Langenbeck's Archives of Surgery

, Volume 396, Issue 7, pp 1017–1026 | Cite as

Promoter methylation status of hMLH1, hMSH2, and MGMT genes in colorectal cancer associated with adenoma–carcinoma sequence

  • Kyung-Hwa Lee
  • Ji-Shin Lee
  • Jong-Hee Nam
  • Chan Choi
  • Min-Cheol Lee
  • Chang-Soo Park
  • Sang-Woo Juhng
  • Jae-Hyuk LeeEmail author
Original Article



Epigenetic silencing of the DNA mismatch repair genes has been poorly described in colorectal carcinomas showing the classic adenoma–carcinoma pathway of carcinogenesis. The aim of this study was to investigate the methylation status of MutL homolog 1 (hMLH1), MutS homolog 2 (hMSH2), and O-6-methylguanine-DNA methyltransferase (MGMT) in a series of colorectal carcinomas that contain both adenomas and carcinomas.


Promoter methylation of hMLH1, hMSH2, and MGMT was evaluated in normal mucosa, adenoma, and carcinoma samples from 112 colorectal cancer patients. Methylation was assessed by bisulfite modification and methylation-specific PCR. Expression of the gene products was also examined by immunohistochemistry.


Of the 112 adenomas, methylation was detected for hMLH1 (2, 1.8%), hMSH2 (9, 8.0%), and MGMT (38, 33.9%). In the carcinoma samples, methylation was seen in hMLH1 (2, 1.8%), hMSH2 (15, 13.4%), and MGMT (53, 47.3%). In normal mucosa, hMSH2 (6, 5.4%) and MGMT (12, 10.7%) were methylated, whereas hMLH1 was not. Immunohistochemical analysis revealed abnormal hMLH1 (14, 12.5%), hMSH2 (11, 9.8%), and MGMT (53, 47.3%) expression with a significant correlation between aberrant MGMT methylation and a loss of MGMT expression.


These data suggest that CpG island methylation in hMSH2 and MGMT, but not hMLH1, is closely related to carcinogenesis in colorectal carcinomas presenting with a conventional adenoma–carcinoma sequence. Therefore, the detection of hMSH2 and MGMT methylation may have clinical significance in the evaluation of colon cancer patients and in tumor-specific management of the disease.


Methylation DNA mismatch repair hMLH hMSH2 MGMT Colorectal carcinoma 



This study was sponsored by grants from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0720570) and the Brain Korea 21 Project, Center for Biomedical Human Resources at Chonnam National University.

