O6-methylguanine-DNA Methyltransferase Promoter Methylation in Patients with Rectal Adenocarcinoma After Chemoradiotherapy Treatment: Clinical Implications

Balkan Med J 2019;36:283-6 B ie Repo 283 O6-me hylguanine-DNA Me hyl ans e ase P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men : Clinical Implica ions 1Cen e o Cance Resea ch and Cell Biology, Queen’s Uni e si y Bel as , Bel as , UK 2Ins i u e o Biopa hology and Regene a i e Medicine, Cen e o Biomedical Resea ch, Uni e si y o G anada, G anada, Spain 3Depa men o Radia ion Oncology, Uni e si a y Hospi al Vi gen de la Vic o ia, Málaga, Spain 4Depa men o Ana omy and Emb yology, Uni e si y o G anada, G anada, Spain 5Biosani a y Ins i u e o G anada (ibs. GRANADA), SAS-Uni e sidad de G anada, G anada, Spain 6Medical Oncology Se ice, Uni e si a y Hospi al Vi gen de las Nie es, G anada, Spain Jaime A. Oli e 1,2*, Jaime Gómez-Millán3*, Jose A. Medina3, Lau a Cabeza2,4,5, Glo ia Pe azzoli2,5, C is ina Jimenez-Luna2, Ke in Doello6, Raúl O iz2,4,5 Aims: To analyze he clinical ele ance o O6-me hylguanine- DNA me hyl ans e ase in ec al adenoca cinoma ea ed wi h chemo adio he apy ollowed by su ge y. Me hods: Tissue samples om 29 ec al adenoca cinoma pa ien s we e ob ained a e chemo adio he apy. O6-me hylguanine-DNA me hyl ans e ase p omo e me hyla ion s a us was es ablished by me hyla ion-speci ic polyme ase chain eac ion. O6-me hylguanine- DNA me hyl ans e ase p o ein le els we e de e mined by immunohis ochemis y. Clinicopa hologic a iables, including ea men eg ession g ade, ecu ence, lymph node in asion, and s age and di e en ia ion g ade o he umo , we e de e mined. Resul s: The O6-me hylguanine-DNA me hyl ans e ase gene p omo e was me hyla ed in 81.5% o samples. Mos pa ien s (88.9%) showed low O6-me hylguanine-DNA me hyl ans e ase p o ein exp ession. O6-me hylguanine-DNA me hyl ans e ase me hyla ion s a us was no co ela ed wi h any o he clinicopa hological a iables de e mined in ec al adenoca cinomas selec ed o chemo adio he apy. Conclusion: O6-me hylguanine-DNA me hyl ans e ase me hyla ion s a us is no co ela ed wi h clinicopa hologic a iables examined in ec al adenoca cinoma selec ed o chemo adio he apy, al hough i s ole as a bioma ke awai s u he in es iga ion. Keywo ds: Chemo adio he apy, O6-me hylguanine-DNA me hyl ans e ase, ec al adenoca cinoma Pa ien s wi h ec al adenoca cinoma s age II-III a e usually ea ed wi h p eope a i e chemo adio he apy based on 5- luo ou acil o capeci abine. Howe e , li le da a on molecula bioma ke s o he p ognosis and ea men esponse in colo ec al cance has been ob ained (1). The enzyme O6-me hylguanine-DNA me hyl ans e ase (MGMT), which elimina es me hyl g oups in he O6-guanine posi ion a oiding G:C o A:T ansi ions, has also been ela ed o colo ec al cance (2,3). MGMT p e en s cell dea h due o cy o oxic d ugs by epai ing DNA, bu i can be silenced by epigene ic me hyla ion (4). Loss o MGMT exp ession has been de ec ed in colo ec al cance and associa ed wi h G o A ansi ion in he p53, K- as, and PIK3CA genes (5). P e ious s udies sugges ed ha MGMT p omo e me hyla ion s a us was ela ed o glioblas oma ea men ailu e (6). In his s udy, MGMT exp ession and MGMT p omo e me hyla ion s a us we e e alua ed in ec al adenoca cinoma pa ien s a e