethodstissue preparationthe protocols in this study were approved by the researchethics committee of lanzhou university tumor sampleswere collected from patients after obtaining written informed consent a total of patients with oral squamous cell carcinoma oscc were registered atthesecond hospital of lanzhou university between january and march among these samples sampleseach showed high moderate and low levels of differentiation the experimental group comprised males and females aged years average years all patientswere diagnosed with oscc based on surgery andfig paraffin pathological sections of tissues a d g 4fold b e h 10fold c f i 40fold 0czhang bmc cancer page of fig immunohistochemical staining was performed to detect the expression of trop2 at different stages of osccpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded cancer and cervical lymph nodetissues were collected after maxillofacial surgery all specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding among them and fig average optical density of trop2 poorly differentiated squamous cell carcinoma showed high expressionp 0czhang bmc cancer page of table correlation between trop2 expression and clinicopathological characteristicscharactersntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvascular invasionnoyespearson x2p value highpatients exhibited no and cervical lymph node metastasesrespectively clinical tnm staging was performed according to the 7th edition tnm staging classification standardjointly developed by the international union for cancercontrol and american joint committee on cancer and world health anization guidelines hematoxylin and eosin he stainingoscc tissues were fixed overnight using neutralformalin solarbio beijing china paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol the sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing solarbiobeijing china sections were visualized and imagedusing the olympus bx53 at magnifications of and sealing and neutral 0czhang bmc cancer page of immunohistochemistryhe sections were subjected to the sp method to detecttrop2 expression the sections were incubated overnight with the primary antibody against trop2 abcam usa at °c followed by incubation withbiotinlabeled goat antirabbitigg abcamusa at °c for h the sections were then developed using dab beijing zhongshan golden bridgebiotechnology china dehydrated transparentizationand film and neutral resin sealed the prepared sections were visualized using microscopy olympusbx53 japanafmfixed tissues were placed in petri dishes containingphosphatebuffered saline all analyses for mechanical properties were performed using the biologicalatomic force microscope bioafm nanowizard iiibruker usa silicon afm probes from the pointprobe®constant of nmcoating nanoworld usa werecontrreflexused the spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm afm was performed using theseries with a khzforcetheresonancefrequencyofcontact model and a scan rate of hzs in airforcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms six random sites wereselected for each sample and each site was measured times we used the modified hertz contact modelto analyze forcedistance curves and calculateyoungs modulus and roughness of oscc tissueswith varying differentiationstatistical analysisstatistical analyses were performed using spss statistical product and service solutions ibm forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the tukeykramerhsd test for pairwise comparisons pearson chisquaretest was used to analyze clinical features and trop2 expression based on the calculated odds ratios ors and confidence intervalci survival was evaluatedusing kaplanmeier curves and the difference was analyzed using the logrank test p was consideredstatistically significantfig trop2 total survival curve using kaplanmeier survival curves low blue line high green line 0czhang bmc cancer page of resultstissue morphology and trop2 expression across theclinical stages of oscctumor cells from poorly differentiated oscc samples exhibited characteristic atypia poor differentiation and irregular morphology fig howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated oscc as compared to those inpoorly differentiated cells trop2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells we observed that low differentiation and high malignancy of oscc was associated with higher trop2 expression fig theaverage optical density of trop2 among the lowmedium and highly differentiated oscc tissues were ± ± and ± respectively fig table trop2 expression risk factors with clinicopathological featurescharacteristicsntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvscular invasionnoyesnote a well vs moderate b moderate vs poor c well vs poorp valueor cihigh 005a 0001b 0001c 0czhang bmc cancer page of association between trop2 expression and clinicalcharacteristics of osccwe analyzed the clinicopathological characteristics of patients with oscc with varying degree of differentiationdifferential expression of trop2 was associated with patient age tumor differentiation tumor size tnm stagepercutaneous nerveinvasiontable p patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high trop2 expressionp however there was no association between theexpression of trop2 and patient gender tumor locationlymph node metastasis or distant metastases p and vascularfiltrationtrop2 expression and patient survivalusing kaplanmeier survival curves we observed that anincrease in trop2 expression negatively correlated withthe overall survival of patients fig and lowno oftrop2 expression groups 3years survival rate was a for high expression group and 5years ratewere and respectively trop2 expression wasassociated with patient age p or ci[] tumor differentiation well vs moderatep or ci [] moderate vspoor p or ci []well vs poor p or ci [] tumor size p or ci[] tnm stage p or ci[] vascular invasion p or ci [] and peripheral nerve invasionp or table high trop2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher tnm staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival thus trop2 may be a prognostic indicator for survival in oscc patientsfig surface morphology of oscc tissue sections via afm detection irregular morphology appeared in the low differentiation 0czhang bmc cancer page of surface morphology and roughness of oscc tissuesthe surface morphologies of oscc tissues with varying degrees of differentiation were analyzed directtopographical imaging using bioafm figure showsthe representative image from each tissue acquiredduring the cantileverbased afm nano indentationtest the tissue interface varied with tumor differentiationindicating that highly differentiated oscc tissues had a regular and flat morphology oscc tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization figure summarizes the roughness ofoscc tissues with varying differentiation the average surface roughness of low medium and highly differentiated oscc tissues were ± ± and ± nm respectively roughness ofthe tissue surface was enhanced with increasing differentiation of oscc tissuesyoungs modulus of oscc tissueswe used bioafm to determine youngs modulusbased on the mechanical properties of oscc tissues with varying degrees of differentiation we randomly selected six contact points from each slice andeach contact point was measured times forcedistance curves were generated for each slice and thejpk data processing software version was usedto calculate youngs modulus figure shows theaverage variation in stiffness within individual tissuesin the range of kpa in the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples p fig surface roughness results are express as mean ± sem nm 0czhang bmc cancer page of fig afm test average tissue stiffness youngs modulus e was thus confirmed to be a parameter of cell hardness for various cells and tissuepa p thus tissue differentiation was positively associated with its stiffness fig association between mechanical properties and trop2expression in osccthe pearson coefficient showed a negative associationbetween the stiffness of oscc tissues and trop2 expression fig r p thus we detectedan increase in stiffness with varying differentiation in thetumor samplesdiscussiontrop2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines studieshave shown that overexpression of trop2 is associatedwith tumorigenesis and malignancy []in thisstudy trop2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness the relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaimmunohistochemical analysis revealed that the expression of trop2 in poorly differentiated oscc tissueswas significantly higher than that in welldifferentiatedoscc tissues additionally trop2 upregulation wascorrelated with tumors of advanced tnm iii iv staging and poor differentiation than that in tumors withlow tnm i ii staging thus the abnormal expressionof trop2 may be associated with the occurrence anddevelopment of tongue malignancies furthermore hightrop2 expression predicted low survival as comparedto that in the tumors with low trop2 expression previous research has also demonstrated the correlation between shorter patient lifespan and high levels of trop2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of trop2 trop2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis upregulated trop2 correlates with the poor prognosis of thyroid papillary carcinoma colon cancer liver cancer and other malignanciesthere have been an increasing number of studies on thebiological role of trop2 at the molecular level trop2induces the downregulationloss of pten thereby stimulating pi3kakt signaling and tumor development pten is a wellknown tumor suppressor that is a phosphatase and affects the pi3kpkbakt signaling axisduring the dephosphorylation of pip2 and pip3 pi3k signaling is important in regulating tumor cell proliferation migration and invasion [ ] thus pten is anegative regulator of cancer [ ] li have shownthat trop2 activates epithelialmesenchymal transitionvia pi3kakt signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer similarly trop2 expression stimulates the proliferation migration and invasion of osteosarcoma cells hou demonstrated that trop2 regulates jak2stat3 signaling in glioblastoma cells 0czhang bmc cancer page of fig correlation analysis between changes in mechanical stiffness of oscc tissues and trop2 expression note changes have statisticalsignificance p and show a certain negative correlation r functional differentiation oftissues influences themicromorphology and mechanical stiffness of oscccells we detected low surface roughness on oscc tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion poorlydifferentiated oscc tissues are softer than highly differentiated oscc tissues pi3k is an important celladhesion molecule trop2 triggers the synthesis of proteins with homologous domains such as pleckstrinrac tiam and vav tiam and vav activate rac thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion the underlying mechanisms involved in the alterationof micromechanical properties of oscc samples and occurrence development metastasis and invasion ofoscc tumors remain to be elucidated he staining isthe gold standard for tumor diagnosis with the development of biomechanics in the past two decades the mechanical properties of tissues need to be investigated based on biomedical and physical parametersin this study we have assayed the changes in mechanicalproperties at the micronanometer level using afm anddetermined the association between the tnm grademetastasis and stiffness of tumor samplesin we have demonstrated the association between differential expression of trop2 and patient agetumor differentiation tumor size tnm stage percutaneousnerve filtration and vascular invasion moreover high levelsof trop2 correlated with poor overall survival in patientshighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness lastly high trop2 expression resulted in reduced tumor stiffness however thisstudy had some limitations first the cohort used in thisstudy was relatively small second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developtrop2 as a specific and efficient prognostic biomarker forosccthese findings could promote new methods for the earlyoscc diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity more detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseabbreviationsoscc oral squamous cell carcinoma trop2 trophoblast cell surfaceantigen afm atomic force microscopyacknowledgementswe thank the individual who participated in this studyauthors contributionsbz sg and rpl are responsible for conception and design data wascollected by ytl rc jyc and ymg data was analyzed by ew and yh klzrevised the all authors have read and approved the manuscriptfundingthis work was supported by the fundamental research funds for thecentral universities no lzujbky2020cd03 baoping zhang doctoralmaster 0czhang bmc cancer page of students of the second hospital of lanzhou university sdkygg17 lan yangand key laboratory of mechanics on disaster and environment in westernchina the ministry of education of china no kailiang zhangavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatewritten informed consent was obtained from each participant before samplecollection the study was approved by the committee for ethical affairs ofschool of stomatology lanzhou universityconsent for publicationnot applicablecompeting intereststhe authors have no conflicts of interestauthor details1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu china 2institute of biomechanics andmedical engineering lanzhou university lanzhou chinareceived april accepted august 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tang q jia l chen y lin l kuai x trop2 increasesgrowth and metastasis of human oral squamous cell carcinomathrough activation of the pi3kakt signaling pathway int j mol medtrerotola m li j alberti s languino lr trop2 inhibits prostate cancer celladhesion to fibronectin through the 1 integrinrack1 axis j cell physiolli t su y yu x mouniir dsa masau jf wei x trop2 guaranteescardioprotective effects of cortical bonederived stem cells on myocardialischemiareperfusion injury cell transplant stoyanova t goldstein as cai h drake jm huang j witte on regulatedproteolysis of trop2 drives epithelial hyperplasia and stem cell selfrenewalvia betacatenin signaling genes dev sun x xing g zhang c lu k wang y he x knockdown of trop2 inhibitsproliferation and migration and induces apoptosis of endometrial cancercells via aktcatenin pathway cell biochem funct lee h jang y seo j nam jm char k nanopfunctionalized polymerplatform for controlling metastatic cancer cell adhesion shape and motilityacs nano 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betweencylinders under inclined forces philos trans a math phys eng sci kowalsky ca faber ms nath a dann he kelly vw liu l rapid fineconformational epitope mapping using comprehensive mutagenesis anddeep sequencing j biol chem zeng p chen mb zhou ln tang m liu cy lu ph impact of trop2expression on prognosis in solid tumors a systematic review and metaanalysis sci rep calvo a xiao n kang j best cj leiva i emmertbuck mr alterationsin gene expression profiles during prostate cancer progression functionalcorrelations to tumorigenicity and downregulation of selenoproteinp inmouse and human tumors cancer res ju x jiao x ertel a casimiro mc di sante g deng s vsrc oncogeneinduces trop2 proteolytic activation via cyclin d1 cancer res cubas r li m chen c yao q trop2 a possible therapeutic target for latestage epithelial carcinomas biochim biophys acta zargari n mokhtari m evaluation of diagnostic utility ofimmunohistochemistry markers of trop2 and hbme1 in the diagnosis ofthyroid carcinoma eur thyroid j zhao p zhang z tnfα promotes colon cancer cell migration and invasionby upregulating trop2 oncol lett sin stk li y liu m yuan yf ma s guan xy downregulation of trop2predicts poor prognosis of hepatocellular carcinoma patients hepatolcommun zhang y zhang r luo g ai k long noncoding rna snhg1 promotes cellproliferation through pi3kakt signaling pathway in pancreatic ductaladenocarcinoma j cancer sai j owens p novitskiy sv hawkins oe vilgelm ae yang j pi3kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antipd1 responses clin cancer res chen x pang b liang y xu sc xin t fan ht overexpression of zhang xr wang sy sun w wei c isoliquiritigenin inhibits proliferation andepcam and trop2 in pituitary adenomas int j clin exp pathol metastasis of mkn28 gastric cancer cells by suppressing the pi3kaktmtor signaling pathway mol med rep 0czhang bmc cancer page of wise hm hermida ma leslie nr prostate cancer pi3k pten and prognosisclin sci lond yuan b zou m zhao y zhang k sun y peng x upregulation of mir130b3p activates the ptenpi3kaktnfκb pathway to defense againstmycoplasma gallisepticum hs strain infection of chicken int j mol sci pii e2172li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep li x teng s zhang y zhang w zhang x xu k trop2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating pi3kakt pathway and inducing emt oncotarget gu qz nijiati a gao x tao kl li cd fan xp trop2 promotes cellproliferation and migration in osteosarcoma through pi3kakt signalingmol med rep hou j lv a deng q zhang g hu x cui h trop2 promotes theproliferation and metastasis of glioblastoma cells by activating the jak2stat3 signaling pathway oncol rep rivard n phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function front biosci landmark ed pankova d jiang y chatzifrangkeskou m vendrell i buzzelli j ryan a rassf1a controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma embo j 20193813e100532 wullkopf l west av leijnse n cox tr madsen cd oddershede lb cancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential mol biol cell publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as san 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 peoples 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 natural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new eight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiangs 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 teams 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 αdaraf1residues 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 yangs 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 | 0 |
" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and its mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ ]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [] sbt oil which contains richfatty acids tocopherols Ï3 and Ï6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturers protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ²reverse ² ggccatcagcaacaacataa ²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining thickness in hestained tissue mast cell number in tb stained tissue c tb staining epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65leung dym new insights into atopic dermatitis j clin investig kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol feinberg h trimeric structure of langerin j biol chem zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889 natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunolpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a | 0 |
as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is conï¬rmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors mnx1 was ï¬rstly foundto be expressed in lymphoid and pancreatic tissues and deï¬nedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia furthermore recent studiesconï¬rmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these ï¬ndingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression proï¬ling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at ¦c in a humidiï¬ed incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the aï¬liated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were conï¬rmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university aï¬liated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraï¬nembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was conï¬rmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the ï¬nalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturers protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcrampliï¬ed and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the cell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at ¦c and ï¬xed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with à clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of à hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using ï¬uorescencemicroscopy and proliferation cell ratios were counted fromï¬ve random ï¬elds in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were ï¬xedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artiï¬cial scratch on a conï¬uent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were ï¬lled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were ï¬xed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from ï¬ve random ï¬elds onevery ï¬lter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension à cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the cell index valuecell cycle analysiscells were digested with trypsinedta and ï¬xed with ethanol for h at ¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at ¦c a facscalibur ï¬ow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0g1 s and g2m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientiï¬c and scraped into a centrifuge tube the cells were centrifugedfor min at g at ¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at à g ¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at ¦c the chip elution buï¬ercontaining proteinase k was used for dna puriï¬cation thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr ampliï¬cation with primersspeciï¬c to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region bp wasampliï¬ed and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice weeks old were purchased from nanjingmedical university school of medicines accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group à exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the speciï¬cation of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length à width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression proï¬ling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage iii vs iiiiv p figure 1f t stage t1 vst2t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two speciï¬c sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression eï¬ciency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 conï¬rmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3cg of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in transcriptionand metabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been conï¬rmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no signiï¬cance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines cf two speciï¬c sirna si1 and si2 of mnx1 were designed and the transfection efï¬ciencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot gi the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefï¬ciencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespeciï¬c dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by ï¬ow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no signiï¬canceaacaataaat and bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efï¬ciency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments bd the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efï¬ciency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacriï¬ced and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacriï¬ced andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no signiï¬cancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection eï¬ciency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6bf and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown eï¬ciency wasconï¬rmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7ce ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7hkfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identiï¬ed mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourï¬ndings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentiï¬ed to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebraï¬sh and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also conï¬rmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identiï¬ed as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identiï¬ed as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i | 0 |
"assess the antioxidative activity of seleniumenriched ChrysomyiaMegacephala Fabricius C megacephala larvae powder SCML and its impact on the diversity and structure ofintestinal microflora in a mouse model of Dgalactose Dgalinduced oxidative damageMethods Sixty male ICR mice were equally randomized to a normal control NC group a model group a positivegroup a lowdose SCML LSCML group a middose SCML MSCML group and a highdose SCML HSCMLgroup Animals in NC and model groups received water animals in the positive group received mgKg vitamin EVE and those in the three SCML groups received SCML which include and μgKg selenium Serespectively An oxidative damage model induced by subcutaneous injection of Dgal for weeks via the neck wasestablished Serum oxidative stress levels and tissue appearance were evaluated Tissues oxidative stress levels weredetected by commercially available kit Nuclear erythroid 2related factor Nrf2 and gut microbiota weredetermined by western blot and high throughput sequencing 16S rRNA gene respectivelyResults An oxidative damage model was established successfully as represented by a significant elevation ofmalondialdehyde MDA and protein carbonylation and inhibition of the antioxidants including superoxide dismutaseSOD glutathione peroxidase GSHPx total antioxidant capacity TAOC and glutathione GSH It was found thatoxidative damage and histological alterations were attenuated the expression of Kelchlike ECHassociated proteinKeap1 was decreased and the expression of Nrf2 and hemeoxygenase1 HO1 was increased after SCML treatmentIn addition significant changes were observed in the gut microbiota including Proteobacteria and the ratio ofBacteroidetes to Firmicutes at the phylum level as well as Helicobacter Clostridium and Lactobacillus at the genus levelContinued on next page Correspondence jiangzcmueducn Dandan Xie and Liqin Jiang contributed equally to this work1College of Pharmaceutical Science Zhejiang Chinese Medical University Binwen Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Complementary Medicine and Therapies Page of Continued from previous pageConclusion SCML exerted an antioxidative effect in vivo probably by increasing the antioxidant activity and reducingthe production of oxidation products via the Nrf2 signaling pathway SCML could also redress the intestinal floraimbalance induced by oxidative stress All these findings suggest that SCML could serve as a functional food andnatural drug additive to protect the human body against oxidative damageKeywords Seleniumenriched Chrysomyia Megacephala Fabricius larvae powder SCML Antioxidant activity Nrf2Intestinal microbiota In vivoBackgroundAging is a natural process that involves the gradual loss ofphysiological functions causing enhanced morbidity andmortality due to various diseases This process is closelyrelated to oxidative stress [] One prevalent theory toexplain aging is the theory of the oxygen free radical []This theory posits that the macromolecules such as nucleic acids lipids sugars and proteins that make up cellsand tissues are subjected to oxidative stress induced bysuperoxide and other free radicals These macromoleculesthen undergo different degrees of oxidation which initiates oxidative damages and ultimately leads to an function impairment and aging [ ] Changes in the level ofoxidative stress affect the microbial environment in the intestine and lead to intestinal flora disorder [] Disorderedintestinal flora may affect the antioxidant activity and lipidmetabolism [] Hence it may be possible to inhibit oxidative stress by regulating the composition and structure ofthe gut flora To prevent oxidative stressassociated cellular damage it is therefore important to keep prooxidantantioxidant balance by supplementation or induction ofcellular antioxidants A high dose of dgalactose is converted to aldose and hydrogen peroxide by dgalactoseoxidase The products then generate reactive oxygen species through oxidative metabolism and glycosylation leading to oxidative stress The accumulation of oxidationproducts further exacerbates the oxidative damage to tissues and cells which then accelerates the aging process[] Therefore dgalactose overload has been used to establish animal models used to conduct aging related metabolic dysfunction and oxidative stress [ ]Selenium Se is an essential trace element for humanbody and other animals The role of Se is reported to beclosely associated with antioxidant activityimmune response and chemoprevention [] Se is mainly presentin the active site of enzymes in the form of selenocysteineMultiple Secontaining proteins such as GSHPx and thioredoxin reductase play important roles in preventing oxidativeimportance of Sesupplementation in boosting up the internal antioxidativedefense has been highlighted in recent years Studies haveshown that anic Se supplements can improve tissue Sedeposition antioxidant level and gene expression whereasSe deficiency may result in cardiac muscular osseous and[] Thereforeinjurytheimmune disturbances [ ] Therefore the healthrelatedbenefits of Se including the type of selenium supplementsand optimal dosage remain to be exploredThe importance of Se has inspired researchers to usebioenrichment to prepare high Se compounds [ ] Cmegacephala larvae is a traditional Chinese medicine witha wide range of pharmacological actions including antioxidant antibacterial and antiinflammatory activitieswhich has been widely applied in agriculture and medicine[] Seenriched C megacephala larvae SCML isgenerated from C megacephala larvae by biological transformation and enrichment of Se Our previous workshowed that SCML was an effective anic Se sourcewith low toxicity and high Se content [] Yet no studyhas reported the antioxidant activity of SCML in vivo andits impact on the gut microbiota which is susceptible toundergo alterations under oxidative stressThe objective of the present study was to evaluate theantioxidant activity of SCML in vivo explore the underlying mechanism as well as evaluate its impact on thegut microbial diversity and structure hoping that the results could provide a scientific basis for a comprehensiveutilization of SCMLMethodsMaterials and chemicalsSCML was provided by Beijing Ershang Biological Technology Co Ltd Beijing China Vitamin E was purchasedfrom Archer Daniels Midland Dictor USA DgalactoseDgal of ¥ purity was purchased from Aladdin Industrial Corporation Shanghai China GSHPx SOD TAOC GSH MDA and protein carbonyl assay kits werepurchased from Nanjin Jiancheng Bioengineering InstituteNanjin China RNA trizol reagent and FastStart Universal SYBR Green Master Rox were purchased from Servicebio Wuhan China The primers for Nrf2 SOD1GSHPx and GAPDH were synthesized and purified byWuhan Servicebio Technology Co LTD Wuhan ChinaThe kits for Revert Aid First Strand cDNA synthesis andHyPure¢Molecular Biology Grade Water were purchasedfrom Thermo Waltham USA and HyClone LoganUSA respectively Keap1 Nrf2 and HO1 polyclonal antibodies were obtained from Proteintech Chicago USARIPA Actin bicinchoninic acid BCA assay kit 0cXie BMC Complementary Medicine and Therapies Page of Western Lightening¢ PlusECL Enhanced chemiluminescence substrate assay kit and the secondary goat antimouse horseradish peroxides HRP were from ServicebioWuhan China All other chemicals and reagents used inthe study were of analytical grade Water used in the experiments was ultrapureDetermination of the compositions of SCMLCompositions of SCML including protein crude fat andmoisture content were analyzed according to methodGB5009 of China National Food Safety StandardSe content was detected by Inductively Coupled PlasmaICP according to Vu with minor modifications[] The results are shown in Table Animal experimentsSixty ICR male mice aged weeks and weighing ± gwere purchased from SinoBritish SIPPRBK Lab Animal Ltd Approval No SCXK HU Theanimal experiments were performed in accordance withthe guidelines of the Laboratory Animal Center of Zhejiang Chinese Medical University Permit No SYSKZHE Allthe experimental procedureswere strictly conducted according to the internationalstandards and nationallegislation on animal care anduse The mice were kept under controlled light conditions h lightdark cycle with free access to food andwater normal light circadian rhythm and 7day adaptivefeeding in a quiet environmentAfter oneweek acclimatization mice were equallyrandomized to six groups normal controlNCgroup model group positive group receiving mgKg·d vitamin E VE group lowdose SCML LSCML group receiving SCML μgKg·d Se middose SCML MSCML group receiving SCML μgKg·d Se and highdose SCML HSCML group receiving SCML μgKg·d Se Except for the mice inNC group animals in the other five groups were givensubcutaneous injection of mgKg·d Dgal for weeksinto the neck to prepare oxidative stress model Animalsin NC and model groups received water and animals inthe other groups as previously described received VE orSCML by intragastric gavage for weeks The experiments were conducted at A certain amountof SCML and gellan gum were weighed precisely anddissolved in purified water heated slightly to a suspension There were three different concentrations and μgmL Se Meanwhile VE was dissolved in purified water containing gellan gum which became aTable Compositions of SCMLsuspension mgmL Dgal was dissolved in physiological saline mgmLThe mice were weighed throughout the experimentThe appearance appetite mental condition and behavioral activity of the mice during the experiment werealso observed and recorded Stool samples were collected at weeks after treatment Blood samples wereobtained from the retrobulbar venous plexus at weeksafter treatment The mice were sacrificed by cervical dislocation and the liver kidney heart brain and caecumwere stripped The dissected ans were divided twoparts one for histological analysis and the other for biochemistry analysis Samples for analysis were thawed onice homogenized with mL cold buffer mM potassium phosphate with mM EDTA pH per gram oftissue and centrifuged at Ãg for min at °CThe supernatants were collected for analysisAnalysis of serum oxidative stress indexesSerum oxidative stress indexes GSHPx SOD and MDAwere determined by using the respective commercial kitsaccording to the manufacturers instructionsAnalysis of tissue oxidative stress indexesThe oxidative stress indexes were determined by measuring GSHPx SOD TAOC GSH MDA and proteincarbonylation ofthe tissue homogenate supernatantusing the commercial kits according to the manufacturers instructionsHistological analysisFor histological analysis the animal tissues were fixed in paraformaldehyde for h dehydrated in alcoholparaffin embedded sliced into μm thick sectionsstained with hematoxylineosin HE and finally photographed under a microscope à objective lensRNA extraction and realtime quantitative PCRexperimentsTotal RNA was extracted from the liver and kidney tissues using Trizol reagent RNA was reverse transcribedinto cDNA using RevertAid First Strand cDNA SynthesisKit Realtime quantitative PCR qRTPCR was performed using FastStart Universal SYBR Green MasterRox and the ABI7900Faxt Sequence Detection systemThe thermal cycle condition was cycle at °C for min followed by cycles of amplification at °C for s and then °C for 30s And the dissolution curvestarted from °C then ascending to °C at °C15ContentProtein g100 gCrude Fat g100 gMoisture g100 gSe μggSCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of s All samples were run in triplicate in each experimentValues were normalized to that for GAPDH The sequences of the primers used are shown in Table Theresults were calculated by using the 2ÎÎCT methodliverandtissueskidneyWestern blot analysisTotal protein and nuclear protein were extracted from mgusing RadioImmunoprecipitation Assay RIPA lysis solution and anuclearcytoplasm protein extraction kit The concentrations of protein lysates were quantified using a BCA protein kit Samples containing an equal amount of protein μg were mixed with the loading buffer containing 2mercaptoethanol heated for min at °C andloaded onto a SDSPAGE gel The proteins fromthe electrophoresing gel were then transferred ontopolyvinylidenethenblocked with milk and Tween in Trisbuffered saline and incubated overnight at °C withantiKeap1 antiNrf2 antiHO1 and actin Then the appropriate horseradish peroxideconjugated secondary antibody wasadded to the membranes at room temperature Finallythe proteins were detected with chemiluminescent substrate Gray semiquantitative analysis was performed byImage J The protein bands were quantified using densitometry Values are expressed as the fold change withrespect to betaactindifluoride membranes whichIntestinal microbiota analysisThe stool samples were sent to BGI Co Ltd WuhanChina for sequencing of the 16S rRNA gene Total genomic DNA of the gut microbiome was extracted and theV3V4 region of the 16S rRNA gene from the sample wassubjected to PCR amplification After normalization of thegenome DNA to ng per PCR reaction V3V4 dualindex fusion PCR primer cocktail and PCR master mixwere added and then a PCR was performed The PCRproducts were purified with Agencourt AMPure XP beadsto remove the unspecific products Pairedend sequencingTable Primers for realtime PCR analysesAccession NoGeneNrf2NM_0109023SOD1GSHPxGAPDHNM_0114341NM_0081606NM_0080842was performed on the Illumina Hiseq platform and theobtained data were subjected to bioinformatics analysisTo obtain clean reads the clean pairedend reads withoverlap were merged to tags using FLASH fast lengthadjustment of short reads v1211 Then the tags wereclustered to operational taxonomic units OTUs at sequence similarity by scripts of software USEARCHv701090 The RDP classifier v22 was used to compare OTUs with the database to comment on the OTUsspecies Finally intestinal microbial diversity and structure were analyzed based on OTUs and taxonomic ranksusing software R v311Statistical analysisAll data are expressed as the means ± SD or means ± SEand analyzed using Statistical Analysis Software SPSS The experimental values were analyzed by oneway ANOVA followed by the Duncans multiplerangetests and Pvalue were considered to be statistically significantResultsEffects of SCML on daily behavior and weight gain inmiceUsual performance of the mice was observed and recorded and no abnormal phenomenon found duringthe experimentincluding antifeeding and vomitingSymptoms such as slow movement and listlessnesswere obviously observed in model groupindicatingthat the oxidative stress model induced by subcutaneous injection of Dgal was successfully establishedHowever the above symptoms receded in varying degrees in VE and SCML groups The weight gain ofthe mice is exhibited in Fig Compared with NCgroup body weight of the mice in model group significantly increased slowly P and increasedsteadily in drug treatment groups MSCML μgKg Se group showed a significant difference compared to the model group P Primer Sequences CTGGCTGATACTACCGCTGTTCAGGTGGGATTTGAGTCTAAGGAGATGTGACTGCTGGAAAGGACGCGCAATCCCAATCACTCCACCCAGGAGAATGGCAAGAATGAGGAAGGTAAAGAGCGGGTGACCTCGTCCCGTAGACAAAATGTGAGGTCAATGAAGGGGTCGTProduct Sizebp bp bp bp bp 0cXie BMC Complementary Medicine and Therapies Page of tissues were decreased significantly compared to NCgroup P except for SOD in the heart as well asGSHPx in the liver and brain After administration ofVE or SCML the activity of GSHPx and SOD as wellas the content of TAOC and GSH were increased gradually As shown in Fig 3a the activity of GSHPx in thekidney and heart was increased significantly comparedto model group P except for the heart in VEgroup and LSCML μgKg Se group The activityof GSHPx in the liver and brain remained unchangedsignificantly compared to model group except for VEgroup in the liver As shown in Fig 3b the activity ofSOD in the kidney and brain was increased significantlycompared to model group P except for the brainin LSCML μgKg Se group However the activityof SOD in the liver and heart remained unchanged significantly compared to model group except for the liverin VE group As seen in Fig 3c the content of TAOCin the liver kidney and heart was increased significantlycompared to the model group P except for theliver in LSCML μgKg Se group The content ofTAOC in the brain was not significantly altered compared to model group except for VE group As shownin Fig 3d the content of GSH in liver kidney and brainof VE group and in the kidney of HSCML μgKgSe and MSCML μgKg Se groups was increasedsignificantly compared to model group P As shown in Fig 3e the MDA level in model group wasincreased significantly compared to NC group P and decreased significantly after VE or SCML treatmentcompared to model group P except for LSCML μgKg Se group in the kidney In Fig 3f the proteincarbonylation level in the liver kidney and brain of