Jul 16, 2014 - ... 38 KDa adaptor protein XRCC4 that is activated by XLF/Cernunnos ..... tumours with MMR deficiency have genomic instability and MSI, ...
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Predictive Biomarkers of Radiation Sensitivity in Rectal Cancer
by Thein Ga TUT MBBS, M.Sc
Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy
Discipline of Pathology Western Sydney University
2016
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TABLE&OF&CONTENTS& TABLE!OF!CONTENTS………………………………………………………………………………….......ii! LIST!OF!FIGURES………………………………………………………………………………………….....xii! LIST!OF!TABLES………………………………………………………………………………………….......xv! ABSTRACT………………………………………………………………………………………………….......xix! STATEMENT!OF!ORIGINALITY……………………………………………………………………......xxi! ACKNOWLEDGEMENTS………………………………………………………………………………....xxii! PUBLICATIONS!ARISING!FROM!THIS!RESEARCH…………………………………………...xxiii! ABBREVIATIONS…………………………………………………………………………………………..xxv! CHAPTER&1:&INTRODUCTION……………………………………………………………………........1& 1.1&Overview………………………………………………………………………………………………......2& 1.2&Anatomy&and&histology&of&the&colorectum………………………………………….........2& !!1.2.1!Distinctive!anatomy!and!histology!of!the!colorectum…………………………….....3! 1.3&General&aspects&of&rectal&cancer………………………………………………………….......5& !!1.3.1!Epidemiology…………………………………………………………………………………….......5! !!!!1.3.1.1!Incidence………………………………………………………………………………………......5! !!!!1.3.1.2!Mortality……………………………………………………………………………………….......6! !!!!1.3.1.3!Survival…………………………………………………………………………………………......7! !!!!1.3.1.4!Screening………………………………………………………………………………………......7! !!!!1.3.1.5!Risk!factors……………………………………………………………………………………......8! !!!!1.3.1.6!Statistics…………………………………………………………………………………………....9! !!1.3.2!Clinical!and!pathological!features………………………………………………………….10! !!!!1.3.2.1!Pathological!types……………………………………………………………………………10! !!!!1.3.2.2!Differentiation.………………………………………………………………………………...11! !!!!1.3.2.3!Prognostic!pointers………………………………………………………….......................13! !!!!!!1.3.2.3.1!Colorectal!cancer!staging…………………………………………………………......13! !!!!!!1.3.2.3.2!Management…………………………………………………………...............................15! !!!!!!1.3.2.3.3!Colorectal!cancer!adjuvant!radiotherapy.......................................................16! !!!!!!1.3.2.3.4!DXT!Regime!options…………………………………………………………................17!
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!!!!!!1.3.2.3.5!DXT!and!clinical!outcomes…………………………………………………………....18! !!!!!!1.3.2.3.6!Histological!assessment!of!DXT!response.......................................................18! 1.4&Literature&review.....................................................................................................................19& &&1.4.1&PoloMlike&kinase&1&(Plk&1)!…………………………………………………………............19& !!!!1.4.1.1!Different!types!of!Plks…………………………………………………………..................21! !!!!1.4.1.2!The!Structure!of!Plks…………………………………………………………....................26! !!!!!!1.4.1.2.1!Plk1!activation!and!polo!box!domain!mediated!targeting......................28! !!!!!!1.4.1.2.2!(A)!Plk!function!at!G2/M!transition..................................................................30! !!!!!!1.4.1.2.2!(B)!Plk!function!at!DNA!damage!checkpoint.................................................31! !!!!!!1.4.1.2.2!(C)!Plk1!and!chromosome!segregation...........................................................32! !!!!!!1.4.1.2.3!Plk1!degradation…………………………………………………………......................36! !!!!1.4.1.3!Roles!of!Plk1…………………………………………………………....................................36! !!!!!!!!1.4.1.3.1!Role!of!Plk1!in!mitosis!and!malignancy........................................................37! !!!!1.4.1.3.2!Role!of!FoxM1!and!Plk1!in!mitosis!and!malignancy....................................39! !!!!1.4.1.3.3!Role!of!Plk1!in!ATM,!ATR!pathway!and!malignancy...................................39! !!!!1.4.1.3.4!Role!of!p53!in!downregulation!of!Plk1.............................................................41! !!1.4.1.4!Possible!link!between!Plk1!and!sensitivity!to!DXT...........................................43! !!1.4.1.5!Evaluation!of!Poloalike!kinase!1!(Plk1)!as!biomarker!for!effectiveness!of!DXT! on!rectal!cancer………………………………………………………………………………………........44! 1.5&DXT,&ionising&radiation&(IR),&DNA&damage&response&(DDR)&and&γH2AX.48& !!1.5.1!Principles!of!DXT……………………………………………………….....................................48! !!1.5.2!Ionizing!radiation!(IR)!……………………………………………………….........................48! !!1.5.3!Different!forms!of!DNA!damage………………………………………………………........49! !!1.5.4!General!scheme!of!DNA!damage!response!(DDR)..................................................50! !!1.5.5!DDR!in!radiation!injury............………………………………………………………….........53! !!!!1.5.5.1!Cell!cycle!arrest!in!radiation!injury………………………………………………......53! !!!!1.5.5.2!Restorative!mechanisms!for!DNA!DSBs…………………………………………....54! !!!!!!1.5.5.2.1!Homologous!recombination............…………………………………………….....54! !!!!!!1.5.5.2.2!Nonahomologuous!endajoining!(NHEJ)…………………………………….......54! !!!!!!1.5.5.3!DSB!repair!.............................……………………………………………….......................55!
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!!!!!!!1.5.5.3.1!DSB!repair!through!Homologous!recombination....................................................55! !!!!!!!!!1.5.5.3.1.1!DSBR!pathway...................................................................................................................55! !!!!!!!!!1.5.5.3.1.2!SDSA!pathway...................................................................................................................57! !!!!!!!1.5.5.3.2!DSB!repair!through!NHEJ...................................................................................................57!
1.6&Histone&H2AX…………………………………………………………...........................................60& !!1.6.1!Histone!H2AX!and!its!structure………………………………………………………........60! !!1.6.2!Histone!H2AX!phosphorylation………………………………………………………........64! !!1.6.3!Possible!link!between!H2AX!and!sensitivity!to!DX................................................64! !!1.6.4! Evaluation! of! γH2AX! as! a! possible! biomarker! for! radiosensitivity! of! rectal! cancer……………………………………………………………………………………………………..........65! !!!!1.6.4.1!Detection!of!Phosphorylated!histone!H2AX.......................................................66! !!!!1.6.4.2!DDR!involvement!in!γH2AX!biomarker................................................................67! !!!!1.6.4.3!ATM!and!ATR!involvement!in!DDR…………………………………………………...69! !!!!1.6.4.4!Upstream!ATM!and!Chk2………………………………………………………...............71! !!!!1.6.4.5!Downstream!CDK2!and!FOXO1………………………………………………………...75! 1.7&Molecular&biology&of&Microsatellite&Instability&(MSI)........................................76! !!1.7.1!Model!of!colorectal!tumorigenesis………………………………………………………..77!! !!1.7.2!MSI!in!CRC…………………………………………………………………………………………..79! !!!!1.7.2.1!DNA!mismatch!repair!(MMR)!………………………………………………………....79! !!!!1.7.2.2!DNA!MMR!and!MSI………………………………………………………...........................80! !!1.7.3! Assessment! of! whether! rectal! cancers! with! MSI! show! increased! sensitivity! to! preoperative!deep!X!ray!therapy………………………………………………………..................81! !!1.7.4!Clinicopathological!features!of!MSI……………………………………………………....82! !!1.7.5!Assessment!of!MSI!status………………………………………………………....................83! !!1.7.6!Prognostic!and!predictive!implication!in!CRC.........................................................84! !!1.7.7!Possible!link!between!MSI!and!sensitivity!to!radiotherapy..............................85! !!1.7.8!Discovering!predictive!markers!of!radiation!response!to!DXT!in!CRC........87! 1.8&Summary&M&Outline&of&Thesis…………………………………………………………..........89! !!1.8.1!Aim…………………………………………………………………………………………………….89!
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!!1.8.2!Hypotheses………………………………………………………………………………………...90! 1.9&Overview&of&contents…………………………………………………………..........................91! ! CHAPTER&2:&MATERIALS&AND&METHODS…………………………………………………...93& 2.1&Overview…………………………………………………………....................................................94& 2.2&Ethics&statement…………………………………………………………....................................94& 2.3&In&vitro&(cell&line)&study………………………………………………………….....................95& !!2.3.1!Radiation!sensitivity…………………………………………………………..........................95! !!!!2.3.1.1!Selection!of!cell!lines…………………………………………………………....................95! !!!!2.3.1.2!Conditions!of!cell!culture!and!storage…………………………………………….....96! !!!!2.3.1.3!Thawing!the!cultured!cells……………………………………………...........................97! !!!!2.3.1.4!Trypsinizing!the!cultured!cells……………………………………………...................97! !!!!2.3.1.5!Freezing!the!cultured!cells……………………………………………............................98! !!!!2.3.1.6!Cell!counting!in!Haemomocytometer…………………………………………….......99! !!!!2.3.1.7!Radiation!exposure……………………………………………........................................100! !!!!2.3.1.8!Clonogenic!Assay!or!Colony!Forming!Assay…………………………….............101! !!!!!!2.3.1.8.1!Plating!Efficiency!and!Surviving!Fraction....................................................102! !!!!!!2.3.1.8.2!Analysis!of!the!radiation!dose!survival!curves!by!Linear!Regression.103! !!!!!!2.3.1.8.3!Linear!Regression!for!“dose”!&!“D2”.................................................................103! !!!!!!2.3.1.8.4!Linear!regression!for!“Colo320DM!Vs.!SW48”!&!“T84!Vs.!HCT116”...104! !!!!!!2.3.1.8.5!Radiation!dose!survival!curve……………………………………………...............105! 2.4&In&Vivo&(Patient&cohort)&Study……………………………………………...........................105& !!2.4.1!Patient!selection……………………………………………......................................................106! !!2.4.2!Clinical!treatments…………………………………………….................................................106! !!2.4.3!Preaoperative!radiotherapy!(RT)!……………………………………………...................106! !!2.4.4!Surgery…………………………………………….……………………………………………........107! 2.5&Histopathological&examination&of&rectal&cancer&resections..........................107&
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!!2.5.1!Fixation!of!tissue!specimen!and!macroscopic!sampling.....................................108! !!2.5.2!Tissue!processing……………………………………………...................................................108! !!2.5.3!Haematoxylin!and!Eosin!(H&E)!staining…………………………………………….....109! !!2.5.4!Light!microscopic!examination…………………………………………….......................109! !!2.5.5!Tumour!differentiation!grade……………………………………………..........................110! !!2.5.6!Tumour!Regression!Grade!(TRG)!……………………………………………..................110! !!2.5.7!Pathological!(p)TNM!staging……………………………………………............................112! !!2.5.8!Other!Variables…………………………………………….......................................................113! 2.6&Patient&Follow&Up…………………………………………….....................................................113& 2.7&Tissueµarray&(TMA)!……………………………………………...................................114& !!2.7.1!Construction!of!tissue!microarrays!(TMAs)!………………………………………….114! !!2.7.2!Treatment!of!TMA…………………………………………….................................................117! 2.8&Immunohistochemistry&(IHC)!……………………………………………..........................117& !!2.8.1!Manual!IHC!staining!of!Plk1!and!γH2AX!biomarkers……………………………..118! !!!!2.8.1.1!Quantification!of!IHC!staining……………………………………………....................120! !!!!2.8.1.2!Quantification!of!Plk1!and!γH2AX!proteins……………………………………....121! !!!!2.8.1.3!Controls……………………………………………...............................................................121! !!2.8.2!Automated!IHC!staining!of!MLH1,!MSH2,!MSH6!&!PMS2!biomarkers.........122! !!!!2.8.2.1!Quantification!of!IHC!staining……………………………………...............................123! !!!!2.8.2.2!Quantification!of!MLH1,!MSH2,!MSH6!&!PMS2!proteins..............................123! !!!!2.8.2.3!Controls……………………………………………................................................................123! 2.9.1&In&Vitro&Study……………………………………………..........................................................124& !!2.9.1.1!Comparison!of!cell!survival…………………………………….......................................124! 2.9.2&In&Vivo&Study……………………………………………............................................................124& !!2.9.2.1!Correlation!between!rectal!cancer!IHC!and!Plk1,!γH2AX................................124!! !!2.9.2.2!Correlation!between!TRG!and!rectal!cancer!IHC!for!DNA!MMR!proteins125! 2.9.3&Survival&Analysis……………………………………………...................................................125&
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!!2.9.3.1!Assessment!of!patients!and!pathological!characteristics................................126! !!2.9.3.2!KaplanaMeier!(KM)!and!Cox!regression!procedures.........................................127! ! CHAPTER&3:&RADIATION&SENSITIVITY&OF&CRC&CELL&LINES......................................129& 3.1&Introduction............................................................................................................!....................130& 3.2& Radiation& sensitivity& of& microsatellite& stable& (MSS)& and& microsatellite& unstable&(MSI)&CRC&cell&lines......................................................................................................132& 3.3&Radiation&sensitivity&of&CRC&cell&lines&results..........................................................133& !!3.3.1! Surviving! Fractions! (SF)! of! COLO320DM,! SW48,! T84! and! HCT116! after! radiation..................................................................................................................................................133! !!3.3.2! Mean! SF! of! Colo320DM,! SW48,! T84! and! HCT116! following! variable! dosage! of! ionizing!radiation..............................................................................................................................135! 3.4&Clonogenic&assay&for&radiation&sensitivity................................................................137& 3.5&Radiation&survival&curves...................................................................................................137& !!3.5.1!Survival!curve!of!Colo320DM!(MSS),!SW48!(MSI)!cells,!T84!(MSS)!and!HCT116! (MSI)!cells.............................................................................................................................................137! 3.6&Discussion....................................................................................................................................139& 3.7&Conclusion...................................................................................................................................143& & CHAPTER& 4& RESULTS:& CLINICOPATHOLOGICAL& PARAMETERS& (CPP)& AND& CORRELATION&TO&RADIOTHERAPY&OUTCOMES..........................................................145! 4.1&Introduction...............................................................................................................................146& 4.2&Clinicopathological¶meters&(CPPs)!....................................................................146& !!4.2.1!Clinicopathological!parameters!of!whole!rectal!cancer!patients....................146! !!4.2.2! Univariate! correlations! of! clinicopathological! parameters! and! RFS! both! of! whole! rectal! cancer! patients! and! rectal! cancer! patients! without! metastasis! cohorts....................................................................................................................................................149! !!4.2.3!Univariate!correlations!of!clinicopathological!parameters!and!OS!of!whole!rectal! cancer!patients’!cohort...................................................................................................................157!