chemo adio he apy ea men in o de o de e mine hei s a us and ele ance as p ognos ic bioma ke s. MATERIALS AND METHODS Clinical his o y and issue samples Twen y-nine ec al adenoca cinoma pa ien s (s age II-III) who we e candida es o p eope a i e chemo adio he apy we e ec ui ed Add ess o Co espondence: Raúl O iz, Ins i u e o Biopa hology and Regene a i e Medicine (IBIMER), School o Medicine, Uni e si y o G anada, 18100 G anada, Spain Phone: +34-958-249322 e-mail: oquesa@ug .es ORCID: o cid.o g/0000-0001-8409-5235 Recei ed: 25 Decembe 2018 Accep ed: 11 June 2019 • Bo h au ho s con ibu ed equally o his a icle • DOI: 10.4274/balkanmedj.galenos.2019.2018.12.93 A ailable a www.balkanmedicaljou nal.o g Ci e his a icle as: Oli e JA, Gómez-Millán J, Medina JA, Cabeza L, Pe azzoli G, Jimenez-Luna C, e al. O6-me hylguanine-DNA Me hyl ans e ase P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men : Clinical Implica ions. Balkan Med J 2019;36:283-6 ©Copy igh 2019 by T akya Uni e si y Facul y o Medicine / The Balkan Medical Jou nal published by Galenos Publishing House. a e hey ga e in o med consen (Biomedical In es iga ion E hic Commi ee; Se icio Andaluz de Salud). All pa ien s we e e alua ed be o e ea men (physical examina ion wi h a digi al ec al examina ion, colonoscopy and biopsy, ches X- ay, abdominopel ic scan and/o endo ec al ul asound, and magne ic esonance image o he pel is). These pa ien s we e ea ed wi h pel ic adio he apy (46-50 Gy in 2 Gy ac ions) and in a enous 5- luo ou acil (5-day cycles o 500 mg/m2 5- luo ou acil e e y 21 days) o capeci abine (4 cycles o 1250 mg/m2 capeci abine e e y 12 h o 14 days) ollowed by su ge y ( o al meso ec al excision) 6 weeks a e chemo adio he apy. Tumo samples we e ob ained om each pa ien om endoscopic biopsy be o e chemo adio he apy. The chemo adio he apy esponse was s aged his opa hologically on he basis o umo eg ession g ade (Manda d’s classi ica ion: g ade I and II = comple e/pa ial eg ession and g ade III, IV, o V = no eg ession) (7). Two expe pa hologis s e alua ed an in a-ope a i e sample a e chemo adio he apy. Demog aphic da a (sex and age) we e ob ained. In addi ion, clinicopa hological a iables, including umo di e en ia ion g ade, umo s age, ea men eg ession g ade, ecu ence, and lymph node in asion, we e analyzed. Me hyla ion-speci ic polyme ase chain eac ion and immunohis ochemis y DNA was ex ac ed om pa a in-embedded issues by using a Chemagic MSM I obo (Chemagen, Ge many, Baesweile ). Me hyla ion pa e ns in CpG islands o he MGMT p omo e we e de e mined by me hyla ion-speci ic polyme ase chain eac ion as p e iously desc ibed (8). Samples we e classi ied as me hyla ed (ampli ica ion p oduc wi h M o bo h M and UM p ime s) and unme hyla ed (ampli ica ion wi h UM p ime s only). Immunohis ochemical analysis was pe o med wi h a Dako Au os aine EnVision™ FLEX Sys em ki (Agilen Technologies) and he esul s e alua ed by wo expe ienced pa hologis s. MGMT (1:50, San a C uz Bio echnology, Inc., Heidelbe g, Ge many) mAb was used as he label and 3.3'-diaminobenzidine as he ch omogen subs a e. Coun e s aining was pe o med wi h hema oxylin (blue). As p e iously desc ibed by Oli e e al. (2), MGMT s aining we e sco ed and g ouped as low (<50%) and high exp ession (≥50%). S a is ical