modelgroup was increased significantly compared to NC groupP However the level decreased significantly inthe mice treated with SCML or VE except for in the liverand brain of LSCML μgKg Se group compared tomodel group P And compared with model groupFig Percentage of weight gain in mice at weeks Valuesrepresent means ± SD n and evaluated by oneway ANOVAfollowed by the Duncans multiplerange tests Compared with NCP Compared with Model P SCML SeleniumenrichedC megacephala larvae powderEffects of SCML on serum oxidative stress indexes in miceAs shown in Fig the serum antioxidative enzyme activities in model group were decreased significantly andthe MDA content was increased significantly comparedto NC group P As shown in Fig 2a the GSHPxactivities in animals treated with SCML or VE were increased significantly P As shown in Fig 2b theactivities of SOD in MSCML μgKg Se and VEgroups were significantly increased compared to modelgroup P As shown in Fig 2c the MDA levels inanimals treated with SCML or VE were decreased significantly P Effects of SCML on tissue oxidative stress indexes in miceAs illustrated in Fig after 6week subcutaneous injection of Dgal the activity of the antioxidative enzymesand the content of the antioxidants in different miceFig Oxidative stress level indexes of the mice serum a GSHPx activity in the mice serum b SOD activity in the mice serum c MDA content inthe mice serum Values represent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by theDuncans multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig Oxidative stress indexes of the mice tissue a GSHPx activity in the mice tissue b SOD activity in the mice tissue c TAOC content in themice tissue d GSH content in the mice tissue e MDA content in the mice tissue f protein carbonylation content in the mice tissue Valuesrepresent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by the Duncans multiplerangetests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powderFig Optical micrographs of mice tissue sections HE staining à Black arrow derangement of hepatic cord cells Red arrow infiltration ofinflammatory cells White arrow pyknosis Blue arrow cavitation and deformation Orange arrow atrophy and breakage of the villus Yellow arrowthinning of the intestinal wall Scale bar50 μm SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of HSCML μgKg Se group in heart also significantlydecreased in protein carbonylation levelEffects of SCML on histopathological changes in miceThe histopathological results are shown in Fig Normal histological architectures were observed in the tissuesections in NC group However the liver tissue sectionsin model group showed that the number of double nuclei was increased the hepatic cords were disarrangedliver cells expanded widely and infiltration oflargenumbers of inflammatory cells was observed Comparedto NC group kidney histopathology in model groupshowed that the glomeruli became atrophic or even disappeared the number of epithelial cells was reduced therenal proximal tubules were dilated Histologically theheart tissue was seen abnormally structured in modelgroupincluding cavitation and deformation in somemyocardial cells nuclear pyknosis and inflammatory cellinfiltration In model group the brain tissue was alsoseen abnormally structured including nuclear pyknosisand incomplete dissolution of nerve fibers The caecallesions including atrophy and breakage of the villus irregular cell arrangement and thinning of the intestinalwall were observed in model group SCML or VE treatment significantly attenuated these abnormal histologicalchanges of the tissues induced by DgalEffects of SCML on oxidative stress gene expression inmiceThe Nrf2 pathway maintains the redox homeostasis exerts antioxidant activity by regulating its multiple downstream cytoprotective genes thereby plays a vital role incell survival The effect of SCML on oxidative stressgene expression is shown in Fig As shown in Fig 5athe Nrf2 expression in model group in liver was lowerthan that of NC group P Except for LSCML μgKg Se group in the liver the Nrf2 expression in liverwas increased all other drug treatment groups comparedwith model group P The Nrf2 expression was increased in the kidney of in HSCML μgKg Se groupcompared to model group P As shown in Fig 5bthe expression of GSHPx mRNA in the liver of modelgroup was decreased P After SCML treatment theGSHPx mRNA expression in the liver was significantly increased compare to model group P and MSCML μgKg group and HSCML μgKg Se group inkidney was increased significantly compared to model groupP As shown in Fig 5c the expression of SOD1mRNA was decreased in model group especially in the kidney compared to NC group P However the expression was obviously increased in the liver of HSCML group μgKg Se and MSCML group μgKg Se compared to model group P Significant change was alsoobserved in SOD1 mRNA expression in the kidney of HSCML group μgKg Se and MSCML μgKgSe group compared to model group P Effects of SCML on oxidative stress protein expression inmiceTo determine whether Nrf2 activation played a role inSCML protection against Dgal induced oxidative stressthe expression of Keap1 Nrf2 and HO1 in the mouseliver and kidney was detected As shown in Fig compared with NC group the western blot results showedthat the Nrf2 and HO1 protein expression in modelgroup was significantly decreased P while theKeap1 protein expression was increased in model groupAfter SCML or VE treatment the Keap1 expression inthe treatment groups was decreased though the difference was not statistically significant Compared withmodel groupthe Nrf2 expression in the treatmentgroups was increased significantly P except forLSCML μgKg Se group in liver Compared withmodel group the HO1 expression in SCML groups wasincreased especially in the liver of HSCML μgKgSe group P Sequencing depth and diversityA total of sequences from all intestinal microbiota samples were produced averaging sequencesFig The effect of SCML on the expression of Nrf2 SOD1 and GSHPx mRNA in the liver and kidney of the mice a Nrf2 mRNA relativeexpression in the liver and kidney b GSHPx mRNA relative expression in the liver and kidney c SOD1 mRNA relative expression in the liver andkidney Values represent means ± SD from three independent replicates and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig The effect of SCML on the protein expression of Keap1 Nrf2 and HO1 in the liver and kidney tissue of the mice a Protein strip b Keap1actin relative density c Nrf2actin relative density d HO1actin relative density Values represent means ± SD from three independentreplicates and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Compared with ModelP SCML Seleniumenriched C megacephala larvae powderclusterper sample These sequences resulted in a mean sequencelength of approximately bp Based on the Clean Tagstheanalysis was processed by USEARCHv701090 The sequences were delineated into operational taxonomic units OTUs at similarity Thevalue of coverage for the observed OTUs was above The species accumulation curves showed clear asymptotes and the curve tended to be flat or reached theplateau stage Fig 7a indicating a nearcomplete sampling of intestinal microbial communities of mice Theboxplot of Shannon index showed that the diversity of theintestinal microbiota was decreased in model group compared to NC group and the diversity of VE group and HSCML μgKg Se group was increased compared tomodel group Fig 7b As shown in Fig 7c the contribution value of PC1 and PC2 for the sample difference was and respectively All intestinal microbiotasamples were presented as three distinct groups Thesefindings indicate that the main components of the intestinal microbiota in model group were different from thoseFig Alpha diversity of the gut microbiota and principal component analysis PCA plots based on abundance of operational taxonomic unitsOTUs a Species accumulation curves b Bacterial diversity estimated by the Shannon index c PCA plots SCML Seleniumenriched Cmegacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of in NC group After VE and SCML treatment the components of the intestinal microbiota were different fromthose in model group while there was an insignificant difference between HSCML μgKg Se group and NCgroup BacteroidetesEffects of SCML on species structures in miceThe species profiling histogram was obtained to knowthe community structural composition of differentgroups at phylum and genus levels Fig As shown inFig 8a the most prevalent phyla in all samples were Firmicutesand Proteobacteria There were otherphyla level bacteria with low abundance in the intestinaltract of mice As shown in Fig 8b species were usedto describe the relative abundance ofthe intestinalmicrobiota at the genus level showing that Prevotella Helicobacter and Clostridium were the most abundant followed byOscillospira Bacteroides andLactobacillus Effects of SCML on intestinal bacteria of differentclassification levels in miceAs shown in Table Proteobacteria were increased significantly at the phylum level in model group comparedto NC group P Proteobacteria were decreasedsignificantly and Bacteroidetes were increased significantly in VE and SCML groups compared to modelgroup P In addition VE group MSCML μgKg Se group and HSCML μgKg Segroup showed significant differences in Firmicutes compared to model group P and MSCML μgKg Se group showed a significant difference in Actinobacteria compared to model group P As shown in Table there was not significant alteration in Bacteroides Lactobacillus Oscillospira Prevotella and Sutterella at the genus level in model groupcompared to NC group Compared with NC group Helicobacter and Clostridium were increased significantlyand Ruminococcus was decreased significantly in modelgroup P Clostridium Helicobacter and Oscillospira were decreased significantly in VE and SCMLgroups compared to model group P while VEgroup and LSCML μgKg Se group showed a significant difference in Bacteroides P In additionLactobacillus in MSCML μgKg Se group and HSCML μgKg Se group Prevotella in VE groupand Sutterella in MSCML μgKg Se group wereall increased significantly compared to model group P There were not significant alterations in Ruminococcus in VE and all SCML groups compared to modelgroupCorrelation analysis of changes in flora abundance andserum biochemical indexesIn order to explain the relationship between the intestinal flora abundance changes of mice and serum biochemicalindexes Spearman correlation analysis wasperformed to analyze correlation between serum biochemical indexes and Clostridium and Helicobacter theabundance of which were significant difference in eachgroup The change of Clostridium abundance was foundto be negatively correlated with GSHPx and SOD andpositively correlated with MDA There was not significant correlation in Helicobacter and serum biochemicalindexes The specific correlation analysis results wereshown in the Table DiscussionThe results of the present study showed that the dailybehaviors of the mice in model group were differentfrom those of the mice in NC group In addition the tissues of the modeled mice underwent significant pathological changes The antioxidant system parametersincluding GSHPx SOD TAOC and GSH in the antissues or serum were decreased while the MDA andcarbonylated protein levels were increased All these results indicated that the Dgalinduced oxidation mousemodel was successfully established in the present studyVE the monomer of which is often used as the positivecontrol for the studies of aging in mice induced by DgalFig Taxonomic composition of the gut microbiome in the mice a Phylumlevel b Specieslevel SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Table Oneway ANOVA test of species differences at the phylum and species level BacteriaNCModelVEPhylumBacteroidetes ± Proteobacteria ± Firmicutes ± GenusActinobacteriaTenericutesBacteroidesClostridiumHelicobacterLactobacillusOscillospiraPrevotellaRuminococcus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SCML μgKg Se ± ± ± ± ± ± ± ± ± ± ± ± Sutterella ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values represent means ± SE n and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Comparedwith Model P SCML Seleniumenriched C megacephala larvae powder ± ± ± ± [ ] Studiesshowed that mice subcutaneouslyinjected with Dgal in the neck exhibited a significantbody weight declined [] In this study Dgal was foundto significantly inhibit weight gain in mice howeverSCML and VE could increase the body mass in varyingdegrees indicating that SCML and VE could effectivelyenhance the constitution of aging mice Oxidative damage appears in body ans to a large extent Our resultsshowed that Dgalinjection for weeks for mice resulted in severe histopathological changes in the antissues However SCML and VE could alleviate these Dgalinduced pathological damages in an tissues ofmice Recent research work has demonstrated that senescent cells accumulated in various tissues of age anddisease [] Cellular senescence is associated with agerelated phenotypes causally and decreasing senescentcells can retard tissue dysfunction and extend healthspan[] The results suggested SCML c | 2 |
sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were ï¬rst described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneï¬cial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing ï¬ve patients with gscin terms of the imaging ï¬ndings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of ï¬ve patients wereidentiï¬ed as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory ï¬ndings clinical interventions and the histologicï¬ndings of the ï¬ve biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma ï¬eldof view fov mm matrix mm and sectionthickness mm finally a 20ml saline ï¬ush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassiï¬ed as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where obviousenhancement was deï¬ned as hu moderate enhancementas hu and mildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingï¬ndings were compared with the postoperative pathologicalï¬ndings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm cm cm cm cm cmcm cm cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm cm cmand cm cm the remaining specimen was a soft massmeasuring cm cm cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched ï¬xed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaï¬n embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl years years years cm cm cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspeciï¬c symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory ï¬ndings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the ï¬ve gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalnarrnnonenonerc yespresentpositive noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the ï¬ve gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickeningintracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclear yespresentpositive noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed inï¬ltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic ï¬gures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2ci all ï¬ve tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures signiï¬cant atypical nuclei pleomorphic nuclei and giant nuclei mitotic ï¬gures visible tumor cells showed inï¬ltrative growth cells were stained withhematoxylin and eosin stain magniï¬cation a b by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the ï¬nal diagnosis was sc [magniï¬cation ci ]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct ï¬ndings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was conï¬rmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identiï¬ed as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region bd contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and signiï¬cantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a speciï¬c line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patientstumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could ï¬nd only sixprevious reports in the english literature table between and patients with sc conï¬rmed by pathologywere retrospectively analyzed with only ï¬ve tumors occurringin the stomach the average age of the reported patientswas years range and that in our series was years range a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious speciï¬cityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmassnenenenenehypernenonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed yespresentpositive noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports in the actual diagnosis processthe diagnosis of sc has always been diï¬cult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termssarcomatoid carcinoma and carcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct ï¬nding sc in the stomach have notbeen previously scientiï¬c reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinï¬ltrated the gastric serosa the hepatic ï¬exure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in of all reviewed cases tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass theï¬nal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot signiï¬cantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these ï¬ndingsreï¬ect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion inï¬ltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the ï¬ndingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct ï¬ndings may only reï¬ect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedï¬uorescence in situ hybridization fish to conï¬rm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no speciï¬c national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the eï¬cacy ofchemotherapy remain controversial domblides ï¬rstevaluated the eï¬cacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be conï¬ned to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at theï¬rst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identiï¬able images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe ï¬nal manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg ï¬uorescence insituhybridization conï¬rms epithelial origin histopathology doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkins lymphoma of the gastrointestinal tract radiographic ï¬ndings ajram j roentgenol doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v eï¬cacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic signiï¬canceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright liu liang feng chen yue ji li zhao and gao this is anopenaccess distributed under the terms of the creative commons attributionlicense cc by the use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice no use distribution or reproduction is permitted which does not complywith these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | 0 |
"This result may have been caused by direct exposure of the lung tissue to the increased temperature through inhalation of the hot air. .0090818.g004 FTSJ2 mRNA expression after in vivo and in vitro heat shock treatments. Porcine Ftsj2 (A) and Hsp70.2 (B) mRNA expressions in piglets which were raised at 25°C 30°C or 35°C for 1 week. Human FTSJ2 (C) and HSP70.2 (D) mRNA expressions in A549 lung cells after 1 hour of heat shock at 42°C or 45°C followed by 0 3 or 6 hours of recovery at 37°C. Porcine Hsp70.2 or human HSP70.2 mRNA expression was used as the heat shock response control. The values are equal to?=?the means±SE; n?=?4. The bars with different letters represent a significant difference (P<0.05). The Level of Human FTSJ2 mRNA Expression Increases after Heat Shock in A549 Cells Our results showed that Ftsj2 mRNA was overexpressed in the porcine lung after heat shock; therefore a human lung adenocarcinoma epithelial cell line (A549) was used to further confirm the heat shock response in vitro to eliminate the systematic effects observed in the piglets. A549 cells were first grown at 37°C and 5% CO2. For the heat shock treatment the cells were grown at 42°C or 45°C 5% CO2 for 1 hour and then returned to 37°C for 03 or 6 hours respectively. Human FTSJ2 mRNA was detected by real-time RT-PCR. The results revealed that after both the 42°C and 45°C heat shock treatments the hFTSJ2 mRNA expression increased by more than 50% (50.6% and 52.6% at 3 hours and 0 hours respectively) (P<0.05) (C) compared with the non-heat shock control. The up-regulation of the HSP70.2 mRNA indicated a positive heat shock response after the 42°C and 45°C treatments in the A549 cells (D). FTSJ2 Inhibits Cancer Cell Migration and Invasion In a recent study in clinical samples of NSCLC the human FTSJ2 gene was located in a novel oncogenic locus of NSCLC. These results indicate that FTSJ2 may also be involved in the growth of cancer cells. To evaluate the roles of FTSJ2 in cancer the gene expressions in the NSCLC cell lines were detected. A human lung adenocarcinoma cell line (CL1) which was isolated from an adenocarcinoma from the lung of a 64-year-old man has been cloned and passed on for more than 60 generations. Using the Transwell invasion chamber the CL1 cell line was separated into six sublines according to their metastatic ability (CL1-0 to CL1-5 from lowest to highest invasiveness) [41]. The hFTSJ2 mRNA expression was detected in two of these sublines (CL1-0 and CL1-5). Surprisingly the more invasive CL1-5 cells showed a 50% decrease in the hFTSJ2 mRNA expression than the less invasive CL1-0 cells (Figures 5B and 5C). .0090818.g005 Comparison of the hFTSJ2 mRNA expression levels in two lung cancer sublines (CL1-0 and CL1-5). (A) Morphology of CL1-0 and CL1-5 cells. (B) Determination of the hFTSJ2 mRNA expression levels in the CL1-0 and CL1-5 cells in triplicate. (C) Relative quantification of the hFTSJ2 mRNA expression. GAPDH mRNA was used as an internal control. The values are equal to?=?the means±SE; n?=?3; **P<0.01 vs. the non-heat shock group. In addition to the down-regulation FTSJ2 increased cell invasion in the CL1-5 cells. To further evaluate the abilities of FTSJ2 to influence cell migration and metastasis the hFTSJ2-overexpressed cell line (TE671-hFTSJ2) previously mentioned was used in the cell migration and invasion assay and was compared with the non-transfected TE671 cells. The results of our wound healing assay showed that at 12 hours after wounding the migration area of the TE671-hFTSJ2 cells was significantly decreased (P<0.01) compared with the non-transfected TE671 cells (Figures 6A and 6B). The same results were also observed in our invasion assay in which the quantity of invaded TE671-hFTSJ2 cells per Trans-well was significantly lower than that of the non-transfected TE671 cells (Figures 6C and 6D). These results indicate that FTSJ2 is involved in the inhibition of cancer cell migration and invasion. .0090818.g006 Inhibition of cell migration and invasion upon the over-expression of hFTSJ2 in TE671 cancer cells. (A) The wound healing assay showing that the TE671-hFTSJ2 cells had a reduced migration compared with the untransfected TE671 cells at 12 hours after wounding. (B) Cell migration area at 12 hours after wounding ([healing area/wounding area]100%). (C) Invasion assay showing that the TE671-hFTSJ2 cells had a reduced invasion compared with the untransfected TE671 cells. The cells that penetrated the Trans-well membrane are shown in purple. (D) Quantity of the cells that invaded the Trans-well membrane ([Giemsa positive area/total area]100%). The values are equal to?=?the means±SE; n?=?3; *P<0.05 vs. untransfected TE671 cells. Discussion In this study we characterized the mammalian FTSJ2 protein which we presumed to be an ortholog of E. coli RrmJ. RrmJ is known as a 2?-O-ribose MTase which methylates U2552 in the A-loop of the peptidyl transferase center in the 23S rRNA [5]. Um2552 is one of the four 2?-O-methylated nucleotides in rRNA [6]. In a previous study a lack of U2552 methylation has been found to influence the tertiary interactions of U2552 U2555 and C2556; to reduce the conformational dynamics of the A-loop [8]; and to subsequently decrease the ribosome stability and translation efficiency [7] [9] [37]. " | 1 |
" 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 patients 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 dhebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall dhebron 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 | 0 |
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phylogenetic trees and networks nucleic acids res w573w579 101093nargks48 letunic i bork p interactive tree of life itol v4 recent updates and new developments nucleic acids res w256w259 saeed a i tm4 a free opensource system for microarray data management and analysis biotechniques 10214403342 mt01 weber t antismash 30a comprehensive resource for the genome mining of biosynthetic gene clusters nucleic acids res 101093nargkv43 battistuzzi f u feijao a hedges s b a genomic timescale of prokaryote evolution insights into the origin of methanogenesis phototrophy and the colonization of land bmc evol biol acknowledgementstiara padayachee and nomfundo nzuza thank the department of science and technologynational research foundation dstnrf south africa for masters scholarships grant numbers mnd190619448759 and mnd190626451135 respectively khajamohiddin syed expresses sincere gratitude to the nrf south africa for a research grant grant number and university of zululand grant number c686 the 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Epidemiologic and clinical features of patients with COVID19 in BrazilCaractersticas epidemiolgicas e clnicas dos pacientes com COVID19 no BrasilVanessa Damazio Teich1 Sidney Klajner1 Felipe Augusto Santiago de Almeida1 Anna Carolina Batista Dantas1 Claudia Regina Laselva1 Mariana Galvani Torritesi1 Tatiane Ramos Canero1 Ot¡vio Berwanger1 Luiz Vicente Rizzo1 Eduardo Pontes Reis1 Miguel Cendoroglo Neto1 Hospital Israelita Albert Einstein S£o Paulo SP Brazil 1031744einstein_journal2020AO6022 Objective This study describes epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil Methods In this retrospective singlecenter study we included all laboratory confirmed COVID19 cases at Hospital Israelita Albert Einstein S£o Paulo Brazil from February until March Demographic clinical laboratory and radiological data were analyzed Results A total of patients with a confirmed diagnosis of COVID19 were included in this study Most patients were male with a mean age of years A history of a close contact with a positivesuspected case was reported by of patients and had a history of recent international travel The most common symptoms upon presentation were fever nasal congestion cough and myalgiaarthralgia Chest computed tomography was performed in patients and of those showed abnormal results Hospitalization was required for patients and were admitted to the Intensive Care Unit Regarding clinical treatment the most often used medicines were intravenous antibiotics chloroquine and oseltamivir Invasive mechanical ventilation was required by of Intensive Care Unit patients The mean length of stay was days for all patients and days for patients requiring or not intensive care respectively Only one patient died during followup Conclusion These results may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemicKeywords Communicable diseases Lung diseasesepidemiology SARSCoV2 COVID19 Coronavirus infections Epidemiology RESUMOObjetivo Descrever as caractersticas epidemiolgicas e clnicas de pacientes com infec§£o confirmada pelo SARSCoV2 diagnosticados e tratados no Hospital Israelita Albert Einstein que admitiu os primeiros pacientes com essa condi§£o no Brasil Mtodos Neste estudo retrospectivo de centro ºnico inclumos todos os casos com confirma§£o laboratorial de COVID19 no Hospital Israelita Albert Einstein em S£o Paulo SP de fevereiro a mar§o de Foram analisados dados demogr¡ficos clnicos laboratoriais e radiolgicos Resultados Foram includos pacientes com diagnstico confirmado de COVID19 A maioria dos pacientes era do sexo masculino com mdia de idade de anos Foi relatada histria de contato prximo com um caso positivosuspeito por dos pacientes e tinham histria de viagens internacionais recentes Os sintomas mais comuns foram febre congest£o nasal tosse e mialgiaartralgia A tomografia computadorizada de trax foi realizada em pacientes e deles apresentaram How to cite this Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Epidemiologic and clinical features of patients with COVID19 in Brazil einstein S£o Paulo 202018eAO6022 httpdx1031744einstein_journal2020AO6022Corresponding authorVanessa Damazio Teich Avenida Albert Einstein MorumbiZip code S£o Paulo SP Brazil Phone Email vanessateicheinsteinbrReceived onJuly Accepted onJuly Conflict of interest noneCopyright This content is licensed under a Creative Commons Attribution International LicenseORIGINAL ISSN eISSN 23176385Official Publication of the Instituto Israelita de Ensino e Pesquisa Albert Einsteineinstein S£o Paulo 0cresultados anormais A hospitaliza§£o foi necess¡ria para pacientes e foram admitidos na Unidade de Terapia Intensiva Quanto ao tratamento clnico os medicamentos mais utilizados foram antibiticos intravenosos cloroquina e oseltamivir A ventila§£o mec¢nica invasiva foi necess¡ria em dos pacientes na Unidade de Terapia Intensiva O tempo mdio de interna§£o foi dias para todos os pacientes e dias para pacientes que necessitaram ou n£o de cuidados intensivos respectivamente Apenas um paciente morreu durante o acompanhamento Conclus£o Estes resultados podem ser relevantes para o Brasil e outros pases com caractersticas semelhantes que come§aram a lidar com essa pandemiaDescritores Doen§as transmissveis Pneumopatiasepidemiologia SARSCoV2 COVID19 Infec§µes por coronavrus Epidemiologia INTRODUCTIONSince December several cases of pneumonia of unknown origin have been reported in Wuhan China1 The pathogen was further identified as a novel RNA coronavirus currently named as severe acute respiratory syndrome coronavirus SARSCoV22 Huang et al reported the first cases in China with a common clinical presentation of fever cough myalgia fatigue and dyspnea with an dysfunction eg acute respiratory distress syndrome ARDS shock acute cardiac and kidney injuries and death in severe cases3Afterwards in January the World Health anization WHO declared the outbreak a Public Health Emergency of International Concern PHEIC and next in March it was characterized as a pandemic4 As of April a total of cases had been reported in countries and regions across all five continents with deaths worldwide5 More recently the Chinese Center for Disease Control and Prevention published data on patients with classified as confirmed cases of coronavirus disease COVID19 Most patients were aged to years with mild clinical presentation ie nonpneumonia and mild pneumonia and overall casefatality rate of increased in elderly population with casefatality rate of in those aged years and older6On February the first Brazilian patient had a confirmed diagnosis of COVID19 at Hospital Israelita Albert Einstein HIAE Hospital Israelita Albert Einstein is a philanthropic hospital in the city of S£o Paulo SP Brazil with twelve health care units including a quaternary hospital with beds and four outpatient emergency care units By the end of this study on March of patients with confirmed COVID19 in Brazil had been diagnosed at HIAE Given the rapid spread of the COVID19 clinical and epidemiological data of several countries are being published on a daily basis79 However no studies have been reported to date presenting the characteristics of COVID19 patients diagnosed in Brazil OBJECTIVETo describe epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil METHODSStudy design and oversightThis was a retrospective observational singlecenter study which included all consecutive patients with a confirmed diagnosis of COVID19 at HIAE between February and March The study was supported by an internal grant from HIAE and designed by the investigators The study was approved by the Research Ethics Committee of the anization protocol number CAAE and the National Commission for Research Ethics PatientsThe diagnosis of the COVID19 disease was performed according to the WHO interim guidance10 A confirmed case of COVID19 was defined as a positive result of realtime reverse transcriptase polymerase chain reaction RTPCR assay of nasal and pharyngeal swab specimens11 All cases included in the current analysis had laboratory confirmationData sourcesThe data were obtained from patients electronic medical records EMR including inpatients and outpatients with laboratoryconfirmed COVID19 Data collected included demographic clinical laboratorial and radiological information and was anonymized so that patients could not be identified Demographic characteristics included age sex tobacco smoking weight and body mass index BMI Clinical information included medical travel Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cand exposure history signs symptoms underlying comorbidities continuous medication use and treatment measures ie antiviral therapy steroid therapy respiratory support and kidney replacement therapy Laboratory assessment consisted of complete blood count assessment of renal and liver function and measurements of electrolytes Ddimer procalcitonin lactate dehydrogenase Creactive protein and creatine kinase Radiologic abnormality was defined based on the medical report documented in the EMR Disease duration from onset of symptoms hospital and Intensive Care Unit ICU length of stay LOS were also documented Statistical analysis Continuous variables were expressed as means with standard deviations medians minimum and maximum values Categorical variables were summarized as counts and percentages No imputation was made for missing data All statistics are deemed to be descriptive only considering that the cohort of patients in our study was not derived from random selection All analyses were performed using Microsoft Excel RESULTSDemographic and clinical characteristicsBetween February and March a total of patients were diagnosed with COVID19 at HIAE This study included patients for whom data regarding demographics clinical symptoms laboratory and imaging findings were available in the EMR The remaining patients had only used the hospital laboratory facilities and were followedup by physicians not working in our service network Patients demographic and clinical characteristics are shown in table A total of had a recent international travel history and had been at the same marriage celebration in Bahia a state in the Northeast region of Brazil patients had a history of close contact either with a positive or suspected case of COVID19 Most patients were male and the mean age was years Only of patients were younger than years old and were older than yearsFever was present in only of patients upon admission but had a reported history of fever followed by nasal congestion cough Table Clinical and epidemiological characteristicsCharacteristicAge years Mean±SDMedianMinimumMaximumNumber of patientsAge distribution years ¥Sex MaleFemaleTravel historyTotal patients n510Total patientsNonhospitalized patients n438Hospitalized patients n72±±± European Union and United Kingdom United States of America and Canada Middle East and IranChina and Japan Latin America Other countries Bahia Brazilian stateNo travel history Exposure source of transmission contact with confirmed or suspected casesExposureNo exposureHealthcare professionalYesNoSmoking historyCurrent smokerFormer smokerNever smokedFever on admission YesNoMedianTemperature distribution on admission°C °C°C°CSymptomsNasal congestionHeadacheCoughSore throatSputum production continueEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cContinuationTable Clinical and epidemiological characteristicsContinuationTable Clinical and epidemiological characteristicsCharacteristicFatigueDyspneaNausea or vomitingDiarrheaMyalgia or arthralgiaChillsFeverConjunctival congestionOther symptomsNo symptomsSymptoms duration daysMean±SDMedianMinimumMaximumSigns of infectionThroat congestionTonsil swellingSkin rashOther alterationsNo alterationsCoexisting disordersAny coexisting disorderAsthma or chronic pulmonary obstructive disorderDiabetesHypertensionCoronary heart disease or other heart conditionsCerebrovascular diseaseHepatitis B C HIV or other immunodeficiencyCancerChronic renal diseasean transplantPregnancy Other coexisting disordersNo coexisting disordersMean BMI±SDChronicuse medicationsAny medicationStatinMultivitaminAntidepressantAntihypertensiveAntiplatelet or anticoagulantThyroid hormonesTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 ± ± ± ±± ± continueCharacteristicAntidiabeticPain killersAntibioticsCorticosteroidInhaled medicationsOther medicationsNo use of medicationsTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 Chronicuse medications number of medications distributionOnly type of medication types of medications types of medications or more types of medications polypharmacy ESI on arrival Destiny after first evaluation Discharge to homeAdmission on general wardAdmission on ICU Return to the emergency room after first evaluation SimN£oResults expressed by total nn if not otherwise indicatedSD standard deviation BMI body mass index ESI Emergency Severity Index ICU intensive care unitand myalgia or arthralgia The mean duration of symptoms was days which was the same for patients hospitalized or not Upon admission the majority of patients had no significant changes on physical examination Considering all included patients had at least one comorbidity This rate however was far higher in the hospitalized group when compared with the nonhospitalized group the most common comorbidities were hypertension and diabetes The distribution of patients in the Emergency Severity Index ESI differs between the two groups analyzed with the hospitalized group showing a higher rate of ESI indicating that the initial severity was greater in this group since the onset of symptoms Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cRadiologic and laboratory findingsTable demonstrates the radiologic and laboratory findings upon admission Only of patients were initially evaluated with chest radiographs whereas were submitted to computed tomography CT Of the radiographs performed had some abnormality while of CT scans showed abnormal results The most common patterns on chest CT were groundglass opacity and bilateral patchy shadowing Table Radiologic and laboratory findingsCharacteristicsRadiologic findings in chest radiographChest radiograph performedAbnormalities on chest radiograph Groundglass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalities Radiologic findings in chest CT Chest CT performed Abnormalities on chest CT Ground glass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalitiesLaboratory findingsMedian PaO2FiO2 ratio IQRWhite blood cell countMedian per mm3 Distribution per mm3 Lymphocyte countMedian per mm3 Distribution per mm3 Platelet countTotal number of patients n510 Total number of patientsNonhospitalized patients n438Hospitalized patients n72 Median per mm3Distribution per mm3 Median hemoglobin gdLDistribution of other findings Creactive protein 5mgL Procalcitonin 05ngmLLactate dehydrogenase 214UL Aspartate aminotransferase 40UL Alanine aminotransferase 40UL Total bilirubin 12mgdL Creatine kinase UL Creatinine 1mgdLDdimer 500ngmL Mean sodium mmolLMean potassium mmolL Results expressed by total nn if not otherwise indicatedCT computer tomography PaO2FiO2 oxygen partial pressurefractional inspired oxygen IQR interquartile rangeleukopenia Upon admission lymphocytopenia was identified in of patients thrombocytopenia in and in Most patients had elevated levels of both Creactive protein and lactate dehydrogenase Less common findings were elevated levels of Ddimer aspartate aminotransferase and alanine aminotransferase The hospitalized group had more patients with higher levels of Creactive protein procalcitonin and lactate dehydrogenase The other results do not show any major difference between groups A viral panel was collected in patients and it was positive for rhinovirus in nine cases influenza B in two cases and influenza A in one caseTreatment and complicationsAs shown in table patients had been hospitalized at HIAE by the time of the analysis Among Table Treatments complications and clinical outcomesTotal number of patients n510 CharacteristicDisease severitySevereNot severeIntensive care use during hospital stayYesNoHospital treatments medicationsIntravenous antibioticsOseltamivirLopinavir and ritonavirChloroquineCorticosteroidsHospital treatments support treatmentsOxygen therapyMechanical ventilationInvasiveNoninvasiveExtracorporeal membrane oxygenationContinuous renal replacement therapyComplicationsSeptic shockAcute respiratory distress syndromeAcute kidney injuryPneumoniaMean length of stay daysLOS all patientsPatients requiring ICU daysICUInpatients unitsPatients not requiring ICU inpatients units daysResults expressed by total nn if not otherwise indicated LOS length of stay ICU intensive care unitEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cthose patients required intensive care during their hospital stay in that were referred from the emergency room to the ICU and eight presented worsening of the clinical