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!!4.2.4!Univariate!correlations!of!clinicopathological!parameters!and!OS!of!whole!rectal! cancer!patients!without!metastasis!cohort..........................................................................168! !!4.2.5! Univariate! correlations! of! clinicopathological! parameters! and! RFS! of! rectal! cancer!patients!with!lymph!node!involvement!(N1/2)!cohort....................................175! !!4.2.6! Univariate! correlations! of! clinicopathological! parameters! and! OS! of! rectal! cancer!patients!with!lymph!node!involvement!(N1/2)!cohort....................................180! !!4.2.7! Univariate! correlations! of! clinicopathological! parameters! and! RFS! of! rectal! cancer!patients!with!neoadjuvant!treatment!cohort........................................................187! !!4.2.8! Univariate! correlations! of! clinicopathological! parameters! and! OS! of! rectal! cancer!patients!with!neoadjuvant!treatment!cohort........................................................190! 4.3& Clinicopathological& parameters& (CPPs)& and& radiotherapy& (neoadjuvant& treatment)&subgroup&analysis&results................................................................................193& !!4.3.1!Correlation!of!clinicopathological!parameters!and!radiotherapy!response!(TRG)! ..................................................................................................................................................................193! !!4.3.2!Correlation!of!clinicopathological!parameters!and!TRG!0,!1,!2,!3..................196! 4.4& Discussion:! Correlations! of! clinicopathological! parameters! and! Radiotherapy........................................................................................................................................198! 4.5&Conclusion...................................................................................................................................202& ! CHAPTER&5:&PLK1&AND&RADIATION&SENSITIVITY&IN&RECTAL&CANCER...........203& 5.1&Immunohistochemical&expression&of&Plk1..............................................................204& !!5.1.1!Plk1!expression!in!normal!rectal!tissue.....................................................................204! !!5.1.2!Plk1!expression!in!rectal!cancer!centre.....................................................................204! !!5.1.3!Plk1!expression!in!rectal!cancer!periphery..............................................................204! !!5.1.4!Plk1!expression!in!involved!lymph!nodes.................................................................205! !!5.1.5!Plk1!expression!in!hyperplastic!polyps......................................................................205! !!5.1.6!Plk1!expression!in!adenomas.........................................................................................205! 5.2&Correlation&of&Plk1&expression&with&clinicopathological¶meters....210& 5.3&Univariate&correlation&of&Plk1&expression&with&survival&outcomes.........213& !!5.3.1!Whole!rectal!cancer!cohort.............................................................................................213!
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!!5.3.2! Subacohort! of! rectal! cancer! patients! without! metastasis! (M0! disease! only).......................................................................................................................................................215! !!5.3.3! Subacohort! of! rectal! cancer! patients! with! lymph! node! involvement! (N1/2! disease!only).......................................................................................................................................220! !!5.3.4! Subacohort! of! rectal! cancer! patients! who! received! neoadjuvant! treatment.............................................................................................................................................225! 5.4&Multivariate&correlation&of&Plk1&expression&with&survival&outcomes....229! !!5.4.1!Whole!rectal!cancer!cohort............................................................................................230! !!5.4.2! Subacohort! of! rectal! cancer! patients! without! metastasis! (M0! disease! only)......................................................................................................................................................233! !!5.4.3! Subacohort! of! rectal! cancer! patients! with! lymph! node! involvement! (N1/2! disease!only)......................................................................................................................................235! !!5.4.4! Subacohort! of! rectal! cancer! patients! who! received! neoadjuvant! treatment.............................................................................................................................................237! 5.5&Discussion..................................................................................................................................239& 5.6&Conclusions...............................................................................................................................243& & CHAPTER&6:&γH2AX&AND&RADIATION&SENSITIVITY&IN&RECTAL&CANCER.....244& 6.1&Immunohistochemical&expression&of&γH2AX.......................................................245& !!6.1.1!γH2AX!expression!in!normal!rectal!tissue..............................................................245! !!6.1.2!γH2AX!expression!in!rectal!cancer!centre..............................................................245! !!6.1.3!γH2AX!expression!in!rectal!cancer!periphery.......................................................245! !!6.1.4!γH2AX!expression!in!involved!lymph!nodes.........................................................246! !!6.1.5!γH2AX!expression!in!hyperplastic!polyps..............................................................246! !!6.1.6!γH2AX!expression!in!adenomas..................................................................................246! 6.2&Correlation&of&γH2AX&expression&with&clinicopathological¶meters250& 6.3&Univariate&correlation&of&γH2AX&expression&with&survival&outcomes....252& !!6.3.1!Whole!rectal!cancer!cohort.............................................................................................252!
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!!6.3.2! Subacohorts! of! rectal! cancer! patients! without! metastasis! (M0! disease! only),! with! lymph! node! involvement! (N1/2! disease! only)! and! who! received! neoadjuvant! treatment..............................................................................................................................................255! 6.4&Multivariate&correlation&of&γH2AX&expression&with&survival&outcomes255& !!6.4.1!Whole!rectal!cancer!cohort.............................................................................................256! 6.5&DISCUSSION...............................................................................................................................258& 6.6&Conclusions...............................................................................................................................261& CHAPTER& 7:& MSI& MARKERS& AND& RADIATION& SENSITIVITY& IN& RECTAL& CANCER................................................................................................................................................262! 7.1&Immunohistochemical&expression&of&MSI&markers&(MSH2,&MSH6,&MLH1&and& PMS2)...................................................................................................................................................263& &&7.1.1&MSH2&expression............................................................................................................263& !!!!7.1.1.1!MSH2!expression!in!normal!rectal!tissue..........................................................263! !!!!7.1.1.2!MSH2!expression!in!rectal!cancer!centre..........................................................263! !!!!7.1.1.3!MSH2!expression!in!rectal!cancer!periphery...................................................263! !!!!7.1.1.4!MSH2!expression!in!involved!lymph!nodes.....................................................264! !!!!7.1.1.5!MSH2!expression!in!hyperplastic!polyps..........................................................264! !!!!7.1.1.6!MSH2!expression!in!adenomas..............................................................................264! &&7.1.2&MLH1&expression............................................................................................................267& !!!!7.1.2.1!MLH1!expression!in!normal!rectal!tissue..........................................................267! !!!!7.1.2.2!MLH1!expression!in!rectal!cancer!centre..........................................................267! !!!!7.1.2.3!MLH1!expression!in!rectal!cancer!periphery...................................................267! !!!!7.1.2.4!MLH1!expression!in!involved!lymph!nodes......................................................267! !!!!7.1.2.5!MLH1!expression!in!hyperplastic!polyps...........................................................267! !!!!7.1.2.6!MLH1!expression!in!adenomas..............................................................................268! &&7.1.3&MSH6&expression............................................................................................................271& !!!!7.1.3.1!MSH6!expression!in!normal!rectal!tissue..........................................................271! !!!!7.1.3.2!MSH6!expression!in!rectal!cancer!centre..........................................................271!
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!!!!7.1.3.3!MSH6!expression!in!rectal!cancer!periphery...................................................271! !!!!7.1.3.4!MSH6!expression!in!involved!lymph!nodes......................................................271! !!!!7.1.3.5!MSH6!expression!in!hyperplastic!polyps...........................................................271! !!!!7.1.3.6!MSH6!expression!in!adenomas..............................................................................272! &&7.1.4&PMS2&expression.............................................................................................................275& !!!!7.1.4.1!PMS2!expression!in!normal!rectal!tissue...........................................................275! !!!!7.1.4.2!PMS2!expression!in!rectal!cancer!centre...........................................................275! !!!!7.1.4.3!PMS2!expression!in!rectal!cancer!periphery....................................................275! !!!!7.1.4.4!PMS2!expression!in!involved!lymph!nodes.......................................................275! !!!!7.1.4.5!PMS2!expression!in!hyperplastic!polyps............................................................275! !!!!7.1.4.6!PMS2!expression!in!adenomas...............................................................................276! 7.2& Correlation& of& MSI& proteins& (MSH2,& MLH1,& MSH6,& PMS2)& expression& with& clinicopathological¶meters...........................................................................................279& 7.3& Univariate& correlation& of& MSI& proteins& (MSH2,& MLH1,& MSH6,& PMS2)& expression&with&survival&outcomes...................................................................................280& !!7.3.1!MSH2!expression!with!survival!outcomes..............................................................280! !!7.3.2!MLH1!expression!with!survival!outcomes..............................................................282! !!7.3.3!MSH6!expression!with!survival!outcomes..............................................................283! !!7.3.4!PMS2!expression!with!survival!outcomes...............................................................286! 7.4& Multivariate& correlation& of& MSI& proteins& (MSH2,& MLH1,& MSH6,& PMS2)& expression&with&survival&outcomes....................................................................................288& !!7.4.1!MSI!markers!expression!with!RFS...............................................................................288! !!7.4.2!MSI!markers!expression!with!OS.................................................................................289! 7.5!Discussion....................................................................................................................................290! 7.6!Conclusion...................................................................................................................................293! CHAPTER&8:&CONCLUSIONS......................................................................................................294& REFERENCES....................................................................................................................................300& APPENDIX.........................................................................................................................................318&
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LIST&OF&FIGURES& Figure& 1.1! WHO! Colorectal! Tumour! Classification! of! Rectal! Adenocarcinoma! Histology!………………………………………………………….................................................................12! Figure!1.2!The!Human!Plk!family…………………………………………………………..................24! Figure!1.3!Cellular!functions!of!Plks…………………………………………………………............25! Figure!1.4!Key!features!of!Plks………………………………………………………….......................27! Figure!1.5!Plk1!activation!and!polo!box!domain!mediated!targeting............................29! Figure!1.6!Plk1!autoinhibition!released!by!phosphopeptide!binding...........................29! Figure!1.7!Plk1,!Cdk1!regulation!at!the!G2/M!transition....................................................31! Figure!1.8!Plk1,!Cdk1!regulation!at!DNAadamage!checkpoint..........................................32! Figure!1.9!(a)!Plk1!at!the!active!spindle!checkpoint!&!(b)!Plk1!at!the!inactive!spindle! checkpoint…………………………………………………………..............................................................34! Figure!1.9!(c)!APC/CCdc20!Regulates!Anaphase...................................................................35! Figure!1.10!Interacting!pathways!of!Plk1…………………………………………………………38! Figure!1.11!Plks!and!different!tumour!suppressor!proteins!implicated!in!DNA!damage! response!checkpoint!control………………………………………………………….........................47! Figure! 1.12! IR! induced! DDR,! involvement! in! cell! cycle,! restorative! mechanism! for! DDR………………………………………………………………………………………………………………..50! Figure!1.13!DNA!damage!response!(DDR)................................................................................52! Figure!1.14!The!MRE11!complex!involvement!in!DDR!and!cell!cycle!arrest.............53! Figure!1.15A!The!DSBR!and!SDSA!pathways!..........................................................................56! Figure!1.15B!Molecular!function!of!DSBs!Repair!through!NHEJ.....................................59! Figure!1.16!Nucleosomal!structure!of!histone!octamer......................................................61! Figure!1.17!Protein!structure!of!histone!H2AX.......................................................................62! Figure! 1.18! The! crystallographic! structure! of! histone! nucleosome! core! particle! of! chromatin…………………………………………………………................................................................63! Figure!1.19!γH2AX!in!ATMaChk2!components!and!CDK2aFOXO1!components.......68! Figure!1.20!Autophosphorylation!of!ATM!on!serine1981!after!IR.................................71! Figure!1.!21!Functional!domains!of!Chk2…………………………………………………………..72! Figure!1.22!Functional!domains!of!human!Chk1!……………………………………………….73! Figure! 1.23! Model! of! colorectal! tumorigenesis! (precursor! adenoma! to! carcinoma! through!chromosomal!instability)!…………………………………………………………..............78! Figure!1.24!DNA!mismatch!repair!(MMR)!in!eukaryotes...................................................80! Figure!1.25!Roles!of!DNA!MMR!proteins!in!the!pathway!of!DDR....................................87! ! Figure&2.1!Haemocytometer…………………………………………….........................................100! Figure!2.2!Histological!appearances!of!rectal!cancer!with!TRG!0,!1,!2!and!3..........111! Figure! 2.3! Schematic! representation! of! manual! construction! of! tissue! microarray! (TMA)!.....................................................................................................................................................116! ! Figure& 3.3.1.1! Comparative! Surviving! Fractions! (SFs)! after! ionizing! radiation! at! 0.5Gy,!2.0Gy!&!5.0Gy!in!MSS!and!MSI!cell!lines!...................................................................134! Figure!3.3.2.1!Trend!in!Mean!SF!of!Colo320DM!and!SW48!cell!lines!after!exposure!to!0,! 0.5Gy,!2.0Gy!&!5.0Gy!of!ionizing!radiation.............................................................................135! Figure! 3.3.2.2! Trend! in! Mean! SF! of! T84! and! HCT116! cell! lines! after! exposure! to! 0,! 0.5Gy,!2.0Gy!&!5.0Gy!of!ionizing!radiation..............................................................................136! Figure! 3.5.1! Radiation! Survival! Curve! of! Colo320DM! (MSS),! SW48! (MSI),! T84! (MSS)! and!HCT116!(MSI)!cells!immediately!after!radiation!(IR)...............................................138!