analysis SPSS e sion 15.0 (IBM, Chicago, IL) was used o da a analyses. Associa ions be ween p omo e gene me hyla ion s a us and clinicopa hologic a iables we e analyzed by Fishe ’s exac es . Resul s we e conside ed s a is ically signi ican i p<0.05. RESULTS The clinical pa ien cha ac e is ics a e summa ized in Table 1. The mean age was 64.43±12.24 yea s ( ange, 33-83 yea s); 75.9% (22/29) o pa ien s we e male and 24.1% (7/29) we e emale. The median ollow-up pe iod was 20.53±9.07 mon hs. No pa ien died due o ec al cance , and disease ecu ence was obse ed in 13.8% (4/29). MGMT p omo e me hyla ion s a us could be de e mined in 93.1% o specimens (27/29). O he 27 pa ien s, he MGMT gene p omo e was me hyla ed 81.5% (22/27) (Figu e 1). Immunohis ochemical analysis (Figu e 2) showed low MGMT p o ein exp ession in mos pa ien s (88.9%). Only 11.1% o pa ien s showed high exp ession o MGMT. We also examined he associa ion be ween MGMT p omo e me hyla ion and clinicopa hologic ea u es. MGMT p omo e me hyla ion s a us was no associa ed wi h sex, umo di e en ia ion, o umo s age. Fu he mo e, no associa ion be ween MGMT me hyla ion and he clinicopa hologic a iables examined was de ec ed (Table 2). 284 Balkan Med J, Vol. 36, No.5, 2019 Oli e e al. MGMT P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men TABLE 1. Cha ac e is ics o ec al cance pa ien s All pa ien s (n=29) Sex Male 22 (75.9%) Female 7 (24.1%) Age ≥50 yea s 27 (93.1%) <50 yea s 2 (6.9%) Tumo di e en ia ion g ade Well-mode a ely 27 (93.1%) Poo ly 2 (6.9%) Tumo s age II 10 (34.5%) III 19 (65.5%) Lymph node me as asis Yes 19 (65.5%) No 10 (34.5%) Recu ence Yes 4 (13.8%) No 25 (86.2%) FIG. 1. Me hyla ion-speci ic polyme ase chain eac ion analysis o he O6-me hylguanine- DNA me hyl ans e ase p omo e in ec al adenoca cinoma issue samples. DNA was ex ac ed by using a Chemagic MSM I obo (Chemagen, Ge many, Baesweile ), dena u ed, and pu i ied wi h an EpiTec Bisul i e ki (Qiagen, USA, Ma yland). P ime sequences o he unme hyla ed eac ion we e 5`-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3` ( o wa d p ime ) and 5`-AACTCCACACTCTTCCAAAAACAAAACA-3` ( e e se p ime ) and o he me hyla ed eac ion we e 5`-TTTCGACGTTCTAGGTTTTCGC-3` ( o wa d p ime ) and 5`-GCACTCTTCCGAAAACGAAACG-3` ( e e se p ime ). Polyme ase chain eac ion-ampli ied p oduc s we e elec opho esed on 3% aga ose gels, isualized by s aining wi h e hidium b omide, and examined unde ul a iole illumina ion. The ep esen a i e image depic s O6-me hylguanine-DNA me hyl ans e ase p omo e me hyla ion analysis o 18 samples. Pa ien s wi h me hyla ed p omo e s showed ampli ica ion in bo h unme hyla ed and me hyla ed lanes o he me hyla ed lane alone. The lack o a band in he lane co esponding o me hyla ion-speci ic p ime s o ec al cance sample 2, 6 o 18 e lec s he absence o O6-me hylguanine-DNA me hyl ans e ase p omo e me hyla ion. M: me hyla ed; UM: unme hyla ed DISCUSSION The ela ionship be ween MGMT and colo ec al cance emains unclea , and esul s ha e been con adic o y. Whe eas Nilsson e al. (9) ound a lowe isk o ecu ence in 5- luo ou acil- ea ed colo ec al cance pa ien s wi h a me hyla ed e sus unme hyla ed MGMT p omo e , Shima e al. (3) concluded ha nei he MGMT p omo e me hyla ion no loss o MGMT exp ession is a use ul p