condition at inpatients units and were transferred to the ICU The majority of patients received intravenous antibiotic therapy received chloroquine and oseltamivir Oxygen therapy was necessary in of hospitalized patients required mechanical ventilation invasive and noninvasive and extracorporeal membrane oxygenation ECMO was used in only one case Considering patients admitted to the ICU invasive mechanical ventilation was required by of them During hospital admission most patients were diagnosed with pneumonia followed by acute kidney injury and ARDS The mean LOS was days considering only patients requiring intensive care the mean ICU LOS was days and the mean total LOS was days whereas for patients not admitted to the ICU the mean LOS was days Only one patient died in this series that is mortality rate DISCUSSION It took months from the first diagnosed case of COVID19 in China until diagnosis of patient zero in Brazil on February at HIAE During days after the first diagnosis all cases had a history of recent international travels On March the first case of local transmission was confirmed also at HIAE A relevant proportion of all patients with confirmed COVID19 infection had been diagnosed at HIAE by the time of the analysisThe patients in our series had a mean age of years and were mostly male The studies describing demographic characteristics in the infected general population showed a median age of years712 and the proportion of males was in the Chinese report7 and in the Singapore report The respiratory symptoms were similar to those of patients described in reports from China United States and Europe7913 However the mean days of symptoms was far lower in our series days versus days in Singapore12 days in the United States13 and days in China3 Although fever was reported by the majority of patients it was only present in of patients at the initial assessment at hospital suggesting not only it might not be considered to determine severity of illness but also that diagnostic algorithms using fever for testing may mask the total number of cases and delay diagnosis The prevalence of chronic diseases was far higher in the hospitalized group as compared to nonhospitalized group This prevalence was even higher in the subgroup admitted to the ICU The mean age of hospitalized patients was higher than nonhospitalized patients versus years and the required hospitalization increased with age for patients aged to years for to years and for patients older than years In this Brazilian case series hospitalization was required for patients and of them demanded critical care accounting for of total admissions a number far greater than the Chinese series in which only required ICU7The majority of patients were admitted to the ICU because of acute hypoxemic respiratory failure that required ventilatory support Invasive mechanical ventilation was needed in of ICU patients of total hospitalizations whereas were managed with noninvasive mechanical ventilation The necessity of invasive mechanical ventilation was similar to an ICU series reported from the United States of Washington13 lower than that reported in an Italian publication of Lombardy9 but higher than the Chinese reports and of Wuhan half of these treated with extracorporeal membrane oxygenation31415 Considering the use of noninvasive ventilation the rate was again similar to that reported in Washington and lower than the rates in China and of Wuhan including patients receiving highflow nasal cannula31415 A total of three patients of patients admitted to the ICU developed acute kidney injury and required continuous renal replacement therapy Among those only one patient had chronic kidney disease The prevalence of chronic kidney disease was among hospitalized patients in the Chinese report14 and among patients admitted to the ICU in the series from the United States This study has important limitations First part of the cases had incomplete information documented in the medical records and patient clinical history documentation was not homogeneous among all patients This is a common limitation in retrospective observational studies taking into account that data Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cgeneration was clinically driven and not in systematic fashion Second since many patients remained at the hospital and the outcomes were unknown at the time of data collection we censored the data regarding their clinical outcomes as of the time of the analysis Third only patients hospitalized at HIAE were included in the hospitalization group and there is no documentation of hospital admissions outside of our service network Finally this study only included patients attended as outpatients or inpatients at HIAE therefore asymptomatic and mild cases who did not seek medical care were not considered Hence our study cohort may represent more severe COVID19 cases CONCLUSIONTo date there is no study in Brazil reporting the characteristics of patients diagnosed with COVID19 Brazil is the country in the south hemisphere with the highest number of confirmed cases this disease and Hospital Israelita Albert Einstein is the center where the first patient was diagnosed with a representative sample of all confirmed COVID19 cases in the country The results presented in this study may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemic CONTRIBUTION OF AUTHORSData were analyzed and interpreted by the authors All authors reviewed the manuscript and checked the exactness and completeness of data AUTHORS INFORMATIONTeich VD httporcid0000000285396037Klajner S httporcid0000000341201047 Almeida FA httporcid0000000171310039 Dantas AC httporcid0000000195056784 Laselva CR httporcid0000000182859633 Torritesi MG httporcid0000000236236475 Canero TR httporcid0000000273994718Berwanger O httporcid0000000249722958Rizzo LV httporcid0000000199499849 Reis EP httporcid000000015110457X Cendoroglo Neto M httporcid0000000281634392 REFERENCES Lu H Stratton CW Tang YW Outbreak of pneumonia of unknown etiology in Wuhan China The mystery and the miracle J Med Virol Zhu N Zhang D Wang W Li X Yang B Song J Zhao X Huang B Shi W Lu R Niu P Zhan F Ma X Wang D Xu W Wu G Gao GF Tan W China Novel Coronavirus Investigating and Research Team A novel coronavirus from patients with pneumonia in China N Engl J Med Huang C Wang Y Li X Ren L Zhao J Hu Y et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Erratum in Lancet Jan World Health anization WHO Coronavirus disease COVID19 outbreak [Internet] Geneva WHO [cited July ] Available from httpswwwwhointwesternpacificemergenciescovid19 The Johns Hopkins Coronavirus Resource Center CRC COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University [Internet] CRC USA [cited July ] Available from httpscoronavirusjhuedumaphtml Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China Summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA Feb 101001jama20202648 Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical characteristics of coronavirus disease in China N Engl J Med Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H Spitters C Ericson K Wilkerson S Tural A Diaz G Cohn A Fox L Patel A Gerber SI Kim L Tong S Lu X Lindstrom S Pallansch MA Weldon WC Biggs HM Uyeki TM Pillai SK Washington State 2019nCoV Case Investigation Team First case of novel coronavirus in the United States N Engl J Med Grasselli G Zangrillo A Zanella A Antonelli M Cabrini L Castelli A Baseline characteristics and outcomes of patients infected with SARSCoV2 admitted to ICUs of the Lombardy region Italy JAMA World Health anization WHO Clinical management of severe acute respiratory infection when novel coronavirus 2019nCoV infection is suspected interim guidance [Internet] Geneva WHO [cited July ] Available from httpswwwwhointdocsdefaultsourcecoronaviruseclinicalmanagementofnovelcovpdf Brasil Ministrio da Saºde Centro de Opera§µes de Emergªncias em Saºde Pºblica Coronavirus Covid19 Boletim Di¡rio [Internet] Braslia DF Ministrio da Saºde [citado Jul ] Disponvel em httpswwwsaudegovbrimagespdf2020marco2929COVIDpdf Young BE Ong SW Kalimuddin S Low JG Tan SY Loh J Ng OT Marimuthu K Ang LW Mak TM Lau SK Anderson DE Chan KS Tan TY Ng TY Cui L Said Z Kurupatham L Chen MI Chan M Vasoo S Wang LF Tan BH Lin RT Lee VJ Leo YS Lye DC Singapore Novel Coronavirus Outbreak Research Team Epidemiologic features and clinical course of patients infected with SARSCoV2 in Singapore JAMA Mar 101001jama20203204 Bhatraju PK Ghassemieh BJ Nichols M Kim R Jerome KR Nalla AK Covid19 in critically Ill patients in the Seattle region Case series N Engl J Med Wang D Hu B Hu C Zhu F Liu X Zhang J Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA Feb 101001jama20201585 Yang X Yu Y Xu J Shu H Xia J Liu H Clinical course and outcomes of critically ill patients with SARSCoV2 pneumonia in Wuhan China a singlecentered retrospective observational study Lancet Respir Med Erratum in Lancet Respir Med 202084e26Epidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0c' | 2 |
"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as San 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 Peoples Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 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 natural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new eight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiangs 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 teams 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 JournalofEthnopharmacology26420211132472 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 JournalofEthnopharmacology26420211132473 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 αDAraf1residues 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 JournalofEthnopharmacology26420211132474 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 JournalofEthnopharmacology26420211132475 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 JournalofEthnopharmacology26420211132476 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 a | 2 |
" Control Asbestos Mesothelioma P TOL (mmol H2O2 equiv./L) 105.9 ± 92.5 145.1 ± 71.9b 196.3 ± 221.1cd 0.001 TAC (mmol Trolox equiv./L) 0.73 ± 0.34 1.27 ± 0.16c 0.8 ± 0.3f <0.001 OSI 159.3 ± 160.9 112.8 ± 48.3c 898.6 ± 129.7bf 0.007 CRP (mg/dL) 2.8 ± 1.7 4.4 ± 3.1 75.6 ± 54.8af <0.001 ?-1 antitrypsin (mg/dL) 146.7 ± 47.3 213.9 ± 33.9c 262.4 ± 94.9cf <0.001 Transferrin (g/L) 216.9 ± 62.3 282.8 ± 50.1c 171.2 ± 44.5cf <0.001 Ferritin (ng/mL) 112.3 ± 43.7 85.4 ± 110.6 370.7 ± 267.8cf <0.001 Copper (?g/mL) 0.8 ± 0.2 0.7 ± 0.3 1.2 ± 0.3cf <0.001 Ceruloplasmin (mg/L) 36.6 ± 8.6 40.1 ± 9.7 42.9 ± 8.2cd 0.001 TOL: total oxidant level; TAC: total antioxidant capacity; OSI: oxidative stree index; CRP: C-reactive protein; asignificantly different from control (P < 0.05) bsignificantly different from control (P < 0.01) csignificantly different from control (P < 0.001) dsignificantly different from asbestos (P < 0.05) esignificantly different from asbestos (P < 0.01) and fsignificantly different from asbestos (P < 0.001). Comput Math Methods Med Comput Math Methods Med CMMM Computational and Mathematical Methods in Medicine 1748-670X 1748-6718 Hindawi Publishing Corporation 24987451 4058834 10.1155/2014/401201 Research Automatic Lung Tumor Segmentation on PET/CT Images Using Fuzzy Markov Random Field Model Guo Yu 1 Feng Yuanming 1 2 Sun Jian 2 http://orcid./0000-0002-1559-111X Zhang Ning 1 Lin Wang 1 Sa Yu 1 Wang Ping 2 * 1Tianjin Key Lab of BME Measurement Tianjin University Tianjin 300072 China 2Department of Radiation Oncology Tianjin Medical University Cancer Institute and Hospital Tianjin 300060 China *Ping Wang: wangpingtjmuch.com Academic Editor: Lei Chen 2014 29 5 2014 2014 401201 28 3 2014 12 5 2014 Copyright 2014 Yu Guo et al. 2014 This is an open access distributed under the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. The combination of positron emission tomography (PET) and CT images provides complementary functional and anatomical information of human tissues and it has been used for better tumor volume definition of lung cancer. This paper proposed a robust method for automatic lung tumor segmentation on PET/CT images. The new method is based on fuzzy Markov random field (MRF) model. The combination of PET and CT image information is achieved by using a proper joint posterior probability distribution of observed features in the fuzzy MRF model which performs better than the commonly used Gaussian joint distribution. In this study the PET and CT simulation images of 7 non-small cell lung cancer (NSCLC) patients were used to evaluate the proposed method. Tumor segmentations with the proposed method and manual method by an experienced radiation oncologist on the fused images were performed respectively. Segmentation results obtained with the two methods were similar and Dice's similarity coefficient (DSC) was 0.85 ± 0.013. It has been shown that effective and automatic segmentations can be achieved with this method for lung tumors which locate near other ans with similar intensities in PET and CT images such as when the tumors extend into chest wall or mediastinum. http://dx../10.13039/501100001809 National Natural Science Foundation of China 81171342 http://dx../10.13039/501100001809 National Natural Science Foundation of China 81201148 Tianjin Research Program of Application Foundation and Advanced Technology 14JCQNJC09500 National Research Foundation for the Doctoral Program of Higher Education of China 20130032120070 National Research Foundation for the Doctoral Program of Higher Education of China 20120032120073 Independent Innovation Foundation of Tianjin University 60302064 Independent Innovation Foundation of Tianjin University 60302069 1. Instruction Combination of positron emission tomography (PET) and CT images provides complementary functional and anatomical information which has been used for tumor volume definition in radiation treatment (RT) planning for lung cancer patients [1]. Automatic methods for tumor segmentation on PET/CT images are highly desired to avoid the inter- and intraobserver variability caused by manual method. Many automatic tumor segmentation techniques for identification and delineation of cancerous tissues have been reported such as for brain tumor [2] lung tumor [3] and prostate tumor [4]. The segmentation can be performed either on a single image set such as CT [5] PET [6] or MRI images [7] or on the fused image set of different image modalities such as CT/PET [810] or multiparametric MRI images [2 11]. Different types of tumors and image modalities have different image features and thus different segmentation strategy should be developed for effective and accurate tumor segmentations. CT images provide anatomical information with high spatial resolution. However for the lung tumors abutting or involved in adjacent structures such as chest wall mediastinum or diaphragm which show intensities similar to those of tumors on the images it is difficult to distinguish them from the adjacent tissues with commonly used autosegmentation algorithms. Lung tumors can be distinguished from the adjacent tissues on PET images but the segmentation accuracy is still limited due to the coarser spatial resolution of the image data and motion artifacts as the result of time-consuming procedure of data acquisition. Therefore one of the key points of lung tumor segmentation on PET/CT images is to combine the advantages of the two image modalities effectively. Several methods [810] are proposed for lung tumor segmentation on PET/CT images. Most of the reported methods fuse different features extracted from PET and CT images inside one single N-dimensional vector. In this paper we propose a new strategy for fusing PET and CT information. The method is based on fuzzy Markov random field (MRF) model which has shown effective performance for unsupervised image segmentation [12 13]. Different from traditional fuzzy MRF method the proposed method designs a new joint posterior probabilistic model for effective combination of PET and CT image information. The new method was evaluated using image data of 7 patients with lung cancer in this study and experimental results showed its good performance in automatic tumor delineation. This paper is anized as follows. We first present the basic theory about image segmentation using fuzzy MRF model in Section 2.1. The framework of lung tumor segmentation on CT/PET images using fuzzy MRF model is then described in Section 2.2. The evaluation of the proposed method and quantification results are shown in Section 3. Finally discussion and are presented at the end. 2. Materials and Methods 2.1." | 1 |
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu | 0 |
" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1 mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2 in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9to this end the central premise put forth in this study is that focused ultrasound fusa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbeccos modified eagles medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1 mice were obtained from the jackson laboratory 4t1 or e0771 cells were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume lengthwidth22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a sham treatment consisting of exposure to the °c degassed water bath exposure for min following sham or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to sham or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of vehicle treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day early αpd1 or day delayed αpd1t cell depletionst cell depletion antibodiesanti cd8 clone bio x cell and anti cd4 gk15 clone bio x cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1bc the exceptionally small focus of this system rendered a low ablation fraction of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcsand cd86 dcs in particularsuggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c sham mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test fh or two way analysis of variance followed by tukey multiple comparison correction ik nsnot significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2bcby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly and reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2de in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytesin particular cd8 and cd4 t cellsplay an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3bc and ef moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3bc and ef from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6ab consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cellsa reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells bc absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to ef absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to ik percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6 fus monotherapy n4 gem monotherapy n9 and combinatorial fusgem therapy n6 groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fishers least significant difference lsd without multiple comparisons correction for ik significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fishers lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6cd these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantationthat is days subsequent to final gem administrationrevealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4bc moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4de however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1 model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1 mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1 mice relative to their wt counterparts with average 4t1 tumor volume in rag1 mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1 mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1 settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1 setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ | 0 |
" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects bodys immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHOs Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles 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 articles We obtained a total of eligible published research articles 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 particles 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 Morans 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 article 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 Ï2 p A μgm3 increase in PM25 was associated with a increase in fatality rate Ï2 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 seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearmans 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 corr | 2 |
" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and its mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ ]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [] sbt oil which contains richfatty acids tocopherols Ï3 and Ï6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturers protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ²reverse ² ggccatcagcaacaacataa ²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining thickness in hestained tissue mast cell number in tb stained tissue c tb staining epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65leung dym new insights into atopic dermatitis j clin investig kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol feinberg h trimeric structure of langerin j biol chem zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889 natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunolpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a | 0 |
"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t 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 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyers patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials []f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2 mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1nod2 mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ ] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti | 0 |
Genetic alterations in the 3q263132 locus conferan aggressive prostate cancer phenotypeBenjamin S SimpsonSusan Heavey Jason Pitt5 Caroline M Moore6 Hayley C Whitaker Niedzica Camacho234 Hayley J Luxton Hayley Pye Ron Finn1Largescale genetic aberrations that underpin prostate cancer development and progressionsuch as copynumber alterations CNAs have been described but the consequences ofspeciï¬c changes in many identiï¬ed loci is limited Germline SNPs in the 3q2631 locus areassociated with aggressive prostate cancer and is the location of NAALADL2 a gene overexpressed in aggressive disease The closest gene to NAALADL2 is TBL1XR1 which is implicated in tumour development and progression Using publiclyavailable cancer genomic datawe report that NAALADL2 and TBL1XR1 gainsampliï¬cations are more prevalent in aggressivesubtypes of prostate cancer when compared to primary cohorts In primary disease gainsampliï¬cations occurred in CI and CI for NAALADL2 and TBL1XR1 respectively increasing in frequency in higherGleason grade and stage tumours Gainsampliï¬cations result in transcriptional changes andthe development of a proproliferative and aggressive phenotype These results support apivotal role for copynumber gains in this genetic region Molecular Diagnostics and Therapeutics Group Research Department of Targeted Intervention Division of Surgery Interventional Science UniversityCollege London London UK Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center New York NY USA MarieJoseand Henry R Kravis Center for Molecular Oncology Memorial Sloan Kettering Cancer Center New York NY USA Department of Pathology MemorialSloan Kettering Cancer Center New York New York for Genomics Research Discovery Sciences Biopharmaceutical RD AstraZeneca Cambridge UK Cancer Institute of Singapore National University of Singapore Singapore Singapore Department of Urology UCLH NHS Foundation Trust London UKemail HayleyWhitakeruclacukCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xProstate cancer PCa is the most common noncutaneouscancer in developed countries12 and is deï¬ned by dynamicgenome alterations and both its pathological and geneticheterogeneity3 An important pathological predictor of prostatecancer aggressiveness is Gleason grade used to assess risk ofprogression and stratify patients for treatment however theunderlying genomic changes which accompany more aggressivetumours remains incompletely deï¬nedOverall copynumber alteration CNA burden has been linkedto poorer prognosis in prostate cancer associating with Gleasongrade biochemical recurrence and prostate cancer speciï¬c deathhowever the exact mechanism driving these prognostic changes isunknown and thought to be primarily driven by general chromosomal instability4 Changes in speciï¬c loci have also beenlinked to aggressiveness in particular gains in proliferative geneseg MYC 8q24 and loss of tumour suppressors PTEN 10q23and NKX31 8p2178 Many genetic alterations have been linkedwith prostate cancer such as point mutations in SPOP FOXA1and IDH19 Largescale oncogenic structural rearrangementstranslocations and copynumber changes are also common oftenleading to the coordinated dysregulation of multiple elements forexample the loss of 21q which is associated with the TMPRSSERG fusion rearrangement and the subsequent rearrangement ofSMAD410 Improved understanding of the mechanisms governing disease pathogenesis and progression may allow for bettertherapeutic exploitation for example genetic alterations in theDNA repair machinery have been linked to susceptibility toPARP inhibitors in a range of tumour types and alterations in ARconfer sensitivity or resistance to androgen deprivation therapy inmetastatic castrate resistant prostate cancer mCRPC11NAALADL2 is located on 3q2631 and is a member of theglutamate carboxypeptidase II family along with the widely studied PCa marker PSMA NAALAD112 and its expression haspreviously been associated with prostate tumour stage andgrade13 with expression predicting poor survival following radicalprostatectomy13 A large genomewide association study GWASof prostate cancer cases found rs78943174 a SNP withinthe 3q2631 NAALADL2 locus was associated with high Gleasonsum score14 A further rs10936845 SNP was identiï¬ed within aGATA2 motif that increases NAALADL2 expression in prostatecancer patients where increased expression also predicted biochemical reccurence15 The same study showed even higherbinding preference to HOXB13 and FOXA1 to this site suggestingcooccupancy by these important transcription factors both ofwhich have been shown to be involved in AR cistromereprogramming1516functionsAdjacent to NAALADL2 in the genome is TBL1XR1 a corecomponent of nuclear receptor corepressor NCoR complex thatacts as a coregulator of nuclear receptors uencing severalcellularandammation17 TBL1XR1 is also an androgen receptor AR coactivator18 Expression of TBL1XR1 has been associated withpoor prognosis in several cancers predicting poor overall survivaland lymph node metastasis in gastric19 and ovarian cancers20 andrecurrence in colorectal21 breast22 and liver cancers23antiapoptosisincludinggrowthHere we utilise largescale publicly available genomic data tobetter characterise the broad somatic copynumber changesoccurring within the 3q263132 locus particularly centredaround gainsampliï¬cations in NAALADL2 and TBL1XR1 andlinking them to the clinical characteristics of aggressive prostatecancerResults3q263132 gain frequency is increased in aggressive PCaCopynumber alterations often alter the expression of the gene inwhich they occur with gene dosage known to correlate withmRNA expression Genetic structural variants are also known toalter transcriptional regulation by altering cisregulatory elementssuch as promotors and enhancers resulting in differentialexpression2425 Increased NAALADL2 and TBL1XR1 expressionhave previously been linked to poor prognosis in cancers leadingus to examine the frequency of somatic copynumber gains inthese genes across various prostate cancer subtypes19Alteration frequency was assessed using data from cBioportalFig 1a and all study data was processed using a standardisedpipeline to ensure comparable results Alteration frequency wasassessed in a total of patients samples in nonoverlapping studies Appendix eleven studies focused onprimary prostate cancer four on metastatic prostate cancer andone on neuroendocrine and castrateresistant cancers Signiï¬cantcopynumber increases above a derived background thresholdwere categorised as gains and copynumber decreases as deletions Overall the distribution of NAALADL2 and TBL1XR1alterations were significantly different between disease subtypesto that which is expected Chisquared goodnessofï¬ttestFig Somatic alteration frequency of NAALADL2 and TBL1XR1 across prostate cancer subtypes in publically available genomic studies n a NAALADL2 genetic alteration frequency across different subtypes of prostate cancer b TBL1XR1 genetic alteration frequency across differentsubtypes of prostate cancer P primary prostate cancer M metastatic prostate cancer NE neuroendocrine prostate cancer and castrate resistantprostate cancer CRPC All annotations were assigned using Genome Nexus and CNAs are called using GISTIC or RAE algorithms Pvalues show theresults of a Chisquared goodnessofï¬t test to determine if the number of observed patients with each alteration type is different from that which isexpected across each cancer subtype Results detailed in Supplementary data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xp and p Fig 1a b with gains beingmost frequent in castrateresistant prostate cancer and respectively followed by neuroendocrine and metastatic and then primary prostatecancer and Fig 1a b3q263132 gains extend across an oncogenerich region ofChr3 As CNAs are known to associate with more aggressivesubtypes of prostate cancer we investigated their association withclinical characteristics to establish if changes can be detected earlyin the life history of cancer predicting more aggressive diseaseWe utilised copynumber data from primary an conï¬neddisease from both the UK and Canadian International CancerGenome Consortium ICGC cohorts and The Cancer GenomeAtlas TCGA These studies use intermediatehigh risk prostatecancer patients with no treatment prior to radical prostatectomyTo allow comparisons between the studies data were reanalysedusing the Genomic Identiï¬cation of Signiï¬cant Targets in Cancer GISTIC2 methodfavoured by the broad institute andTCGA27 as it distinguishes between lowlevel copy numberincreases gains and highlevel copynumber increases ampliï¬cations Within the three cohorts we found that copynumbergains across both genes were frequent with gains in NAALADL2ranging from Canada to UK and between for ampliï¬cations Table TBL1XR1 had an almostidentical CNA frequency of between UK to Canada Table We ï¬tted a randomeffects model to more accurately estimatethe frequency of NAALADL2 and TBL1XR1 gainampliï¬cationscombining the data from all three cohorts which estimated thetrue frequencies to be CI and CI for NAALADL2 and TBL1XR1 respectively Supplementary Fig Leaveone out analysis and adiagnostic plots revealed that the ICGC Canada study was asignificant source of heterogeneity thereforethe study wasremoved and the model reï¬t The ï¬nal estimated frequency ofNAALADL2 and TBL1XR1 gainsampliï¬cations was CI and CI respectivelyin primary prostate cancerDue to their close proximity in the genome we investigated ifgainsampliï¬cations in NAALADL2 and TBL1XR1 cooccurred inthe same patients using a genomewide Fishers exact test with afalse discovery rate correction NAALADL2 and TBL1XR1significantly coampliï¬ed in all three cohorts ICGC UK p ICGC Canada p 158e and TCGA p testingconï¬rmed that widespanning gainsampliï¬cations occurred inneighbouring regions in the majority of patients In the ICGC UKSupplementary Fig Additionallycohort n there was a significant cooccurrence of somaticcopynumber gainsampliï¬cations in NAALADL2 with TBL1XR1FDRcorrected Fishers exact test Fig 2a Gainsin this region also significantly correlated with two regionsspanning chromosomes and both gains previously describedas being abundant in prostate cancer Supplementary Data The Canadian cohort n showed a similar pattern of cooccurrence with gainsampliï¬cations spanning the region surrounding NAALADL2 and TBL1XR1 3p253 to 3q29 Fig 2bThere was also a significant cooccurrence with gains in thebeginning of chromosome as well as some sporadic cooccurrence across the genome Fig 2b Supplementary Data These results were supported by the outcome of the same analysisin the TCGA cohort n although several large spikes ofcooccurrence were also observed in regions nottoNAALADL2 and TBL1XR1 as these spikes were not present inthe other two cohorts they most likely represent artefacts Fig 2cSupplementary Data Overall across the three cohorts therewere was a consistent coampliï¬cation in region spanning genes between 3p141 and 3q29 While a number of patients hadmultiple CNAs we found no consistent cooccurrence withcommon CNAs such as MYC gain FGFR1 gain PTEN loss RB1loss or NKX31 loss FDRcorrected Fishers exact test p The 3q26 region where NAALADL2 and TBL1XR1 are locatedis rich in oncogenes such as PIK3CA SOX2 ECT2 and PRKCIwhich may act to drive tumorigenesis29 We determined thenumber of known oncogenes within this deï¬ned region bycomparing the overlapping genes that coampliï¬ed withNAALADL2 and TBL1XR1 in alltheNetwork of Cancer Genes database30 This revealed that of genes are known oncogenes including BCL6 ATRand PI3K family members Supplementary Data These resultsconï¬rm that a high proportion of prostate cancer patientsdevelop large copynumber gains across multiple oncogenes inthis genetic regionthree cohorts againstlocalGains in 3q263132 associate with adverse clinical featuresCommon prostate cancer CNAs such as those in MYC andPTEN are known to associate with higher Gleason grade31Consistent with these ï¬ndings we also found NAALADL2 andTBL1XR1 ampliï¬cations were highly correlated with GradeGroup GG showing that the frequency of NAALADL2 andTBL1XR1 gains tripling between GG1 and GG2 lesions and morethan doubling between GG2 and Table A Chisquaredgoodnessofï¬t test showed that the distribution of gainsampliï¬cations between Grade groups was significantly different to thedistribution of diploid patientsfor both NAALADL2 andTBL1XR1 p and p WhenTable Alteration frequency of NAALADL2 and TBL1XR1 called via the GISTIC2 method in three nonoverlapping primary anconï¬ned radical prostatectomy cohorts from the International Cancer Genome Consortium ICGC and The Cancer GenomeAtlas TCGAICGC UKICGC CANADATCGANAALADL2TBL1XR1NAALADL2TBL1XR1NAALADL2TBL1XR1AlterationDeep DelShallow DelDiploidGainAmpliï¬cationTotalnnnnnnThe degree of copy number alteration is discretised into ï¬ve categories ampliï¬cation gain representing low and high level copy number increase diploid no significant CNA and shallow and deepdeletion representing low and high level copy number lossCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xand compared to those without and Moreover ofthe patients who had their lymph nodesexamined the percentage of patients with lymph node positivitydeï¬ned through positivity on haematoxylin and eosin stainingHE was more than double in patients with NAALADL2 orTBL1XR1 gains and compared to those withouta gain and Chisquared goodnessofï¬t test p and p Finally while only one man in the cohorthad evidence of positive ï¬ndings in his bone scan we did observea significant between the number of equivocal bone scans inpatients with gains and compared to and in those patients without gainsChisquaredgoodnessofï¬t test p and p for NAALADL2and TBL1XR1 respectively however the number of expectedcases in each of these categories was less than adding someuncertainty to this result We found no significant difference inthe mean age between patients with different copynumbers ofNAALADL2 or TBL1XR1 KruskallWallis rank sum test p and As gainsampliï¬cations in NAALADL2 and TBL1XR1 coincidewith a cluster of known oncogenes and coincide with clinicalvariables linked to more aggressive disease we also compareddiseasefree survival Comparing patients with gainsampliï¬cations in NAALADL2 and TBLXR1 to those with diploid copies weobserved no significant association in the ICGC UK cohort n although there was a trend towards reduced diseasefreesurvival Supplementary Fig 4A In the larger TCGA cohortn there was a significant reduction in diseasefree survivalin patients with a gain in either NAALADL2 Logrank MantelCox p or TBL1XR1 Logrank MantelCox p Supplementary Fig 4BUnivariable Cox regression conï¬rmed that carrying a gainampliï¬cation in NAALADL2 and TBL1XR1 in the TCGA cohortresulted in reduction in diseasefree survival hazard ratio HR CI p Forreference weperformed a similar analysis of patients with PTEN deletion orMYC gains two common copy number alterations with provenin prostate cancer3233association with diseasefree survivalWhen patients were stratiï¬ed solely by CNA status and survivalcompared using the KaplanMeier method those patients withMYC gain or PTEN deletion homo or hemizygous showed nosignificant difference in diseasefree survival Logrank MantelCox p and p respectively while those stratiï¬ed byNAALADL2 gain TBL1XR1 gain or both NAALADL2 andTBL1XR1 gain showed significant differences in survival Logrank MantelCox p Supplementary Fig 5AE Univariable Cox regression estimated the hazard ratios for thesecopynumber alterations as CI CI and CI for MYCPTEN and NAALADL2TBL1XR1 respectively We also comparedthe diseasefree survival of patients with only a copynumberalteration in each of the four genes where each group wasmutually exclusive Supplementary Fig 5F G This showed thaton the whole patients with CNAs in NAALADL2TBL1XR1 hadreduced or equal diseasefree survival as those with either onlyMYC gain or only PTEN loss Patients with copy number gains inboth had a worse prognosis All clinical data is available inSupplementary Data Fig Genomewide cooccurrence with NAALADL2 and TBL1XR1 gainsampliï¬cations The Y axis shows log10 qvalues from a Fishers exact testbetween gainampliï¬cations in NAALADL2 and cooccuring genes Thedotted line represents the threshold for statistical signiï¬cance aftercorrection for multiple testing a Signiï¬cantly cooccurring gains across thegenome in the ICGC UK cohort b Signiï¬cantly cooccurring gains acrossthe genome in the ICGC Canada cohort c Signiï¬cantly cooccurring gainsacross the genome in the TCGA cohort NAALADL2 and TBL1XR1 cytobandpositions are labelled All Fisher tests use NAALADL2 gain or ampliï¬cationas the altered group Full results are detailed in Supplementary Data compared to common CNAs such as PTEN loss and MYC gainthe alteration frequency of NAALADL2 and TBL1XR1 was morecorrelated with higher Gleason grade groups Spearmans rho was p p for NAALADL2 and TBL1XR1and p for PTEN and MYC respectivelySupplementary Fig 3AMoreover we also noted the same pattern ofincreasingfrequency of gains with T stage Chisquared goodnessofï¬t testp and p respectively Table Patients with gains exhibited differences in the location of thetumour within the prostate with and of thosewith NAALADL2 and TBL1XR1 gains having tumoursinoverlapping and multiple zones compared to just and for those without gains Chisquared goodnessofï¬t testp and p There was also an increased relativenumber of positive surgical margins Chisquared goodnessofï¬ttest p and p in patients with gains As CNAs in NAALADL2 and TBL1XR1 were associated withclinical characteristics such as Gleason grade group and T stagewe used multivariable Cox regression models to conï¬rm that anychanges in survival were driven by these associations and foundthat copy number gains in NAALADL2 and TBL1XR1 were nolonger significant once corrected for Gleason grade and T stagep Supplementary Data These results suggest thatthe differences in diseasefree survival seen when stratiï¬ed byCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xTable The frequency of NAALADL2 and TBL1XR1 gainampliï¬cations by Gleason Grade Group in the TCGA cohortGrade groupGG1ObservedExpected within GGGG2ObservedExpected within GGGG3ObservedExpected within GGGG4ObservedExpected within GGGG5ObservedExpected within GGTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalGrade groups deï¬ned as Grade Group Gleason score ¤ Grade Group Gleason score Grade Group Gleason score Grade Group Gleason score Grade Group Gleason scores and Displayed are the numbers of patients observed with gain or without diploid a gainampliï¬cation in this region in each Grade Group Additionally the expected number ofpatients estimated to be within each category is also shown along with the percentage of each Grade Group which is made up by patients with or without a gain Bold values indicate the overallpercentage of the group with a given copynumber state All clinical data detailed in Supplementary Data Table The frequency of NAALADL2 and TBL1XR1 gainampliï¬cations by T stage in the TCGA cohortT stageT2aObservedExpected within T stageT2bObservedExpected within T stageT2cObservedExpected within T stageT3aObservedExpected within T stageT3bObservedExpected