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! Figure& 4.1!Univariate!correlations!of!clinicopathological!parameters!&!RFS!of!whole! rectal! cancer! patients! and! rectal! cancer! patients! without! metastasis! cohorts! .............................................................................................................................................................152a156! Figure!4.2!Univariate!correlations!of!clinicopathological!parameters!and!OS!of!whole! rectal!cancer!patients’!cohort...............................................................................................160a167! Figure! 4.3! Univariate! correlations! of! clinicopathological! parameters! and! OS! of! rectal! cancer!patients!without!metastasis!cohort....................................................................170a174! Figure!4.4!Univariate!correlations!of!clinicopathological!parameters!and!RFS!of!rectal! cancer!patients!with!lymph!node!involvement!(N1/2)!cohort.............................177a179! Figure! 4.5! Univariate! correlations! of! clinicopathological! parameters! and! OS! of! rectal! cancer!patients!with!lymph!node!involvement!(N1/2)!cohort.............................182a186! Figure!4.6!Univariate!correlations!of!clincopathological!parameters!and!RFS!of!rectal! cancer!patients!with!neoadjuvant!treatment!cohort................................................188a189! Figure! 4.7! Univariate! correlations! of! clincopathological! parameters! and! OS! of! CRC! rectal!cancer!patients!with!neoadjuvant!treatment!cohort.................................................191a 192! Figure!4.8!Correlations!of!positive!lymph!nodes,!AJCC!&!TRG........................................195! Figure!4.9!Correlations!of!AJCC!and!TRG..................................................................................196! ! Figure& 5.1! IHC! expression! of! Plk1! in! different! tissue! types,! showing! nuclear! and! cytoplasmic!staining.................................................................................................................207a209! Figure! 5.2! Univariate! correlation! of! Plk1! expression! (low! vs.! high)! combined! from! tumour!centre!and!periphery!(p!=!0.006)!and!in!the!involved!lymph!node!(p!=!0.011)! with!OS!in!the!whole!rectal!cancer!cohort......................................................................213a214! Figure! 5.3! Univariate! correlation! of! Plk1! expression! (low! vs.! high)! in! tumour! periphery!(p!=!0.009),!combined!from!tumour!centre!and!periphery!(p!=!0.005)!and!in! involved! lymph! node! (p! =! 0.039)! with! OS! in! M0! rectal! cancer! subacohort! ...........................................................................................................................................................217a218! Figure!5.4!Univariate!correlation!of!Plk1!expression!(low!vs.!high)!in!involved!lymph! node!(p!=!0.032)!and!combined!from!tumour!centre!and!periphery!(p!=!0.05)!and!OS! in!the!N1/2!rectal!cancer!subacohort...............................................................................222a223! Figure!5.5!Multivariate!correlations!of!Plk1!expression!in!tumour!periphery!and!RFS! in!the!whole!rectal!cancer!cohort!(p!=!0.03)..........................................................................231! Figure! 5.6! Multivariate! correlations! of! Plk1! expressions! in! tumour! periphery! (p! =! 0.005)! and! combined! from! tumour! centre! and! periphery! (p! =! 0.007)! with! OS! in! the! whole!rectal!cancer!cohort............................................................................................................232! Figure!5.7!Multivariate!correlation!of!Plk1!expression!in!tumour!periphery!(p!=!0.03)! with!RFS!in!the!M0!rectal!cancer!subacohort........................................................................233! Figure! 5.8! Multivariate! correlations! of! Plk1! expressions! in! tumour! periphery! (p! =! 0.005)!and!combined!from!tumour!centre!and!periphery!(p!=!0.009)!with!OS!in!the!M0! rectal!cancer!subacohort......................................................................................................234a235! Figure!5.9!Multivariate!correlation!of!Plk1!expression!in!tumour!periphery!(p!=!0.018)! with!OS!in!the!N1/2!rectal!cancer!subacohort.....................................................................237! Figure! 5.10! Multivariate! correlation! of! Plk1! expression! in! tumour! periphery! (p! =! 0.024)!and!OS!in!rectal!cancer!neoadjuvant!therapy!subacohort...............................238! & Figure&6.1!IHC!expression!of!γH2AX!in!different!tissue!types,!showing!predominantly! nuclear!staining!when!positive.........................................................................................247a249! & !
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Figure& 7.1! IHC! expression! of! MSH2! in! different! tissue! types,! showing! nuclear! staining...............................................................................................................................................265a266! Figure! 7.2! IHC! expression! of! MLH1! in! different! tissue! types,! showing! nuclear! staining...............................................................................................................................................269a270! Figure! 7.3! IHC! expression! of! MSH6! in! different! tissue! types,! showing! nuclear! staining...............................................................................................................................................273a274! Figure! 7.4! IHC! expression! of! PMS2! in! different! tissue! types,! showing! nuclear! staining...............................................................................................................................................277a278! Figure! 7.5! The! loss! of! MSH6! expression! in! involved! lymph! node! and! neoadjuvant! therapy.........................................................................................................................................................280! Figure!7.6!Univariate!correlation!of!MSH6!expression!(positive!vs.!negative)!in!tumour! centre!(p!=!0.04),!involved!lymph!node!(p!=!0.04)!and!RFS........................................283a284! Figure! 7.7! Univariate! correlation! of! MSH6! expression! in! tumour! centre! (p! =! 0.02),! involved!lymph!nodes!(p!=!0.02)!and!OS.......................................................................................285! Figure! 7.8! Univariate! correlation! of! PMS2! expression! in! tumour! periphery! and! OS! .........................................................................................................................................................................287!
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LIST&OF&TABLES& Table!2.1!Colorectal!Cancer!(CRC)!cell!lines!used!in!this!thesis………………….............95! Table!2.2!Tumour!Regression!Grades!(TRG)!……………………………………………..........111! Table!2.3!Patients!and!pathological!variables!that!were!dichotomized!for!analyses! ....................................................................................................................................................................126! ! Table 3.1.1 Colo320DM Vs SW48 colonies................................................................131 Table 3.1.2 T84 Vs HCT116 colonies..........................................................................131 Table 3.2.1 Colo320DM Vs SW48 Surviving Fraction (SF).......................................132 Table 3.2.2 T84 Vs HCT116 Surviving Fraction (SF) ................................................132 Table 3.3 Comparison of mean Surviving Fractions (SFs) between COLO320DM (MSS) and SW48 (MSI) CRC cell lines & T84 (MSS) and HCT116 (MSI) CRC cell lines ......................................................................................................................................133 Table 3.3.1.1 Comparative Surviving Fractions (SFs) after ionizing radiation at different doses (0.5Gy, 2.0Gy, 5.0Gy) of radiation in MSS and MSI cell lines........................134 Table 3.3.2.1 Mean SF of Colo320DM, SW48 T84 and HCT116 following variable dosage of ionizing radiation....................................................................................................135 Table 3.3.2.2 D37 values for Colo320DM, SW48, T84 and HCT116 following variable dosage of ionizing radiation........................................................................................136 ! ! Table!4.1!Clinicopathological!variables!of!whole!rectal!cancer!patients’!cohort.148! Table!4.2!Univariate!correlations!of!clinicopathological!parameters!&!cumulative!RFS! in! whole! rectal! cancer! patients! and! rectal! cancer! patients! without! metastasis! cohorts...................................................................................................................................................151! Table!4.3!Univariate!correlations!of!clinicopathological!parameters!and!cumulative!OS! in!whole!rectal!cancer!patients’!cohort..................................................................................159! Table!4.4!Univariate!correlations!of!clinicopathological!parameters!and!cumulative!OS! in!rectal!cancer!patients!without!metastasis!cohort........................................................169! Table! 4.5! Univariate! correlations! of! clinicopathological! parameters! and! cumulative! RFS!in!rectal!cancer!patients!with!lymph!node!involvement!(N1/2)!cohort.........176! Table!4.6!Univariate!correlations!of!clinicopathological!parameters!and!cumulative!OS! of!rectal!cancer!patients!with!lymph!node!involvement!(N1/2)!cohort..................181! Table! 4.7! Univariate! correlations! of! clinicopathological! parameters! and! cumulative! RFS!in!rectal!cancer!patients!with!neoadjuvant!treatment!cohort..............................187! Table!4.8!Univariate!correlations!of!clincopathological!parameters!and!cumulative!OS! in!rectal!cancer!patients!with!neoadjuvant!treatment!cohort...........................................190! Table! 4.9! Correlation! of! clinicopathological! parameters! and! radiotherapy! response! [Favourable!response!(TRG!0,!1,!2)!and!Poor!response!(TRG!3)]...............................194! Table!4.10!Correlations!of!clinicopathological!parameters!and!TRG!0,!1,!2,!3!(N!=!74)! ...................................................................................................................................................................197! & Table!5.1!Plk1!expressions!in!normal!rectal!tissue............................................................206! Table!5.2!Plk1!expressions!in!rectal!cancer!centre............................................................206! Table!5.3!Plk1!expressions!in!rectal!cancer!periphery.....................................................206! Table!5.4!Plk1!expressions!in!lymph!node!metastasis......................................................206! Table!5.5!Plk1!expressions!in!accompanying!hyperplastic!polyp................................207! Table!5.6!Plk1!expressions!in!accompanying!adenoma....................................................207! Table!5.7!Relationship!of!Plk1!expressions!in!different!rectal!tissues.......................210!