Conflicts of interest



  1. 1.
    Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60(5):277–300PubMedCrossRefGoogle Scholar
  2. 2.
    Grady WM, Carethers JM (2008) Genomic and epigenetic instability in colorectal cancer pathogenesis. Gastroenterology 135(4):1079–1099PubMedCrossRefGoogle Scholar
  3. 3.
    Lynch HT, de la Chapelle A (2003) Hereditary colorectal cancer. N Engl J Med 348(10):919–932. doi: 10.1056/NEJMra012242 PubMedCrossRefGoogle Scholar
  4. 4.
    Thiagalingam S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J, Willson JK, Markowitz S, Hamilton SR, Kern SE, Kinzler KW, Vogelstein B (1996) Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet 13(3):343–346. doi: 10.1038/ng0796-343 PubMedCrossRefGoogle Scholar
  5. 5.
    Imai K, Yamamoto H (2008) Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics. Carcinogenesis 29(4):673–680PubMedCrossRefGoogle Scholar
  6. 6.
    Pegg AE (1990) Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res 50(19):6119–6129PubMedGoogle Scholar
  7. 7.
    Esteller M, Risques RA, Toyota M, Capella G, Moreno V, Peinado MA, Baylin SB, Herman JG (2001) Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis. Cancer Res 61(12):4689–4692PubMedGoogle Scholar
  8. 8.
    Harris LC, Potter PM, Tano K, Shiota S, Mitra S, Brent TP (1991) Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene. Nucleic Acids Res 19(22):6163–6167PubMedCrossRefGoogle Scholar
  9. 9.
    Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J, Houlihan PS, Krouse RS, Prasad AR, Einspahr JG, Buckmeier J, Alberts DS, Hamilton SR, Issa JP (2005) MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst 97(18):1330–1338PubMedCrossRefGoogle Scholar
  10. 10.
    Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller CG, Morrow M (eds) (2002) AJCC cancer staging manual. Springer, New YorkGoogle Scholar
  11. 11.
    Moskaluk CA, Kern SE (1997) Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections. Am J Pathol 150(5):1547–1552PubMedGoogle Scholar
  12. 12.
    Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93(18):9821–9826PubMedCrossRefGoogle Scholar
  13. 13.
    Shi SR, Cote RJ, Yang C, Chen C, Xu HJ, Benedict WF, Taylor CR (1996) Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections. J Pathol 179(3):347–352. doi: 10.1002/(SICI)1096-9896(199607)179:3<347::AID-PATH559>3.0.CO;2-L PubMedCrossRefGoogle Scholar
  14. 14.
    Klarskov L, Ladelund S, Holck S, Roenlund K, Lindebjerg J, Elebro J, Halvarsson B, von Salome J, Bernstein I, Nilbert M (2010) Interobserver variability in the evaluation of mismatch repair protein immunostaining. Hum Pathol 41(10):1387–1396PubMedCrossRefGoogle Scholar
  15. 15.
    Strand M, Prolla TA, Liskay RM, Petes TD (1993) Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365(6443):274–276. doi: 10.1038/365274a0 PubMedCrossRefGoogle Scholar
  16. 16.
    Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, Markowitz S, Willson JK, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB (1998) Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 95(12):6870–6875PubMedCrossRefGoogle Scholar
  17. 17.
    Esteller M, Corn PG, Baylin SB, Herman JG (2001) A gene hypermethylation profile of human cancer. Cancer Res 61(8):3225–3229PubMedGoogle Scholar
  18. 18.
    Kim YH, Petko Z, Dzieciatkowski S, Lin L, Ghiassi M, Stain S, Chapman WC, Washington MK, Willis J, Markowitz SD, Grady WM (2006) CpG island methylation of genes accumulates during the adenoma progression step of the multistep pathogenesis of colorectal cancer. Genes Chromosomes Cancer 45(8):781–789. doi: 10.1002/gcc.20341 PubMedCrossRefGoogle Scholar
  19. 19.
    Psofaki V, Kalogera C, Tzambouras N, Stephanou D, Tsianos E, Seferiadis K, Kolios G (2010) Promoter methylation status of hMLH1, MGMT, and CDKN2A/p16 in colorectal adenomas. World J Gastroenterol 16(28):3553–3560PubMedCrossRefGoogle Scholar
  20. 20.
    Bai AH, Tong JH, To KF, Chan MW, Man EP, Lo KW, Lee JF, Sung JJ, Leung WK (2004) Promoter hypermethylation of tumor-related genes in the progression of colorectal neoplasia. Int J Cancer 112(5):846–853. doi: 10.1002/ijc.20485 PubMedCrossRefGoogle Scholar
  21. 21.
    Zhang H, Fu WL, Huang Q (2006) Mapping of the methylation pattern of the hMSH2 promoter in colon cancer, using bisulfite genomic sequencing. J Carcinog 5:22PubMedCrossRefGoogle Scholar
  22. 22.
    Belvederesi L, Bianchi F, Galizia E, Loretelli C, Bracci R, Catalani R, Amati M, Cellerino R (2008) MSH2 missense mutations and HNPCC syndrome: pathogenicity assessment in a human expression system. Hum Mutat 29(11):E296–E309. doi: 10.1002/humu.20875 PubMedCrossRefGoogle Scholar
  23. 23.
    Seifert M, Reichrath J (2006) The role of the human DNA mismatch repair gene hMSH2 in DNA repair, cell cycle control and apoptosis: implications for pathogenesis, progression and therapy of cancer. J Mol Histol 37(5–7):301–307. doi: 10.1007/s10735-006-9062-5 PubMedCrossRefGoogle Scholar
  24. 24.
    Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S, Li GM, Drummond J, Modrich PL, Sedwick WD, Markowitz SD (1998) Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A 95(15):8698–8702PubMedCrossRefGoogle Scholar