within T stageT4ObservedExpected within T stageTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalDisplayed are the numbers of patients observed with gain or without diploid a gainampliï¬cation in this region in each T stage Additionally the expected number of patients estimated to be withineach category is also shown along with the percentage of each T stage which is made up by patients with or without a gain Bold values indicate the overall percentage of the group with a given copynumber state All clinical data detailed in Supplementary Data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xgainampliï¬cation status are driven by strong association withthese clinical variablesIn the ICGC cohortsindividuals with somatic singlebasealterations in NAALADL2 also associated with reduced diseasefree survival in a combined ICGC cohort as well as associatingwith reduced diseasefree and overall survival in an early onsetprostate cancer cohort ICGC EOPC Denmark Singlebasesubstitutions in TBL1XR1 were only associated with diseasefreesurvival in the ICGC EOPC cohort Supplementary Fig Singlebase alterations did not occur with a frequency greater than onein any single base in NAALADL2 or TBL1XR13q263132 gains cooccur with proproliferative transcriptionTo determine the potentialfunctional consequences of gainswithin the NAALADL2 and TBL1XR1 amplicon mRNA expression proï¬les were explored using the TCGA RNAseq dataDESeq2 was used to determine differentially expressed genesbetween patients with copynumber gains for both NAALADL2and TBL1XR1 compared to those without For NAALADL2 therewere differentially expressed genes DEGs and DEGsfor TBL1XR1 when the two groups were compared FDR Supplementary Data Our previous study on NAALADL2identiï¬ed nine genes which were reciprocally regulated by overexpression or knockdown of NAALADL226 Of these nine wefound that three cancer antigen XAGE1B adhesionmotiliy regulator SPON2 and AR regulator HN1 were significantly differentially expressed p in patients with a NAALADL2 gainand in the same direction as the overexpression model2634When comparing the DEGs between patients with and withoutin either NAALADL2 or TBL1XR1 wegainsampliï¬cationsobserved that of the DEGs overlapped between NAALADL2 and TBL1XR1 Fig 3a of the geneswere located within the locus we identiï¬ed as coampliï¬ed withNAALADL2 and TBL1XR1 and were differentially expressedconsistent with a mechanism of selfregulating expression2425TBL1XR1 was one ofsignificant overlapping DEGsNAALADL2 was just on the boundary of statistical signiï¬canceFDR corrected Wald test p Supplementary Fig theNAALADL2 has been shown to be coexpressed with numberof androgen regulated proteins and contains a number of ARbinding sites and TBL1XR1 is an AR coactivator and may beinvolved in AR cistrome reprogramming18263738 We thereforelooked at overlap between androgen regulated genes with ARbinding sites full or partial and genes demonstrated to beandrogen regulated following R1881 stimulation in at least twoindependent studies3739 shared genes were differentiallyexpressed in patients with NAALADL2 and TBL1XR1 gainsampliï¬cations that contained AR binding sites and demonstratedandrogen regulation by R1881 genes had either aAR binding motif were androgen regulated in two or morestudies or both Fig 3bOf the overlapping DEGs a total of were knownoncogenesSupplementary Data which may drive anaggressive clinical phenotype Of note was PI3K family membersPIK3C2G PIK3CA PIK3CB PIK3R4 Mucin family membersMUC1 MUC4 and MUC6 and other prostate cancer associatedgenes such as SMAD4 SOX9 and SPOP794041 Additionallyseveral genes which form commercial prognostic assays were alsodifferentially expressed such as the Decipher assay NFIB LASP1ZWILCH THBS2 COL1A2 and COL5A142 Oncotype DX assaySFRP4 COL1A1 KLK2 TPX24344 and the Prolaris assayASPM BUB1B CENPF and FOXM145We inspected of the top most significant shared DEGs usingunsupervised hierarchal clustering Fig 3b SupplementaryData DEGs mostly displayed upregulation consistent with agenedosage effect Fig 3b24 Enrichment for biological processes was assessed by Geneset enrichment analysis GSEA forNAALADL2 and TBL1XR1 gainsseparately and by overrepresentation analysis ORA on the shared DEG list usingWebGestalt46GSEA on the individual lists of DEGs showed that despite a largeoverlap the enriched biological processes did differ between the twogenes patients with a gain in NAALADL2 showed enrichment inprocesses related to NADH dehydrogenase complex assemblyFDR mitochondrial respiratory chain complex assemblyFDR translational initiation FDR cytochromecomplex assembly FDR protein localisation toendoplasmic reticulum FDR and cytoplasmic translationFDR Supplementary Data Patients with a gain inTBL1XR1 showed enrichment in mitotic cell cycle phase transitionchromosome segregation actin ï¬lamentbased movement microtubule cytoskeleton anisation involved in mitosis regulation ofcell cycle phase transition cell cycle G1S phase transition FDR as well as a number of other processes SupplementaryData To understand the combined effect of gainsampliï¬cation inthese genes we investigated overrepresentation of processes in theDEGs which were common to both NAALADL2 and TBL1XR1In the shared DEG list the significantly enriched Gene OntologyGO biological processes were all involved in the cell cycle cyclepathway including mitotic regulation and chromosome segregation Fig 3c Supplementary Data These ï¬ndings support ahypothesis whereby gains in NAALADL2 and TBL1XR1 concomitantly bring about mRNA expression changes which supportan aggressive proproliferative phenotype in primary prostatecancerDiscussionIn this study we present evidence that somatic copynumber gainsin NAALADL2 and TBL1XR1 are more frequent in high gradeand aggressive forms of prostate cancer These results are bolstered by studies which have identiï¬ed CNAs in this region inmCRPC however to our knowledge this is the ï¬rst time thesegains have been reported in neuroendocrine disease47 We alsodemonstrate that NAALADL2 and TBL1XR1 gains occur in anearlier setting cooccurring with gains in neighbouring genes Amajor barrier to the adoption of CNA based tests in the clinic isthe reliance on expensive NGS approaches as well as sufï¬cientsequencing depth and coverage to assess overall copynumberburden The discovery of smaller clinically significant loci couldallow for cheaper quicker targeted approaches particularly if asingle loci can elude to gainsampliï¬cations in a larger regionsurvivalto diseasefreeIn primary prostate cancer Gainsampliï¬cations in this regionassociated with Gleason grade tumour stage number of positivelymph nodes bone scan results and as these variables contributetostratiï¬ed byNAALADL2TBL1XR1 status also have altered diseasefree survival times Our work is supported by previous studies that haveeluded to the clinical signiï¬cance of this locus particularly asgermline SNPs within this locus have been associated with higherGleason grade tumours and more aggressive disease14 This alsosupports smaller studies such as those by HeselmeyerHaddadet al who identiï¬ed two out of seven patients with gains inTBL1XR1 in recurrent prostate cancer48 However these studiesinvestigated these genes in isolation na¯ve to the larger context inwhich these alterations occur Here we have found that gainsampliï¬cations atthis locus not only coamplify with otherdescribed oncogenes but associate with much larger transcriptional changes which are consistent with the observed aggressiveclinical phenotypetimepatientsCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xFig Transcriptomic changes in patients with NAALADL2TBL1XR1 gains a Venn diagram showing the number and percentage of overlapping DEGsbetween patients with NAALADL2 gainampliï¬cation and TBL1XR1 gainampliï¬cation overlap b Venn diagram showing the number ofNAALADL2 and TBL1XR1 DEGs and genes with identiï¬ed AR binding sites determined through ChIPSeq and AR knockdown and genes shown to beandrogen regulated following R1881 stimulation c Unsupervised hierarchal clustering of the top most significant DEGs bar beneath upper dendrogramshows copynumber status of patients where red is patients with a gain in both NAALADL2 and TBL1XR1 and grey represents those without gainampliï¬cation in these genes Heatmap represents meancentred z scores derived from RKPM values d Chord diagram showing significantly overrepresented GO biological processes and key genes within these processes All clinical data detailed in Supplementary DataCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xOverall changes in copynumber burden have been shown to beindicative of genetically unstable tumours and predict prostatecancer relapse5 Many single CNAs have already been describedthat predict PSA recurrence after radical prostatectomy includingPTEN loss cooccurrence of PTEN FAS 10q2331 and PAPSS210q23210q2331 loss a loss of 16q with or without a loss ofPTEN a loss within 6q 13q gains in MYC 11q1317 7q and aconcurrent loss of 8p22 with a gain of 8q2487 Compared towellknown CNAs such as PTEN loss and MYC ampliï¬cation wehave observed that Gainsampliï¬cations in NAALADL2TBL1XR1equally or better segregate patients who will have reduced diseasefree survivalThe gainsampliï¬cations in NAALADL2TBL1XR1 also corresponded to a significantincrease in both NAALADL2 andTBL1XR1 mRNA supporting previous studies that have described upregulation of these genes and linked them to poor prognosis in various cancers19 This suggests that gains in thesegenes may cause increased expression of NAALADL2 andTBL1XR1 in cancers We also noted a number of the differentiallyexpressed genes between patients with and without a gainampliï¬cation in NAALADL2TBL1XR1 have been shown to beandrogen regulated however further work is required to determine if gainsampliï¬cations in this region cause changes in ARtranscriptional regulation through cis regulatory elements or as adirect consequence of the genes altered in this region1837In those patients with these gains we noted transcriptionalchanges in several genes associated with aggressive prostatecancer including differential expression of genes appertaining toprognostic assays such as Decipher Oncotype DX and Prolaris aswell as families such as mucins50 This may explain theaggressive clinical phenotype observed in these patients We alsoobserved that when weighted individually there were differencesin enrichment of biological processes between those with NAALADL2 gains and TBL1XR1 gains suggesting that each generesults in some unique cellular changesOur ï¬nding that gains in the 3q26 locus result in concurrentexpression of oncogenes located within this region and theirdownstream targets identiï¬es multiple potential therapeutic avenues warranting further investigations This study centred aroundtwo genes NAALADL2 and TBL1XR1 both of which areattractive therapeutic targets with TBL1XR1 previously suggestedas a potential cancer target operating via the TGFβ signallingpathway and potentially regulating AR signalling5354 Additionally the tumour speciï¬city of NAALADL2 and basal membranouslocalisation makes it potentially accessible using antibodydrugconjugates13 This approach may be feasible if like other familymembers such as PSMA antibody binding results in subcellularinternalisation12 Moreover several of the oncogenes in whichgains cooccur as well as the downstream oncogenes activatedfrom gains in the 3q26 region such as ATR PI3K family members PIK3C2G PIK3CA PIK3CB PIK3R4 MUC4 BCL6 SOX9can be therapeutically targeted or have been suggested as therapeutic targets in cancer5155 In the PI3K pathway PIK3CBspeciï¬c inhibitors may have utility in patients with mutationsampliï¬cations andor fusion of this gene59 These ï¬ndings mayhave clinical relevence as it has been reported by de Bono et althat many individuals who had durable year responses toPIK3CBspeciï¬c inhibition harboured activating mutation orampliï¬cation in PIK3CB60 and phase II trials of ipatasertib anAkt inhibitor targeting the PI3KAkt axis has shown promise inlate stage mCRPC61 Together our results suggest that largescalegenomic gainsampliï¬cations occur in the 3q26 region in a hi | 2 |
craigpattersontelecaretoj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per loncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatientssaï¬f al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset ogorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseandimplicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 commonduringadversetheoutbreakconï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | 0 |
neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing antiammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these ï¬nd ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the beneï¬t of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as insilico drug re purposing in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to ï¬nd out a new se ries of nep inhibitors to the best of our knowledge this is the ï¬rst study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of Ëa prior to docking and simulation studies the biological unit of protein was prepared using protein preparation wizard in schrodinger suite during the process of protein preparation the protein was subjected to import and reï¬ne review and modify and minimize processes in protein preparation wizard missing side chains and residues were ï¬lled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond Ëa were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force ï¬eld and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workï¬ow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force ï¬eld in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding aï¬nities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer ï¬le of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force ï¬eld to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme proï¬le the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coeï¬cient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of ï¬ve were calculated induced ï¬t docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedï¬t docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifdsp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which reï¬nement of all residues within Ëa of the ligands pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedï¬t protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula prime_energy glide score ifd score glide_ecoul molecular dynamics md simulation the ï¬exibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede ï¬ned spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol Ëa balanced at k tem perature and bar pressure via npt ensemble in the ï¬nal step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme proï¬le further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workï¬ow was used and the cubic box of speciï¬c dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the ï¬nal torsional reï¬nement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were ï¬ltered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identiï¬ed for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this ï¬nding indicates that the selected drugs may act as nep inhibitors induced ï¬t docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced ï¬t docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the ï¬exible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications ï¬rst it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd conï¬rmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme proï¬le was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of ï¬ve given in table all the selected drugs obey the lip inski rule of ï¬ve molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited ï¬exibility which is insuï¬cient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and lig ï¬t prot for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be Ëa and Ëa respectively the rmsd values were found to be in the acceptable range Ëa but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are Ëa and Ëa for protein and ligand respectively for complex the rmsd values were found to be Ëa for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of Ëa for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signiï¬ 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of ï¬ve sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is antiammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding aï¬nity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are ï¬rst it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drugs activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identiï¬ed based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0 | 0 |
"Oral cancer is one of the most common noncommunicable diseases worldwide This paper presentsan evaluation of the trends and geographical distributions of oral cancers in the Saudi Arabian populationMethods Data from Saudi Cancer Registry reports were used in this analysis which assessed the period between and All cancer cases are recorded in these reports as well as the age gender region and histologicalcancer sites for each patient Agestandardised and agespecific incidence rates were calculated in these reportsFor the purposes of this paper only cancers of the lips tongue and mouth were considered oral cancersResults Between and the Saudi Cancer Registry identified cancer cases in total Of these were oral cancer The mean agestandardised rate of oral cancer for the study period was per peoplefor females it was and for males it was The incidence of oral cancer varied by region with Jazan displayingthe highest agestandardised rate and Hail displaying the lowest A positive correlation was observed between oralcancer incidence and ageConclusion The overall trend of the agestandardised rate for both sexes remained constant from to However the oral cancer incidence in Saudi Arabia varies by region Studying this variation in more detail will helpto guide awareness programmes in the regions that are most in needKeywords Cancer epidemiology Cancer prevention and control Oral neoplasmBackgroundCancer is an intractable global health problem and theleading cause of death in the developed world in the developing worldit is the secondleading cause [] In the most recent year for which information fromthe International Agency for Research on Cancer IARCis available approximately million new cancer caseswere diagnosed and million people died from cancerworldwide [] In this same year new cases oflip and oral cavity cancers were reported representing of all cancer casesA review of the global prevalence of oral cancer revealsa wide variation in distribution among countries []Correspondence bmalshehrinuedusaDepartment of Clinical Laboratory Faculty of applied Medical SciencesNajran University PO Box Najran Kingdom of Saudi ArabiaTwothirds of the estimated incidence of oral cancer occurred in developing countries with up to of allnew oral cancer cases in Sri Lanka India Pakistan andBangladesh [] Converselyin France which has thehighest rate of oral cancer incidence in the EuropeanUnion only oral cancer cases were reported in representing just of all cancer cases [] Inthe USA the American Cancer Society estimated that in approximately people were diagnosed withoral cavity or oropharyngeal cancer and will dieof these cancers [] In Arab countries the prevalence oforal cancer is concentrated between western and southeast Asia [] While this type of cancer is relatively uncommon across Arab gulf countries Saudi Arabia andYemen are notable exceptions [] No studies have beenpublished discussing the epidemiological parameters andgeographic distribution of oral cancer cases or any of its 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 0cAlshehri World Journal of Surgical Oncology Page of subtypes in the Saudi Arabian population Thereforethis study analysed and discussed oral cancer trends inthe Saudi population by using the most recent dataavailableAccording to the International Classification of Diseases 10th revision ICD10 oral cancer is classifiedinto six sites mucosal lip ICD10 C00 tongue ICD C02 gum ICD10 C03 mouth floor ICD10C04 palate ICD10 C05 and mouth ICD10 C06However examining trends in oral cancer incidencerates that include all oral sites can be misleading Thedata analysed in this study only include cancers of thelip tongue and mouth ICD10C00C06 which formthe majority of oral cancers moreover they have severalrisk factors in common and share a similar biology []Thus those accounting for a minority of oral cancercases were excludedMaterials and methodsDataThis retrospective descriptive epidemiological study analysed oral cancer cases in a Saudi population that hadbeen diagnosed from January through December The study used a method of analysis similar tothat used by Alshehri [] Their analyses incorporated male and female data on lip tongue and mouthICD10C00C06 cancer cases to evaluate disease patterns in the Saudi population Data for the present studywere obtained from the Saudi Cancer Registry SCR apopulationbased registry established in by theMinistry of Health in Saudi Arabia This data can onlybe obtained from the reports published by the SCRSince the SCR has been publishing reports oncancer in Saudi Arabia with the primary objective of defining populationbased cancer incidences The presentstudy was conducted using these reports to derive a descriptive epidemiology of oral cancer in Saudi Arabia InSCR reports agestandardised ASR and agespecificAIR incidence rates were calculated with a focus ongenderspecific and regional differencesThe analysis included cases recorded in the SCR filesfrom January to December totalling cancer cases overall approximately of which wereoral cancerData analysisThe GraphPad Prism6 software was used to analyse thedata Descriptive analyses of epidemiological data wereconducted by calculating the mean of the percentagesand ASR stratified by age sex region and year of diagnosis The ASR was calculated in the SCR reports byadjusting all Saudi regions populations mathematicallyto have the same age structure On the other hand theAIR was calculated by summation of the number ofcancer cases occurring during the year in a regionspopulation among specific age and sex groups dividedby the midyear population of these age and sex groupsUsing these two standardised rates is important because age is a basic element of the risk of developingcancer globally [] Using summary measure tools suchas the ASR and AIR which represent the schedule ofagespecific rates in different regions and across timewill give us a more representative picture of the characteristics in question and enable comparisons of cancerincidences between several populations of Saudi regionsthat differ with respect to ageResultsIncrease in the number of oral cancer casesThe total number of cancer cases identified by the SCRfrom to was with males and females Of this total cases were oral cancer The number of registeredoral cancer cases increased gradually from MF in to a peak of MF in however only cases were reported in MF Table Table Number of oral cancer cases in Saudi Arabia for theperiod from to YearNumber of female casesNumber of male casesTotal 0cAlshehri World Journal of Surgical Oncology Page of The percentage of cases representing oral cancers was for females and for males Fig in These percentages decreased to for females and for males in Fig The percentage curve fororal cancer out of all cancer types for males and femalescorrelated with increases and decreases over the studyperiod apart from the years and Fig ASR of oral cancer fluctuated over the study periodBetween and the ASR per male casesfluctuated in it was trending downwards to alow of in and peaking at in beforedropping again to in Fig The female ASRper increased from in to a peak of in decreasing again to in Fig For bothsexes ASR curves like oral cancer percentages correlateto increases and decreases over the study period apartfrom the years and Fig and generally remained constant from to ASR of oral cancer varies by regionThe ASR data for oral cancer cases of all persons demonstrated a wide variation across Saudi regions TheASR means per people for the period from to ranged from in Hail to in Jazan with anational average of per Fig The Jazan region had the highest male ASR mean at followed by the Najran and Tabuk regions at each Fig Conversely Qassim Baha Hail and theNorthern province reported the lowest ASR averages at and per respectively Fig Male and female ASR data were generally equivalentin terms of region rankings with the Jazan region posting the highest overall ASR of as an average valueof both genders followed by the Makkah region at and the Najran region at Fig Similarly the HailBaha Qassim and Madinah regions posted the lowestASR averages at and respectively FigAIR of oral cancer increases with ageThe AIR data from to showed a positive correlation between oral cancer incidence and age withmost cancer cases occurring in the older age groups Figure shows the AIR of oral cancer increasing noticeablywith age up until age More than of cases werediagnosed after the age of Some AIR differences were found between the sexesacross age groups From ages to rates of oral cancer were higher in females than in males however thistrend had reversed to favour males in the 75andoverage group The overall AIR per showed onlyslight differences between the sexes at for femalesand for males Fig DiscussionA review of oral cancer data in Saudi Arabia for theperiod from to showed an overall increasingtrend in the numbers of oral cancer patients Despitethis rise ASR data trends for oral cancer remained constant from to Fig This curve stabilised inthe face of a substantial Saudi Arabian population increase from million in to million in [] Many accumulative factors could be contributed tothis stability First the significant increased access tohealth services in Saudi regions has contributed to thedissemination of oral health awareness and early diagnosis of some cases of metaplasia that were discovered before they could develop into cancerous tumours SecondFig Consistency in percentage curves for oral cancer out of all cancer types from to The percentage curve for oral cancer out of allcancer types for males and females are correlated with overall increases and decreases over the period from to with the exception ofyears and 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer fluctuated over the study period Between and the male ASR was per in and dropped to in The female ASR fluctuated between and per the increased level of public health in the Kingdom isusually linked to an increase in the economic level of thecountry and individuals may have contributed to thisconstancy as many infectious factors such as virusesand fungi have been linked to oral cancers Third SaudiArabia is a majority Islamic country wherein many oralcancer risk factors such as alcohol consumption andcigarette smoking are forbidden by Islamic law Islamiclaw may thus mediate the lower number of oral cancercases in Saudi Arabia compared to the rest of the world[] Thus based on the IARC data for eight ofthe nine world regions whose ASR of oral cancer isabove the global rate [ ] were located in nonMuslimcountries [] with Melanesian regions having the highest rate [] In contrast most of the regions locatedwithin Muslim countries were ranked below the globalASR [] Further investigation of this aspect could therefore be valuable to cancer prevention effortsThe ASR data revealed that more females than maleswere diagnosed with oral cancer in Saudi Arabia at for men and for women This finding is in contrastwith global data showing that men are more likely to develop oral cancer than women [] In the most recent year for which IARC information is available theglobal ASR of oral cancer was for men and forwomen [] While these rates do not match the globalFig Agespecific incidence rates AIR of oral cancer increases with age The total AIR of oral cancer increased noticeably with up until patientswere and over More than of the cases were diagnosed after the age of 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer varies by region in Saudi Arabia For all persons the ASR means per peoplefor the period from to ranged from in the Hail region to in the Jazan regionsex distribution oral cancer in Saudi Arabia has a relatively low overall ASR when compared to the globalaverage as discussed aboveResults also revealed consistency in the ASR oral cancer curve for both sexes Fig potentially due to thepresence of common risk factors for oral cancer in malesand females This finding could be used as a startingthreshold for studying the risk factors of oral cancer inthe Saudi population through studying the common factors between the sexesAs with many other types of cancer the present studyfound a correlation between the occurrence of oral cancer and age with of cases diagnosed after the age of years In the USA the average age at diagnosis of oralcancer is years and twothirds of individuals with thisdisease are over the age of [] Ageing is accompaniedby increased susceptibility to cancercausing geneticmaturations due to accumulated exposures to environmental and behavioural risk factors Avoiding these riskfactors could greatly reduce the role that ageing plays incancerThis study found a wide variation in the incidence oforal cancers among Saudi regions Such differencescould indicate that regional environmental factors andlifestyle habits affect oral cancer incidence The resultsreviewed above found that the Jazan region possessedthe highest ASR of people with oral cancer In contrastthe Northern province presented the lowest ASR Severalstudies have focused on investigating why the Jazan region has such a high incidence of oral cancer []Ibrahim and others focused on the association ofcertain eating habits and lifestyle behaviours with the development of oral cancer especially the abuse ofshamma a form of smokeless tobacco and the chewingof khat Catha edulis leaves These substances havebeen classified as carcinogens especially in relation tooral cancer Studies by these researchers found that consuming shamma increased the odds of developing oralcancer 29fold suggesting a strong link between oralcancer and diet and lifestyle choicesAccording to the above poor dietary habits related totobacco use and its derivatives are one of the main reasons for the high incidence of oral cancer in some citiesand not others Other factors such as variations in thegenetic background of the Saudi regions citizens cannotbe excluded especially because most of the populationin the Kingdoms regions is tribal so consanguineousmarriages are highly common Thus genomic sequencing can provide information on genetic variants thatmay be present in citizens of these regions and that maybe linked with increased or decreased rates of oral cancer development Populationbased genetic testing issuggestedConclusionDespite the presence of yeartoyear changes in the incidence of oral cancer in the Saudi population there wasoverall no noticeable change in the incidence of oralcancer in the Saudi Arabian population for the periodbetween and In contrast to some international findings females were somewhat more likelythan males to be diagnosed with oral cancer in SaudiArabia The positive correlation between ageing and theincidence of oral cancer for both males and femalesdemonstrates that oral cancer is mainly a disease of theelderly both in Saudi Arabia and across the globe Thewide variation in the incidence rates among Saudi regions raises an important research question concerning 0cAlshehri World Journal of Surgical Oncology Page of World Bank World Development Indicators Washington DC WorldBank Access online via httpwwwirieduarpublicaciones_irianuariocd_anuario_2014Economia4bpdf Albar MA Islamic teachings and cancer prevention J Family CommunityMed Ibrahim EM Satti MB Al Idrissi HY Higazi MM Magbool GM Al QA Oralcancer in Saudi Arabia the role of alqat and alshammah Cancer DetectPrev Alsanosy RM Mahfouz MS Gaffar AM Khat chewing among students ofhigher education in Jazan region Saudi Arabia prevalence pattern andrelated factors Biomed Res Int Quadri MFA Alharbi F Bajonaid AMS Moafa IHY Al Sharwani A AlamirAHA Oral squamous cell carcinoma and associated risk factors in JazanSaudi Arabia a hospital based case control study Asian Pacific J CancerPrev Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspotential causes that need to be investigated furtherThe knowledge produced by this study must be translated into interventions by performing indepth analysesof regional differences This will contribute to the effortsof preventing oral cancer in Saudi ArabiaAbbreviationsAIR Agestandardised incidence rates ASR Agestandardised specific ratesIARC International Agency for Research on Cancer SCR Saudi CancerRegistry ICD10 International Classification of Diseases 10th revisionAcknowledgementsI express my thanks and gratitude to the Saudi Ministry of Health forproviding me with the Saudi Cancer Registry reportsAuthors contributionsI certify that I have participated sufficiently in the intellectual contentconception and design of this work analysis and interpretation of the dataas well as the writing of the manuscript to take public responsibility for itand have agreed to have my name listed as a contributor The author readand approved the final manuscriptFundingThis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsAvailability of data and materialsThe data that support the findings of this study are available from SaudiMinistry of Health but restrictions apply to the availability of these datawhich were used under authorization for the current studyEthics approval and consent to participateThis study was approved by the Research Ethics Committee at NajranUniversity The ethical document reference No ETConsent for publicationA secondary data analysis was conducted in this retrospective study byusing a published dataCompeting interestsThe author declares that he is the only author for this work No other authorcontributed to this work He is also in agreement with the content of themanuscript He declares no conflict of interestReceived June Accepted August ReferencesJemal A Bray F Center MM Ferlay J Ward E Forman D Global cancerstatistics CA Cancer J Clin Bray 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 Warnakulasuriya S GloWarnakulasuriya S Global epidemiology of oral andoropharyngeal cancer Oral Oncology httpsdoi101016joraloncology200806002bal epidemiology of oral andoropharyngeal cancer Oral Oncol Rick A Afsaneh B Cancer Facts Figures American Cancer Society Access online via httpswwwcancercontentdamcancerresearchcancerfactsandstatisticsannualcancerfactsandfigures2017cancerfactsandfigures2017pdfAlJaber A AlNasser L ElMetwally A Epidemiology of oral cancer in Arabcountries Saudi Med J Ariyawardana A Johnson NW Trends of lip oral cavity and oropharyngealcancers in Australia overall good news but with rising rates inthe oropharynx BMC Cancer Alshehri B Descriptive epidemiological analysis of thyroid cancer in the Saudipopulation Asian Pacific J Cancer Prev Armitage P Doll R The age distribution of cancer and a multistage theoryof carcinogenesis Br J Cancer 0c" | 2 |