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Table! 5.8! Clinicopathological! parameters! showing! correlation! with! Plk1! expression! .................................................................................................................................................................211! Table!5.9!Plk1!expressions!(low!vs.!high)!in!different!tissues!and!RFS!in!whole!rectal! cancer!cohort!.................................................................................................................................212! Table!5.10!Plk1!expressions!(low!vs.!high)!in!different!tissues!and!OS!in!whole!rectal! cancer!cohort..................................................................................................................................213! Table!5.11!Plk1!expression!(absent!vs.!present)!in!different!tissues!and!RFS!in!whole! rectal!cancer!cohort....................................................................................................................214! Table! 5.12! Plk1! expression! (absent! vs.! present)! in! different! tissues! and! OS! in! whole! rectal!cancer!cohort...................................................................................................................215! Table!5.13!Plk1!expression!(low!vs.!high)!in!different!tissues!and!RFS!in!the!M0!rectal! cancer!subacohort.......................................................................................................................216! Table!5.14!Plk1!expression!(low!vs.!high)!in!different!tissues!and!OS!in!the!M0!rectal! cancer!subacohort......................................................................................................................216! Table!5.15!Plk1!expression!(absent!vs.!present)!in!different!tissues!and!RFS!in!the!M0! rectal!cancer!subacohort.........................................................................................................219! Table!5.16!Plk1!expression!(absent!vs.!present)!in!different!tissues!and!OS!in!the!M0! rectal!cancer!subacohort..........................................................................................................220! Table! 5.17! Plk1! expression! (low! vs.! high)! in! different! tissues! and! RFS! in! the! N1/2! rectal!cancer!subacohort..........................................................................................................221! Table!5.18!Plk1!expression!(low!vs.!high)!in!different!tissues!and!OS!in!the!N1/2!rectal! cancer!subagroup........................................................................................................................222! Table! 5.19! Plk1! expression! (absent! vs.! present)! in! different! tissues! and! RFS! in! the! N1/2!rectal!cancer!subacohort...............................................................................................224! Table! 5.20! Plk1! expression! (absent! vs.! positive)! in! different! tissues! and! OS! in! N1/2! rectal!cancer!subacohort...........................................................................................................225! Table! 5.21! Plk1! expression! (low! vs.! high)! in! different! tissues! and! RFS! in! the! rectal! cancer!subacohort!with!neoadjuvant!therapy..................................................................226! Table! 5.22! Plk1! expression! (low! vs.! high)! in! different! tissues! and! OS! in! the! rectal! cancer!subacohort!with!neoadjuvant!therapy..................................................................227! Table! 5.23! Plk1! expression! (absent! vs.! present)! in! different! tissues! and! RFS! in! the! rectal!cancer!subacohort!with!neoadjuvant!therapy.....................................................228! Table! 5.24! Plk1! expression! (absent! vs.! present)! in! different! tissues! and! OS! in! the! rectal!cancer!subacohort!with!neoadjuvant!therapy......................................................229! Table! 5.25! Multivariate! correlations! of! Plk1! expression! in! different! tumour! tissues! and!RFS!in!the!whole!rectal!cancer!cohort..........................................................................230! Table!5.26!Multivariate!correlation!of!Plk1!expression!in!different!tumour!tissues!and! OS!in!the!whole!rectal!cancer!cohort.....................................................................................231! Table!5.27!Multivariate!correlation!of!Plk1!expression!in!different!tumour!tissues!and! RFS!in!the!M0!rectal!cancer!subacohort.............................................................................233! Table!5.28!Multivariate!correlation!of!Plk1!expression!in!different!tumour!tissues!and! OS!in!the!M0!rectal!cancer!subacohort.............................................................................234! Table!5.29!Multivariate!correlation!of!Plk1!expression!in!different!tumour!tissues!and! RFS!in!the!N1/2!rectal!cancer!subacohort......................................................................236! Table!5.30!Multivariate!correlation!of!Plk1!expression!in!different!tumour!tissues!and! OS!in!the!N1/2!rectal!cancer!subacohort.........................................................................236! Table!5.31!Multivariate!correlation!of!Plk1!expression!in!tumour!periphery!and!RFS!in! the!rectal!cancer!neoadjuvant!therapy!subacohort....................................................238! Table!5.32!Multivariate!correlation!of!Plk1!expression!tumour!periphery!and!OS!in!the! rectal!cancer!neoadjuvant!therapy!subacohort............................................................238! !
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& Table!6.1!γH2AX!expression!in!normal!rectal!tissue..........................................................246! Table!6.2!γH2AX!expression!in!rectal!cancer!centre..........................................................247! Table!6.3!γH2AX!expression!in!rectal!cancer!periphery...................................................247! Table!6.4!γH2AX!expression!in!lymph!node!metastasis....................................................247! Table!6.5!γH2AX!expression!in!accompanying!hyperplastic!polyp..............................247! Table!6.6!γH2AX!expression!in!accompanying!adenoma.................................................248! Table!6.7!Clinicopathological!parameters!showing!correlation!with!γH2AX!expression! ..........................................................................................................................................................251a252! Table!6.8!γH2AX!expression!(low!vs.!high)!in!different!tissues!and!RFS!in!whole!rectal! cancer!cohort.........................................................................................................................................253! Table!6.9!γH2AX!expression!(low!vs.!high)!in!different!tissues!and!OS!in!whole!rectal! cancer!cohort.........................................................................................................................................253! Table! 6.10! γH2AX! expression! (absent! vs.! present)! in! different! tissues! and! RFS! in! whole!rectal!cancer!cohort..............................................................................................................254! Table!6.11!γH2AX!expression!(absent!vs.!present)!in!different!tissues!and!OS!in!whole! rectal!cancer!cohort............................................................................................................................254! Table! 6.12! Multivariate! correlation! of! γH2AX! expression! in! different! tumour! tissues! and!RFS!in!the!whole!rectal!cancer!cohort...............................................................................256! Table! 6.13! Multivariate! correlation! of! γH2AX! expression! in! different! tumour! tissues! and!OS!in!the!whole!rectal!cancer!cohort.................................................................................256! Table! 6.14! Multivariate! correlation! of! γH2AX! expression! in! different! tumour! tissues! and!RFS!in!the!whole!rectal!cancer!cohort...............................................................................257! Table! 6.15! Multivariate! correlation! of! γH2AX! expression! in! different! tumour! tissues! and!OS!in!the!whole!rectal!cancer!cohort.................................................................................257! & Table! 7.1! shows! the! distribution! of! percentage! of! MSH2! positive! and! negative! cases! amongst!the!above!described!tissue!types...............................................................................264! Table! 7.2! shows! the! distribution! of! percentage! of! MLH1! positive! and! negative! cases! amongst!the!above!described!tissue!types................................................................................268! Table! 7.3! shows! the! distribution! of! percentage! of! MSH6! positive! and! negative! cases! amongst!the!above!described!tissue!types.!..............................................................................272! Table! 7.4! shows! the! distribution! of! percentage! of! PMS2! positive! and! negative! cases! amongst!the!above!described!tissue!types.................................................................................276! Table!7.5!Clinicopathological!parameters!showing!correlation!with!expression!of!MSI! proteins.....................................................................................................................................................279! Table! 7.6! MSH2! expression! (negative! vs.! positive)! in! different! tissues! and! RFS! in! whole!rectal!cancer!cohort...............................................................................................................281! Table!7.7!MSH2!expression!(negative!vs.!positive)!in!different!tissues!and!OS!in!whole! rectal!cancer!cohort.............................................................................................................................281! Table! 7.8! MLH1! expression! (negative! vs.! positive)! in! different! tissues! and! RFS! in! whole!rectal!cancer!cohort...............................................................................................................282! Table!7.9!MLH1!expression!(negative!vs.!positive)!in!different!tissues!and!OS!in!whole! rectal!cancer!cohort.............................................................................................................................282! Table! 7.10! MSH6! expression! (negative! vs.! positive)! in! different! tissues! and! RFS! in! whole!rectal!cancer!cohort...............................................................................................................283! Table! 7.11! MSH6! expression! (negative! vs.! positive)! in! different! tissues! and! OS! in! whole!rectal!cancer!cohort...............................................................................................................284! Table! 7.12! PMS2! expression! (negative! vs.! positive)! in! different! tissues! and! RFS! in! whole!rectal!cancer!cohort................................................................................................................286! !
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Table! 7.13! PMS2! expression! (negative! vs.! positive)! in! different! tissues! and! OS! in! whole!rectal!cancer!cohort.............................................................................................................287! Table! 7.14! Multivariate! correlation! of! MSI! markers! expression! in! different! tumour! tissues!and!RFS!in!the!whole!rectal!cancer!cohort..............................................................289! Table! 7.15! Multivariate! correlation! of! MSI! markers! expression! in! different! tumour! tissues!and!OS!in!the!whole!rectal!cancer!cohort................................................................289! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
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ABSTRACT Colorectal cancer (CRC) is the third most common cancer in the world. Australia, New Zealand, Canada, the United States, and parts of Europe have the highest incidence rates of CRC. China, India, South America and parts of Africa have the lowest risk of CRC. CRC is the second most common cancer in both sexes in Australia. Even though the death rates from CRC involving the colon have diminished, those arising from the rectum have revealed no improvement. The greatest obstacle in attaining a complete surgical resection of large rectal cancers is the close anatomical relation to surrounding structures, as opposed to the free serosal surfaces enfolding the colon. To assist complete resection, pre-operative radiotherapy (DXT) can be applied, but the efficacy of ionising radiation (IR) is extremely variable between individual tumours. Reliable predictive marker/s that enable patient stratification in the application of this otherwise toxic therapy is still not available. Current therapeutic management of rectal cancer can be improved with the availability of better predictive and prognostic biomarkers. Proteins such as Plk1, γH2AX and MMR proteins (MSH2, MSH6, MLH1 and PMS2), involved in DNA damage response (DDR) pathway may be possible biomarkers for radiation response prediction and prognostication of rectal cancer. Serine/threonine protein kinase Plk1 is overexpressed in most of cancers including CRC. Plk1 functional activity is essential in the restoration of DNA damage following IR, which causes DNA double strand break (DSB). The earliest manifestation of this reparative process is histone H2AX phosphorylation at serine 139, leading to γ-H2AX. Colorectal normal mucosa showed the lowest level of γH2AX with gradually increasing levels in early adenoma and then in advanced malignant colorectal tissues, leading to the possibility that γH2AX may be a prospective biomarker in rectal cancer management. There are numerous publications regarding DNA mismatch repair (MMR) proteins, the insufficiency of which is characteristic of CRCs with microsatellite instability (MSI). MSI may enable unlimited replicative potential of malignant cell that leads to radiation injury resistance. Therefore, these proteins were characterized in both CRC cell lines (MMR proteins) and different (core and invasive front) rectal cancer tissues (Plk1, γH2AX and MMR proteins) exposed to radiation. Histopathological grading of tumour regression was performed following radiotherapy in rectal cancer as a marker of radiotherapy response and a surrogate indicator of patient prognosis. Though MMR protein expressions correlated with improved in vitro cell survival following radiation, these findings could only be partially replicated in patient
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tissue samples. This may not be entirely unexpected, given intratumoural heterogeneity in genetic profiles and oxygenation between individual cancer cells, their interaction with stromal environment and a multitude of other factors that cannot be adequately replicated in cell line experiments. In our rectal cancer patient cohort, histopathological regression following radiotherapy did appear to correlate with better clinical outcome, but certainly no replacement for the routine pTNM staging with which it was compared. Overexpression of Plk1 in the primary rectal cancer also correlates with poor tumour regression and reduced overall survival. High level of γH2AX correlates with higher tumour stage, perineural invasion and vascular invasion. However, interpretation of the results is limited by the small number of positivity amongst the cohort, with respect to γH2AX and MMR proteins. The combined analysis of all the proteins examined in this thesis revealed no interactions, possibly suggesting these biomarkers act individually within the DDR pathway, rather than in a demonstrably interdependent manner. Though our results are mixed, finding biomarkers predictive of radiation response is nonetheless critical. Enhancing the radiosensitivity of cancers through manipulating the functional activity and/or expression of prospective biomarkers could conceivably enhance tumour response to the level that the extent of consequent surgical resection can be minimized. & & & & & & & &
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Statement of Authentication The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. !
! Thein Ga Tut Discipline of Pathology School of Medicine Western Sydney University Locked Bag 1797, Penrith NSW 2751 New South Wales Australia ! ! ! ! & & &
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ACKNOWLEDGEMENTS My sincere gratitude, and heartfelt thanks to Principal supervisor Professor Cheok Soon Lee, a guide and a philosopher, for the inspiration, his vigilant supervision, guidance, continued support and encouragement in making my doctoral dreams come true. He has taken me in as his student and taught me a great deal in many areas beyond the confines of this thesis, including how to be a better doctor and a good scientist. I am grateful and indebted to Associate supervisor Dr Joo-Shik Shin, for giving me the confidence, encouragement, dedicated supervision, and motivation to realize the “impossible”. I would also like to thank Mrs Dianne Gourlie, Executive Assistant to Professor Soon, Mrs Sandra Pereira, Administrative Assistant to Professor of Colorectal Surgery, Department of Surgery, Mr. Askar Abubakar, Scientific Officer, Discipline of Pathology, School of Medicine, Western Sydney University, Dr Irani Dissanayake, Pathologist, Mr Joseph Descallar, Biostatistician, Ingham Institute and Conjoint Lecturer, South Western Sydney Clinical School, University of New South Wales and Dr. Stephanie Hui-Su Lim, Medical Oncology Research Fellow, Ingham Institute of Applied Medical Research for their time, expertise and resources which helped to achieve my goals. Special thanks to Higher Degrees Research Director Dr David A Mahns, Senior Lecturer in Integrative Physiology in School of Medicine, Western Sydney University, for his encouragement and great help whenever I had problems, histopathologists and staff at Liverpool Hospital, especially Professor Jim Yong. A grateful thanks to the Department of Education, Commonwealth Government and Western Sydney University for awarding me The Australian Postgraduate Award (APA) scholarship. To my friends and peers: “thank you for your enthusiastic support and assistance in every way”. Last but not the least, I would like to express sincere gratitude to my parents and my supportive, cheerful wife Khin Sandar Soe, for their perpetual support and understanding, my beloved elder daughter Aurea Tut for helping me when needed and my dear younger daughter Sorcha Tut for understanding me during this course.