ognos ic bioma ke . Sinha e al. (10) obse ed ha MGMT me hyla ion was associa ed wi h s age III in spo adic colo ec al cance cases. Recen ly, he me hyla ion s a us o MGMT has been co ela ed wi h pa hologic comple e esponse in colo ec al cance pa ien s (11). Shalaby e al. (12) showed a good co ela ion be ween MGMT me hyla ion and down egula ion o i s mRNA exp ession. Al hough hese au ho s p oposed MGMT me hyla ion as a new bioma ke o di e en ia e benign and malignan ec al umo s, no ela ion be ween MGMT me hyla ion and clinicopa hological ea u es was de ec ed. Sun e al. (13) showed ha e en he MGMT p omo e me hyla ion s a us o plasma-cell- ee DNA was associa ed wi h a be e umo esponse. In addi ion, MGMT me hyla ion le els in he blood we e simila o hose in ec al cance issues (12). Ou esul s showed ha he me hyla ion s a us o he MGMT p omo e was no associa ed wi h a be e ea men esponse. Howe e , he ole o MGMT p omo e me hyla ion s a us as an ea ly bioma ke o colo ec al cance has no ye been es ablished, despi e se e al s udies in colo ec al adenoma and adenoca cinoma (14,15). In ac , Balkan Med J, Vol. 36, No.5, 2019 Oli e e al. MGMT P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men 285 TABLE 2. Co ela ion be ween MGMT me hyla ion s a us and demog aphic and clinicopa hologic a iables Va iables MGMT me hyla ion s a us: n (% o pa ien s) Unme hyla ed Me hyla ed Sex Male 3 (11.1) 17 (63) Female 2 (7.4) 5 (18.5) Tumo di e en ia ion g ade Well-mode a ely 4 (14.8) 21 (77.8) Poo ly 1 (3.8) 1 (3.8) Tumo s age II 1 (3.8) 9 (33.3) III 4 (14.8) 13 (48.1) Recu ence Yes 3 (11.1) 20 (74) No 2 (7.4) 2 (7.4) T ea men eg ession g ade I, II 1 (3.7) 9 (33.3) III, IV, V 4 (14.8) 13 (48.1) Lymph node me as asis Yes 4 (14.8) 13 (48.1) No 1 (3.8) 9 (33.3) MGMT: O6-me hylguanine-DNA me hyl ans e ase FIG. 2. a-c. Immunohis ochemical s aining o ec al adenoca cinoma issue samples wi h a mouse monoclonal an ibody agains human O6-me hylguanine-DNA me hyl ans e ase p o ein. Fo malin- ixed, pa a in-embedded ec al cance samples we e s ained wi h an an ibody agains O6-me hylguanine-DNA me hyl ans e ase (see Me hods). O6- me hylguanine-DNA me hyl ans e ase s aining o umo cells was sco ed and g ouped as low exp ession (<50%) (-, +, and ++ sco es) and high exp ession (≥50%) (+++ and ++++ sco es). The in ensi y o O6-me hylguanine-DNA me hyl ans e ase s aining was sco ed as low o high. The igu e shows ep esen a i e pho omic og aphs o slides illus a ing di e en pe cen ages o O6-me hylguanine-DNA me hyl ans e ase exp ession. A umo wi h no de ec able O6-me hylguanine-DNA me hyl ans e ase exp ession (a); A posi i e umo wi h low O6-me hylguanine-DNA me hyl ans e ase exp ession (˂50%) (b); A posi i e umo wi h high O6-me hylguanine-DNA me hyl ans e ase exp ession (>50%) (20× magni ica ion) (c). Side is e al. (16) showed ecen ly ha he e was no associa ion be ween he s a us o MGMT exp ession and pa hological ea u es, including esponse o neo-adju an he apy. Fu u e esea ch will be needed o elucida e he ela ionship be ween hese bioma ke s and ec al cance ea men . Con lic o In e es : No con lic o in e es was decla ed by he au ho s. Financial Disclosu e: No inancial disclosu e was decla ed by he au ho s. REFERENCES 1. Huh JW, Lee JH, Kim HR. P e ea men exp ession o 13 molecula ma ke s as a p edic o o umo esponses a e neoadju an chemo adia ion in ec al cance . Ann Su g 2014;259:508-15. 