" trophoblast cell surface antigen trop2 is overexpressed in many squamous cell carcinomas andpromotes tumor development and invasion the association between trop2 expression and occurrence anddevelopment of oral squamous cell carcinoma oscc remains to be understoodmethods we investigated the role of trop2 in oscc patients using a combination of biophysical approaches atotal of oscc patient specimens with varying degrees of differentiation were subjected to hematoxylin andeosin staining immunohistochemistry kaplanmeier survival curve analysis and atomic force microscopy to analyzetrop2 expression morphology and mechanical properties of oscc tissuesresults trop2 was overexpressed in of poorly differentiated oscc samples high levels of trop2 wereassociated with survival rate lower than and patient age odds ratio [or] p confidence interval [ci ] tumor size or p ci [] and tnm stageor p ci [] average surface roughness of low medium and highly differentiatedoscc tissues were ± ± and ± nm respectively the pearson coefficient revealed anegative association between tumor stiffness and trop2 expression r p overexpression of trop2 negatively associated with patient survival degree of tumor differentiationand tissue mechanics taken together our findings demonstrated that trop2 may be an indicator of osccdifferentiation leading to the altered mechanical properties of oscc tissueskeywords oral squamous cell carcinoma trop2 tissue stiffness differentiation survival oral squamous cell carcinoma oscc is a commonsubtype of head and neck and other malignant tumors[ ] the past few decades have shown increased incidence of oscc that is expected to rise further in the future thereforeimperative to determineisit correspondence zhangkllzu163com baoping zhang shuting gao and ruiping li contributed equally to thiswork1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu chinafull list of author information is available at the end of the biological factors associated with the early diagnosis andtreatment of oscchuman trophoblast cell surface antigen trop2 alsocalled tumorassociated calcium signaltransduction2tacstd2 is a surface glycoprotein encoded by tacstd that has extracellular domains a single transmembrane domain and a short tail [ ] trop2 is overexpressed in many human cancers including ovarian [ ]gastric [ ] colorectal pancreatic and laryngealcancers inhibiting trop2 expression has shownpromise in clinical applications [ ] trop2 regulates 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 bmc cancer page of tumorigenic properties including cancer cell adhesion invasion and migration tang have recentlyshown that trop2 impacts growth and metastasis byactivating pi3kakt signaling this phenomenon hasalso been observed in gallbladder cancer amongtheinvolved intumorigenesis the role of catenin has been studiedextensively [ ] this has shed light on the biological functions of trop2 and its use as a prognostic biomarker for osccvarious biochemical mechanismsatomic force microscopy afm is a powerful toolthat generates surface topographical images with magnifications that range between macro and nanoscalesafm has been used to determine the mechanical properties of tumor tissues in a variety of cancers such asthose of the breast liver and lung parameters for tissue stress such as mechanical phenotypeindex correlate with cancer development and invasion advancements in technology used for determiningbiophysical properties have facilitated the nanolevelanalysis of tumor tissuesthis study aims at investigating the correlation between trop2 expression and clinicopathological characteristics of oscc we have demonstrated the tissuemorphology and mechanics of oscc samples duringtumor development using afm we believe our findingswill help develop trop2 in accurately diagnosing osccin tumors with different grades of differentiationmethodstissue preparationthe protocols in this study were approved by the researchethics committee of lanzhou university tumor sampleswere collected from patients after obtaining written informed consent a total of patients with oral squamous cell carcinoma oscc were registered atthesecond hospital of lanzhou university between january and march among these samples sampleseach showed high moderate and low levels of differentiation the experimental group comprised males and females aged years average years all patientswere diagnosed with oscc based on surgery andfig paraffin pathological sections of tissues a d g 4fold b e h 10fold c f i 40fold 0czhang bmc cancer page of fig immunohistochemical staining was performed to detect the expression of trop2 at different stages of osccpathology patients did not undergo radiotherapy chemotherapy or immunotherapy before surgery pathologicalanalysis after tumor biopsy was performed by two experienced pathologists after which the diagnosis of other diseases including inflammation at other sites and secondarytumors were excluded cancer and cervical lymph nodetissues were collected after maxillofacial surgery all specimens were sampled from typical areas of the lesion andfixed with neutralbuffered formalin followed by conventional paraffin embedding among them and fig average optical density of trop2 poorly differentiated squamous cell carcinoma showed high expressionp 0czhang bmc cancer page of table correlation between trop2 expression and clinicopathological characteristicscharactersntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvascular invasionnoyespearson x2p value highpatients exhibited no and cervical lymph node metastasesrespectively clinical tnm staging was performed according to the 7th edition tnm staging classification standardjointly developed by the international union for cancercontrol and american joint committee on cancer and world health anization guidelines hematoxylin and eosin he stainingoscc tissues were fixed overnight using neutralformalin solarbio beijing china paraffin embeddedsliced into 4μm thick sections dewaxed using xyleneand rehydrated using different concentrations of ethanol the sections were stained with hematoxylin for min and hydrochloric acidethanol and eosin for min followed by gradient dehydration transparentizationresin sealing solarbiobeijing china sections were visualized and imagedusing the olympus bx53 at magnifications of and sealing and neutral 0czhang bmc cancer page of immunohistochemistryhe sections were subjected to the sp method to detecttrop2 expression the sections were incubated overnight with the primary antibody against trop2 abcam usa at °c followed by incubation withbiotinlabeled goat antirabbitigg abcamusa at °c for h the sections were then developed using dab beijing zhongshan golden bridgebiotechnology china dehydrated transparentizationand film and neutral resin sealed the prepared sections were visualized using microscopy olympusbx53 japanafmfixed tissues were placed in petri dishes containingphosphatebuffered saline all analyses for mechanical properties were performed using the biologicalatomic force microscope bioafm nanowizard iiibruker usa silicon afm probes from the pointprobe®constant of nmcoating nanoworld usa werecontrreflexused the spring constant ofthe probe was calibrated using builtin thermal vibration before measuringandthickness of μm afm was performed using theseries with a khzforcetheresonancefrequencyofcontact model and a scan rate of hzs in airforcedistance curves are generated when the probecontacts the tissue following whichthe structuremorphology and mechanical properties of samplesare measured at μms six random sites wereselected for each sample and each site was measured times we used the modified hertz contact modelto analyze forcedistance curves and calculateyoungs modulus and roughness of oscc tissueswith varying differentiationstatistical analysisstatistical analyses were performed using spss statistical product and service solutions ibm forcespectrum data were expressed as mean ± standard errorand statistical comparisons were performed using oneway analysis of variance followed by the tukeykramerhsd test for pairwise comparisons pearson chisquaretest was used to analyze clinical features and trop2 expression based on the calculated odds ratios ors and confidence intervalci survival was evaluatedusing kaplanmeier curves and the difference was analyzed using the logrank test p was consideredstatistically significantfig trop2 total survival curve using kaplanmeier survival curves low blue line high green line 0czhang bmc cancer page of resultstissue morphology and trop2 expression across theclinical stages of oscctumor cells from poorly differentiated oscc samples exhibited characteristic atypia poor differentiation and irregular morphology fig howeverthe number volume atypia nuclear pyknosis andmitotic structures decreased in tumor cellsfromhighly differentiated oscc as compared to those inpoorly differentiated cells trop2 primarily localizedin the cytoplasm of tumor cells but not in adjacentnormal epithelial cells we observed that low differentiation and high malignancy of oscc was associated with higher trop2 expression fig theaverage optical density of trop2 among the lowmedium and highly differentiated oscc tissues were ± ± and ± respectively fig table trop2 expression risk factors with clinicopathological featurescharacteristicsntrop2 expressionlow or nototalgendermalefemaleage¥ localizationmucosatonguedifferentiationwellmoderatepoortumor sizet1 cm cmt2 cmt34cmt4blymph node metastasesn0nxdistant metastasesm0m1tnm stagei iiiii ivperineural infiltrationnoyesvscular invasionnoyesnote a well vs moderate b moderate vs poor c well vs poorp valueor cihigh 005a 0001b 0001c 0czhang bmc cancer page of association between trop2 expression and clinicalcharacteristics of osccwe analyzed the clinicopathological characteristics of patients with oscc with varying degree of differentiationdifferential expression of trop2 was associated with patient age tumor differentiation tumor size tnm stagepercutaneous nerveinvasiontable p patients with poorly differentiated tumors were more likely than patients with well and moderate differentiated tumors to have high trop2 expressionp however there was no association between theexpression of trop2 and patient gender tumor locationlymph node metastasis or distant metastases p and vascularfiltrationtrop2 expression and patient survivalusing kaplanmeier survival curves we observed that anincrease in trop2 expression negatively correlated withthe overall survival of patients fig and lowno oftrop2 expression groups 3years survival rate was a for high expression group and 5years ratewere and respectively trop2 expression wasassociated with patient age p or ci[] tumor differentiation well vs moderatep or ci [] moderate vspoor p or ci []well vs poor p or ci [] tumor size p or ci[] tnm stage p or ci[] vascular invasion p or ci [] and peripheral nerve invasionp or table high trop2 expressionwas detected in older patients with low degree of differentiation larger tumor volume higher tnm staging andvascular and peripheral nerve invasion thereby resultingin lower overall survival thus trop2 may be a prognostic indicator for survival in oscc patientsfig surface morphology of oscc tissue sections via afm detection irregular morphology appeared in the low differentiation 0czhang bmc cancer page of surface morphology and roughness of oscc tissuesthe surface morphologies of oscc tissues with varying degrees of differentiation were analyzed directtopographical imaging using bioafm figure showsthe representative image from each tissue acquiredduring the cantileverbased afm nano indentationtest the tissue interface varied with tumor differentiationindicating that highly differentiated oscc tissues had a regular and flat morphology oscc tissueswith low differentiation exhibited an overall irregularmorphology with distinct modulation and loose tissueanization figure summarizes the roughness ofoscc tissues with varying differentiation the average surface roughness of low medium and highly differentiated oscc tissues were ± ± and ± nm respectively roughness ofthe tissue surface was enhanced with increasing differentiation of oscc tissuesyoungs modulus of oscc tissueswe used bioafm to determine youngs modulusbased on the mechanical properties of oscc tissues with varying degrees of differentiation we randomly selected six contact points from each slice andeach contact point was measured times forcedistance curves were generated for each slice and thejpk data processing software version was usedto calculate youngs modulus figure shows theaverage variation in stiffness within individual tissuesin the range of kpa in the low differentiationsamples we observed low stiffness as compared tothat in high or medium differentiation samples p fig surface roughness results are express as mean ± sem nm 0czhang bmc cancer page of fig afm test average tissue stiffness youngs modulus e was thus confirmed to be a parameter of cell hardness for various cells and tissuepa p thus tissue differentiation was positively associated with its stiffness fig association between mechanical properties and trop2expression in osccthe pearson coefficient showed a negative associationbetween the stiffness of oscc tissues and trop2 expression fig r p thus we detectedan increase in stiffness with varying differentiation in thetumor samplesdiscussiontrop2 belongs to the family of genes involved in calcium signaling associated with tumorigenesis and foundin human trophoblast and chorionic cell lines studieshave shown that overexpression of trop2 is associatedwith tumorigenesis and malignancy []in thisstudy trop2 expression was observed to be a highlysensitive and specific marker of tongue squamous cellcarcinoma and tissue stiffness the relative thickness ofsamples helped accurately diagnose and determine thestaging of tongue squamous cell carcinomaimmunohistochemical analysis revealed that the expression of trop2 in poorly differentiated oscc tissueswas significantly higher than that in welldifferentiatedoscc tissues additionally trop2 upregulation wascorrelated with tumors of advanced tnm iii iv staging and poor differentiation than that in tumors withlow tnm i ii staging thus the abnormal expressionof trop2 may be associated with the occurrence anddevelopment of tongue malignancies furthermore hightrop2 expression predicted low survival as comparedto that in the tumors with low trop2 expression previous research has also demonstrated the correlation between shorter patient lifespan and high levels of trop2as compared to that in patients with laryngeal squamouscell carcinoma and low levels of trop2 trop2possesses sites for tyrosineserine phosphorylation thatregulate signal transduction or its expression and activity thereby rendering cancer cells resistant to apoptosis upregulated trop2 correlates with the poor prognosis of thyroid papillary carcinoma colon cancer liver cancer and other malignanciesthere have been an increasing number of studies on thebiological role of trop2 at the molecular level trop2induces the downregulationloss of pten thereby stimulating pi3kakt signaling and tumor development pten is a wellknown tumor suppressor that is a phosphatase and affects the pi3kpkbakt signaling axisduring the dephosphorylation of pip2 and pip3 pi3k signaling is important in regulating tumor cell proliferation migration and invasion [ ] thus pten is anegative regulator of cancer [ ] li have shownthat trop2 activates epithelialmesenchymal transitionvia pi3kakt signaling thereby promoting proliferationmigration and metastasis in gallbladder cancer similarly trop2 expression stimulates the proliferation migration and invasion of osteosarcoma cells hou demonstrated that trop2 regulates jak2stat3 signaling in glioblastoma cells 0czhang bmc cancer page of fig correlation analysis between changes in mechanical stiffness of oscc tissues and trop2 expression note changes have statisticalsignificance p and show a certain negative correlation r functional differentiation oftissues influences themicromorphology and mechanical stiffness of oscccells we detected low surface roughness on oscc tissues with loose structure reduced hardness and enhanced cell adhesion migration and invasion poorlydifferentiated oscc tissues are softer than highly differentiated oscc tissues pi3k is an important celladhesion molecule trop2 triggers the synthesis of proteins with homologous domains such as pleckstrinrac tiam and vav tiam and vav activate rac thatleads to reanization of the actin cytoskeleton cellrecognition and adhesion the underlying mechanisms involved in the alterationof micromechanical properties of oscc samples and occurrence development metastasis and invasion ofoscc tumors remain to be elucidated he staining isthe gold standard for tumor diagnosis with the development of biomechanics in the past two decades the mechanical properties of tissues need to be investigated based on biomedical and physical parametersin this study we have assayed the changes in mechanicalproperties at the micronanometer level using afm anddetermined the association between the tnm grademetastasis and stiffness of tumor samplesin we have demonstrated the association between differential expression of trop2 and patient agetumor differentiation tumor size tnm stage percutaneousnerve filtration and vascular invasion moreover high levelsof trop2 correlated with poor overall survival in patientshighly differentiated cancer tissues exhibited increasedsurface roughness and stiffness lastly high trop2 expression resulted in reduced tumor stiffness however thisstudy had some limitations first the cohort used in thisstudy was relatively small second we did not employ molecular methods of analysis such as western blotting orenzymelinked immunosorbent assay thus using a largerpatient cohort and multiple techniques in molecular andcell biology will help validate our findings and developtrop2 as a specific and efficient prognostic biomarker forosccthese findings could promote new methods for the earlyoscc diagnosis depend on the stage of cancer and developing screening methods with high sensitivity andspecificity more detailed studies are needed to determine the feasibility and therapeutic benefit of testing tissue stiffness in human diseaseabbreviationsoscc oral squamous cell carcinoma trop2 trophoblast cell surfaceantigen afm atomic force microscopyacknowledgementswe thank the individual who participated in this studyauthors contributionsbz sg and rpl are responsible for conception and design data wascollected by ytl rc jyc and ymg data was analyzed by ew and yh klzrevised the all authors have read and approved the manuscriptfundingthis work was supported by the fundamental research funds for thecentral universities no lzujbky2020cd03 baoping zhang doctoralmaster 0czhang bmc cancer page of students of the second hospital of lanzhou university sdkygg17 lan yangand key laboratory of mechanics on disaster and environment in westernchina the ministry of education of china no kailiang zhangavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable requestethics approval and consent to participatewritten informed consent was obtained from each participant before samplecollection the study was approved by the committee for ethical affairs ofschool of stomatology lanzhou universityconsent for publicationnot applicablecompeting intereststhe authors have no conflicts of interestauthor details1department hospital of stomatology lanzhou university donggang westroad lanzhou gansu china 2institute of biomechanics andmedical engineering lanzhou university lanzhou chinareceived april accepted august 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trop2 induced epithelialmesenchymal transitionthrough mediated betacatenin in gastric cancer cancer med zhao w jia l zhang m huang x qian p tang q the killing effect ofnovel bispecific trop2pdl1 cart cell targeted gastric cancer am jcancer res jordheim lp chettab k crosperrial e matera el dumontet c unexpectedgrowthpromoting effect of oxaliplatin in excision repair crosscomplementation group transfected human colon cancer cellspharmacology nishimura t mitsunaga m sawada r saruta m kobayashi h matsumoto n photoimmunotherapy targeting biliarypancreatic cancer withhumanized antitrop2 antibody cancer med wang xd wang q chen xl huang jf yin y da p trop2 inhibitionsuppresses the proliferation and invasion of laryngeal carcinoma cells viathe extracellular signalregulated kinasemitogenactivated protein kinasepathway mol med rep wanger tm dewitt s collins a maitland nj poghosyan z knauper vdifferential regulation of trop2 release by pkc isoforms through vesiclesand adam17 cell signal tang g tang q jia l chen y lin l kuai x trop2 increasesgrowth and metastasis of human oral squamous cell carcinomathrough activation of the pi3kakt signaling pathway int j mol medtrerotola m li j alberti s languino lr trop2 inhibits prostate cancer celladhesion to fibronectin through the 1 integrinrack1 axis j cell physiolli t su y yu x mouniir dsa masau jf wei x trop2 guaranteescardioprotective effects of cortical bonederived stem cells on myocardialischemiareperfusion injury cell transplant stoyanova t goldstein as cai h drake jm huang j witte on regulatedproteolysis of trop2 drives epithelial hyperplasia and stem cell selfrenewalvia betacatenin signaling genes dev sun x xing g zhang c lu k wang y he x knockdown of trop2 inhibitsproliferation and migration and induces apoptosis of endometrial cancercells via aktcatenin pathway cell biochem funct lee h jang y seo j nam jm char k nanopfunctionalized polymerplatform for controlling metastatic cancer cell adhesion shape and motilityacs nano 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betweencylinders under inclined forces philos trans a math phys eng sci kowalsky ca faber ms nath a dann he kelly vw liu l rapid fineconformational epitope mapping using comprehensive mutagenesis anddeep sequencing j biol chem zeng p chen mb zhou ln tang m liu cy lu ph impact of trop2expression on prognosis in solid tumors a systematic review and metaanalysis sci rep calvo a xiao n kang j best cj leiva i emmertbuck mr alterationsin gene expression profiles during prostate cancer progression functionalcorrelations to tumorigenicity and downregulation of selenoproteinp inmouse and human tumors cancer res ju x jiao x ertel a casimiro mc di sante g deng s vsrc oncogeneinduces trop2 proteolytic activation via cyclin d1 cancer res cubas r li m chen c yao q trop2 a possible therapeutic target for latestage epithelial carcinomas biochim biophys acta zargari n mokhtari m evaluation of diagnostic utility ofimmunohistochemistry markers of trop2 and hbme1 in the diagnosis ofthyroid carcinoma eur thyroid j zhao p zhang z tnfα promotes colon cancer cell migration and invasionby upregulating trop2 oncol lett sin stk li y liu m yuan yf ma s guan xy downregulation of trop2predicts poor prognosis of hepatocellular carcinoma patients hepatolcommun zhang y zhang r luo g ai k long noncoding rna snhg1 promotes cellproliferation through pi3kakt signaling pathway in pancreatic ductaladenocarcinoma j cancer sai j owens p novitskiy sv hawkins oe vilgelm ae yang j pi3kinhibition reduces mammary tumor growth and facilitates antitumor immunityand antipd1 responses clin cancer res chen x pang b liang y xu sc xin t fan ht overexpression of zhang xr wang sy sun w wei c isoliquiritigenin inhibits proliferation andepcam and trop2 in pituitary adenomas int j clin exp pathol metastasis of mkn28 gastric cancer cells by suppressing the pi3kaktmtor signaling pathway mol med rep 0czhang bmc cancer page of wise hm hermida ma leslie nr prostate cancer pi3k pten and prognosisclin sci lond yuan b zou m zhao y zhang k sun y peng x upregulation of mir130b3p activates the ptenpi3kaktnfκb pathway to defense againstmycoplasma gallisepticum hs strain infection of chicken int j mol sci pii e2172li jw wang xy zhang x gao l wang lf yin xh epicatechin protectsagainst myocardial ischemiainduced cardiac injury via activation of theptenpi3kakt pathway mol med rep li x teng s zhang y zhang w zhang x xu k trop2 promotesproliferation migration and metastasis of gallbladder cancer cells byregulating pi3kakt pathway and inducing emt oncotarget gu qz nijiati a gao x tao kl li cd fan xp trop2 promotes cellproliferation and migration in osteosarcoma through pi3kakt signalingmol med rep hou j lv a deng q zhang g hu x cui h trop2 promotes theproliferation and metastasis of glioblastoma cells by activating the jak2stat3 signaling pathway oncol rep rivard n phosphatidylinositol 3kinase a key regulator in adherens junctionformation and function front biosci landmark ed pankova d jiang y chatzifrangkeskou m vendrell i buzzelli j ryan a rassf1a controls tissue stiffness and cancer stemlike cells in lungadenocarcinoma embo j 20193813e100532 wullkopf l west av leijnse n cox tr madsen cd oddershede lb cancer cells' ability to mechanically adjust to extracellular matrix stiffnesscorrelates with their invasive potential mol biol cell publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
chronic respiratory diseases are highly prevalent worldwide and will continue to rise in theforeseeable future despite intensive efforts over recent decades the development of novel and effectivetherapeutic approaches has been slow however there is new and increasing evidence that communities ofmicroanisms in our body the human microbiome are crucially involved in the development andprogression of chronic respiratory diseases understanding the detailed mechanisms underlying this crosstalk between host and microbiota is critical for development of microbiome or hosttargeted therapeuticsand prevention strategies here we review and discuss the most recent knowledge on the continuousreciprocal interaction between the host and microbes in health and respiratory disease furthermore wehighlight promising developments in microbiomebased therapies and discuss the need to employ moreholistic approaches of restoring both the pulmonary niche and the microbial communityreceived nov accepted after revision april copyright ers this version is distributed under the terms of the creative commons attribution noncommercial licence 10118313993003023202019eur respir j 0crespiratory basic science r gosens introductionin the past decade we have learned that the lungs previously considered sterile in fact harbour a dynamicecosystem of diverse bacteria fungi and viruses this lung microbiota is detectable in health [ ]altered in disease predictive of disease outcomes [ ] and correlates with variations in host immunity[ ] recent insights based on studies in both humans and mice are that the baseline immunetone of the lungs even in health is closely linked to the local microbial milieu this hypothesis thatthe immune tone in the airways and alveoli is at least in part regulated by the microbiota is a radicaldeparture from our conventional dichotomous understanding of lung immunity dormant in healthactivated in infection next to known changes in the inflammatory milieu of the lungs most lung diseasessuch as asthma copd cystic fibrosis idiopathic pulmonary fibrosis ipf andrecently lung cancer have been associated with a microbial dysbiosis in the lung however it isunknown if changes in the bacterial composition drive disease pathogenesis or if they are rather areflection of alterations of the ecological niche [ ] thus it is of utmost importance to understand theunderlying molecular mechanisms at the hostmicrobiome interface to develop novel targeted therapies orpreventative approacheshere we discuss the impact of hostmicrobiome crosstalk on respiratory health and disease whilefocusing on how the local microbiota and the host interact at the epithelial surface furthermore wereview current therapeutic approaches and suggest a more holistic approach for treating lung disease inthe future this review is a followup of presentations and discussions at and after the ers researchseminar crosstalk in the lung microenvironment implications for understanding chronic lung diseaseberlin february the mucosal niche in the lungall external interfaces of the human body such as gut skin reproductive and respiratory tracts arecolonised by a distinct microbial flora the microbial communities differ at the various body sites dueto local factors eg oxygen carbon dioxide ph nutrients host defence factors inhaled pollutants thatshape the niche as examples the lumen of the lower gastrointestinal tract represents a lowoxygennutrientrich environment and is thus commonly populated by highabundance communities of anaerobicanisms by contrast the skin is a lownutrient environment directly exposed to environmental oxygenand is thus more commonly populated by relatively sparse communities of oxygentolerant bacteria thusthe local environment is a crucial determinant of the formation of microbial communities early indevelopment but also at later stages antimicrobial peptidesand otherclearance production ofconsequently the lung microenvironment creates a special ecological niche for microbes that differs fromother body sites and will presumably influence nichespecific colonisation accordingly airway epithelialcells are a principle contributor in shaping this niche as they are strategically located to be the first contacttissues various mechanisms arebetween inhaled substances including microanisms and hostemployed by the airway epithelium in host defences against infectionincluding its barrier functionmucociliaryandantiinflammatory mediators and ability to transport eg polymeric iga and igm from the basal to theapical side of the epithelium through the polymeric immunoglobulin receptor pigr [] it is likelythat similar mechanisms contribute to the formation of nichespecific communities along the respiratorytract and such local conditions are crucial for allowing beneficial microbiota to persist at epithelialsurfaces the involvement of such hostmicrobiota interactions in disease is wellillustrated in cysticfibrosis in cystic fibrosis mutational dysfunction of the cystic fibrosis transmembrane regulator cftrprotein results in a reduction of anion mostly bicarbonate and chloride exchange across the epithelialsurface resulting in a dehydrated surface and sticky mucus this mucus cannot be readily cleared from theairways which helps to explain why cftr mutations are associated with alterations of the residentmicrobiota including frequent colonisation with staphylococcus aureus and pseudomonas aeruginosa asrecently reviewed in however we are still lacking knowledge on the underlying mechanisms of theniche alterations in more complex genetic lung diseases such as asthma and copdsubstances proadditionallyallowing it to mount appropriate responses or develop tolerance [ ]the epithelium transmits environmental and microbial signals to the immune systemvarious mechanisms of sensing microbial presence include pattern recognition receptors such as thetolllike receptors and other processes not mediated via classical receptormediated signalling egthe integrated stress response upon sensing microbial environmental or endogenous challengesepithelial cells mount active responses by increasing defence molecules such as antimicrobial peptidescytokines and chemokines these enhance the defence against respiratory pathogens while simultaneouslychanging the ecological niche of complex microbial communities indicating that the properties of theecological niche encountered by microanisms changes as a result of environmental exposures10118313993003023202019 0crespiratory basic science r gosens anatomical differences along the respiratory tract such as differences in epithelial cell composition shapethis ecological niche while the epithelium contributes to local conditions that shape the microbiotaepithelial exposure to microbes and their products has marked effects on its function consequentlyhuman epithelia and the microbiota have developed interactions that are of mutual benefit responding topathogens and tolerating innocuous substances this concept is important to understand how inhaledenvironmental triggers regulate immunity since microbial components contribute to the response toenvironmental exposures such as farm and geogenic earthderived dust however how theepithelium integrates these microbial and nonmicrobial signals into a finetuned response is incompletelyunderstoodin order to transmit signals from the environment epithelial cells use sophisticated communicationsystems with other lung cell types the airway epithelium and that of gut and skin plays key roles intransmitting signals from microanisms and environmental stimulito instruct antigenpresentingdendritic cells to direct immunity towards inflammation or tolerance epithelial cells interact not onlywith other immune cells such as macrophages neutrophils innate lymphoid cells and tcells but alsowith structural cells such as fibroblasts airway smooth muscle cells and endothelial cells via a plethora ofdifferent mechanisms figure [ ] this array of interactions with environmental triggersmicroanisms and lung cells allows epithelial cells to orchestrate host defence and immunity and tomaintain epithelial integrity and mediate pathologic airway remodelling figure establishment and maintenance of the lung microbiotathe establishment of the lung microbiota likely occurs in the first days and weeks of life although earlyhighprofile reports suggested the presence of a placental microbiome that could influence prepartumlung development subsequent wellcontrolled studies have failed to detect bacterial signals distinctfrom contaminating dna present in negative controls the lung microbiota of newborn mice isbelow the limit of detection via quantitative pcr and increase in total burden in the following days andweeks [ ] in human infants the composition of the respiratory microbiome seems to mature in apredictable wellcharacterised pattern during the first year oflife besides the special nichecharacteristics of the lung earlylife respiratory microbiota are influenced by mode of delivery vaginalversus caesarean method of feeding breast versus bottle and exposures siblings daycare attendance provocatively earlylife respiratory microbiota may predict subsequent susceptibility to respiratorytract infections [ ] suggesting roles of the local microbes in immune homeostasis and resistance topathogens in contrast being exposed to an increased microbial diversity during childhood promotes thedevelopment of balanced immunity and is protective against inflammatory responses to allergens andasthma development [ ] this protection is partly associated with distinct farm dust which in vitroincreases epithelial barrier function and antiviral defences once established the composition of the lung microbiome is determined by three ecological factorsimmigration elimination and relative growth rates of community members figure in health lungcommunities are sparse and dynamic largely determined by the equilibrium between immigration viamicroaspiration and elimination via cough mucociliary clearance and immune defences littleevidence supports the presence of resident lung bacteria in health that reproduce and survive selectivepressures [ ] nevertheless the transient and dynamic communities detected in health are largelyviable and exert a detectable effect on the immune constitution of the lower respiratory tract [ ]the establishment of a diverse respiratory microbial flora is influenced by many different factors there isa finetuned balance between tolerance of commensal or beneficial bacteria at the epithelial surface andthe development of active immune responses to pathogens notably this balance is regulated by both hostand microbederived signals sudden shifts in this tightly controlled equilibrium such as outgrowth ofspecific pathogens or for example virally induced damage to the airway epithelium destroying the barrierand inducing local immune responses can have detrimental effects on both the host and the microbiomeand might therefore contribute to the pathogenesis of respiratory diseasesrole of the environment in shaping the lung microbiomeenvironmental influences play pivotal roles in the development of lung diseases this might be due to directeffects on the host epithelial barrier and immune responses but most known risk factors such as cigarettesmoke air pollution [ ] viral infections and di so directly impact the microbiota [ ]thus a combination of both might be critical for disease developmentfor example one of the most studied inhaled toxins cigarette smoke supposedly has a detrimental effecton both the host and microbial communities prolonged cigarette smoke exposure contributes to increasedbaseline inflammation in the airways and epithelial remodelling towards goblet cell hyperplasia and areduction in cilia and ciliary activity and can reduce the antimicrobial defence provided by the airway10118313993003023202019 0crespiratory basic science r gosens mucociliaryclearingmicroaspirationairway regionbacterial metabolitesouter membrane vesiclesquorumsensing moleculesalveolar regionantimicrobial peptidesciliary beatingmucuscytokineschemokinesgrowth factorsextracellular vesiclescritically altered in chronic lung diseasebacteriarespiratory virusclub cellciliated cell goblet cell alveolar type cell alveolar type cellfibroblastmucus layer basal celldendritic cell macrophagesmooth muscle cellcapillaryfigure hostmicrobiome crosstalk in the lung microenvironment the lung microenvironment consists of different cell types depending onthe location in the proximaltodistal airway tree the epithelial layer in larger airways is constantly exposed to a variety of different microbes ofthe local microbiota while the composition of the latter is influenced by host factors such as elimination via mucociliary clearance it alsodepends on the competition among the microbial inhabitants it is now evident that there is a complex crosstalk between host and microbes inthis environment accordingly bacterial metabolites or outer membrane vesicles can influence the host status while antimicrobial peptides orcytokines can shape the composition of the microbiota as virtually all of these single factors are altered in chronic respiratory disease it is ofutmost importance to appreciate and assess the complexity of this system in future studiesepithelium in patients with copd it has been shown to be associated with reduced lung bacilli andincreased haemophilus influenzae and streptococcus pneumoniae but the largest study to date inhealthy smokers has not found a significant effect of cigarette smoke on the lung microbiome thus it is intriguing to speculate that cigarette smoke primarily affects the host system which over timeand upon disease copd development in susceptible individuals may also affect the microbiomeanother example of environmental influence on respiratory disease is diet which has profound effects onboth microbiota and health even in the short term the intake of dietary fibre induces similarbeneficial microbiota changes in the lung and gut while lowfibre diets change the gut microbialcommunity over multiple generations in mice highfibre diets have beneficial effects in pregnant miceand suppress allergic airway disease aad in mothers and their offspring this also highlights theimportance of crosstalk between the gut and lung highfibre diets induce the production of shortchainfatty acids by gut bacteria which are transported systemically to the lung where they exertantiinflammatory actions and ameliorate aad in mice in contrast a lipidrich di ters themicrobiota and promotes metabolic inflammation and has been associated with premetastatic nichedevelopment in lung cancer however the detailed mechanisms of action remain unclearalong with bacteria common major respiratory viruses including respiratory syncytial virus rhinoviruscoronaviruses influenza and adenoviruses are part of the respiratory microbiome and contribute tothe pathogenesis of chronic respiratory diseases this may result from complex interactions of viruses withthe hosts immune system and the microbiota including other pathogens these interactions can influencethe prevalence of bacterial pathogens by increasing the expression of adhesion molecules on therespiratory epithelium damaging respiratory epithelial cells compromising barrier functionimpairingmucociliary clearance and altering host immunity and the lung microenvironment [] interestinglyit has been suggested that viruses and bacteriophages induce consistent and reproducible changes inrespiratory microbiomes in chronic disease but not the healthy state and have been shown to increasemicrobial diversity in the nasopharynx [ ] furthermore fungi such as aspergillus spp contributeto the pathogenesis of chronic respiratory diseases as pathogens on their own but also by activation of theimmuneinteractions with microbiota particularly withnontuberculous mycobacteria [ ]through theirsystemand probablyimportantly the absence of certain members from the microbiome can have detrimental influences on lunghealth this is illustrated by the relative disappearance of parasitic worms which have been a constant partof our gut microbiome and have even been found in fossils from the period of jawed fish in fact our10118313993003023202019 0crespiratory basic science r gosens modern immune system developed in the continuous presence of helminths which may explaintheir important regulatory role in immunity typically worms induce type2 immune responses which areconsidered instrumentalin host defence against these parasitic infections however worm infectionsinduce regulatory responses that are aimed to suppress immune responses directed at worm antigenswhich allows worms to live in their host for years additionally this regulatory response has a bystandereffect by promoting allergen tolerance this is illustrated by studies in mouse models in whichvarious helminth infections have been shown to provide protection against the development of allergicasthma the range of helminthinduced protective mechanisms includes the inhibition of interleukinil33 in the airways and the induction of regulatory tcells [ ] bcells [ ] andmacrophages intriguingly the presence of helminths also affects the composition of the bacterialcommunity which might be important for the protection against allergic airway disease in mice niche and microbiome alterations in respiratory diseasein disease the ecology of the lower respiratory tract changes dramatically the airways and alveolinormally inhospitable to bacterial reproduction are radically altered by the influx of nutrientrichoedema and mucus establishment of stark oxygen gradients surge of bacterial growthpromotinginflammatory responses [ ] and impairment of local host defences [ ] thus it is unsurprisingthat crosssectional studies have identified altered lung microbiomes in established lung diseases aschanges in ecological niches supposedly result in different microbial communities due to selective pressurethe composition of the lung microbiome then becomes increasingly determined by the relative growthrates of its constituentswhile there are some studies on the role of the microbiome in ipf and lung cancer mostknowledge is based on studies of chronic inflammatory respiratory diseases such as asthma copd andcystic fibrosis generally those patients have increased susceptibility to infections and exacerbations thatagain affect the microbiome [ ] h influenzae moraxella catarrhalis and s pneumoniae areassociated with the development of severe asthma and copd they modify the lung microbiome anddrive inflammation oxidative stress symptoms and exacerbations [ ] which may form a viciouscircle perpetuating the disease m catarrhalis and hinfluenzae in particular induce neutrophilicinflammation more severe disease and steroid resistance [] furthermorein asthma alteredrespiratory microbiome profiles are associated with asthma phenotype and severity responses toallergens and treatment in cystic fibrosis and bronchiectasis haemophilus and pseudomonas spp increasingly dominate the lungmicrobiota [ ] genetic association studies in phe508del cystic fibrosisaffected homozygousindividuals have shown associations of single nucleotide polymorphisms with important disease featuresrelated to bacterial colonisation for example gene variants of human leukocyte antigen class ii genesimplicating a role for tcell and bcell immunity associate with age of onset of persistent p aeruginosainfection indicating the role of the hosts immune system in selecting a distinct microbiota like the bacterial composition ofthe lung microbiome antiviral responses are altered in chronicrespiratory diseases in asthma where airway epithelial repair in response to viral infection is aberrant dueto changes in genes regulating epithelial barrier function and repair the airway epithelium hasreduced innate immunity to common viruses such as rhinovirus [ ] this is due to reduced type iand iii interferon and increased mirna levels [ ] transforming growth factor tgfβ a cytokinecommonly upregulated in asthma suppresses airway epithelialinnate immune responses throughsuppressor of cytokine signalling socs1 and socs3 contributing to impaired antiviral immunity this impaired antiviral immunity may also alter the bacterial microbiome and contribute to coinfectionsby bacteria rhinovirus infection can also impair the phagocytosis of bacteria by alveolarmacrophages which can lead to bacterial outgrowth in copd murine studies showed thatinfluenzainfected epithelial progenitors of the distal lung exhibited severely impaired renewal capacity dueto virusinduced blockade of βcatenindependent fgfr2b signalling this could contribute toimpaired repair of the distal lung in copd although its involvement in human disease is still unclearthus viral and bacterial infections and the inability to resolve them may aggravate impaired airway andalveolar repair combined with a heightened airway andor alveolar immune tone aberrant repairmechanisms following pathogen exposures in vulnerable individuals might represent a mechanism for howhostmicrobiome interactions contribute to disease progression it is currently unclear if alterations in thelung microbiome can also precede lung structural changes and inflammation and thereby contribute toonset of diseaseas discussed earlier the relationship between lung dysbiosis and lung inflammation is bidirectionaldisordered lung communities trigger epithelial and luminal inflammation further altering growth conditionsof the lung microenvironment chronic inflammation and perpetuating dysbiosis [ ] a common10118313993003023202019 0crespiratory basic science r gosens isthe outgrowth oftheinflammatory lung conditionsfinding acrossinflammationassociatedproteobacteria phylum and in mice the lung microbiome has been shown to be associated withpulmonary levels of the innate cytokine il1α additionally alterations in the microbiome of the gutmight influence the immune tone of the lung which is shown by the development of more severeexperimental asthma in germfree mice [ ] or mice with disturbed microbiomes due to earlylifeantibiotic treatment [ ] in contrast the absence of microbiota in germfree mice or due toantibiotic treatment has recently been shown to protect mice with kras mutations and p53 loss from lungcancer development according to the authors this effect was due to the induction of il1β andil23 through the microbiota that led to il17 production by γδt cells and tumorigenesisin humans copd patients are two to three