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PUBLICATIONS ARISING FROM THIS RESEARCH Recent Publications (1) Maxine Revoltar, Joo-Shik Shin, Stephanie H Lim, Tut Thein-Ga, Irani Dissanayake, Joseph Descallar, Vincent Ho, Wei Chua, Weng Ng, Mark Lee, Christopher Henderson, Paul De Souza, Matthew Morgan, C. Soon Lee. Early marker of DNA damage response, ATM, as a predictor of clinical outcome following radiotherapy in rectal cancer patients. Pathology 48:S153 · February 2016 DOI: 10.1016/j.pathol.2015.12.417 (Citation Index: Impact Factor: 2.188) (2) Tut TG, Lim SH, Dissanayake IU, Descallar J, Chua W, Ng W, de Souza P, Shin JS, Lee CS. Upregulated Polo-Like Kinase 1 Expression Correlates with Inferior Survival Outcomes in Rectal Cancer. PLoS One. 2015 Jun 5;10(6):e0129313. doi: 10.1371/journal.pone.0129313. (Citation Index: Impact Factor 3.534 - 2014) (3) Shin JS, Tut TG, Ho V, Lee CS. Predictive markers of radiotherapy-induced rectal cancer regression. J Clin Pathol. 2014 Aug 12. pii: jclinpath-2014-202494. doi: 10.1136/jclinpath-2014-202494. [Epub ahead of print] Review. (Citation Index: Impact Factor 2.551 - 2013) (4) Shin JS, Tut TG, Yang T, Lee CS. Radiotherapy response in microsatellite instability related rectal cancer. Korean J Pathol. 2013Feb; 47(1): 1-8.doi: 10.4132/KoreanJPathol.2013.47.1.1. Epub 2013 Feb 25. Abstracts & Poster Publications (1) Maxine Revoltar, Joo-Shik Shin, Stephanie Lim, Thein-Ga Tut, Irani Dissanayake, Joseph Descallar, Vincent Ho, Wei Chua, Weng Ng, Mark Lee, Christopher Henderson, Paul De Souza, Matthew Morgan, C. Soon Lee. Early marker of DNA damage response, ATM, as a predictor of clinical outcome following radiotherapy in rectal cancer patients. Commendations in The 40th Annual Scientific Meeting of Australasian Division of International Academy of Pathology (IAP 3rd June - 5th June 2015) as Poster 107 Australasian Division of International Academy of Pathology (IAP) NEWS September 2015 Issue (2) Thein Ga Tut, Stephanie Lim, Irani Dissanayake, Joseph Descallar, Wei Chua, Weng Ng, Paul De Souza, Les Bokey, Joo-Shik Shin, C. Soon Lee “Upregulated PLK1 expression confers radiation resistance and poor patient survival outcomes in rectal cancer” Second Poster Award at 39th Annual Scientific Meeting of the Australasian Division of the International Academy of Pathology Limited (IAP) May 30 - June 1, 2014. Brisbane. Abstract published in Pathology.01/2015;47: S104-S105. DOI:10.1097/01.PAT.0000461623.74546.8a (Impact Factor:2.62) (3) Stephanie Hui-Su Lim, J-S Shin, T G Tut, Wei Chua, I U Dissanayake, Kevin Spring, Weng Ng, Paul De Souza, C S Lee “Polo-like kinase 1 as a biomarker in rectal cancers” !
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Poster Abstract (No: e14542) published in. J Clin Oncol 32, 2014 (suppl; abstr e14542) for 50th Annual Meeting of American Society of Clinical Oncology (ASCO) May 30 June 3, 2014, McCormick Place, Chicago, Illinois (4) S. H. Lim, J.-S. Shin, T. G. Tut, W. T. W. Ng, W. Chua, I. U. Dissanayake, K. J. Spring, E. L. Bokey, W. Ng, P. de Souza, C. S. Lee. “POLO-LIKE KINASE 1 AS A BIOMARKER IN RECTAL CANCER” Poster Abstract (page 60) published in AsiaPacific Journal of Clinical Oncology, WILEY-BLACKWELL Volume 10, Issue Supplement S6, pages 52–68, August 2014; Article first published online: 16 JUL 2014 DOI: 10.1111/ajco.12250 for the Medical Oncology Group of Australia Incorporated Annual Scientific Meeting. Integrating Molecular and Immunologic Advances into Practice, 6-8 August 2014, Hilton Sydney, Sydney, New South Wales. (5) TUT TG, Dissanayake IU, Lim SH, Descallar J, Ng W, Chua W, De Souza Paul, Bokey EL, Shin JS, Lee CS “Biomarkers Of Radiation Sensitivity in Rectal Cancer” Poster Abstract published in 2013 Ingham Institute (Applied Medical Research) 8Th Annual Research & Teaching Showcase on 29 November 2013 & & & & & & & & & & & & & & !
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ABBREVIATIONS AACR
Australian Association of Cancer Registries
APR
Abdomino-perineal resection
ACIM
Australian Cancer Incidence and Mortality
ACPS
Australian clinico-pathological staging
AIHW
Australian Institute of Health and Welfare
AJCC
American Joint Committee on Cancer
AR
Anterior resection
APC/C
Anaphase promoting complex/cyclosome
APC
Adenomatous polyposis coli
ATCC
American Tissue Culture Collection
ATM
Ataxia telangiectasia mutated (also known as TEL1)
ATP
Adenosine triphosphate
ATR
Ataxia telangiectasia and RAD-3 related
ATRIP
ATR-interacting protein
ASD
Autism spectrum disorder
BAX
Bcl-2-associated X protein
BRCA1
Breast cancer susceptibility type 1
BRCA2
Breast cancer susceptibility type 2
Bub
Budding uninhibited by benzimidazole
CA 19-9
Carbohydrate antigen 19-9
!
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CA 242
Sialylated Lewis carbohydrates
Cak
CDK-activating kinase
CAP
College of American Pathologists
Cdc4
Cell division control protein 4
Cdc20
Cell-division cycle protein 20
Cdc25
Cell-division cycle protein 25
CDE
Cell cycle-dependent element
Cdh1
Cdc20homologue-1
CDK
Cyclin dependent kinase
cDNA
complementary DNA
CEA
Carcinoembryonic antigen
CHK
Checkpoint effector kinase
CHR
Cell cycle genes-homology region
CI
Chromosomal instability
CIMP
CpG island methylator phenotype
CHO cells
Chinese Hamster Ovary cells
CMT
Combined modality therapy
Colo320DM
Human Dukes' type C, colorectal adenocarcinoma cells
CpG
5'—Cytosine—phosphate—Guanine—3'
CPPs
Clinicopathological parameters
CRC
Colorectal cancer
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CREs
Consensus response elements
CRM
Circumferential resection margin
CT
Computed Tomography
Da
Daltons
DBD
DNA-binding domain
DDR
DNA damage response
DFS
Disease free survival
DM
Diabetes mellitus
DMEM
Dulbecco's Modified Eagle Medium
DMEM/F12
Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 Medium
DMSO
Dimethylsulfoxide
DNA
Deoxyribonucleic acid
DNAPKcs
DNA dependent protein kinase catalytic subunit
DPBS
Dulbecco's Phosphate-Buffered Saline
DSB
Double strand break
DXT
Deep X-ray therapy/radiotherapy
ECACC
European Collection of Cell Culture
EDTA
Ethylene diamine tetra acetic acid
EGFR
Epidermal growth factor receptor
EXO1
Exonuclease 1
FAP
Familiar Adenomatous Polyposis
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FBS
Fetal Bovine Serum
FHA
Forkhead associated domain
Fnk
Fibroblast-growth factor-1 inducible kinase
FOBT
Faecal occult blood test
FoxM1
Forkhead Box protein M1
FOXO1
Forkhead box O transcription factor1
FU
Fluorouracil
γH2AX
Gamma Histone 2AX
G1
Growth 1 (phase of cell cycle)
G2
Growth 2 (phase of cell cycle)
Gy
Grays
H2 O2
Hydrogen peroxide
HCC
Hepatocellular carcinoma
HCT116
Human colorectal carcinoma cell lines
HCT15
Human Dukes' type C, colorectal adenocarcinoma
H&E
Haematoxylin and eosin
HNPCC
Hereditary non-polyposis colorectal cancer
HR
Hazard ratio
HT29
Human colorectal adenocarcinoma cell line with epithelial morphology
hPrk
!
human proliferation-related kinase
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IBD
Irritable Bowel Disease
IGF2R
Insulin like growth factor-2 receptor
IgG
Immunoglobulin G
IHC
Immunohistochemistry / immunohistochemical
IMRT
Intensity-modulated radiotherapy
IP
Immediate plating
IR
Ionising radiation
KM
Kaplan-Meier
KRAS
Kirsten rat sarcoma viral oncogene homolog (also V-Ki-ras2)
kDa
Kilo Dalton
LARC
Locally advanced rectal cancer
LCR
Long course radiotherapy regime
LN
Lymph nodes
M
Distant metastasis
Mad
Mitotic arrest defective
Mat1
Menage a trios-1
MDC1
Mediator of DNA damage checkpoint protein 1
MDM2
Mouse double minute 2 homolog
MIR
Mortality-to-incidence ratio
MLH1
MutL Homolog 1
MMR
Mismatch repair
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mAb
Monoclonal antibody
mH2A
MacroH2A
Mre11
Meiotic recombination 11
mRNA
messenger RNA
MSH2
MutS Homolog 2
MSH6
MutS Homolog 6
MSI
Microsatellite instability
MSI-H
Microsatellite instability – high
MSI-L
Microsatellite instability - low
MSS
Microsatellite stable
MP
Muscularis propria
Mps
Monopolar spindle
Myt1
Myelin transcription factor 1
N
Lymph node involvement
NBS1
Nijmegen breakage syndrome 1
NF-κB
Nuclear factor kappa B
NF-Y
Nuclear factor Y
NHEJ
Non-homologous end joining
NIH3T3
Primary mouse embryonic fibroblast cells, 3-day transfer, inoculum 3 x 105 cells
NSCLC
!
Non-small cell lung cancer
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NSW
New South Wales
ORF
Open reading frame
OS
Overall survival
PBD
POLO-Box domain
PBs
POLO boxes
PCNA
Proliferating cell nuclear antigen
pCR
Pathological complete response
pN
Nodal status
pM
Metastatic status
pT
Tumour status
Pc
POLO-BOX cap
PE
Plating Efficiency
PMS2
Postmeotic Segregation Increased 2
p21Waf1
also known as p21Cip1, cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1
p53
Tumour protein 53 kilodalton (TP53)
PBS
Phosphate buffered saline
PCNA
Proliferating cell nuclear antigen
PCR
Polymerase chain reaction
PIKK
Phosphatidylinositol 3-kinase-like
PI3Ks
Phosphoinositide 3-Kinases
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Plk1
Polo-like Kinase 1
PME
Partial mesorectal excision
PNKP
Polynucleotide kinase phosphatase
pRb
Retinoblastoma protein
pTNM
pathological TNM staging
Rad50
DNA Double strand break repair protein
RCT
Radiochemotherapy
RE1&RE2
p53 responsive elements 1 & 2
RFS
Recurrence free survival
RNA
Ribonucleic acid
RPA
Replication protein A
RPMI1640
Roswell Park Memorial Institute 1640 Medium
RT
Radiotherapy
RT-PCR
Reverse transcription polymerase chain reaction
S
Synthesis (phase of cell cycle)
SAC
Spindle assembly checkpoint
Sak
Serum akin kinase
Scc1
Sister-chromatid cohesion 1
SCD
Ser-Gln/Thr-Gln cluster domain
SCR
Short course radiotherapy regime
SE
Standard Error
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SF
Surviving fraction after x Gy of ionizing radiation
Snk
Serum inducible kinase
SPSS
Statistical Package for the Social Sciences
SQ
Serine-Glutamine
SSB
Single strand breeak
ssDNA
Single-stranded DNA
Stk18
serine/threonine kinase 18
SW48
Human Dukes' type C, grade IV, colorectal adenocarcinoma cells
T
Primary Tumour
TBS-T
Tris-Buffered Saline with Tween 20
TGFβ
Transforming growth factor-β
TGFβR
Transforming growth factor-β receptor
TIMP1
Tissue inhibitor of metalloproteinase 1
TILs
Tumour infiltrating lymphocytes
TMA
Tissue microarray
TME
Total mesorectal excision
TNF
Tumour necrosis factor
TNFR
Tumour necrosis factor receptor
TNM
Tumour-node-metastasis
TQ
Threonine-Glutamine
TRG
Tumour regression grade
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TRUS
Trans-rectal ultrasound
T84
Human colorectal carcinoma cells
VEGF
Vascular endothelial growth factor
Wee1
Serine/Theronine family nuclear kinase
WHO
World Health Organisation
X2
Chi squared test
53BP1
Tumour suppressor p53–binding protein 1
95% CI
95 percent confidence interval
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Ch 1. Introduction
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CHAPTER 1
INTRODUCTION
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1!