  25. 25.
    Goel A, Nagasaka T, Arnold CN, Inoue T, Hamilton C, Niedzwiecki D, Compton C, Mayer RJ, Goldberg R, Bertagnolli MM, Boland CR (2007) The CpG island methylator phenotype and chromosomal instability are inversely correlated in sporadic colorectal cancer. Gastroenterology 132(1):127–138PubMedCrossRefGoogle Scholar
  26. 26.
    Jass JR (2007) Molecular heterogeneity of colorectal cancer: implications for cancer control. Surg Oncol 16(Suppl 1):S7–S9PubMedCrossRefGoogle Scholar
  27. 27.
    Oh K, Redston M, Odze RD (2005) Support for hMLH1 and MGMT silencing as a mechanism of tumorigenesis in the hyperplastic adenoma–carcinoma (serrated) carcinogenic pathway in the colon. Hum Pathol 36(1):101–111PubMedCrossRefGoogle Scholar
  28. 28.
    Ahlquist T, Lind GE, Costa VL, Meling GI, Vatn M, Hoff GS, Rognum TO, Skotheim RI, Thiis-Evensen E, Lothe RA (2008) Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers. Mol Cancer 7:94PubMedCrossRefGoogle Scholar
  29. 29.
    Petko Z, Ghiassi M, Shuber A, Gorham J, Smalley W, Washington MK, Schultenover S, Gautam S, Markowitz SD, Grady WM (2005) Aberrantly methylated CDKN2A, MGMT, and MLH1 in colon polyps and in fecal DNA from patients with colorectal polyps. Clin Cancer Res 11(3):1203–1209PubMedGoogle Scholar
  30. 30.
    Jung SH, Kim HC, Kim JS, Choi J, Yu CS, Kim JC (2006) Efficacy of hMLH1/hMSH2 immunohistochemical staining as representative index for microsatellite instability status in sporadic colorectal cancer. J Korean Soc Coloproctol 22(3):184–191Google Scholar
  31. 31.
    Shaw RJ, Liloglou T, Rogers SN, Brown JS, Vaughan ED, Lowe D, Field JK, Risk JM (2006) Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br J Cancer 94(4):561–568PubMedCrossRefGoogle Scholar
  32. 32.
    Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG (1999) Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59(4):793–797PubMedGoogle Scholar
  33. 33.
    Park JW, Chang HJ, Park S, Kim BC, Kim DY, Baek JY, Kim SY, Oh JH, Choi HS, Park SC, Jeong SY (2010) Absence of hMLH1 or hMSH2 expression as a stage-dependent prognostic factor in sporadic colorectal cancers. Ann Surg Oncol 17(11):2839–2846. doi: 10.1245/s10434-010-1135-8 PubMedCrossRefGoogle Scholar
  34. 34.
    Rigau V, Sebbagh N, Olschwang S, Paraf F, Mourra N, Parc Y, Flejou JF (2003) Microsatellite instability in colorectal carcinoma. The comparison of immunohistochemistry and molecular biology suggests a role for hMSH6 [correction of hMLH6] immunostaining. Arch Pathol Lab Med 127(6):694–700PubMedGoogle Scholar
  35. 35.
    Salahshor S, Koelble K, Rubio C, Lindblom A (2001) Microsatellite Instability and hMLH1 and hMSH2 expression analysis in familial and sporadic colorectal cancer. Lab Invest 81(4):535–541PubMedGoogle Scholar
  36. 36.
    Shia J, Klimstra DS, Nafa K, Offit K, Guillem JG, Markowitz AJ, Gerald WL, Ellis NA (2005) Value of immunohistochemical detection of DNA mismatch repair proteins in predicting germline mutation in hereditary colorectal neoplasms. Am J Surg Pathol 29(1):96–104PubMedCrossRefGoogle Scholar
  37. 37.
    Zaidi NH, Liu L, Gerson SL (1996) Quantitative immunohistochemical estimates of O6-alkylguanine-DNA alkyltransferase expression in normal and malignant human colon. Clin Cancer Res 2(3):577–584PubMedGoogle Scholar
  38. 38.
    Overbeek LI, Ligtenberg MJ, Willems RW, Hermens RP, Blokx WA, Dubois SV, van der Linden H, Meijer JW, Mlynek-Kersjes ML, Hoogerbrugge N, Hebeda KM, van Krieken JH (2008) Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting. Am J Surg Pathol 32(8):1246–1251PubMedCrossRefGoogle Scholar
  39. 39.
    Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72:141–196PubMedCrossRefGoogle Scholar
  40. 40.
    Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21(2):163–167. doi: 10.1038/5947 PubMedCrossRefGoogle Scholar
  41. 41.
    Kim HC, Roh SA, Ga IH, Kim JS, Yu CS, Kim JC (2005) CpG island methylation as an early event during adenoma progression in carcinogenesis of sporadic colorectal cancer. J Gastroenterol Hepatol 20(12):1920–1926PubMedCrossRefGoogle Scholar
  42. 42.
    Banerjea A, Hands RE, Powar MP, Bustin SA, Dorudi S (2009) Microsatellite and chromosomal stable colorectal cancers demonstrate poor immunogenicity and early disease recurrence. Colorectal Dis 11(6):601–608PubMedCrossRefGoogle Scholar
  43. 43.
    Kim YH, Lee HC, Kim SY, Yeom YI, Ryu KJ, Min BH, Kim DH, Son HJ, Rhee PL, Kim JJ, Rhee JC, Kim HC, Chun HK, Grady WM, Kim YS (2011) Epigenomic analysis of aberrantly methylated genes in colorectal cancer identifies genes commonly affected by epigenetic alterations. Ann Surg Oncol. doi: 10.1245/s10434-011-1573-y Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Kyung-Hwa Lee
    • 1
  • Ji-Shin Lee
    • 1
  • Jong-Hee Nam
    • 1
  • Chan Choi
    • 1
  • Min-Cheol Lee
    • 1
  • Chang-Soo Park
    • 1
  • Sang-Woo Juhng
    • 1
  • Jae-Hyuk Lee
    • 1
    Email author
  1. 1.Department of PathologyChonnam National University Medical SchoolGwangjuSouth Korea

Personalised recommendations