times more likely to have crohns colitis and of peoplewith inflammatory bowel disease also have pulmonary inflammation [] the gastrointestinal tracthas by far the highest microbial content and thus an interaction of gut microbiota and their metaboliteswith the gastric mucosa affects systemic immunity which may in turn impactthe lung consequently alterations in gut microbiota and physiology might contribute to respiratory disease andvice versa possibly via the gutlung axis however it is important to note that germfree breeding and tosome extent antibiotic treatment will affect both the gut and the lung microbiome so it is still notcompletely clear whether these effects can fully be explained by gutlung crosstalk or if they areinfluenced by aberrant local microbiotaimmune crosstalk along this line in mice the lung immune tonehas been suggested to be more correlated to lung bacteria than gut bacteria in contrast in mice colitisinduced pulmonary pathology was associated with increased inflammation andgramnegative bacterial endotoxins in the lung that probably emanate from the gut furthermorecigarette smokeinduced experimental copd models [] indicate that reduced gas exchange andhypoxia in the lung is associated with hypoxia in the gut causing colon remodelling cell deathinflammation and impaired barrier function additionally activation of nodlike receptors bybacteria in the gut increase production of reactive oxygen species by alveolar macrophages suggesting thatthe gastrointestinal microbiome contributes to oxidative stress in the lung conversely oralapplication of beneficial probiotic bacteria bifidobacterium and lactobacillusbased reduced airwayinflammation and emphysema in cigarette smokeexposed mice a large canadian study reported that four bacterial genera lachnospira veillonella faecalibacterium androthia were reduced in the faeces of human infants that subsequently developed asthma and inoculating ahuman faecal microbiome supplemented with these four taxa into germfree mice partially protected theirf1 progeny from experimentally induced aad thus there are encouraging studies in mousemodels highlighting cause and effect of the gutlung axis in respiratory disease however findings firstneed to be validated in humans before proposing this crosstalk as a treatable trait for respiratory diseasein summary at the hostmicrobiome interface disordered communities are probably both cause andeffects of host inflammation however given the close reciprocal interactions between the microbiome andhost at all times it might be impossible to determine the real cause of the very first changes in diseasedevelopment as host and microbiome factors are coinciding and closely intertwinedimplications for the development of novel therapiesdue to the strong and dynamic interdependency between host and microbiome in local niches it isunsurprising that most drugs used in clinical practice that were designed to target the host also affect themicrobiome accordingly inhaled corticosteroids and proton pump inhibitors affect the lung microbiota[] as well as subsequent pneumonia risk [ ] macrolide antibiotics broadly effective acrosschronic lung conditions such as copd affect both lung microbiota and host immunity perhapsmost provocatively baseline differences in lung microbiota appear to predict patient responsiveness totherapies such as inhaled corticosteroids suggesting that variation in lung microbiota may representan untapped phenotype of precision medicine in the lung this opens new possibilities to exploit thisimportant crosstalk in therapeutic interventions but in order to do so we first need to improve ourunderstanding of the molecular mechanismsincreasing evidence of the importance of the microbiome raises the concept of restoring diseasedmicrobiomes to prevent or treat diseases using microbiotadirected therapies or hosttargeted therapiessuch as probiotics metabolites lung microbiota transplantation or vitamin d therapy which we discusshere in more detailmany clinical studies have investigated the efficacy of probiotic bacteria which are supposedly beneficialfor the host to prevent chronic diseases such as asthma or allergic rhinitis however these studies havelargely produced contradictory outcomes which might be due to the fact that the used probioticswere not selected based on potential mechanistic effects and may not be ideal the microbiome is a10118313993003023202019 0crespiratory basic science r gosens complex ecosystem comprised of a variety of different inhabitants and influenced by many externalhostderived factors thus the addition of single strains may not make a profound difference thus thetransfer of whole microbiomes via faecal microbiota transplantation fmt could be a more promisingapproach the introduction of healthy microbiota into diseased hosts has restored immunity andphysiology demonstrating that intestinal microbiota and their products can modulate host immunitylocally and systemically and that fmt can replace diseaserelated microbiomes with healthy ones fmt is remarkably successfulin treating antibioticresistant clostridium difficileinducedcolitis and is now being used as treatment in selected patients [ ] the new microbiomeengrafts quickly and lasts for at least a month indicating a potential difficulty in inducing longtermbeneficial changes in the microbiome via only targeting the microbial side while there are encouragingdata questions remain whether fmt may also affectlung health and whether lung microbiotatransplantation is feasible furthermore recently severe complications of fmt have been reported due totransfer of drugresistant bacteria thus it is essential to determine whether such approaches that sofar only transiently change the microbiome can be used for the required longterm treatments of chroniclung diseases that coincide with a variety of structural changes aberrant mucociliary clearance and manymore such as[]specific microbial moleculeslipopolysaccharide lps andalong with living bacteriapeptidoglycan can induce or modulate inflammatory responsesin addition culturesupernatants of probiotic bacteria display antiinflammatory effects which have been ascribed to thepresence of secreted immunemodulatory metabolites for example culture supernatants of certainprobiotic bifidobacterium species decreased the secretion of type cytokines from immune cell lines andthe expression of costimulatory molecules on primary dendritic cells a likely mechanism is quorumsensing quorum sensing is a means of communication among bacteria of the same species to coordinateeffector functions such as biofilm formation sporulation or toxin secretion the bestdescribed quorumsensing molecules are acyl homoserine lactones ahls several ahls are targeted by the host tointerfere with growth of pathogens and are in turn exploited by bacteria to regulate host geneexpression for their benefit some ahls can bind to distinct bitter taste receptors expressed on the airwayepithelium and innate and adaptive immune cells [ ] thereby modulating barrier andimmune functions the moststudied ahl 3oc12hsl can activate phagocytesto increasephagocytosis expression of adhesion receptors and chemotaxis [ ] but is itself cleaved andinactivated by airway epithelial cells another example of bacterialderived modulators of the hosts immune responses are outer membranevesicles omvs omvs are spherical bilayered membrane vesicles released from the surface of bothgramnegative and grampositive bacteria and contain much of the biological material from the parentbacterium but in a nonreplicative form [ ] evidence suggests that the release of omvs providesbacteria with competitive advantages when exposed to acute and chronic hostassociated stressors innate and adaptive immune responses [] antimicrobialthey may protect bacteria againstpeptides and antibiotics [ ] moreover omvs contain factors eg siderophores aiding in theacquisition of nutrients in an environment devoid of crucial elements such as iron besidessupporting the survival of the parent bacteria omvs may also play role in the progression of pulmonarydiseases bacteria frequently associated with copd exacerbations are known to release omvs furthermore macrophages stimulated with omvs derived from prominent airway pathogens such asp aeruginosa h influenzae or m catarrhalis release higher amounts of tumour necrosis factorα and il6 legionelladerived omvs significantly enhanced bacterial replication in macrophages andbacteriafree p aeruginosa omvs have been shown to potently induce pulmonary inflammation in mice strengthening the idea that omvs exert diseasepromoting activities in addition omvs have beenshown to induce tolerance and hyporesponsivenessthereby facilitating bacterial adherence to andinternalisation by macrophages which may contribute to clearance of the infection [ ] thusdespite our increasing knowledge on omvs and their potential role in interkingdom communicationthere is a need for further research to better understand their pathogenic properties and possibletherapeutic or prophylactic implications eg novel vaccinesan interesting alternative to fmt or probiotic bacteria might be to use immunemodulatory microbialmetabolites or beneficial omvs such chemically defined bacterial substances could be produced at largescale under controlled conditions applied in defined effective doses both systemically or locally and mayhave fewer adverse sideeffects ie in immunocompromised patients compared to live bacteria several studies have already used defined bacterial metabolites to treat aad in mice lps from escherichiacoli o111 bacterial polysaccharide a oligodeoxynucleotides with bacterial cpg motifs flagellin b shortchain fatty acids dtryptophan and the neutro | 0 |
cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull 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 give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1pccn by eq for a large n we can approximatethe mean μ and standard deviation Ï of pccn1 pccn as and pcc2nn respectively pccchange pccn1 pccnz score pccchange μpccchangeÏ pccchange pccchangepcc2nnthe edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ¥ and 01pcc are correlatedgained gene pairs and those with zscore ¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is correlationgained positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr cid2 ppiexprwcnv mcnv à ppicnv wmethyl cid2 ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene is expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene is expression level on cnv and methylation respectively βmirnais the regression coefficientof gene is expression level on its mirna regulator jsexpression leveland βmethyl mexpr mmethylcid3 à cid2cid3 à cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is correlationgained positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyi¡¢¢£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44¤¥¥¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and st¤ à §ªªªª¨ªªªª©if v is a nonseed nv ¥ αif v is a nonseed nv αif v is a seednvnvenvα à nvnvdv st λ à st à w λ à d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k à mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ¼and dna methylation data auc ¼ alonewas lower than that with mrna expression data auc ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ ] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev verma m personalized medicine and cancer j personalized medbarabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum | 0 |
"The main complexity of modeling and interpreting such phenomena lies in the additional temporal dimension needed to express the association as the risk depends on both intensity and timing of past exposures. This type of dependency is defined here as exposurelagresponse association. In this contribution I illustrate a general statistical framework for such associations established through the extension of distributed lag non-linear models originally developed in time series analysis. This modeling class is based on the definition of a cross-basis obtained by the combination of two functions to flexibly model linear or nonlinear exposure-responses and the lag structure of the relationship respectively. The methodology is illustrated with an example application to cohort data and validated through a simulation study. This modeling framework generalizes to various study designs and regression models and can be applied to study the health effects of protracted exposures to environmental factors drugs or carcinogenic agents among others. 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. latency distributed lag models exposurelagresponse delayed effects splines 1. Introduction In biomedical research it is commonly appreciated that an exposure event produces effects lasting well beyond the exposure period with an increase in risk occurring from few hours to many years later depending on the physiological processes linking the exposure and the health outcome. The problem is made even more complicated in the presence of protracted time-varying exposures when the health effect measured at a given time can be described as the result of multiple exposure events of different intensities sustained in the past. This phenomenon common to various research fields has been associated for example with peak 1 or chronic exposures 2 to environmental stressors drug intake 34 or occupational exposures to carcinogenic substances 5. The main complexity of modeling and interpreting such dependencies lies in the additional temporal dimension needed to express the association beyond the usual exposureresponse relationship as the risk depends on both intensity and timing of past exposures. Nonetheless the appropriate representation of the temporal pattern of risks may provide further insights on the association of interest in particular regarding the underlying pathophysiological mechanisms and prevent biases in estimates and predictions. Revising previous terminology 6 I define these dependencies as exposurelagresponse associations. In particular this issue has been debated in cancer epidemiology 79. Analytical approaches extend simple indices such as cumulative exposure in order to accommodate the temporal variation in risk because of protracted exposures. In particular the pioneering work by Thomas 106 helped develop sophisticated statistical methods on the basis of weighting past exposures through specific functions whose parameters are estimated by the data. Vacek 11 Langholz and colleagues 12 and Richardson 13 provided interesting applications in case-control studies with weights represented through simple parametric functions. The methodology was improved by Hauptmann and colleagues in a series of papers 1416 by using flexible and smooth spline functions. Sylvestre and Abrahamowicz 17 and Abrahamowicz and colleagues 18 extended the spline methods to the analysis of time-to-event data with a cohort design and presented their applications in pharmaco-epidemiology. The main limitation of the statistical techniques described in these papers is the assumption of a linear exposureresponse relationship. Models for nonlinear dependencies introduce further nontrivial complexities from both statistical and interpretational perspectives as the problem becomes inherently bidimensional. Abrahamowicz and Mackenzie 19 proposed a model for analyzing the nonlinear time-dependent effects of fixed exposures while Vacek 11 and Berhane and colleagues 20 extended this scheme to the case of protracted time-varying exposures. However the modeling techniques illustrated in these other papers still face some limitations as they are based on complex estimation routines with convergence issues and problems in producing uncertainty measures such as standard errors and confidence intervals. Interestingly equivalent approaches were previously established in time series analysis on the basis of distributed lag models (DLMs) a methodology originally formulated in econometrics 21 then applied in epidemiological research 22. These models involve the definition of a distributed lag function analogous to the weighting function described before. In particular Armstrong 23 generalized the method to distributed lag non-linear models (DLNMs) a class of models with different options for the functions applied to model nonlinearity and distributed lag effects. The theory of DLMs and DLNMs have been recently re-evaluated 24 offering a well-grounded statistical tool and a comprehensive scheme for interpretation. In this paper I aim to establish a general conceptual and statistical framework for modeling exposurelagresponse associations built upon the paradigm of DLMs and DLNMs. This modeling class extended beyond time series analysis provides a unified methodology applicable in different study designs data structures and regression models including most of the previous methods as specific cases. Also the statistical framework is defined by completely parametric functions and fitted through standard regression methods with measures of uncertainty and fit statistics easily available. The R package dlnm originally developed for time series data 25 is extended in parallel offering a easy-to-use implementation of the modeling approach. The manuscript is structured as follows. The development and algebraic definition of the modeling framework is described in Section 2. As an illustrative example in I apply the method for investigating the relationship between occupational exposure to radon and lung cancer mortality by using the data from the Colorado Plateau miners cohort. The modeling framework is then validated in a simulation study n. A final discussion is provided in. Information on data and software implementation is included in. The R code and data are included in the supporting information together with additional details making the results of the illustrative example and of the simulation study entirely reproducible. 2. Modeling framework The modeling skeleton is derived by extending the class of DLNMs beyond the time series context. This extension provides a neat algebraic representation and a comprehensive statistical definition. The focus is on a function here defined s(xt) which describes the dependency in terms of the exposure history to x evaluated at time t. The function s(xt) is commonly included in regression models in order to estimate the association while controlling for potential confounders. Although the regression model varies depending on the study design and the type of data the definition of s(xt) provided later and the related modeling framework generally apply. 2.1. Models for linear exposureresponse relationships Previous studies on the topic have defined the function s(xt) by using slightly different algebraic formulae 1026111417. Assuming a linear exposureresponse relationship a general notation can be given by (1a) (1b) (1c) In (1a) the increase in risk at time t is defined as the integral of the instantaneous exposure intensity xu over the period ?t = [t0t1] with t0 and t1 representing the times of the first and last relevant exposures. Here w(t ? u) is the weighting function previously described in which assigns weights to past exposures experienced at time t ? u on the basis of their contribution to the risk at time t. The model can be reparameterized as in (1b) where the risk is now expressed along the lag with ? ? [?0L]. Here L ? ?0 = t1 ? t0 is interpreted as the lag period over which an exposure to x is assumed to affect the risk at time t usually with ?0 = 0. This parameterization offers the advantage that the function w is now directly defined in the new dimension of lag ? and it is independent of the time axis chosen for t which may represent different time scales depending on the study design. The function w(?) termed from here on as the lagresponse function models the lagresponse curve associated with exposure x. Finally for computational purposes the integral is approximated in (1c) by a sum of terms derived by partitioning the lag interval in equally spaced discrete units and assuming the protracted exposure as a sequence of exposure events xt ? ? at lags ? = ?0 ¦ L. A statistical model for (1) can be defined by expressing the lagresponse function w(?) as a linear combination of terms obtained through basis transformation with related parameters. By using matrix notation let the vector qxt of exposure history be defined by (2) Such exposure history changes along time depending on the time t at which the vector qxt is computed. Given (2) the cumulative function s(xt) in (1) can be written using a compact and general matrix notation as (3) The (L ? ?0 + 1) à v? matrix C is obtained from the transformation of the lag vector ? = [?0 ¦ ? ¦ L]T by choosing a specific basis with dimension v? for w(?) which defines the related basis functions. In this parameterization the function s(xt) representing the integral of x · w(?) over the interval [?0L] is defined as a lagbasis function with parameters ?. Interestingly the equation in (3) is almost identical to that defining DLMs 24 Eq. (4). The different indexing in the original version reflects the specific application in time series where the data are perfectly ordered in time and the matrix Q has a structure such that qt? ? qt + 1? + 1. However this is a specific case of the general representation in (2)(3). The theory and software already developed for DLMs can be therefore extended in parallel. Alternative lagbasis functions for representing s(xt) are derived through different lagresponse functions w(?) in (1). In particular the traditional index of unweighted cumulative exposure is a specific case of (3) where reduces to with w(?) equal to a constant c. This is obtained by specifying C as an (L ? ?0 + 1)-dimensional vector of 1's with v? = 1. More sophisticated models with splines or other functions such as those illustrated in publications cited in only require the application of different bases for deriving C but are nevertheless represented by 3). 2.2. Extension to nonlinear exposureresponse relationships The extension to the nonlinear case presents further complexities as anticipated earlier. The model in (1) can be extended by defining an additional exposureresponse function f(x) to express the potentially nonlinear exposureresponse curve along the dimension of the predictor. An intuitive generalization of (1) is: (4) with f(x) as the standard exposureresponse function. However the function f(x) · w(?) in 4) previously proposed 1119 is not easily represented as a linear combination of basis variables and generates models that are not linear in their parameters and thus require ad hoc optimization routines. More importantly this representation is based on the strong assumption of independency between f(x) and w(?) namely that the exposureresponse shape is the same at each lag ? and vice versa that the lag structure is the same at each value of x. This assumption can be relaxed by expressing s(xt) as a truly bivariate function with the more flexible representation: (5) Here the bidimensional function f · w(x?) is defined as the exposurelagresponse function and models simultaneously the exposureresponse curve along x and lagresponse curve along ? namely an exposurelagresponse surface. Differently from 4) the exposurelagresponse function in 5) can be expressed as a linear combination of basis variables and related parameters through a special tensor product. As anticipated earlier Armstrong 23 proposed the same approach for time series data within the DLNM framework generalizing this tensor product parameterization through the concept of cross-basis. Specifically two sets of basis functions are independently chosen to represent f(x) and w(?) respectively. The cross-basis is the bidimensional space of functions obtained by the combination of the two sets integrated over the lag dimension and represents the core of DLNMs. The algebraic representation has been previously presented 24 and a revised version is proposed here. Briefly the simpler lag-basis for DLMs in 3) can be extended by choosing an additional basis with dimension vx for representing f(x). The application of the related basis functions to the vector of exposure history qxt obtained by 2) generates a (L ? ?0 + 1) à vx matrix Rxt. Let Axt be: (6) with 1v as a v-dimensional vector of 1's and C defined in 3). The cross-basis function s(xt;?) can be defined as (7) In this case the dimension of the cross-basis is determined by the product of the dimensions of the bases for the two spaces and the association is expressed through vx · v? values W and related parameters ?. The cross-basis function s(xt) represents the integral of f · w(x?) over the interval [?0L] cumulating the contributions of events representing the exposure history. In spite of the relatively complex algebraic form the definition of cross-basis and the specification of DLNMs only amount to the choice of the bases for the functions f(x) and w(?). These can be independently selected between several options such as splines linear threshold or piecewise constant (step) functions. The DLNM modeling class comprises the simpler DLMs from Section 2.1. For example the bidimensional exposurelagresponse function f · w(x?) in 5) reduces to a non-linear function for un-weighted cumulative exposure f(x) · c when w(?) is a constant function c and to the lagresponse function x · w(?) in (1) when f(x) is simply an linear function of the untransformed x. The model proposed by Berhane and colleagues 20 can be written in the form of 6)7) when both f(x) and w(?) are cubic B-splines. 2.3. Estimation and prediction Although the lag-basis and cross-basis functions in (1)(3) and (5)(7) involve a nonstandard parameterization in terms of exposure histories DLMs and DLNMs do not require specialized estimation procedures. The association is entirely expressed by the vx à v? parameters ? of the cross-basis values W. The computation of the exposure history in (2) can be extended to all N observations with x measured at time t producing an N à (L ? ?0 + 1) matrix of exposure histories Q. The matrix of transformed variables W in (3) and (7) is consequently derived. This matrix can be included in the design matrix of standard regression models to estimate the parameters ?. In the completely parametric development proposed here the number of coefficients vx à v? represents the degrees of freedom (df) used to model the association. Inference on the parameters ? and interpretation of the estimated association is aided by the prediction of specific risk measures. For simpler DLMs that assume a linear exposureresponse relationship this step reduces to the computation of a series of estimated risk contributions at lag ?p with ?0 ? ?p ? L and the associated (co)variance matrix . The series of risk contributions is provided by (8) with Cp obtained from the vector of lag ?p used for prediction by applying the same basis functions for w(?) used for estimation. These estimated risk contributions compose the lagresponse curve and can be interpreted using either a forward or backward perspective. Namely represents the risk contribution at time t + ?p in the future from a unit increase in exposure x at time t or the contribution from a unit increase in exposure x occurring at time t ? ?p in the past to a given risk measured at time t. The estimated risk contributions associated with different exposure increases are easily derived. The equations in (8) only apply to DLMs with lag-bases as defined in (3). For DLNMs the association is allowed to vary nonlinearly in the space of x. Moreover the specification in (5)(7) allows the lag-response curve to change depending on the level of the exposure. The prediction of risk contributions corresponding to a specific exposure intensity xp at lag ?p involves a more complex procedure. First let be the (L ? ?0 + 1)-dimensional vector of exposure history with constant exposure xp. The related matrices and are derived from (6) substituting qxt and C with and Cp by applying the same two sets of basis functions for f · w(x?) chosen for estimation. The exposure-specific risk contributions and associated (co)variance matrix are provided by (9) The estimated risk contributions may be interpreted as a lag-response curve similar to in (8) but this time associated with a specific exposure level xp instead of a unit increase. These measures may be used to define a grid of predicted risk contributions defined within the ranges of the exposure x and the lag ? thus obtaining a bi-dimensional representation of the association. From this grid besides above it is also possible to derive the vector of lag-specific risk contributions expressing the exposure-response curve for lag ?p. As noted in Section 2.2 the truly bivariate definition of (7) allows both the lag-response curve and exposure-response curve defined by and respectively to change depending on the specific exposure and lag values xp and ?p. The grid is interpreted as a risk surface along x and ? representing the exposurelagresponse. In addition predictions in (8)(9) may be extended to a generic exposure history qh. Substituting it into in (9) provides the vector of lag-specific risk contributions for each exposure that occurred within the lag period. The overall cumulative effect of such exposure history with associated (co)variance matrix may be computed with: (10) The Equation (10) can be used to estimate the predicted cumulative risk for a given pattern of exposure qh. This method can also be applied to investigate how the risk progressively evolves along an exposure profile computing the cumulative risk at each time associated with the time-varying exposure history qh. 2.4. Identifiability and constraints The tensor product structure of the cross-basis defined in (5)(7) poses some identifiability issues. In particular each of the vx basis variables in R is multiplied by each of the v? basis variables in C. If an intercept is included in f(x) the related matrix of cross-basis variables W is not of full rank and the parameters of the regression model are not identifiable even when a common intercept is not included. Therefore the cross-basis in (7) should always be defined without an intercept in the basis functions for x. Also these basis functions can be centered on a specific exposure value x0 which will represent the reference for the risk summaries computed by (8)(10). The bidimensional shape of the exposurelagresponse can be constrained to follow a prespecified pattern. In particular a priori assumptions on the lag structure can be imposed through functional constraints on the basis for the space of ?. Left and right constraints on the extremes of the supporting interval ?0L are particularly meaningful for smooth functions. A left constraint can be imposed by excluding the intercept from the basis. This step will force the lagresponse curve to predict a null risk at the beginning of the lag period. A right constraint on a B-splines basis can be produced by excluding specific basis variables as previously described for linear exposureresponse relationships 17. The constraint produces a smooth dependency which approaches a null risk at the end of the lag period. Such constraints are particularly useful in the presence of sparse data in order to limit the flexibility of the model under specific assumptions about the lagresponse curve. However biases can be introduced if these assumptions are not met. Additional information is provided in Section D1 of the supporting information. The functional constraints discussed in this section can be specified without introducing customized optimization methods for estimating the parameters ? in (3)(7). More sophisticated methods are required for example to constrain the lagresponse curve to be non-negative in the whole lag period L. These approaches have been previously proposed for linear dependencies 141718 and introduce further complexities in the bidimensional context of DLNMs. This development is not pursued here. 2.5. Model selection and inferential procedures The framework described in Sections 2.12.2 includes a fairly large number of models defined by different functions for each of the two dimensions and by different choices regarding each function such as number and location of knots in splines. This raises the issue of selecting the optimal model for describing the exposurelagresponse association. Previous studies on temporal dependencies have proposed selection procedures on the basis of profile likelihood 15 AIC 141620 or BIC 17. Simulation studies seems to indicate a better performance of AIC when compared with BIC in this context 18 a result consistent with unpublished simulations performed on time series data for DLNMs. Inference on the models illustrated in the previous sections primarily focuses on the specification of confidence intervals for the risk measures in Section 2.3 and on the definition of tests for a set of null hypotheses. Confidence intervals for lagresponse curves exposureresponse curves and cumulative risks obtained through and can be easily derived from the diagonal of the related (co)variance matrices in (8)(10) assuming a multivariate normal distribution of the estimators. Regarding hypothesis testing two null hypotheses are particularly relevant in this framework. The first one postulates a linear exposureresponse relationship namely H0 : f(x) = x. The second one assumes a constant risk namely H0 : w(?) = c. Tests on constrained models can be also defined. The assumption of independency is not easily tested as the form in (4) cannot be expressed as a model linear in its parameters. However defining general inferential procedures in this setting is not straightforward. First the null hypotheses H0 : f(x) = x and H0 : w(?) = c are not independent and an incorrect assumption about the association in one dimension may bias the test estimator for the hypothesis related to the other space as previously reported 19. In addition estimates are usually conditional on a posteriori selection of a best-fitting model based on the selection methods discussed before. Under these conditions the estimators for the (co)variance matrices in (8)(10) are likely to underestimate the true sampling (co)variance and the distribution of the test statistics may be different from that assumed unconditional on the selection procedure. This may generate undercoverage of confidence intervals and inflated type I error for tests 1727. Given these complexities a general framework for hypothesis testing embedded in the model selection procedure is not provided here. An assessment through simulations of the performance of estimators generated by AIC and BIC-selected models will be presented in. Specifically simulations will provide an empirical evaluation of the ability of the information criteria to identify the correct model between those defining the null or alternative hypotheses about linearity and constant effects and measures of performance such as bias coverage and root mean square error. 3. An application The conceptual and statistical framework of DLNMs described in extended beyond time series data is general and applicable in different study designs. As an illustrative example I propose here an application in survival analysis of time-to-event data. This represents one of the most complex settings as the temporal pattern of risk is produced by exposure histories that vary during the follow-up of each subject. Specifically the methodology is used to investigate the association between occupational exposure to radon and mortality for lung cancer. The analysis is based on data from the Colorado Plateau uranium miners cohort already used in previous methodological contributions 121520. Section A of the supporting information provides a list of the main steps to replicate the analysis in other real-life examples. 3.1. Data The cohort data used in this example were collected by the National Institute for Occupational Safety and Health. Detailed information on the cohort is given elsewhere 12. Briefly subjects were eligible to enter the cohort if they worked in mines within the Colorado Plateau area between 1950 and 1960 and provided demographic personal and occupational information during their working period. Vital status and cause of death were ascertained by linkage with different sources. The data used in this example refer to the follow-up of the cohort on December 311982 including 3347 subjects and 258 lung cancer deaths. Exposure data available in the data set include cumulative measures of radon and smoking in 5-year age intervals. The radon exposure history for each subject expressed in working-level months (WLM) was reconstructed by linking employment information with measured or predicted levels in each mine in each year. The smoking history expressed in the number of cigarettes packs à 100 was reported by each subject during his working period and assumed constant after the last reporting age. A summary of the data is provided in Table I. Table I Descriptive statistics of the Colorado Plateau uranium miners cohort. The data included here refer to the follow-up on December 31 1982. Exposure to radon is measured in working level months (WLM) while smoking is reported as packs of cigarettes/100 Full cohort Lung cancer cases N % N % Subjects 3347 100.0 258 7.7 Deaths (%) 1258 37.6 258 100.0 Ever smokers (%) 2656 79.4 238 92.2 Median Min 25th 75th Max Median Min 25th 75th Max Age at entry 34.0 15.8 25.8 44.0 80.0 41.6 18.6 34.3 48.0 63.9 Follow-up time (years) 23.9 0.1 19.6 25.5 32.5 18.3 0.3 12.9 22.0 30.8 Exposure to radon Exposure period (years) 6.7 0.1 2.7 11.8 53.0 12.8 0.1 7.8 17.6 39.5 Total cumulative exposure (WLM/year) 429.0 0.0 153.5 1016.8 10000.0 1231.9 8.0 553.7 2528.6 10000.0 Yearly exposure (WLM/year) All 60.2 0.1 26.7 122.2 3245.3 81.6 1.0 42.3 165.4 1295.7 Lag 09 52.4 0.1 23.8 102.5 2994.0 61.4 3.9 31.3 144.7 1110.8 Lag 1019 53.8 0.1 24.3 112.5 3245.3 78.3 1.0 42.9 164.0 1295.7 Lag 2029 74.0 0.1 33.0 141.7 3245.3 104.7 4.1 52.2 180.0 1295.7 Lag 3040 95.7 0.2 48.0 151.6 2994.0 104.7 5.5 60.0 175.3 860.2 Smoking Exposure period (years) 38.0 5.0 31.0 46.0 75.0 40.0 14.0 33.0 48.0 72.0 Total cumulative exposure (packs à 100) 131.6 0.4 94.5 174.5 676.3 147.4 21.8 109.5 188.1 567.2 Yearly exposure (packs à 100) 3.6 0.0 2.5 3.6 24.4 3.6 0.0 3.5 4.2 13.4 3.2. Modeling strategy For this illustrative example the analysis is performed through a Cox proportional-hazard model with time-varying covariates by using age as the time axis. Effect measures are reported as a hazard ratio (HR). The model is represented by the following: (11) where the log-hazard log [h(t)] is expressed as a sum of baseline log-hazard log [h0(t)] and contributions of additional covariates. These comprise cross-basis functions sx(xt) and sz(zt) for radon and smoking respectively as defined in (1)(7) and a linear term for calendar time u in order to control for secular trends in lung cancer risk not accounted for by the delayed effects of the two exposures. Radon is the exposure of interest and is modeled with different combinations of bases for f(x) and w(?) in the cross-basis sx(xt). Given the limited information on smoking histories in this analysis the cross-basis sz(zt) is a priori defined with a natural cubic B-spline with one knot at the median of 2.5 yearly packs à 100 for the exposureresponse and a step function with a single cut-off at lag 20 for the lag structure with lag period 240 years. However different cross-basis functions can be applied. The model spends 5 df controlling for confounders and a different amount for modeling the effect of radon depending on the chosen cross-basis sx(xt). Modeling exposurelagresponse associations in time-to-event data assumes the definition of an extended version of continuous time-varying predictors namely the varying exposure history for each subject at the ages he contributes to different risk sets 28. The lag scale is chosen as years with lag 0 identifying the exposure during the last year. The lag period is fixed at 240 assuming no effect of exposure after 40 years and in the last 2 years consistently with previous analyses. Multiple exposure histories are computed for each subject at the ages he contributed to each risk set given his exposure profile reconstructed from the 5-year periods. This step produced matrices of exposure histories Qx and Qz for radon and smoking respectively as defined in (2). These matrices are used to specify the lag-bases or cross-bases matrices Wx and Wz from (3)(7) included in the design matrix of the Cox model." | 1 |
"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 cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant 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 hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l 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 ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj 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 cprds 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 ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu | 0 |
glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research du 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 wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city peoples hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city peoples hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage yearsgendermalefemalegradeiiiiiiivtumor size cmlow n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturers instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq¢ ii takara japan gapdh was the normalized control relative expression was calculated through the δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturers instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturers instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using students ttest or oneway anova survival rate was analyzed by the kaplanmeier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells be proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4cf therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearsons correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1 v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your 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" chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer yet the toxicity of chemotherapy agents such as fluorouracil 5fu limits the effectiveness of chemotherapy ginsenoside rg3 rg3 is an active ingredient isolated from ginseng rg3 has been shown to display anticancer effects on a variety of malignancies yet whether rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer remains unknown the present study was designed to ascertain whether rg3 is able to enhance the anticolon cancer effect of 5fu the results revealed that combined treatment of rg3 and fu significantly enhanced the inhibition of the proliferation colony formation invasion and migration of human colon cancer cells sw620 and lovo in vitro we also found that combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway and arrested the cell cycle of the colon cancer cells in g0g1 by promoting the expression of cyclin d1 cdk2 and cdk4 in addition the pi3kakt signaling pathway in colon cancer cells was suppressed by rg3 and 5fu in vivo rg3 synergized the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude mice similarly combined treatment of rg3 and 5fu altered the expression of colon cancer protein in vivo and in vitro collectively the present study demonstrated that ginsenoside rg3 enhances the anticancer effect of 5fu in colon cancer cells via the pi3kakt pathwaycorrespondence to dr xiangbo chen endoscopy center the quanzhou first hospital affiliated to fujian medical university east street licheng quanzhou fujian pr chinaemail coloboyeahnetkey words ginsenoside rg3 colon cancer 5fluorouracil pi3kaktintroductioncolon cancer is a common malignant tumor of the digestive tract located in the colon which mainly occurs at the junction of the rectum and the sigmoid colon statistics show that the highest incidence of colon cancer is in the age group of years the ratio of male to female is and the incidence of colon cancer ranks third among all cases of gastrointestinal tumors the 5year survival rate of patients with colon cancer is approximately yet the 5year survival rate of patients with advanced stage disease is as low as since the early symptoms of patients with colon cancer are not obvious only about of patients can be diagnosed at the early stage of the disease chemotherapy is one of the most important treatments for patients with advanced colon cancer of which fluorouracil fu is the most widely used 5fu inhibits the proliferation invasion and migration of tumor cells by interfering with the nucleic acid metabolism of tumor cells but it is also toxic to normal cells causing serious adverse reactions even endangering the life safety of patients severely limiting its clinical application previous research has shown that 5fu combined with other agents may reduce the required dosage of 5fu consequently reducing the adverse reactions caused by 5fu without affecting or even improving the efficacy of chemotherapy compared with chemical drugs and biopharmaceuticals multicomponent multitarget and less adverse reactions are unique advantages of traditional chinese medicine for the treatment of diseases in patients with colon cancer chinese medicine can improve patient immunity reduce the side effects of radiotherapy and chemotherapy or enhance drug sensitivity inhibiting the expression of oncogenes helps to inhibit the migration of cancer cells and has a good effect on the treatment of colon cancer ginsenoside rg3 rg3 an active ingredient isolated from ginseng is a tetracyclic triterpenoid saponin that inhibits neovascularization induces tumor cell apoptosis and selectively inhibits tumor cell metastasis and enhances immune function previous studies have shown that rg3 exhibits an inhibitory effect on proliferation invasion and migration of human tumor cells such as lung cancer gastric carcinoma and prostate cancer in colon cancer rg3 was found to activate the ampk signaling pathway to accelerate apoptosis in colon 0chong effects of ginsenoside rg3 on colon cancercancer cell line ht29 in vitro and also to block colon cancer progression by targeting inhibition of cancer stem cells and tumor angiogenesis in vivo although numerous studies have shown that rg3 increases the efficacy and decreases the toxicity of chemotherapeutic drugs and suppresses the chemotherapeutic resistance in cancer its combination with chemotherapeutic agent fu to achieve extra benefits in anticolon cancer treatment warrants detailed investigationin the present study the effects of a combined treatment of rg3 and 5fu on the biological properties of sw620 and lovo cells were investigated in vivo and in vitro we found that a combined treatment of rg3 and 5fu not only enhanced the inhibition of colon cancer cell proliferation migration and invasion but also promoted apoptosis of colon cancer cells and arrested the cells in the g0g1 phase in addition it was also found that rg3 could synergize the capacity of 5fu to inhibit the growth of human colon cancer xenografts in nude mouse and the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt pathway in colon cancer cellsmaterials and methodscell lines and agents sw620 ccl atcc american type culture collection manassas va usa and lovo ccl229 atcc cell lines were cultured with dmem medium cat no thermo fisher scientific inc supplemented with fetal bovine serum fbs cat no thermo fisher scientific inc and penicillinstreptomycin cat no thermo fisher scientific inc the cell lines used in the present study were cultured at Ëc with co2rg3 cat no sigmaaldrich merck kgaa and 5fu cat no sigmaaldrich merck kgaa were dissolved in dmso for the cell experiments the diluted culture solution of rg3 or 5fu was dissolved in dmso to achieve the experimental concentration and was administered to the cells for h for animal experiments pbs diluted rg3 or 5fu was dissolved in dmso to the experimental concentration the experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinamtt assay a total of 2x103 cellswell were inoculated in a 96well culture plate containing the indicated medium dmem plus fbs we evaluated the viability of the sw620 and lovo cells by mtt assay in short after h of culture mtt µl mgml which was dissolved in dmso was added to the cells and incubated the cell supernatant was removed and then µl dmso was added after min the optical density od570 was determined using a plate reader elx808 biotek instrumentscell colony formation assay a total of 2x103 cellsml were seeded in 6well plates with ml mediumwell and medium was exchanged once every days cells were routinely cultured for about weeks when visible clones appeared in the well the culturing was stopped the supernatant culture medium was drawn washed with pbs times and fixed with formaldehyde for min the supernatant was drawn stained with crystal violet for min and slowly rinsed with sterile water plates were placed in a sterile purification table and images were captured after drying the relative proliferation was measured by measuring the absorbance at nm using a plate reader elx808 biotek instrumentswestern blot analysis ripa lysate buffer cat no p0013c beyotime institute of biotechnology shanghai china was used to extract total cellular protein and the bca kit cat no p0009 beyotime institute of biotechnology was used to determine the protein concentration then cell lysates of sw20 and lovo cells were separated by sdspage with µg total protein and transferred to a pvdf membrane the following primary antibodies were selected as follows antincadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution antimmp ab38898 dilution antiactivecaspase antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution antipi3kp85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution antipanakt phospho t308 antibody ab38449 dilution antipanakt antibody ab8805 dilution antipdk1 antibody ab52893 dilution and antigapdh ab9484 dilution the secondary antibody was selected as follows goat antirabbit ab150077 dilution or goat antirat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at Ëc and the secondary antibody was incubated for h at room temperature the beyoecl plus kit cat no p0018s beyotime was used for the chromogenic protein bands with beckman coulter immunoassay system unicel dxi beckman coulter and imagej v2147 national institutes of health was used for the densitometric analysis of protein bands all antibodies were purchased from abcam unless otherwise statedtranswell invasion experiment the cell density was adjusted to 05x106 cellsml and then the cells were added to a 24well transwell upper chamber corning corning ny usa medium containing fbs gibco thermo fisher scientific inc was added into the lower transwell chamber and the transwell was incubated at Ëc for h the transwell was taken out and the medium was removed it was washed twice with pbs methanol was added and dried after being fixed for min after the membrane was dried it was stained with crystal violet for min and the relative migration was determined by measuring the absorbance at nm using a plate reader elx808 biotek instruments inccell scratch test a total of 5x105 cells were placed in a 6well plate mlwell a scratch was made as far as possible perpendicular to the back of a horizontal line by using tips against a ruler tips should be vertical and cannot be tilted the cells were washed with pbs for three times and the scratched cells were removed and serumfree dmem was added cells were cultured at Ëc in a co2 incubator for h and images were captured in and h using an ckx41 olympus inverted microscope magnification x100 olympus corp 0concology reports figure effect of the combined treatment of rg3 and 5fu on proliferation of colon cancer cells in vitro sw620 and lovo cells were treated with different doses of a rg3 mmoll or b 5fu µmoll and then the mtt assay was used to detect cell viability c and d after treatment with rg3 mmoll or 5fu µmoll or their combination the colony formation of the colon cancer cells was photographed wt group was used as a baseline for cell viability and cell colony formation three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the wt group fu fluorouracil rg3 ginsenoside rg3 flow cytometric analysis cells that had been treated in different manners were collected and precooled ethanol prechilled pbs and waterfree configuration was added at Ëc overnight then the cells were washed with pbs and stained with propidium iodine pi for cell cycle macsquant® analyzer flow cytometer miltenyi biotec was used to detect the cell cycle and the annexin v fitcpi kit invitrogen thermo fisher scientific inc was used for flow cytometry to detect apoptosisanimal experiment human colon cancer cells 5x10602 ml in the logarithmic phase were selected a total of female nude mice weeks of age g shanghai lingchang biological technology co ltd that were adaptive for feeding [room temperature of Ëc half day light and night dark cycle air humidity of ] for one week were selected mice were anesthetized [ sodium pentobarbital mgkg intraperitoneal ip] and then the lateral skin of the nude mice was selected as a cell inoculation site to inoculate 5x10602 ml human colon cancer cells at the logarithmic phase of growth when the tumor tissue grew to a volume of approximately mm3 then the mouse were randomly assigned to the solvent group equal amount of pbs dmso rg3 group mgkg gavage administration once every two days 5fu group mgkg ip injection once every two days and rg35fu group combined rg3 and 5fu group administration after weeks of treatment the mice were sacrificed using cervical dislocation and breathing and heartbeat for min were observed to determine death and tumor tissues were extracted and weighed with an analytical balance bsa124s beijing sartorius instruments ltd beijing china all animal experiments were approved by the ethics committee of quanzhou first hospital affiliated to fujian medical universitystatistical analysis all data are expressed as mean ± standard deviation and spss ibm corp was used to analyze the data student's ttest was used to compare differences between two groups and multiple groups were compared with oneway anova followed by duncan test as a post hoc test p005 was assigned to indicate that a difference was statistically significantresultscombined treatment of rg3 and fu enhances inhibition of cell proliferation after treatment with different doses of rg3 or 5fu mtt assay was used to measure the cell viability the results revealed that the cell viability of sw620 and lovo cells was significantly and gradually decreased with an increasing dose of rg3 thus we chose mmoll rg3 for subsequent experiments fig 1a as shown in fig 1b the proliferative activity of the colon cancer cells in the combined treatment group of rg3 and fu was significantly lower than 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on migration and invasion of colon cancer cells in vitro treatment of colon cancer cells with combined treatment of rg3 mmoll and 5fu µmoll inhibited the migration a and invasion b abilities of the sw620 and lovo cells scale bar µm c western blot analysis was used to detect the expression of emtrelated protein ncadherin ecadherin and mmp9 the solvent group was used as a baseline for the migration and invasion of cells three independent repetitions for each experiment were performed p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 emt epithelialmesenchymal transition mmp matrix metalloproteinase that of the 5fu treatment alone group in addition the cell viability of sw620 and lovo cells gradually decreased with the increasing dose of 5fu however after treatment with the combination of mmoll rg3 and µmoll fu for h the cell viability of sw620 cells was only which was not conducive to subsequent protein detection experiments thus mmoll rg3 and µmoll 5fu were chosen for subsequent experimentationcell clone formation assays were also used to detect in vitro proliferation of colon cancer cells as shown in fig 1c and d the number of colonies formed by the colon cancer cells treated with rg3 and fu was significantly lower than that of rg3 or fu alone these findings indicated that combined treatment of rg3 and 5fu enhanced the inhibition of colon cancer cell proliferation in vitrocombined treatment of rg3 and fu enhances the inhibition of cell migration and invasion the ability of tumor cells to invade and migrate is the key to tumor progression in the present study we compared the effects of different treatment conditions on the invasion and migration of colon cancer cells it was demonstrated that the invasion and migration ability of the colon cancer cells treated with rg3 combined with fu was significantly lower than that of rg3 or 5fu alone fig 2a and b epithelialmesenchymal transition emt is the source of tumor cell ability to acquire higher invasion and migration capacity thus we determined the levels of three emtrelated proteins ncadherin ecadherin and mmp9 and found that the expression of ncadherin and mmp9 protein in the rg35fu group was significantly lower than that of rg3 or fu alone group but 0concology reports figure effect of the combined treatment of rg3 and 5fu on the apoptosis of colon cancer cells in vitro a the percentage of apoptotic sw620 and lovo cells in the different groups b apoptosisrelated proteins [cleaved clcaspase clcaspase and apaf1] were assessed by western blot analysis in sw620 and lovo cells three independent repetitions for each experiment were carried out p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of ecadherin protein was significantly higher fig 2ccombined treatment of rg3 and fu promotes apoptosis of colon cancer cells fist we found that the apoptosis of the colon cancer cells treated with rg3 combined with 5fu was significantly higher than that of rg3 or fu alone fig 3a the levels of apoptosisrelated proteins in the sw620 and lovo cells were assessed by western blot analysis as shown in fig 3b expression levels of apaf1 cleaved clcaspase and clcaspase protein in colon cancer cells sw620 and lovo treated with rg3 were significantly increased and the expression of these apoptosisrelated protein in colon cancer cells following fu treatment was significantly higher than that treated with rg3 more importantly expression levels of these apoptosisrelated proteins in colon cancer cells treated with the combination of rg2 and 5fu were significantly higher than levels treated with rg3 or 5fu alonewe analyzed the cell cycle distribution of the colon cancer cells after treatment with the different agents as shown in fig 4a the percentages of colon cancer cells in the g0g1 phase treated with the rg3 and 5fu combination were significantly higher than the percentages following rg3 or 5fu alone similarly we also detected cell cycleassociated protein by western blot analysis as shown in fig 4b the expression levels of cyclin d1 cdk2 and cdk4 protein in colon cancer cells which were treated with the rg3 and 5fu combination were significantly lower than levels following treatment with rg3 or 5fu alonecombined treatment of rg3 and fu suppresses pi3kakt signaling in colon cancer cells the pi3kakt signaling 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on cell cycle progression of colon cancer cells in vitro a flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with rg3 mmoll or 5fu µmoll or the combination b cell cycleassociated protein cyclin d1 cdk2 and cdk4 were assessed by western blot analysis three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 cdk cyclindependent kinase pathway is a signaling pathway involved in cancer cell proliferation invasion and migration and its abnormal activation can confer high proliferation invasion and migration ability of cancer cells in the present study we found that the expression levels of pp85 p110 ppdk1 and pakt protein in the colon cancer cells which was treated with rg3 and 5fu combination were significantly lower than levels in the cells treated with rg3 or 5fu alone fig these results indicated that the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt signaling pathway in colon cancer cells in vitrocombined treatment of rg3 and fu suppresses tumor growth in nude mice based on the results of in vitro studies we further investigated the effects of the rg3 and 5fu combination on colon cancer cell proliferation and protein expression in nude mice sw620 cells were injected into the armpits of nude mice after weeks of treatment the mice were sacrificed and the weight and volume of tumor tissues were measured it was found that the weight and volume of tumor tissues in the rg35fu group were significantly lower than these parameters in the groups treated with rg3 or 5fu alone fig 6a and bmoreover western blot analysis was used to detect the expression of emtrelated proteins cell cyclerelated proteins and key proteins in the pi3kakt signaling pathway it was found that although the effects of the rg3 and 5fu combination were not as obvious as the in vitro results compared with rg3 of 5fu alone the overall trend in protein expression was consistent fig 6ce these results demonstrated that rg3 0concology reports figure effect of the combined treatment of rg3 and 5fu on pi3kakt signaling in colon cancer cells in vitro a and b western blot analysis was used to detect the expression of key proteins in the pi3kakt signaling pathway after treatment with rg3 mmoll or 5fu µmoll or the combination three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude micediscussionthe anticancer effect of 5fluorouracil 5fu is exerted mainly by interfering with tumor cell dna replication and it is a commonly used antitumor agent for the treatment of advanced colon cancer however since 5fu displays nonspecific cytotoxicity it also causes damage to normal cells causing irreversible renal dysfunction and severe gastrointestinal reactions these adverse effects limit its clinical application and further improvements in the efficacy of chemotherapy are needed therefore it is urgent to discover a drug that can enhance the chemotherapeutic effects of 5fu and reduce the 5fu toxicity when used in combination with 5fuginsenoside rg3 rg3 is one of the main active ingredients extracted from ginseng research has shown that ginsenoside rg3 has certain inhibitory effects on lung cancer breast and prostate cancer the antitumor mechanism of rg3 was that rg3 reduced the neovascularization probability of tumor recurrence proliferation and metastasis in tumors by inhibiting kdrvegf protein expression and blocking hif1αcox2vegf pathway in the present study we found that the combined treatment of rg3 and 5fu promoted the inhibition of colon cancer cell proliferation in vivo and in vitro tumor growth development and metastasis are closely related to cell proliferation the previous study found that rg3 inhibits the proliferation of tumor cells such as rg3induced egfrmapk pathway deactivation was found to inhibit melanoma cell proliferation by decreasing fut4ley expression rg3 was found to inhibit the proliferation of multiple myeloma cells by inducing the secretion of igf1 promoting tumor cell apoptosis is also a method of inhibiting tumor cell proliferation in the present study we found that the combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway in the mitochondrial pathway of apoptosis apoptosisrelated signals release cytochrome c by stimulating the mitochondrial outer membrane cytochrome c enters the cytoplasm which activates caspase9 by binding with apaf1 activation of caspase9 further activates caspase3 while the activated caspase3 can activate caspase leading to apoptosis in addition we also found that the rg3 and 5fu combination enhanced the number of g0g1 phase colon cancer cells and decreased expression of cyclin d1 cdk2 and cdk4 the cell cycle refers to the whole process that the cell undergoes from the completion of one division to the end of the next division and 0chong effects of ginsenoside rg3 on colon cancerfigure effects of the combined treatment of rg3 and 5fu on tumor growth and protein expression of colon cancer cells in vivo after weeks of treatment the mice were sacrificed tumor tissues were excised and the weight a and volume b of tumor tissues were measured ce total protein was extracted from the colon cancer tumor tissues and the expression of proteins was detected by western blot analysis five nude mice in each group and at least tumor tissues were used to evaluate protein expression p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 the regulation of the cell cycle is mainly achieved by the retention of the g1 phase when a cell is in the g1 phase there is an important node regulating the cell cycle the r point when the cell cycle is before the r point the cell needs the external growth factor to achieve the normal operation of the cell cycle after the cell cycle crosses the r point the cell cycle becomes a process that is controlled autonomously by the cell and no longer depends on the presence of external cytokines cyclin d1 is a g1sspecific cyclin and its main function is to promote the cell cycle from g1 to s by binding and activating the cyclindependent kinase cdk24 a unique cyclindependent kinase of g1 so as to promote cell proliferation invasion and migration of tumor cells are the most important features of malignant tumors and the important causes of death in patients with malignant tumors ncadherin ecadherin and mmp9 are three proteins that play important roles in cell epithelialmesenchymal transition emt whereas emt provides cells the ability to transfer and invade promoting tumor cell emt can inhibit the expression of intercellular junction protein resulting in decreased intercellular connectivity which is beneficial to the invasion and migration of tumor cells to surrounding healthy tissues previous studies have found that rg3 not only inhibits metastasis and invasion of lung cancer cells by inhibiting emt induced by transforming factor 1 but also inhibited the metastasis of prostate pc3m cells by downregulating the expression of aqp1 by downregulating mmp rg3 affected the metastasis and invasion ability of melanoma cells the present study demonstrated that the combined treatment of rg3 and fu significantly suppressed the invasion and migration ability of human colon cancer cell in vitro by altering emtrelated proteinfurthermore we also found that rg3 and 5fu combination inhibited the conduction of the pi3kakt signaling pathway in vivo and in vitro many studies have shown that the occurrence and development of tumors are the result of multifactor multigene and multipathway processes and the cell signal transduction pathway is crucial in the process of tumor development invasion and metastasis the phosphatidylinositol 3kinaseserinethreonine kinase b pi3kakt signaling pathway plays an important role in the regulation of solid tumors [eg liver cancer breast cancer colon cancer gastric cancer neuroblastoma ] and blood tumors [eg leukemia ] pi3k acts as a bridge molecule for the relationship between extracellular signals and cellular responses under the influence of a series of upstream or bypass signaling molecules it acts on the downstream of the effects of a variety of molecules thus promotes cell migration 0concology reports inhibits cell apoptosis accelerates the process of the cell cycle and promotes cell proliferation many previous studies have shown that traditional chinese medicine or traditional chinese medicine monomers can play an antitumor role by inhibiting the pi3kakt signaling pathway in conclusion rg3 enhances 5fu inhibiting proliferation invasion and migration of colorectal cancer cells and helps 5fu block g1 phase induced apoptosis in more colorectal cells all in all our study found that rg3 enhanced the anticancer effect of 5fu on colon cancer cell via pi3kakt pathwayacknowledgementsnot applicablefundingno funding was receivedavailability of data and materialsthe datasets used during the present study are available from the corresponding author upon reasonable requestauthors' contributionsxc made substantial contributions to the conception and design of the study and critically revised it for important intellectual content sh contributed to the acquisition of the data wc zh yw xm yh and zl analyzed and interpreted the data all authors read and approved the final manuscriptethics approval and consent to participateall animal and cell experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinapatient consent for publicationnot applicablecompeting intereststhe authors state that they have no competing interestsreferences siegel rl ward em and jemal a trends in colorectal cancer incidence rates in the united states by tumor location 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one e98921 gao y xiao x zhang c yu w guo w zhang z li z feng x hao j zhang k melatonin synergizes the chemotherapeutic effect of fluorouracil in colon cancer by suppressing pi3kakt and nfbinos signaling pathways j pineal res doi 101111jpi12380 wang sf wu my cai cz li m and lu jh autophagy modulators from traditional chinese medicine mechanisms and therapeutic potentials for cancer and neurodegenerative diseases j ethnopharmacol ernst e traditional chinese medicine for cancer br j cancer sun hy lee jh han ys yoon ym yun cw kim jh song ys and lee sh pivotal roles of ginsenoside rg3 in tumor apoptosis through regulation of reactive oxygen species anticancer res tang yc zhang y zhou j zhi q wu my gong fr shen m liu l tao m shen b ginsenoside rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo int j oncol wang j tian l khan mn zhang l chen q zhao y yan q fu l and liu j ginsenoside rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of nfκb mediated epithelialmesenchymal transition and sternness cancer lett joo e ha yw and kim ys abstract lb23 molecular mechanisms of ginsenoside rg3 related to apoptosis in human lung and pancreatic adenocarcinomas cancer res lb23 kim bj nah sy jeon jh so i and kim sj transient receptor potential melastatin channels are involved in ginsenoside rg3induced apoptosis in gastric cancer cells basic clin pharmacol kim sm lee sy cho js son sm choi ss yun yp yoo hs yoon dy oh kw han sb and hong jt combination of ginsenoside rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of nfkappa b eur j pharmacol yuan hd quan hy zhang y kim sh and chung sh 20sginsenoside rg3induced apoptosis in ht29 colon cancer cells is associated with ampk signaling pathway mol med rep liu tg huang y cui dd huang xb mao sh ji ll song hb and yi c inhibitory effect of ginsenoside rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice bmc cancer sun my ye y xiao l duan xy zhang ym and zhang h anticancer effects of ginsenoside rg3 review int j mol med longley db harkin dp and johnston pg fluorouracil mechanisms of action and clinical strategies nat rev cancer hokmabady l raissi h and khanmohammadi a interactions of the fluorouracil anticancer drug with dna pyrimidine bases a detailed computational approach struct chem rateesh s luis sa luis cr hughes b and nicolae m myocardial infarction secondary to fluorouracil not an absolute contraindication to rechallenge int j cardiol e331e333 shan x aziz f tian ll wang xq yan q and liu jw ginsenoside rg3induced egfrmapk pathway deactivation inhibits melanoma cell proliferation by decreasing fut4ley expression int j oncol luo y zhang p zeng hq lou sf and wang dx ginsenoside rg3 induces apoptosis in human multiple myeloma cells via the activation of bcl2associated x protein mol med rep cardone mh roy n stennicke hr salvesen gs franke tf stanbridge e frisch s and reed jc regulation of cell 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"fluoroscopicallyguided interventional FGI procedures by orthopedic surgeons has beenincreasing This study aimed to investigate the occupational radiation exposure among orthopedic surgeons inSouth KoreaMethods A nationwide survey of orthopedic surgeons was conducted in South Korea in October Thedosimetry data of the participants were obtained from the National Dosimetry Registry The orthopedic surgeonswere categorized by job specialty [spine or trauma specialists other orthopedic specialists and residents] anddescriptive statistics for the demographics and workrelated characteristics were presented Multivariable logisticregression analysis was used to evaluate the risk factors for the orthopedic surgeons who were not linked with thedosimetry dataResults Among the total participants n of the orthopedic surgeons spent more than of theirtime working with the FGI procedures when compared with their overall work The average frequency of the FGIprocedures among the orthopedic surgeons was days per month Less than of the participants wereregularly provided with radiation monitoring badges The proportion of subjects who always wore lead aprons andthyroid shields were and respectively The residents group experienced more unfavorable workingconditions of radiation exposure than the other specialists The dosimetry data were not significantly linked amongthe residents odds ratio [OR] confidence interval [CI] and orthopedic surgeons working atsmall hospitals OR CI Conclusions Although orthopedic surgeons often performed FGI procedures they wore protective gear lessfrequently and a large proportion of orthopedic surgeons were not monitored by the national radiation dosimetrysystem As the number of radiation procedures performed by the orthopedic surgeons increases more intensiveapproaches are needed to reduce radiation exposure especially for spine and trauma surgeonsKeywords Fluoroscopy Health professional Occupational exposure Orthopedics Radiation Correspondence leewjkoreaackr2Department of Epidemiology and Health Informatics Graduate School ofPublic Health Korea University Seoul South Korea3Department of Preventive Medicine Korea University College of Medicine Goryeodaero Seongbukgu Seoul South KoreaFull 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 0cKang Journal of Occupational Medicine and Toxicology Page of BackgroundWith the markedly increased use offluoroscopicallyguided interventional FGI procedures during surgerythe risk of exposure to the ionizing radiation has increased for the orthopedic surgeons [ ] Fluoroscopicprocedures have been beneficial for patients undergoingorthopedic surgery because these minimally invasiveprocedures provide better direct visualization for soft tissue dissection spare blood supply and result in fewercomplications than surgeries [ ] However during the fluoroscopic procedures the surgeons may beexposed to the primary beam and scattered radiation [] As the use of fluoroscopy is continuously increasingin orthopedic surgery and orthopedic surgeons are oftenclosely exposed to the radiation source during operations they are considered a highrisk group of occupational radiation exposure []A few epidemiologic studies have reported an increased risk of cancer from radiation exposure amongorthopedic surgeons worldwide [] Diverse harmful effects such as cancer cataracts chromosomal abnormalities and other chronic diseases have also beenreported in physicians performing FGI procedures globally [] In addition orthopedic surgeons have concerns about the hazards of radiation but they have beenshown to demonstrate lower rates of wearing personalprotective equipment and dosimeters [ ] Howeverprevious studies on orthopedic surgeons were mainlylimited by the small sample sizes and limited information on the FGI procedurerelated work practices andoccupational radiation exposureIdentifying the occupational characteristics and radiation exposure could provide scientific evidence andserve as a fundamental step in developing strategies toprotect against occupational radiation exposure Therefore this study aimed to investigate the work practicesrelated to radiation exposure among orthopedic surgeons in South Koreasurgeonsatamong orthopedicMethodsStudy populationWe conducted a field survey using a selfadministeredquestionnairetheconference of the Korean Orthopedic Association inOctober The association includes all memberorthopedic surgeons and the conference is the representative meeting for orthopedic surgeons in SouthKorea A total of orthopedic surgeons participated in this study Written informed consent wasvoluntarily obtained from all study participants priorto enrollment The protocols ofthe study werereviewed and approved by the Institutional ReviewBoard of our university KUIRB1736A2Questionnaire and dosimetryA detailed questionnaire was developed from a previousstudy for interventional medical radiation workers []The questionnaire included demographics date of birthgender workplace address work history job title specialty years since beginning worktotal duration ofwork work practices proportion of interventional procedures performed for the recent year working days permonth working hours per week name of the main procedure performed badge wearing wearing protectiveequipment and concern for developing radiationassociated diseases 5point Likert scale The questionnaire is provided as a supplementary material Supplementary Table The Korea Center for Disease Control and PreventionKCDC has been monitoring the hospital personnel exposed to radiation since it maintains a centralizedNational Dosimetry Registry NDR and implements alifelong followup management system for radiation doseusing a personal thermoluminescent dosimeter TLD[] The NDR has collected dose measurements quarterly by five personnel monitoring centers designated bythe KCDC for all diagnostic radiation workers Thestandard protocol of wearing the NDR badge for alldiagnostic radiation workers is wearing one TLD badgebeneath the apron on the left side of the chest To evaluate the individual radiation dose the survey data werelinked with the NDR up to the second quarter of with respect to the participants name gender date ofbirth and workplace address This effective dose measured in Sievert was derived from the personal doseequivalent at a depth of mm Hp [] The lowestdetectable quarterly level of the NDR is mSv Incases where the dose was below the minimum detectablelevel the dose was considered as half of the detectablelevel owing to the highly skewed distribution []Data analysisStudy participants were classified by job specialty asspine or trauma specialists ST other orthopedic specialists Others and residents based on previous studiesin which spine or trauma surgeons were reported to beexposed to radiation more than the other orthopedicsurgeons [ ] Descriptive statistics for the demographics and workrelated characteristics are presented Thelevel of concern for developing radiationassociated diseases was recategorized as low very unlikely and unlikely medium and high likely and very likely Usingthe chisquare analysis the three job specialty groupswere compared according to the demographics occupational characteristics such as involvement with fluoroscopy and wearing the badges and protective devicesMultivariable logistic regression analysis was used toevaluate the risk factors for the orthopedic surgeons 0cKang Journal of Occupational Medicine and Toxicology Page of linked with the dosimetry data afterwho were notadjusting for agelocation of medical facility and jobspecialty Among the orthopedic physicians who werelinked with the TLD data the individual quarterly badgedoses recorded during the study period were summedand divided by the number of years to obtain the annualeffective doses this was compared according to the jobspecialty using oneway analysis of variance ANOVASTATA version StataCorp College Station TXUSA was used for statistical analysis and pvalues were considered statistically significantResultsA total of ST Others and residents participated in this study Table Most orthopedic surgeonswere aged years and worked at general hospitalsmany were young residents who were in training Therates of high concern for occupational radiation riskwere higher among the ST than the other groups Ourstudy participants comprised of all the members ofthe Korean Orthopedic Association but the residents inour study accounted for of all the members of theassociation Supplementary Table Approximately of the orthopedic surgeons spentmore than of their time working with the FGI procedures when compared with their overall work Table The average work duration of performing FGI procedures was years and the average frequency of interventions was every days per month The specialistsworked with fluoroscopy for longer periods than the residents however within the same period ie workloadper week or month the residents group performedfluoroscopy procedures more frequently than the specialists The proportion of subjects who always woreprotective gearsleadglasses and gloves ranged from to and the residents wore the protective gears less frequently than theother specialiststhyroid shieldslead apronsAmong the orthopedic surgeons who respondedto the survey only were linked with theTLD data Table The odds ratios OR of not beinglinked with the dosimetry data was significantly increased among the surgeons working at small hospitalsOR confidence interval [CI] andresidents group OR CI afteradjusting for potential confounding factors Among theorthopedic doctors who were linked with the dosimetrydata the annual effective dose was higher in the ST mSv than in Others mSv or residents mSv Supplementary Table DiscussionThe orthopedic surgeons in this study often performedfluoroscopy during the course of their work howeverthe rate of wearing the dosimetry badges and protectivedevices has been shown to be low In addition most participants in this study were not linked to the nationaldosimetry data The residents group experienced moreTable Characteristics of the orthopedic surgeons according to the job specialty in South KoreaCharacteristicsTotaln Na SpecialistsSTn N Age group year ¥ Type of the medical facilityGeneral hospitalSmall hospitalLocation of the medical facilityMetropolitanProvince Level of concern for occupational radiation exposureLowMediumHigh ST spine or trauma specialists Others other orthopedic specialistsaNumbers may not reflect the total owing to missing valuesbpvalue for the chisquare test or Fishers exact test Othersn N Residentsn N pvalueb 0cKang Journal of Occupational Medicine and Toxicology Page of Table Occupational characteristics of the orthopedic surgeons according to the job specialty in South KoreaResidentsOccupationaln characteristicsN Totaln Na SpecialistsSTn N Others n N Fluoroscopy workCalendar year began working with fluoroscopy ¥ Years working