Ch 1. Introduction
1.1 Overview This chapter discusses the anatomy and histology of the colorectum as well as the epidemiology and the clinical, pathological and molecular characteristics of colorectal cancer (CRC), particularly those that originated from primary rectal mucosa. Colorectal cancer treatment options such as deep X ray therapy (DXT) that are practically applied in routine management, and affect underlying molecular biological mechanisms strengthening malignant cell proliferation and causing cell death after ionising radiation, are also explained. Polo-like kinase 1, Gama H2AX, DNA mismatch repair proteins (MLH1, MSH2, MSH6 & PMS2) are justified as potential biomarkers for predicting DXT response in rectal malignancy, hence improving patient stratification of management options.
1.2 Anatomy and histology of the colorectum Colorectum is the final part of gastrointestinal tract which starts at the ileocaecal valve in the right iliac fossa (RIF) and the rectum ends with an opening known as the anus in the pelvis. It is embryonally derived from midgut (lower part to the transverse colon) and hindgut (the transverse colon to the anal canal). The colon has four parts (ascending, transverse, descending & sigmoid) and it is about 1 - 1.5 meters (six feet) long. The connection of the sigmoid colon and anus is the rectum, which is about 8 - 15 cm (8 inches long). The part between the rectum and anus is the anal canal (2.5 - 4cm long) (Levine and Haggitt 1989). The main functions of the colorectum are reabsorption of water, inorganic salts and some essential vitamins (Vit K) and secretions of mucus. Mucus reduces friction for intestinal contents movement toward the anus. The rectum
!
2!
Ch 1. Introduction provides the transient storing of waste (faeces) and a place for gut flora/bacteria that aid fermentation. The pH in the colon varies between slightly acidic to neutral (pH 5.5 - 7.0). There are mucosa, submucosa and serosa (adventitia) in the colorectum. The mucosa has columnar epithelium covering lumen & cloverleaf-like crypts, lamina propria, absorptive cells, goblet cells and pyramidal shaped paneth cells secreting eosinophilic granules found in the caecum and proximal right colon (Levine and Haggitt 1989). The Paneth cells can be seen in different parts of the colon inflammation (Symonds 1974). The lamina propria has different types of white blood cells. Lymphoid follicles are scattered widely within the lamina propria or within submocosa. Submucosa has fat cells, similar to those in the lamina propria, and two neural plexuses such as the Messiner’s plexus and Auerbach’s myenteric plexus. The smooth muscle layers are composed of a circular muscle layer and a longitudinal muscle layer. The Auerbach’s neural plexus is found in between these muscle layers. The longitudinal layer of muscle forms three compressed strands known as the taenia coli. There is a subserosal connective tissue in between the muscularis propria and the serosa. The serosa or adventitia is mesothelium that forms little pouches (appendices epiploicae) packed with fatty tissue along the large intestine (Levine and Haggitt 1989).
1.2.1 Distinctive anatomy and histology of the colorectum The sigmoid colon loses its mesentery and gradually evolves into the rectum (15 cm) at the sacral vertebra three (S3). The rectum ends 2.5 cm anteroinferiorly at the coccyx vertebra tip and at the same level as the prostate apex. The upper rectum is wrapped with a free serosa surface on its anterior and lateral aspects. The middle rectum has a peritoneal cloak anteriorly, however the lower rectum lacks peritoneum (Morgan 1936).
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3!
Ch 1. Introduction The male rectum is anteriorly related to the small intestine in the rectovesical pouch above and is posteriorly related to the bladder, prostate, seminal vesicles and ductus deferentes below. The female rectum is related anteriorly to the small intestine in the rectouterine pouch above and posteriorly to the vagina below (Murphy 1983). The rectum sits on the sacrum (composed of 3 sacral vertebrae), coccyx, anococcygeal ligament, pelvic diaphragm, regional blood vessels, the inferior parts of the sympathetic trunk and sacral plexuses. Surgically, the rectum is covered by visceral pelvic fascia. The pelvic fascia is fused to the parietal pelvic fascia surrounding the sacrum. The space between the rectum and fascia is filled with a large amount of fat within which perirectal lymph sinus, retrorectal lymphatic glands, vessels and nerves can be found (Morgan 1936). The parietal pelvic fascia becomes thickened at 2.5 cm above the leavator ani muscle to form two strong fascial supports to the rectum and they are known as the sphincteric portion of rectum. The ampullary portion of the rectum extends from the third sacral to the pelvic diaphragm at the levator ani insertion to form the anococcygeal ligament (Morgan 1936). The superior rectal artery receives arterial blood from the inferior mesenteric artery and supplies the upper part of the rectum and anal canal (Church, Raudkivi et al. 1987). The branches of the internal iliac arteries supply the middle and inferior rectal arteries, which then carry the arterial supply to the middle and lower rectum. The superior and inferior mesenteric veins and its tributaries, the superior rectal vein, bring venous blood from the upper part of the rectum and they flow into the portal circulation. The venous blood from the middle and lower rectum flows into the systemic circulation through the middle and inferior rectal vein which finally empty into the internal iliac vein (Moore 2010).
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4!
Ch 1. Introduction Lymphatics from the colon and proximal rectum are drained into the paraaortic lymph nodes, the lymph from distal rectum and anus drain either to the same route or the internal iliac and superficial inguinal lymph nodes. More of the mucosal goblets cells are histologically found in the caecum than the rectum. However, pyramidal paneth cells with eosinophilic granules are found only in the caecum and proximal right colon. The proximal colorectum has more cells than distal where cloverleaf-like crypts become deeper but scantily dispersed in the rectum. The rectum is a continuation of the sigmoid colon without mesocolon and appendices epiploicae. However, three taeniae coli coalesce in order to continuously support inner circular muscle uniformly when opening the anus during defecation (Saha 2011, Saha 2011).
1.3 General aspects of rectal cancer 1.3.1 Epidemiology 1.3.1.1 Incidence The Australian Institute of Health and Welfare (AIHW), a national health and welfare information & statistics agency collects vital cancer databases regularly. Based on their Australian cancer databases, the Australian Cancer Incidence and Mortality (ACIM) has published a book “About Cancer and Cancer Screening”, in 16 Dec 2010, reporting the various cancer incidences in Australia. Carcinoma of the large intestine and carcinoma of the rectum and anus are collectively known as colorectal cancer (CRC). CRC (14,234 cases) was second to prostate cancer
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Ch 1. Introduction (19,403 cases) reported in 2007 (ACIM 2007). Bowel cancer (15,840) [male (8,760) & female (7,080)] is the second most common cancer reported, following prostate cancer (18,560 cases) in 2012 (ACIM 2012). In males, CRC incidence rate (age-standardised) was 76 per 100,000 in 1991, which increased to 80 per 100,000 in 2000 but has reduced in the following years. It was 73 per 100,000 in 2012 that is a 4.7% reduction from the 1991 rate. In females, CRC incidence rate had lowered to between 51 and 55 per 100,000 from 1991 to 2009, due to behaviour differences that contribute to bowel cancer and also the different effects of obesity on males and females.
1.3.1.2 Mortality Australian Cancer Incidence and Mortality (ACIM) figures reported 42,844 deaths (majority in males 57%) from cancer (an average of 117 cancer deaths every day in 2012 compared to 109 in 2007). The third leading cause of cancer death in 2012 was bowel cancer (among males 2,205; among females 1,777). The greatest statistically significant percentage-point decrease in bowel cancer mortality rate was from 28 to 16 per 100,000. The age-standardised mortality rate of bowel cancer reduced to 41% for males (from 34 to 20 per 100,000) and 45% for females (from 24 to 13 per 100,000) between 1991 and 2010. Faecal occult blood screening detecting pre-cancerous polyps earlier and improved management were the main reasons for mortality reduction in colorectal cancer.
Malignant death due to rectal neoplasia (4,047) is second commonest cause of cancer related deaths with lung cancer (7,626) being the first (ACIM 2007). Increased mortality rate (60%) was reported in 2007 (39,884 deaths) compared to (24,922 deaths) in 1982.
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Ch 1. Introduction However, the mortality rate (age-standardised & all combined malignancy) was reduced by 16% [from (209 deaths) out of 100,000 people in 1982 to (176 deaths) per 100,000 people in 2007] (AIHW 2007).
1.3.1.3 Survival Survival from CRC declined slowly with age (72% for people aged 0-39 and 58% for those aged 80 and over). CRC had one of the largest absolute increased in survival together with prostate cancer, kidney cancer, non-Hodgkin lymphoma, breast cancer in female and melanoma where 5-year survival increased by 17% or more. The mortalityto-incidence ratio (MIR)! for Australia was 0.33 (33 deaths for every 100 new cases of cancer diagnosed in that year). This indicated that CRC patients in Australia have relatively better survival than in African regions, Melanesia and East Asia (MIR 0.70 70 deaths for every 100 new cases of cancer diagnosed in that year). The 5-year relative survival data for all cancers combined displayed females (64%) had a better survival rate than males (58%). However, 1982-1986 relative survival rates for males have improved from 41% to 58% in 1998-2004. A similar increase in survival trends was found in females (53% to 64%) during the same period. [Australasian Association of Cancer Registries (AACR) 2008; AIHW, CA].
1.3.1.4 Screening The National Bowel Cancer Screening Program aims to abate incidence, prevalence and deaths from colon and rectal malignancy. A faecal occult blood test (FOBT) is offered free to all eligible Australians, (people at age over 50 & period between start of 2011 to
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Ch 1. Introduction end of 2014) in the privacy of their own homes by the Australian Government. The World Gastroenterology Organization has published a new conceptual model of cascade for colon and rectal malignancy screening with evidence-based guidelines recently. The ultimate goal is earliest prevention, which can be achieved by identifying and removing the adenoma, a precursor lesion of the colorectal cancer, with a screening tool for early detection (Winawer, Krabshuis et al. 2011). Few countries in the world have effectively established a National Screening Program for the colon and rectal malignancy. Regular FOBT has detected early CRC (91 - 94%) but over diagnosis probability exhibits lower than expected that is around 6% and 9% [with 95%CI (for females) 2.5% - 21.3% and (for males) 1.9% - 44.7%]. This has proved that FOBT effectiveness in detection of CRC is valuable for long term (Luo, Cambon et al. 2012).
1.3.1.5 Risk factors Early detection and effective endoscopic resection of newly developed adenoma is the best possible prevention for colorectal malignancy. The risk factors for CRC have been documented to include people over 50 years, female gender, western type diet such as calorie-dense dietary intake (packaged and processed food, using microwave oven and plastic wares, availability of fast food, consuming carbonated drinks instead of natural water), a lifestyle with a lack of daily physical activities (modern lifestyle increases convenience and decreases motivation for daily exercise, even as simple as walking), family history of the colorectal cancer, Familial Adenomatous Polyposis (FAP) and/or Hereditary Nonpolyposis Colon Cancer (HNPCC) syndromes, adenomatous polyps and Irritable Bowel Disease (IBD), concomitance of diabetes mellitus (DM), central obesity and smoking. Smoking cigarettes is a significant risk for Taiwanese male colon and
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Ch 1. Introduction rectal malignancy. However, betel nut chewing and drinking alcohol are not significant risks among Taiwanese (Wu, Lee et al. 2009). Patients, over 60 years old with extra (synchronous) lesions removed during surgery for primary colon and rectal malignancy with diabetes mellitus (DM), were associated with a newly formed adenoma. Patients having risk factors (at least 3) survived (27.8%) with no adenoma for at least 5 years post surgery. The colonoscopic surveillance should be done at least every year for patients having more than 3 risk factors (Rex, Kahi et al. 2006). Patients without any risk factor have their survival rate about 90.4% and they live without adenomas. Therefore, follow up endoscopic surveillance should be done every 4 years for patients with risk factors (Rex, Kahi et al. 2006, Kawai, Sunami et al. 2012).
1.3.1.6 Statistics CRC is the most prevalent cancer with a five-year survival rate of between 30 - 70%. The improvement of survival depends on the establishment of bowel cancer screening. It is reduced by 1% in 2008 as a proportion of all cancer compared with 2007. It is the 4th most common cancer in people by age 30 - 49, 3rd most common cancer in age 50 - 64, 2nd most common cancer in age 65 and all ages in 2008. The overall malignancy incidence trends (1999 to 2008) showed incidence rates of bowel malignancy (males) have been stable. Incidence rates of breast and bowel malignancy (females) have also been stable too (Tracey, Kerr et al. 2010). The relative survival rate [2000 - 2006 (5 years)] was nearly the same [65% (males) and 66 % (females)] in people who were diagnosed with CRC. The top two cancers in males aged 65 years and over were prostate and bowel cancers, for the women in the same age group; breast and bowel were the major cancer organs. Bowel cancer risk started to
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Ch 1. Introduction increase in all people from age 40 - 44 years. Death rates from bowel cancer were higher in males compared to female. However mortality rates decreased between 1999 and 2008 by about 16.3% in male and 14.9% in female compared to 2007 (p < 0.05)(Tracey, Kerr et al. 2010).