2. Oli e JA, O iz R, Melguizo C, Al a ez PJ, Gómez-Millán J, P ados J. P ognos ic impac o MGMT p omo e me hyla ion and MGMT and CD133 exp ession in colo ec al adenoca cinoma. BMC Cance 2014;14:511. 3. Shima K, Mo ikawa T, Baba Y, Nosho K, Suzuki M, Yamauchi M, e al. MGMT p omo e me hyla ion, loss o exp ession and p ognosis in 855 colo ec al cance s. Cance Causes Con ol 2011;22:301-9. 4. Jacin o FV, Es elle M. MGMT hype me hyla ion: a p ognos ic oe, a p edic i e iend. DNA Repai (Ams ) 2007;6:1155-60. 5. Ros y C, Young JP, Walsh MD, Clendenning M, Sande son K, Wal e s RJ, e al. PIK3CA ac i a ing mu a ion in colo ec al ca cinoma: associa ions wi h molecula ea u es and su i al. PLoS One 2013;8:e65479. 6. Aoki K, Na sume A. O e iew o DNA me hyla ion in adul di use gliomas. B ain Tumo Pa hol 2019;36:84-91. 7. Suá ez J, Ve a R, Balén E, Gómez M, A ias F, Le a JM, e al. Pa hologic esponse assessed by Manda d g ade is a be e p ognos ic ac o han down s aging o disease- ee su i al a e p eope a i e adiochemo he apy o ad anced ec al cance . Colo ec al Dis 2008;10:563-8. 8. Melguizo C, P ados J, González B, O iz R, Concha A, Al a ez PJ, e al. MGMT p omo e me hyla ion s a us and MGMT and CD133 immunohis ochemical exp ession as p ognos ic ma ke s in glioblas oma pa ien s ea ed wi h emozolomide plus adio he apy. J T ansl Med 2012;10:250. 9. Nilsson TK, Lö -Öhlin ZM, Sun XF. DNA me hyla ion o he p14ARF, RASSF1A and APC1A genes as an independen p ognos ic ac o in colo ec al cance pa ien s. In J Oncol 2013;42:127-33. 10. Sinha R, Hussain S, Meh o a R, Kuma RS, Kuma K, Pande P, e al. K as gene mu a ion and RASSF1A, FHIT and MGMT gene p omo e hype me hyla ion: indica o s o umo s aging and me as asis in adenoca cinoma ous spo adic colo ec al cance in Indian popula ion. PLoS One 2013;8:e60142. 11. Jeong JH, Hong YS, Pa k Y, Kim J, Kim JE, Kim KP, e al. Phase 1 S udy o P eope a i e Chemo adia ion The apy Wi h Temozolomide and Capeci abine in Pa ien s Wi h Locally Ad anced Rec al Cance . In J Radia Oncol Biol Phys 2016;96:289-95. 12. Shalaby SM, El-Shal AS, Abdelaziz LA, Abd-Elba y E, Khai y MM. P omo e me hyla ion and exp ession o DNA epai genes MGMT and ERCC1 in issue and blood o ec al cance pa ien s. Gene 2018;644:66-73. 13. Sun W, Sun Y, Zhu M, Wang Z, Zhang H, Xin Y, e al. The ole o plasma cell- ee DNA de ec ion in p edic ing p eope a i e chemo adio he apy esponse in ec al cance pa ien s. Oncol Rep 2014;31:1466-72. 14. Lee KH, Lee JS, Nam JH, Choi C, Lee MC, Pa k CS, e al. P omo e me hyla ion s a us o hMLH1, hMSH2, and MGMT genes in colo ec al cance associa ed wi h adenoma-ca cinoma sequence. Langenbecks A ch Su g 2011;396:1017-26. 15. Kycle W, Sza zyńska B, Loziński C, Ko ski K, Lampe ska K. Analysis o O6- me hylguanine-DNA me hyl ans e ase me hyla ion s a us in spo adic colon polyps. Rep P ac Oncol Radio he 2012;17:13-8. 16. Side is M, Moo head J, Diaz-Cano S, Haji A, Papag igo iadis S. KRAS Mu an S a us May Be Associa ed wi h Dis an Recu ence in Ea ly-s age Rec al Cance . An icance Res 2017;37:1349-57. 286 Balkan Med J, Vol. 36, No.5, 2019 Oli e e al. MGMT P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men