with fluoroscopy ¥ Proportion time spent working with fluoroscopy Working days per month with fluoroscopy ¥ Number of fluoroscopic procedures per week ¥ Working hours per week with fluoroscopy ¥ Badge wearingProvided with the badges regularlyNoYes Proportion of personnel wearing the badges Personal protective equipment useProportion of personnel wearing the lead aprons pvalueb 0cKang Journal of Occupational Medicine and Toxicology Page of Table Occupational characteristics of the orthopedic surgeons according to the job specialty in South Korea ContinuedOccupationalcharacteristicsTotaln Na Proportion of personnel wearing the thyroid shields Proportion of personnel wearing the lead glasses Proportion of personnel wearing the lead gloves SpecialistsSTn N Others n N Residentsn N pvalueb ST spine or trauma specialists Others other orthopedic specialistsaNumbers may not reflect the total owing to missing valuesbpvalue for the chisquare test or Fishers exact testunfavorable working conditions in terms of radiation exposure than the other specialists Among the orthopedicsurgeons who were linked with the dosimetry data theST had higher radiation doses than the other orthopedicsurgeons To the best of our knowledge this study is thefirst attempt to investigate the status of occupational radiation exposure among the orthopedic surgeons inSouth Korea Our findings may contribute to the increasing awareness of the radiation protection and itspotential risks among hospital workersThe rates of use of fluoroscopy and protective devicesin our study were comparable with those in a worldwidestudy on orthopedic surgeons that reported that morethan half of the procedures performed by of thesurgeons involved radiation exposure whereas the ratesof using lead aprons thyroid shields and lead glasseswere and respectively [] According to aUS survey of the subjects reported that lead apronswere not available and the remaining half reported thatthey were not appropriately sized [] The other studyreported that one out of three orthopedic surgery residents were not provided protective gowns in the US[] These rates of personal protective equipment usewere lower among the orthopedic surgeons than amongthe interventional cardiologists particularly the rates ofwearing the lead aprons thyroid protectors and leadglasses were and respectively [] Thepossible reasons for low rates of apron use was not beingproperly provided with aprons and inconvenience whilewearing aprons among the Korean Intern and ResidentsAssociation []The rate of always wearing a badge was generally comparable with those in a worldwide survey that reportedthat about onefifth of the orthopedic surgeons wear adosimeter [] The Irish orthopedic surgeons also reported that the regular use of dosimeters among theorthopedic trainees was [] and only of theorthopedic surgeons were reported to wear the TLD 0cKang Journal of Occupational Medicine and Toxicology Page of Table Odds ratios of not being linked with dosimetry data among the orthopedic surgeons in South KoreaCharacteristics CIORNot linked with the dosimetry n Na Linked with the dosimetry n N Age group year ¥ Type of medical facilityGeneral hospital Small hospital Location of medical facilityMetropolitanProvince Level of concernLowMediumHighJob specialtySTOthersResidents Calendar year began working with fluoroscopy ¥ Years working with fluoroscopy ¥ Proportion time spent working with fluoroscopy Proportion of personnel wearing the badges referencereferencereferencereferencereferencereferencereferencereferencereferenceOR odds ratio AOR adjusted odds ratio ST spine or trauma specialists Others other orthopedic specialists CI confidence intervalsaNumbers may not reflect the total owing to missing valuesbAdjusted for age location of medical facility and job specialtyORb CIreferencereferencereferencereferencereferencereferencereferencereferencereference 0cKang Journal of Occupational Medicine and Toxicology Page of during surgery in Turkey [] A possible reason for theorthopedic surgeons not preferring to wear the dosimeters is that they may believe that it will affect their performance and make them uncomfortable [ ]Another reason may be that if their radiation exposurewas greater than the specified limit they would be prohibited from operating with fluoroscopy for a specifiedperiod of time therefore they do not routinely wear thepersonal dosimetry badges []Approximately threefourth of our participants werenot linked with the national dosimetry data and the riskof not being linked was increased among the residentsgroup and those who worked at smaller medical facilities A possible reason may be owing to the incompleteness by which the radiation safety managers at eachmedical facility select the radiation exposed orthopedicsurgeons In members of the Korean Internand Resident Association responded that of themhad been exposed to fluoroscopy however only ofthem wore the TLD when exposed to radiation duringfluoroscopy [] These results suggest that the monitoring system for the orthopedic surgeons with radiationexposure appears to be unsuccessfulin particular forresidents and for small hospital workersand additionalefforts to improve dosimetry monitoring system are required in South KoreaThe doses monitored in the dosimetry data of theorthopedic surgeons in this study were lower than thosereported in Italy [] The Philippines [] India []and South Korea [ ] This finding supports the notion that the radiation exposure among the orthopedicsurgeons may be widely ranged depending on the workprocedures and experience [] The higher radiationdose among ST in this study may be attributed to longerexposure time and more fluoroscopic shots in ST []than those of other surgeons However the actual exposuremight be underestimated because of the orthopedic surgeons low rate of wearing the badges and protective devices Owing to the irregular and inconsistent use ofdosimeters estimating the radiation dose with personal dosimeters needs to be improved among orthopedic surgeonsMore than onethird of the orthopedic surgeons weregreatly concerned about health problems caused by occupational radiation exposure which is similar to thatreported in a previous worldwide study on orthopedicsurgeons [] Among all the participants the ST surgeons showed a higher rate of concern than the othergroups and this may be related to them performing ahigher proportion of the FGI procedures than other doctors Previously a lack of radiation knowledge andawareness about fluoroscopy were also associated with ahigh level of concern and low rate of wearing the protective devices [ ] therefore education about radiation exposure is warranted for orthopedic surgeonsThis study is the first attempt to investigate the statusof occupational radiation exposure in a relatively largenumber of orthopedic surgeons in South Korea However the study did not represent all members of the Korean Orthopedic Association although the residentsgroup may represent the total population of the orthopedic residents The high proportion of residents in thissurvey may be owing to their high participation in theconference and having greater interests in radiation exposure because of their harsh working conditions Ourfindings represent mainly male orthopedic surgeonsowing to a very small number of female participants Inaddition this study recruited few orthopedic surgeonswho worked at clinics which limited our findings mainlyto orthopedic surgeons at large hosptials Recall biasmay be present because of using a selfadministeredquestionnaire However the participants were a relatively young and highly educated group and the questionnaire items were related to their daily worktherefore the bias should be minimal The informationon selfreported working practices regarding radiationexposure has been reported as reliable among South Korean radiologic technologists []ConclusionsWe reported the occupational characteristics and radiation exposure among orthopedic surgeons in SouthKorea Although many orthopedic surgeons performinterventional FGI procedures unfavorable work characteristicssuch as the low rate of wearing protective devices and dosimetersmay increase the radiation riskAdditionally badge monitoring was not noted for therelatively large proportion of the orthopedic surgeonswho performed FGI procedures The orthopedic surgeons are at a risk of occupational radiation exposureand more intensive approaches are needed to reduce radiation exposure and protect possible workrelatedhealth effectsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1299502000276xAdditional file Supplementary Table Questionnaire used fororthopedic surgeons at the conference of the Korean OrthopedicAssociation in Additional file Supplementary Table Comparison of selectedcharacteristics between the target population and the study participantsAdditional file Supplementary Table Annual effective doses byoccupational characteristics and specialties among orthopedic surgeonsin South KoreaAbbreviationsNDR National Dosimetry Registry TLD Thermoluminescent dosimeterKCDC Korea Centers for Disease Control and Prevention ST Spine or trauma 0cKang Journal of Occupational Medicine and Toxicology Page of specialists Others Other orthopedic specialists OR Odds ratioCI Confidence intervalsAcknowledgmentsWe would like to thank all study participants for their voluntary participationthe Korea Centers for Disease Control and Prevention for providingdosimetry data and the Korean Orthopedic Association for providingdemographic data of their membersAuthors contributionsSK and WJL conceptualized the research SK performed the data analysesand wrote the first draft of the manuscript ESC YJB TWN DL and SYSinterpreted the findings and provided advice on the data analyses WJL andSK revised the final manuscript All authors contributed to the draft revisionand approved the final manuscriptFundingThis work was supported by the National Research Foundation of KoreaNRF grant funded by the Korea government MSIT No2020R1A2C1008891Availability of data and materialsThe datasets generated during the current study are not publicly availablebecause the detailed individual data is restricted for both legal and ethicalconcerns but are available from the corresponding author on reasonablerequestEthics approval and consent to participateThe protocols of the study were reviewed and approved by the InstitutionalReview Board of Korea University KUIRB1736A2 Written informedconsent was voluntarily obtained from all study participants prior toenrollmentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Occupational and Environmental Medicine COMWEL AnsanHospital Korea Workers Compensation Welfare Service GyeonggidoSouth Korea 2Department of Epidemiology and Health Informatics GraduateSchool of Public Health Korea University Seoul South Korea 3Department ofPreventive Medicine Korea University College of Medicine GoryeodaeroSeongbukgu Seoul South Korea 4Department of Policy andAdministration National Radiation Emergency Medical Center Korea Instituteof Radiological and Medical Sciences Seoul South Korea 5Department ofRadiation Effects Research National Radiation Emergency Medical CenterKorea Institute of Radiological and Medical Sciences Seoul South Korea6Department of Orthopedic Surgery Gyeongsang National UniversityHospital Jinju South KoreaReceived February Accepted August Gausden EB Christ AB Zeldin R Lane JM McCarthy MM Trackingcumulative radiation exposure in orthopaedic surgeons and residents whatdose are we getting J Bone Joint Surg Am Jung GH Jang JH Kim JD Kim CK Radiation exposure over the course ofa year from an image intensifier in the orthopaedic operating room JKorean Fract Soc Oddy M Aldam C Ionising radiation exposure to orthopaedic trainees theeffect of subspecialty training Ann R Coll Surg Engl Rampersaud YR Foley KT Shen AC Williams S Solomito M Radiationexposure to the spine surgeon during fluoroscopically assisted pediclescrew insertion Spine Tunçer N Kuyucu E Sayar Å Polat G Erdil Ä° Tuncay Ä° Orthopedic surgeonsknowledge regarding risk of radiation exposition a survey analysis SICOTJ Chou LB Chandran S Harris AH Tung J Butler LM Increased breast cancerprevalence among female orthopedic surgeons J Women's Health Chou LB Lerner LB Harris AH Brandon AJ Girod S Butler LM Cancerprevalence among a crosssectional survey of female orthopedic urologyand plastic surgeons in the United States Womens Health Issues Mastrangelo G Fedeli U Fadda E Giovanazzi A Scoizzato L Saia BIncreased cancer risk among surgeons in an orthopaedic hospital OccupMed Ko S Kang S Ha M Kim J Jun JK Kong KA Health effects fromoccupational radiation exposure among fluoroscopyguided interventionalmedical workers a systematic review J Vasc Interv Radiol Fan G Wang Y Guo C Lei X He S Knowledge deficiency of workrelatedradiation hazards associated with psychological distress among orthopedicsurgeons a crosssectional study Medicine 20179621e6682Joeris A Goldhahn S Kalampoki V Gebhard F Intraoperative radiationexposure of orthopaedic surgeonsmismatch between concerns andprotection Occup Med Health Aff Ko S Chung HH Cho SB Jin YW Kim KP Ha M Occupational radiationexposure and its health effects on interventional medical workers studyprotocol for a prospective cohort study BMJ 2017712e018333Korea Centers for Disease Control Prevention KCDC ReportOccupational Radiation Exposure in Diagnostic Radiology in Korea p httpwwwcdcgokr Accessed Feb Hornung RW Reed LD Estimation of average concentration in the presenceof nondetectable values Appl Occup Environ Hyg Hassan M Patil A Channel J Khan F Knight J Loos M Do we glowEvaluation of trauma team work habits and radiation exposure J TraumaAcute Care Surg Bowman JR Razi A Watson SL Pearson JM Hudson PW Patt JC Whatleads to lead results of a nationwide survey exploring attitudes andpractices of orthopaedic surgery residents regarding radiation safety J BoneJoint Surg Am 20181003e16Jacob S Boveda S Bar O Brézin A Maccia C Laurier D Interventionalcardiologists and risk of radiationinduced cataract results of a Frenchmulticenter observational study Int J Cardiol The Korean Intern and Resident Association Summary of ResidentsRadiation Exposure Experience Survey httpyoungmd Accessed Feb ReferencesRehani M CirajBjelac O Vañó E Miller D Walsh S Giordano B et alRadiological protection in fluoroscopically guided procedures performedoutside the imaging department Ann ICRP Singer G Occupational radiation exposure to the surgeon J Am AcadOrthop Surg Matityahu A Duffy RK Goldhahn S Joeris A Richter PH Gebhard F Thegreat unknowna systematic literature review about risk associated withintraoperative imaging during orthopaedic surgeries Injury Miller DL Overview of contemporary interventional fluoroscopy proceduresHealth Phys Richter P Dehner C Scheiderer B Gebhard F Kraus M Emission of radiationin the orthopaedic operation room a comprehensive review OAMusculoskeletal Med Nugent M Carmody O Dudeney S Radiation safety knowledge andpractices among Irish orthopaedic trainees Ir J Med Sci Kesavachandran CN Haamann F Nienhaus A Radiation exposure of eyesthyroid gland and hands in orthopaedic staff a systematic review Eur JMed Res Klein LW Tra Y Garratt KN Powell W LopezCruz G Chambers C et alOccupational health hazards of interventional cardiologists in the currentdecade results of the SCAI membership survey Catheter CardiovascInterv Rosario M Garduce V Geronilla M Radiation exposure of Filipino orthopedicresidents in tertiary level hospitals Bone Rep Recommendations Mahajan A Samuel S Saran AK Mahajan MK Mam MK Occupationalradiation exposure from C arm fluoroscopy during common orthopaedicsurgical procedures and its prevention J Clin Diagn Res 201593RC01 Bae SY Kim JO Yoo JD Yoon SY Jang JW Radiation exposure fromfluoroscopy during orthopaedic surgical procedures J Korean Frac Soc 0cKang Journal of Occupational Medicine and Toxicology Page of Kim JW Kim JJ Radiation exposure to the orthopaedic surgeon duringfracture surgery J Korean Fract Soc Kim MJ Cha ES Ko Y Chun BC Lee WJ Reliability of selfreportedquestionnaire on occupational radiation practices among diagnosticradiologic technologists Am J Ind Med Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
"Despite recent interest in the use of ketogenic diets KDs for cancer evidence of beneficial effects islacking This study examined the impact of a randomly assigned KD on quality of life physical activity andbiomarkers in patients with breast cancerMethod A total of patients with locally advanced or metastatic breast cancer and without a history of renaldisease or diabetes were randomly assigned to either a KD or a control group for this 12week trial Concurrentwith the first third and fifth chemotherapy sessions quality of life physical activity and biomarkers thyroidfunction tests electrolytes albumin ammonia ALP lactate and serum ketones were assessed Dietary intake wasalso recorded on admission and the end of the treatmentResults No significant differences were seen in quality of life or physical activity scores between the twogroups after weeks however the KD group showed higher global quality of life and physical activityscores compared to the control group at weeks P P Also serum lactate and ALP levelsdecreased significantly in the KD group compared to the control group at the end of the intervention ± vs ± ± vs ± P and P respectively A significant inverse associationwas observed between total carbohydrate intake and serum betahydroxybutyrate at weeks r P No significant differences between groups were observed in thyroid hormones electrolytes albuminLDH or ammonia Compliance among KD subjects ranged from to as assessed by dietary intakeand serum ketones levels of Continued on next page Correspondence khodabakhshiadelehyahoocom6Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranFull 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 0cKhodabakhshi Nutrition Journal Page of Continued from previous pageConclusion According to our results besides a higher global quality of life and physical activity scores compared tothe control group at weeks KD diet combined to chemotherapy in patients with breast cancer does not bringadditional benefit about quality of life and physical activity at weeks However decreases seen in levels of lactateand ALP in the KD group suggest that a KD may benefit patients with breast cancerTrial registration This trial has been registered on Iranian Registry of Clinical Trials IRCT under the identification codeIRCT20171105037259N2 wwwirctirtrial30755Keywords Ketogenic diet Breast cancer quality of life Physical activity Lactate Alkaline phosphatase chemotherapythere are stillIntroductionKetogenic diets KDs are high in fat and very low incarbohydrate They have been used as a dietary treatment in epilepsy for nearly a century [] RecentlyKDs have gained the attention of cancer researchersdue to their potential impact on cancer cell metabolism [] Despite the growing evidence of possibleantitumor benefitssome concernsabout potential adverse effects of KDs in cancer patientsincluding micronutrient deficiencies appetitereduction nausea constipation [] fatigue [] hyperlipidemia and especially unintended weight loss [ ]KDs are perceived as restrictive in nature which mayadd to the burden of cancer patients who already suffer from considerable physical emotional and financialstress all of which are known to negativelyimpact quality of life QoL In addition alterations inphysical and cognitive function during cancer treatment are pervasive It is estimated that of patients undergoingfromcancerrelated fatigue [] Prior studies have foundthat KD may improve physical and mental wellbeing[] Less fatigue has been reported in healthy overweight and obese adults following lowglycemic compared to highglycemic diets [] Results ofthreestudies using the validated European anization forResearch and Treatment core QoL questionnaire tofindings [] Aassess fatigue lacked consistentin advanced cancer patients showed imsmall trialprovementin sleep and emotionalfunction after athreemonth KD intervention [] Other studies havesuggested enhanced cognitive function [ ]cancertreatmentsufferTo date only four studies have assessed QoL in adultpatients with cancer [ ] Hunger is a reported sideeffect of restricted KDs however previous research hasfound that perceived hunger is reduced in low carbohydrate diets compared to low fat diets [] A recent systematic review has highlighted the need for additionallarger investigations on the impact of ketogenic diets onQoL [] The goal of this present trial was to assesswhether a KD had beneficial effects on QoL dietary intake physical activity and specific biomarkersinindividuals with breast cancer while also evaluating compliance to KD guidelines in these patientsThe protocol used in this trial [] and part of the resultsfrom this trial have been previously published [ ]MethodsThe study protocol was approved by the National Nutrition and Food Technology Research InstituteNNFTRI Shahid Beheshti University of MedicalSciencesIRSBMUNNFTRIREC1396187 All participants provided writteninformed consent prior to participating in the studySBMU TehranIranThis trial was a randomized controlled labelclinical trial to breast cancer patients with locally advanced or metastatic disease who were receiving chemotherapy for atleast weeks The studythe medical oncology clinic atwas conducted atShohadaeTajrish hospital Cancer Research CenterTehran Iran from July to October of Participation was to patients to years of ageExclusion criteria screened forsignificant cardiacrenal or neurologic comorbidities symptoms of malnutrition diabetes pregnancy and Karnofsky indexless than Using a block balanced randomizationmethod patients were assigned to the interventionn or controln groups Randomizationwas computergenerated by a statistician who was nota member of the medical team Blinding the participants or study personnel was not deemed feasible inthis dietintervention The project coordinator enrolled the participants and assigned them to their interventions Both the KD and the control diet werecalculated to be eucaloric using the MifflinSt Jeorformula The KD consisted of of calories fromCHO from protein from mediumchain triglyceride MCT oil and from fat A dietitianprovided specific nutritional counseling to each participantfacetoface meetings Patientsengaged in ongoing weekly counseling sessions viaphone WhatsApp or Telegram and were assessed forcompliance and possible adverse effects To furtherenhance compliance dietary recommendations werein individual 0cKhodabakhshi Nutrition Journal Page of individualized and appropriate recipes were providedto patients in the KD group were asked to refrainfrom eating any grains grain products starchy vegetables fruit or sugar Dietary carbohydrates were limited to nonstarchy vegetables and dietary proteinswere obtained primarily from egg meat poultry andfish Small amounts of lower carbohydrate berries andnuts were allowed as long as they did not exceed thecarbohydrate limit in the diet prescription Subjectswere encouraged to increase their fat intake and toselect from a variety of sourcesincluding olive oilbutter and cream cheese Patients were asked tochoose only the foods specified in the diet plan provided to them Patients were also encouraged to usemediumchain triglyceride MCT oil MCT oil anodorless and tasteless saturated fat does not requirebile or pancreatic enzymes for digestion It is easilyconverted to ketones in the liver thereby enhancingketosis Every weeks ml of MCT oilfromNutricia Erlangen Germany was provided to eachsubject in the KD group For better tolerance initialdosage of MCT was kept low and increased daily overa 6day period until maximum tolerable dosage wasachieved Dosage was reduced in a similar steppedprocessThe patients in the control group were instructed tofollow a standard diet consisting of CHO protein and fat Dietary compliance was checked byassessing blood betahydroxybutyrate levels every weeks and dietary intake at baseline and end of thestudyQoL assessmentQoL was assessed using the EORTC QLQC30 version and IORTC QLQBR23 questionnaires developed bythe European anization for Research and Treatmentof Cancer The validity and reliability of the questionnaires has previously been evaluated in Iran [ ]The questionnaires were completed at enrollment at weeks and at the end of the interventionDietary intake assessmentHospital dietitians used a 24h dietary recall 24HR toobtain a total of days intake one weekend day andtwo workdays through telephone and facetoface interviews both at the beginning and end of the study Theamount of each food consumed was estimated usingcommon household containers bowls cups and glassesand standard measuring cups and spoons as referencesThe mean quantity of total energy carbohydrate proteinand fat were estimated from the 24HRDietary intake wasanalyzed by Nutritionist IV software Version USPhysical activity assessmentPhysical activity was measured using the IPAC International Physical Activity questionnaire at baseline at weeks and at the end of the studyBiomarker assessmentFasting blood sampling for serum Na K Ca P lactate Mg LDH albumin ammonia and ALP were performed at baseline midway through the intervention weeks and at weeks T3 T4 and TSH were measuredat baseline and the end of the interventionStatistical analysisConsidering the power and α the sample sizewas calculated as individuals per group Assuming a dropout during the weeks of the study the finalnumber of participants was calculated as patients ineach groupStatistical analysis was carried out according to theintentiontotreat protocol Continuous variables weretested for normal distribution by the KolmogorovSmirnov test and then reported as mean ± standard deviation or median as appropriate Student ttest or MannWhitney U test was used to compare the continuousvariables between the two groups Paired sample ttestor Wilcoxon was used to compare the continuous variables within the two groups The ANCOVA test wasused to eliminate the effect of confounding factorsPearson correlation analyses were used to estimate associations between total carbohydrate intake and serumbetahydroxybutyrateData were analyzed using the SPSS version software Chicago IL USA and Stata version P was considered as statistically significantResultsDetailed patient demographics and a flow diagram werereported previously [] A total of women withbreast cancer were enrolled and randomly assigned to either the intervention n or control n groupsThree patients in the control group withdrew before beginning their assigned diet while10 patients in the KDgroup and patients in the control group withdrewfrom the study after beginning their assigned diet Ultimately patients in each group completed the studyand were included in the analysis No significant differences were seen between the two groups with regard toage cancer type metastasis and marriage or educationstatus P The intervention group included patients with locally advanced disease and patients withmetastatic disease liver bone lung liver andbone while the control group consisted of patientswith locally advanced disease and patients with 0cKhodabakhshi Nutrition Journal Page of metastatic disease bone liver lung liver andbone at other sites P Table Data related to quality of life are shown in Tables and No significant differences were seen in QoL betweenthe two groups after weeks however the KD groupshowed better global QoL compared to the controlgroup at week P Also at week diarrhea increased in the control groupcompared to the intervention P Data on week not shown Using the QoL questionnaire there was awithingroup decrease in reported hunger from baselineto weeks in the KD group P A withingroupdecrease was seen in physical performance measuresfrom baseline to weeks in both groups which was significant only in the KD group P In addition rolefunctioning and socialfunctioning scores significantlydecreased in the control group compared to the baselinebut not in the KD group P P Table Mean dietary intake is shown in Table and Fig The mean caloric and carbohydrate intake decreasedsignificantly at the end of the study compared to control P and P respectively while fatintake increased significantly in the KD group compared to the control group P After adjustingfor total energy intake this difference remained significant When data from both groups was combineda significant inverse association was observed betweentotalserum betaandr P hydroxybutyratealthough this effect was not seen when the KD groupwas analyzed separatelyintakeat weekscarbohydrateWithingroup analysis showed significant decreasesin energy carbohydrate and protein intake in bothgroups compared to the baseline Fat intake increasedsignificantly compared to the baseline in the KDgroup P and decreased significantly in thecontrol group P During the intervention of the subjects in the KDarm limited carbohydrates to g and of subjects consumed of calories from carbohydratesAt weeks of patients in the KD group hadserum ketones mmolL at 6weeks had ketone levels of mmolL As previously reportedserum ketone concentrations increased significantly inthe KD group ± to ± mmollP []At weeks physical activity improved in the KD groupcompared to the control group adjusted for cancer typeand baseline value P but after weeks physicalactivity did not show any significant differences in a between or withingroup analysis Fig No significant difference was observed in a betweenor withingroup analysis of thyroid hormones electrolytes albumin Ammonia and LDH Howeverlactateand ALP decreased significantly after intervention in theKD group compared to the control group P andP respectively ALP is adjusted for baseline valueand cancer type Table Data on thyroid hormones notshownDiscussionThe effect of KD on QoL physical activity dietary intake and biomarkers in patients with locally advancedand metastatic breast cancers was evaluated in thisstudy Based on our findings in the KD group globalQoL was higher at weeks perhaps in part because diarrhea was more frequent in the control group than theKD group No significant differences were seen in theQoL physical activity and biomarkers between the twogroups after the week intervention Lactate and ALPwere lower in the KD group compared to the controlEffect of diet on QoLIn our study in the KD group global QoL was higher at weeks No adverse effects were observed in thoseTable Baseline characteristics in breast cancer patients before interventionScale categoriesCancer TypeLocally AdvancedIntervention Ketogenic dietn Control Ordinaryn ERPRHER2Metastaticpositivenegativepositivenegativepositivenegative ER Estrogen receptor PR Progesterone receptorHER2 Human epidermal growth factor receptor aCalculated by chi square testbCategorical data shown as No p value008a057a043a079a 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before andafter intervention in KD group and control group as measuredby the EORTC QLQC30aFunctioningaPhysical functioningMD CIControlKDpvalueTable Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQC30SymptomsaFatiguepvalueControlKD Week Week pvalue33aNausea and vomitingWeek Week pvalueRole functioningWeek Week pvalue ± 11a ± ± ± Cognitive functioningWeek Week pvalue ± ± Emotional functioningWeek Week pvalueSocial functioningWeek Week pvalue ± ± ± ± Global quality of lifeWeek Week pvalue ± ± ± ± ± ± ± ± ± ± ± ± ± ± After adjusting for baseline value and chemotherapy status no significantdifferences were observedStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the two groupsData shown as mean and SDaThe higher values indicate higher level of functioning and quality of lifeparticipants assigned to the KD compared to the controlgroup after weeks Withingroup analysis showed decreased hunger and physical function in the KD groupcompared to the baseline In the control group role andsocial functioning decreased significantly compared tobaselineResults of a systematic review and metaanalysis haveshown that KDs suppress appetite [] Decrease in hunger or appetite in our study may be due to the high fatcontent of the KD as it decreases the ghrelin releasewhich in turn may reduce appetite High fat intake alsoslows digestion which could also impact the perceptionof hunger Previously we have shown that the KD resultsin weight loss [] As a clinical benefit KDinduced Week Week pvaluePainWeek Week pvalueReduction in appetiteWeek Week pvalueSleep difficultiesWeek Week pvalueDyspneaWeek Week pvalueConstipationWeek Week pvalueDiarrheaWeek Week pvalueFinancial concernsWeek Week pvalue MannWhitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate a higher grade of symptoms Data shown asmedian and quartile 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQBR23aKDControlpvalue aFunctioningFuture perspectiveWeek Week pvaluebSymptomsArmWeek Week pvalueBreastWeek Week pvalue Week Systemic therapy side effects Week pvalueConcerns over hair lossWeek Week pvalue Table Comparison of mean ± SD macronutrient intake atbaseline and 12weeksVariableMD CIpvalueKDMean ± SDControlMean ± SDEnergy KcaldayBefore ± ± ± AfterpvalueCarbohydrate grBefore ± ± ± ± AfterpvalueProtein grBeforeAfterpvalueFat gr ± ± ± ± ± 0001a 041aBefore ± ± ± ± AfterpvalueStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the groupsMD Mean differenceCI Confidence intervalaAncova Adjusted for baseline value and energy0001aMannWhitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate higher level of functioning and quality of lifebThe higher values indicate a higher grade of symptomsData shown as median and quartile decreases in appetite weight and body fat may result infavorable changes in breast cancer patients notably inoverweight or obese women [ ]In contrast with our findings Cohen found that aKD significantly enhanced physical function scores inwomen with ovarian or endometrial cancer comparedto the control group but appetite did not change atthe end of the study compared to the baseline []Part ofstudy andCohens trial may be explained by the design of thestudy While only of the participants in the Cohen study were undergoing chemotherapy all of ourpatients were receiving treatmentthe inconsistency between ourAlso timing of the administration of the questionnaires and whether the participants were in positive ornegative energy balance may have influenced ourfindingsNo significant difference was reported in QoL at theend of study compared to the baseline by TanShalaby [] However a slight decrease in physical androle functioning as well as temporary constipation andfatigue were reported in the KD group in one study []In our study constipation was noted by participants inthe KD arm during the early days which was managedby dietary changesAlso after weeks in the KD group physical activityscores was higher compared to the control group but at weeks differencessignificantbetween the two groupsin scores were notDietary intake and adherenceOur study data showed a significant decrease in carbohydrate intake and a significant increase in fat intake inthe KD group compared to the control Protein intakewas not significantly different between the two groupsbut decreased overall in both groups when compared tobaseline Total daily carbohydrate intake was similar toresults in the Cohen study [] We also assessed serumbetahydroxybutyrateIn the KD group ofpatients at weeks and at 6weekshad serum ketones and patients at weeks and weeks 0cKhodabakhshi Nutrition Journal Page of Fig Mean caloric intake and distribution of macronutrients as percentage of total kilocalories before and after week intervention in breastcancer patients in two groupsFig Comparison of trend changes in physical activity in breast cancer patients in two groups 0cKhodabakhshi Nutrition Journal Page of LBsvskwleuavpitnopdmisvskwleuavpLBsviitnopdmeuavlpinorrefnoBnodesabldetauclacerewseuavlpllaerusaemdetaepeRepytlsisyanAlavretnIecnedifnoCICecnereffiDnaeMDMpuwolloftsalroskeewskeewkeewropuwolloftsitnopdMiepytrecnacdnaeuavlenilesabrofdetsudAjavocnAsnosirapmoclepitlumrofnoitcerroc±±±±skeew±±itnopdMi±±enilesaB±±±±±±smArlairTDKICDMlortnoClortnoCDKlebairaVetatcaLluHDLstneitaprecnactsaerbdetaertDKdnalortnocnislevelrekramoBielbaTICDM±±±±lortnoCDKldgcmianommAICDMICDM±±±±±±lortnoCDK±±±±±±lortnoCDKICDMICDM±±±±±±±±±±±±ICDMlortnoCDKlortnoCDK±±±±±±±±±±lortnoCDKICDM±±ICDMlortnoCDKICDM±±±±±±lortnoCDKLdgmgMldginmubAllqemKlDgmPPLAldgmaClqemaNenilesaBLB 0cKhodabakhshi Nutrition Journal Page of had serum ketones mmoll Cohen reported that of patients had betahydroxybutyrate concentrations mmollA recent systematic study of KDs in adult cancerpatients reported a range of to with a adherence rate overall reported by [] According toour data the level of adherence to the KD interventionsuggests that the diet is a feasible option for women withbreast cancer who are receiving chemotherapyDespite the lack of any restriction in calorie intakein the study design and consistent with findings ofCohen [] the KD group showed a significant reduction in calorie intake compared to the control groupThe decrease in calorie intake may be due to reductions in appetite associated with ketosis as the subjects in the KD arm did not consume all of the fatcalculated for their diet This may also be due in partto customary practices surrounding meal preparationA decrease in appetite and subsequentinadvertentcalorie restriction most often results in weight loss inthe absence of malnutrition or cachexiathis mayhave antiinflammatory and proapoptotic propertieswhich in turn may exert a positive effect on thesehallmarks of cancer Ketosis may also enhance theeffectiveness of chemotherapy while reducing the sideeffects of treatment [ ]Effect of diet on biomarkersConsistent with the outcomes of the previous studiesour results revealed that the KD had no adverse effecton thyroid hormones electrolytes LDH urea and albumin Significant decreases were seen in serum levels oflactate KDs reduce glycolytic activity which in turn mayslow metastases by reducing the acidity of the tumormicroenvironment and lowering the availability of lactate as a substrate for biomass synthesis [] Decreaseswere also seen in ALP High levels of ALP in breast cancer patients is a negative prognostic marker often indicating progression of metastatic disease [] Moreresearch is needed to assess whether lower ALP and lactate as seen in this study contributes to slower rates ofdisease progressionTo our knowledge this is the first randomized controlled trial examining the effects of a KD on QoL inbreast cancer patientsThe primary limitation of this study was the heterogeneous nature of the sample in regards to cancer stageA secondary limitation was the small sample sizeConclusionAccording to our results besides a higher global QoLand physical activity scores compared to the controlgroup at weeks KD diet combined to chemotherapy inpatients with breast cancer does not bring additionalbenefit about QoL and physical activity at weeksWhile many blood biomarkers did not differ significantlybetween the two groups ketosis may still offer benefit tosome patients with breast cancer in part by decreasinglactate and ALPSupplementary informationSupplementary information accompanies this paper at doi101186s1293702000596yAdditional file figure Flow diagram of the patient treatmentprocessAdditional file figure Median confidence interval tyroidhormones in baseline and 12week by two trial arms in breast cancerpatientsAcknowledgmentsWe would like to thank all the patients at our clinic who took part in thisstudyAuthors contributionsKhodabakhshi carried out the conception Methodology performed theexperiments design of the diet and wrote the Davoodi and Seyfriedcollaborated in the design of the study Davoodi supervise on the thesisKalamian and Beheshti collaborated in the design of the diet Kalamian gavecritical review of the manuscript All authors have read and approved thefinal manuscriptFundingNot applicableAvailability of data and materialsData described in the manuscript code book and analytic code will bemade available upon request pendingEthics approval and consent to participateAll participants provided written informed consent prior to participating inthe studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nutrition School of Public Health Kerman University ofMedical Sciences Kerman Iran 2Physiology Research Center KermanUniversity of Medical Sciences Kerman Iran 3Biology Department BostonCollege Chestnut Hill MA USA 4Dietary Therapies LLC Hamilton MT USA5Department of Nutrition and Dietetics Mofid childrens hospital ShahidBeheshti University of Medical Sciences Tehran Iran 6Cancer ResearchCenter Shahid Beheshti University of Medical Sciences Tehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranReceived March Accepted July ReferencesNeal EG Chaffe H Schwartz RH Lawson MS Edwards N Fitzsimmons G The ketogenic diet for the treatment of childhood epilepsy arandomised controlled trial Lancet Neurol Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab 0cKhodabakhshi Nutrition Journal Page of adverse effects on blood lipids a randomized controlled trial Nutr CancerFine EJ SegalIsaacson CJ Feinman RD Herszkopf S Romano MC TomutaN Targeting insulin inhibition as a metabolic therapy in advancedcancer a pilot safety and feasibility dietary trial in patients Nutrition Epub Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic 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