1.3.2 Clinical and pathological features The screening only identifies a quarter of total CRC. Most cases are diagnosed after symptoms have developed. The symptoms may include abdominal pain and/or tenderness, rectal bleeding, diarrhoea, constipation, weight loss, anaemia (Hamilton, Round et al. 2005). World Health Organization (WHO) revised the digestive tumours classification as: neuroendocrine tumour G1 & neuroendocrine tumour G2, neuroendocrine carcinoma (subdivided into small cell type & large cell type) and mixed adenoneuroendocrine carcinoma (mixed tumours) in 2010 (Scoazec and Couvelard 2011).
1.3.2.1 Pathological types Most
carcinomas
in
the
colorectum
are
adenocarcinoma.
Microscopically
adenocarcinoma is very similar to the glandular cells found elsewhere in the gastrointestinal tract. Adenocarcinomas of the rectum are then divided into mucinous, signet-ring cell, adenosquamous and medullary types. Some adenocarcinomas secrete plenty of mucin (> 50% of tumour mass) as extracellular pools, which are mostly called mucinous adenocarcinoma. Signet-ring cells (>50% of tumour cells) adenocarcinomas have large mucin vacuoles that push the nuclei to the side (resembles ring) in the
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Ch 1. Introduction cytoplasm of cells (Hamilton, Rubio et al. 2000, Compton 2003). The rare adenosquamous variant demonstrates the partial squamous differentiation admixed with foci of adenocarcinoma and conspicuous atypia in the tumour cell. The rarer medullary carcinoma, non-gland forming, undifferentiated type consists of a nest of compact polygonal cells or a fine spicules network and/or organ like patterns and characteristic tumour infiltrating lymphocytes (TILs) (Jessurun, Romero-Guadarrama et al. 1999, Compton 2003). It is strongly associated with microsatellite instability (MSI), dysfunctional DNA repair gene and hereditary nonpolyposis colon cancer (HNPCC) (Hamilton, Rubio et al. 2000, Compton 2003).
Undifferentiated carcinoma is almost devoid of histological differentiation but is genetically distinct. Its microsatellite is highly unstable. There are mixed heterologous mesenchymal and carcinomatous elements in carcinosarcoma. Sarcomatoid carcinoma has spindle cells that are focally immunoreactive for cytokeratin. The rarest colorectal carcinomas are giant cell or pleomorphic, choriocarcinoma, pigmented, clear cell, stem cell, and paneth cell-rich or crypt cell carcinoma (Hamilton, Rubio et al. 2000).
1.3.2.2 Differentiation Histological grading for rectal adenocarcinoma is based on the tumour cells differentiation extent of glandular structure. Adenocarcinoma’s differentiation status can be
well
differentiated,
moderately
differentiated,
poorly
differentiated
and
undifferentiated (Compton 2000). The prognostic power of tumour grade can be obtained by applying a two-tiered stratification in most of the multivariable analyses (Compton 1999). Dichotomised low grade is comprised of well differentiated and moderately !
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Ch 1. Introduction differentiated with ≥50% gland formation & high grade is composed of poorly differentiated and undifferentiated with two folds) after 2 hours exposure of low gamma ray (1CGy/H) in irradiated human foreskin fibroblast cells (Zhang, Rohde et al. 2008).
However, the loss of MLH1
expression in human CRC cells (HCT116 – Lacking MLH1 and MSH6) is associated with the defect in activation of Nuclear factor-KappaB (NF-kB), a downstream effector of DNA transcription in DDR pathway (Habraken, Jolois et al. 2003). MSH2 deficient cells are unable to assemble Mre11 & Rad51 (important proteins for Homologous Recombination) after irradiation in the G2 phase of cell cycle (not in the S phase) (Franchitto, Pichierri et al. 2003). Both MLH1 and MSH2 necessitate the effective cell cycle arrest at G2/M phase transition and modulate the accurate base pairing at DSB ends that are restored by erroneous NHEJ process (Franchitto, Pichierri et al. 2003, Zhang, !
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Ch 1. Introduction Sheng et al. 2009). Besides, investigational reports suggested that MLH1 is a molecular target for p53, a central effector of cell cycle arrest and apoptosis in DDR (Chen and Sadowski 2005). It may be directly implicated in the early recognition of DNA damage due to radiation (Zhang, Sheng et al. 2009). Serial analysis of binding elements (SABE) identified PMS2 and MLH1 (p53-response elements within their first intron) that may serve as sensors in DDR (Chen and Sadowski 2005). Figure 1.25 displayed as summary: MLH1 and MSH2 might have impact on the surveillance of DNA damage injury, induction of cell cycle, DNA restoring mechanism activation, actual DNA repair, cell cycle arrest maintenance through programmed cell death (apoptosis), restoration of smaller misrepair and/or intermediate injuries & final integrity check of DNA prior to progression of cell cycle or programmed cell death (Zhang, Sheng et al. 2009).
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Ch 1. Introduction Figure 1.25 Roles of DNA MMR proteins in the pathway of DDR [modified and adapted from (Zhang, Sheng et al. 2009)]
1.7.8 Discovering predictive markers of radiation response to DXT in CRC The main therapeutic modality of colorectal cancer treatment is complete surgical resection. However, large rectal tumours residing in rigid bony pelvis restrict total surgical resection and require ionising radiation therapy as a supplemental preoperative therapy. However, the latter may have significant short term side effects (Marijnen, Kapiteijn et al. 2002) such as nausea, diarrhoea, skin erythema, acute urinary retention, proctitis to thromboembolic disease, lumbosacral plexopathy with pain and neurological symptoms and long term faecal incontinence (Peeters, van de Velde et al. 2005) and
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Ch 1. Introduction reduced sexual dysfunction (Marijnen, van de Velde et al. 2005). The histological regression in post-radiotherapied rectal cancer is always unpredictable. A careful consideration of the possible risks and benefits is essential in the use of preoperative radiotherapy for the management of rectal cancer patients because having undesirable and toxic post DXT effects may later contribute to a significant reduction in the quality of life of patients with CRC. It may be very hard to quantify the actual outcome of pre-operative radiotherapy with the current lack of effective and predictive biomarkers that can reflect likelihood of substantial DXT precise response in large rectal cancer. Exploring and finding the efficient predictive biomarkers for rectal cancer radiation sensitivity has proved exhaustively difficult. The discovery and identification of clinically reliable predictive biomarkers of radiosensitivity may finally enhance radiotherapy individualisation management. Therefore, the need to accomplish the clinically applicable and proven predictive biomarkers is a real necessity. In this thesis, MMR proteins such as MSH2, MSH6, MLH1 and PMS2 will be investigated as possible novel biomarkers in this context in large rectal cancer.
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1.8 Summary - Outline of Thesis Pre-operative DXT ameliorates local disease control and possibly disease free (DFS) and overall survival (OS) in rectal cancer sufferers. These benefits of DXT differ greatly between individuals, and a mean of forecasting their likelihood should facilitate a modified approach that evades the nonessential application of this potentially toxic radiotherapy to a subset of patients. Such predictive markers are currently unavailable.
1.8.1 Aim Preoperative deep X ray therapy is a supplemental treatment prior surgery to reduce tumour size. Curative treatment of the colorectal malignancy can be achieved by surgical resection of the original colorectal tumour mass and regional lymph nodes. Primary carcinomas arising from the rectum are first pre-exposed to ionising radiation (IR) to diminish the actual size of the tumour where satisfactory and complete removal by surgical procedures is extremely difficult in the first instance. Therefore, radiation sensitivity of specific tissue (such as the rectum which lies in the rigid bony pelvic cavity) is the most important gold standard prior to successful surgical intervention. This study will primarily evaluate the molecular biomarkers such as Plk1, γH2AX, MSI 4 markers (MSH2, MSH6, MLH1 & PMS2) that may predict radiosensitivity of rectal cancer tissue. For this purpose, the following steps should be achieved. First, identify the colorectal
cancer
cohort
at
Liverpool
Hospital,
then
set
up
database
for
clinicopathological features and finally establish the tissue microarray construct for rectal cancer.
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Ch 1. Introduction Principal aims of my thesis are (1) To evaluate Polo-Like Kinase 1 (Plk1) as a biomarker for radiosensitivity response of rectal cancer to DXT (2) To assess γH2AX as a biomarker for rectal cancer’s response of radiosensitivity (3) To appraise whether rectal cancers with microsatellite instability (MSI) show increased sensitivity to preoperative deep X ray therapy (4) To analyse the histological changes of rectal cancer after DXT and evaluate their relationship with clinical outcome measures like recurrence of local tumour, survival rates (both RFS & OS) of patients (5) To compare the prognostic usefulness of biomarkers such as Plk1, γH2AX and MMR proteins (MSH2, MSH6, MLH1 & PMS2), histological evaluation of tumour regression (TRG), and the pTNM staging system of the AJCC (6) Linkage of clinical, pathological and experimental data
1.8.2 Hypotheses This thesis will consequently examine the following hypotheses: (1) Rectal cancers with high expression of Polo-like kinase 1 (Plk1) and phosphorylated gamma histone 2AX (γH2AX), show reduced sensitivity to preoperative deep X ray therapy (2) Rectal cancers with microsatellite instability (MSI) display increased sensitivity to preoperative deep X ray therapy
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Ch 1. Introduction (3) DXT induces characteristic histological changes in rectal cancers and these may be predictive of clinical outcome measures such as recurrence of local tumour, survival rates (both RFS & OS) of patients (4) Expression of Plk1, γH2AX and MMR proteins (MSH2, MSH6, MLH1 & PMS2) in vitro and in human rectal cancer tissues may be related to histological changes of tumour regression (TRG), the pTNM staging system of the AJCC, 5 years disease/recurrence free survival (D/RFS) and overall survival (OS)
1.9 Overview of contents This thesis is divided into 8 further chapters. Chapter 2 outlines the materials and methods used. Chapter 3 presents clonogenic assay for radiation sensitivity in MSS and MSI cell lines, association of their sensitivity to different doses of radiation and survival fractions of MSS and MSI cell lines. Chapter 4 describes the clinicopathological features of the whole patient cohort of this thesis, including the grades of histopathological tumour regression (i.e. TRG) assigned to the individual cases and their correlation with outcome recurrence free survival (RFS) and overall survival (OS). Chapter 5 describes the IHC assessment of expression levels of polo-like kinase 1 in different rectal cancer tissues, with subsequent attempts at the association of Plk1 expression and their final outcome recurrence free & overall survival. Chapter 6 details the IHC assessment of expression levels of γH2AX in different rectal cancer tissues, with subsequent attempts at the correlation of γH2AX expression and their final outcome recurrence free & overall survival. These are followed in Chapter 7 by the IHC assessment of expression levels of each of the four DNA MMR proteins MSH2, MSH6, MLH1, and PMS2 and the patients’ final outcome recurrence free & overall survival (OS). Finally, the findings are
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Ch 1. Introduction summarised and concluded in chapter 8. Chapter 8 addresses the important question of whether these biomarkers were indicators of patient overall survival outcome following DXT and surgical resection of these tumours.
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Ch 2. Materials and Methods
CHAPTER 2
MATERIALS AND METHODS
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2.1 Overview The colorectal cancer (CRC) patients were first identified from the databases the Department of Anatomical Pathology, South Western Area Pathology Services and the Cancer Registry of the South Western Sydney Local Health District (SWSLHD). Data sheets in excel format were set up comprising age, sex, clinical and pathological features. The respective patients databases, archived histological slides and tissue wax blocks were examined.
2.2 Ethics statement Ethics approval was obtained on 22nd June 2012 from the Sydney South-West Area Health Service Ethics Review Committee, reference number HREC/12/LPOOL/102. The Cancer Registry of the South Western Sydney Local Health District (SWSLHD), the institutional review board waived the need for written informed consent from the participants as the project was deemed to be in the low or negligible risk category. The Biosafety and Radiation Ethics approval was obtained on 20th June 2012 from the Biosafety and Radiation Safety Committee,!Office of Research Services, University of Western Sydney Penrith (Kingswood campus), Building K.1.48, Locked Bag 1797, Penrith NSW 2751. Participants’ details were de-identified prior to analysis for protection of confidentiality. A retrospective study of patients with locally advanced node-positive, metastatic colorectal cancer diagnosed between 2000 and 2011 was performed. All data were extracted from an electronic database (Mosaiq Version 2.4, Elekta AB, Stockholm, Sweden) and clinical follow-up data were manually obtained. Variables of interest
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included pathological parameters such as tumour size, tumour differentiation, histological subtypes, positive nodes, grade, age and race. Outcomes of interest included progression-free/ disease free /or recurrence free survival (RFS) and overall survival (OS). The primary aim was to compare the expression of Plk1, γH2AX and MSI markers in tumour and normal tissue by immunohistochemistry (IHC) for variables and outcomes of interest, using Fisher’s exact test and Chi square test. The secondary aim was to examine these variables of interest for recurrence free survival & overall survival by performing Kaplan Meier univariate and Cox regression multivariate analyses. Data analysis was generated using IBM SPSS software (Version 21).
2.3 In vitro (cell line) study 2.3.1 Radiation sensitivity 2.3.1.1 Selection of cell lines Four cell lines of human CRCs - two each of microsatellite stable (MSS) such as T84 (MSS), COLO320DM (MSS) & microsatellite instable (MSI) HCT116 (MSI) and SW48 (MSI) were selected for radiation sensitivity, as listed in Table 2.1. Table 2.1 Colorectal Cancer (CRC) cell lines used in this thesis Cell line
Source
Description
T84 (MSS) Colo320DM (MSS) HCT 116 (MSI) SW48 (MSI)
(Cat No - 88021101) ECACC (Cat No - 87061205) ECACC (Cat No - 91091005) ECACC (Cat No - 89012702) ECACC
MSS CRC cell line (Lot 07F017) MSS CRC cell line (Lot 02H017) MSI CRC cell line (Lot 08B012) MSI CRC cell line (Lot 08B012)
ECACC – European Collection of Cell Culture (Sigma)
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2.3.1.2 Conditions of cell culture and storage The selected CRC cell lines were cultured in T75 cm2 flasks as a monolayer. They were maintained in exponential growth phase. CRC cells flasks were placed in 37°C incubator!(The BINDER GMBH - C 150 CO2) with 5% CO2 and 95% air until the cells were confluent enough to be exposed to ionising radiation. The cells were checked 24 to 48 hours after radiation treatment for cells counts and other experiments.! Routine testing for Mycoplasma was performed to exclude contamination. HCT 116 (MSI) cells were cultured in (89%) DMEM supplemented with (10%) foetal bovine serum (FBS) and (1%) antibiotics Penicillin/Streptomycin. SW48 (MSI) cells which grows in (89%) DMEM supplemented with (10%) FBS and (1%) Pen/Strept. T84 (MSS) cells were maintained in (89%) DMEM/F12 supplemented with (10%) FBS and (1%) Pen/Strept. Colo320DM (MSS) cells were seeded in (88%) RPMI1640 supplemented with (1%) L-Glutamine, (10%) FBS and (1%) Pen/Strept. When cells have reached the late log phase, cell density was determined using Haemocytometer cell counter (Thermo Fisher Scientific - SCH81501.01). Then total numbers of cells in the flasks were calculated, and the amount of freezing medium needed was determined. Cells should be resuspended in the freezing medium at 5,000,000 to 20,000,000 cells/mL.! Cells were then centrifuged (Thermo Scientific™ Heraeus Multifuge X3R Refrigerated Bench Top Centrifuge) in a 50 mL falcon tube at 10°C, 1000g for 15 minutes. First few passages of each cell lines were frozen and kept in the liquid nitrogen cryotank!(Taylor Wharton LABS20K).
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2.3.1.3 Thawing the cultured cells First, a vial of frozen cells was taken out from the liquid nitrogen cryotank and transferred to an ice bucket and then into a 37°C water bath (Thermoline 24 Litres Digital Uncirculated Water Bath - TWB-24D) in order to thaw out. Thawing generally takes only 1-2 minutes. Cryotube with the thawed cells can be spun down in Heraeus Multifuge X3R. The cells were resuspended in about 10 ml of an appropriate media [DMEM supplemented with 10% FBS, antibiotics for HCT 116 and SW48 cell lines, DMEM/F12 supplemented with 10% FBS, antibiotics for T84 cell line and RPMI1640 supplemented with 1% L-Glutamine, 10% FBS, antibiotics for Colo320DM cell line].! Cells were rinsed once with 10 - 15mls of appropriate media and spun at 10°C, 1000 rpm, 5 mins [to wash out toxic 10% dimethylsulfoxide (DMSO)]. Afterward, the cells pellet was gently resuspended in an appropriate media. The cells were then seeded in a fresh T25 flask.
2.3.1.4 Trypsinizing the cultured cells When splitting the cells, the culture medium was removed by electronic pipette (Thermo Scientific Matrix) and the residual serum in the cell monolayer was eliminated by rinsing the cell monolayer with 6 ml of the sterile Dulbecco's phosphate buffer saline (DPBS Invitrogen) for at least 2 times.! Trypsin-EDTA solution (2.5 ml of a 0.25%, Invitrogen) was slowly added into the T25 flask and covered the cell monolayer. The T25 flask was incubated at room temperature for 30 - 45 seconds.! T25 flask was then incubated at 37°C for 5 - 10 minutes in the incubator. The flask should not be shaking while waiting for the cells to detach. Appropriate media (10mls) was added into the flask and the cells were then resuspended in the media by the gentle pipette action.
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Hemocytometer was used to count the cells. Cells were seeded into a new T75 culture flask with approximately 106 cells in 15mls of the appropriate media. Cultures were then incubated at 37°C in a 5% CO2/95% air environment of incubator. Cells should be split upon their confluency (always check and renew the media approximately every 3 days).
2.3.1.5 Freezing the cultured cells Cells to be frozen should be in late log phase growth. First, cells were trypsinized and harvested in the normal way in the Bio-cabinet (Safemate 1.2). Cells Count was done in Haemocytometer (200 µl aliquot) and the total cell number was determined. The volume of media (1.8 mL of appropriate media + 200 µL of DMSO) required was calculated to get a final freezing density of 3.0 x 107 cells/mL. The cells were spun in a Heraeus Multifuge X3R centrifuge at 10°C, 1000 rpm for 10 minutes. The supernatant was aspirated off and the cells pellet was resuspended in 1 mL of freezing media prepared. Appropriate media such as DMEM/FBS/Antibiotics for HCT116 and SW48, DMEM/F12/FBS/Antibiotics for T84 and RPMI1640/L-Glutamine/FBS/Antibiotics for Colo320DM were used for the cells to be frozen. The cells were resuspended in 1:1 with prepared freezing media (freshly prepared earlier). The media was added dropwise and mixed well after each addition. The cell suspension was aseptically aliquoted into the sterile freezing vials. Each vial was labelled with the date and cell type/clone number, and placed the vials into a Styrofoam/plastic container. The vial was stored overnight at -80°C Freezer (Thermo Scientific Freezer Model 906) then transferred to the liquid nitrogen cryotank the next day.
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2.3.1.6 Cell counting in Haemocytometer Cells were trypsinized and harvested in the normal way. Trypan Blue stain (0.4% fresh & filtered) in Dulbecco's phosphate buffered saline (DPBS) was used in the cell counting. Equal volumes of trypan blue stain (100µl) and a well-mixed cell suspension (100 µl - not too vigorous) were prepared. Trypan blue/cell mix (approximately 10µl) was pipetted at the edge of the coverslip and allowed for the mix to run under the cover slip on Neubauer (0.100 mm Depth & 0.0025 mm2) glass, the Haemocytometer (Hirschmann Laborgerate – SCH81501.01). The haemocytometer grid was visualised under the Olympus microscope (Olympus CK2 – Optical Co Ltd Japan). Live cells appeared colourless and bright (refractile) under phase contrast. Dead cells stained blue and were non-refractile. Viable (live) and dead cells were counted in one or more large corner squares and cell counts were recorded. It may be necessary to count more than one large corner square.!Average number of cells in one large square was multiplied to dilution factor [usually (2) because 1:1 dilution with trypan blue] and 104 to obtain cell concentration per mL. However, there may be a need to further dilute (or) concentrate cell suspensions. Percentage (%) of viable cells counted was calculated by multiplying with 100.
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Figure 2.1 Haemocytometer
Volume of one large corner square was 1 mm2 x 0.1 mm = 0.1 cm x 0.1 cm x 0.01cm = 10-4 cm3 or 10-4 mL
2.3.1.7 Radiation exposure Cells were irradiated at room temperature with a dose of 0.5, 2.0 & 5.0Gy using Pantak Therapax DXT300 (Pantak Inc, USA – SN300). Radiation doses were delivered using a 300kVp beam (HVL = 3.8 mm Cu) and 50 cm source to focus distance radiation cone in the Liverpool Cancer Therapy Centre, Liverpool Hospital. The Pantak Therapax DXT300 has been calibrated using IPEMB code of practice (Klevenhagen, Aukett et al. 1996) for the determination of absorbed dose for X-rays with an uncertainty in delivery of ±3% and a variation across the field of ~2% giving an overall dose uncertainty of ± 5%. T-75 flasks were irradiated one at a time by placing the flask in between the radiation cone and Certified Therapy Grade Solid Water® (Gammex 457 - CTG, Gammex Inc, !
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USA). Solid water was used as a back scattering material. An Advanced Markus ion chamber (PTW Freiburg, Germany, SN115) was placed 0.5 cm below the solid water surface to check radiation dose constancy. The homogeneity of radiation dose delivered to the flask was estimated at ± 5%. Each cell line was exposed to the different doses of radiation at 0.5, 2.0 and 5.0Gy. Previous results (Shin, Jalaludin et al. 2011) have indicated that any greater level of radiation than the latter is too high a dose to effectively discriminate the subsequent survival levels between the CRC cell lines.
2.3.1.8 Clonogenic Assay or Colony Forming Assay It is an in vitro cell survival (clonogenic/colony forming) assay determining cell reproductive death. The assay examines all cells in the population for their limitless division ability. A colony can arise from a single cell after treating with ionizing radiation. A colony contains at least 50 cells (Franken, Rodermond et al. 2006). Only a minute fraction of the seeded cell preserves the capacity to reproduce colonies. Colorectal cancer cells were grown in the appropriate medium to form colonies in 1-3 weeks (especially 10 days) before and after radiation treatment (Franken, Rodermond et al. 2006). Colonies were then fixed with glutaraldehyde (6% v/v), stained with crystal violet (0.05% w/v) and counted manually under the naked eye. The accurate count of cell is quite necessary to seed with the appropriate growth medium so as to obtain the plating efficiency (PE) after radiation treatment for a proper cell survival calculation.
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The CRC cells (~ 100,000 cells/ flask) were irradiated in 75m2 flasks (at least 3 replicates). Then the cells were trypsinised and replated with three replicates on cell culture dishes (60 x 15mm - 22.1cm2 surface area) with accurate count of cells in each dose of radiation (500 cells - 0Gy, 500 cells - 0.5Gy, 1000 cells - 2.0Gy and 5000 cells 5.0Gy). The replating of cells is done immediately after radiation and it is known as immediate plating (IP). The flasks were kept in 37°C CO2 incubator (Binder GMBH C150) with 5% CO2 and left the cells to grow and form large colonies within 10 days (1-3 weeks).
2.3.1.8.1 Plating Efficiency and Surviving Fraction The plating efficiency (PE) is the ratio of the number of colonies formed to the number of cell plated expressed in percentage. Number of colonies formed PE = ------------------------------------ X 100% Number of cells seeded The surviving fraction (SF) is the ratio of the number of colonies that formed after radiation treatment of the cells to the number of cell plated and plating efficiency (PE the number of cells plated expressed in percentage). Number of colonies formed after radiation treatment SF = -----------------------------------------------------------------Number of cells seeded X PE
2.3.1.8.2 Analysis of the radiation dose survival curves by Linear Regression Survival (S) data after a radiation dose (D) are plotted against by a weighted, stratified, linear regression according to the linear-quadratic formula [S(D)/S(0) = exp (αD+βD2)] (Barendsen 1997, Barendsen, Van Bree et al. 2001, Franken, Van Bree et al.
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2001, Franken and Barendsen 2014). D refers to the radiation dose, S(D) is the surviving fraction of cells exposed to the radiation dose, S(0) symbolizes the survival rate for non-irradiated (0Gy) cells, α (Gy-1) is the initial slope of cell survival curve parameter, β (Gy-1) represents the cumulative damage contribution resulting from interaction of two or more lesional induction by different ionizing particles (Van Oorschot, Oei et al. 2014). The linear parameter (α) and quadratic parameter (β) are determined by creating an SPSS data file for each radiation dose and survival curve separately (without and with radiation treatment) with the following variables such as number of the experiments (at least three separate experiments), doses (0, 0.5, 2.0 & 5.0Gy), number of cells plated (500 cells - 0Gy, 500 cells - 0.5Gy, 1000 cells - 2.0Gy and 5000 cells - 5.0Gy), number of surviving colonies (252 colonies after 0.5Gy radiation) and PEs of that experiment were calculated.
2.3.1.8.3 Linear Regression for “dose” & “D2” In SPSS data file, the following transformations (under transform/compute) were performed to obtain D2 (quadratic term) by transforming Target variable D2 = dose*dose, to obtain S (survival) by transforming Target variable S = Ln (Natural Log) (colonies/cells) – Ln (PE), to obtain W (Weight of colonies found) by transforming Target variable W = colonies*cells/(cells – colonies). After performing the analysis, a linear regression (under analyse/regression/linear) was carried out with “S” as a dependent variable, “dose” and “D2” as independent variables and “W” under WLS weight. Confidence intervals and R2 changed were included in regression analysis (under statistics). The constant in the equation was not included (under option) and
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computed to get the relationship between radiation & dose and survival. The output of model summary and the regression (ANOVA) output hypothesized that there is no relationship (P ≥ 0.05) between radiation dose and survival. If P-value for the output of regression was
