Tumor Biology

, Volume 36, Issue 5, pp 3853–3861 | Cite as

Combined methylation of p16 and hMLH1 (CMETH2) discriminates a subpopulation with better prognosis in colorectal cancer patients with microsatellite instability tumors

  • S. Veganzones
  • M. L. Maestro
  • S. Rafael
  • V. de la Orden
  • M. Vidaurreta
  • B. Mediero
  • M. Espantaleón
  • J. Cerdán
  • E. Díaz-Rubio
Research Article


The aim of this study was to determine the frequency of p16 and hMLH1 genes simultaneous methylation in colorectal cancer patients with Microsatellite Instability (MSI) tumors. We also wanted to analyze the relationship with other clinical features, with BRAF gene V600E mutation and with prognosis. Samples from fifty one patients with MSI positive sporadic colorectal cancer were included. DNA was extracted from tumor samples. Promoter methylation was analyzed using bisulfite modification and was detected by quantitative methylation-specific PCR. BRAF gene was amplified using specific primers and mutations were detected by real time PCR. Simultaneous methylation was transformed in a new variable called CMETH2. Frequency of CMETH2 was analyzed and compared with other clinicopathological variables. 33.3 % of patients were positive for CMETH2 and 25 % had BRAF V600E mutation. CMETH2 was related with proximal location, with poorly differentiated tumors and with BRAF V600E mutation. CMETH2 only showed influence in the overall survival (OS) in patients with distal tumors. However, with regard to the disease free survival (DFS) measure, CMETH2 was independent prognostic factor. We were able to discriminate tumors with high methylation features using a transformation analysis of variables into a new computed one (CMETH2). CMETH2 has demonstrated to be a useful prognostic factor in MSI tumors. The prognostic value of CMETH2 in DFS was independent of other clinicopathological variables. The use of CMETH2 could help in the election of the best therapeutic alternative for CCR patients with MSI tumors.


Colorectal cancer Prognosis p16 methylation hMLH1 methylation MSI 


Conflicts of interest



  1. 1.
    Sweeney C, Boucher KM, Samowitz WS, et al. Oncogenic Tree model of somatic mutations and DNA methylation in colon tumors. Genes, Chromosomes Cancer. 2009;48:1–9.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Tóth E, Serester O, Gallai M, et al. Molecular pathways and pathomorphology of colorectal cancers. Rom J Morphol Embryol. 2011;52:767–73.PubMedGoogle Scholar
  3. 3.
    Miladi-Abdennadher I, Abdelmaksoud-Damak R, Ayadi L, et al. Aberrant methylation of HMLH1 and p16INK4a in Tunisian patients with sporadic colorectal adenocarcinoma. Biosci Rep. 2011;31(4):257–64.CrossRefPubMedGoogle Scholar
  4. 4.
    Weisenberger DJ, Siegmund KD, Campan M, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–93.CrossRefPubMedGoogle Scholar
  5. 5.
    Ogino S, Nosho K, Kirkner GJ, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58(1):90–6.CrossRefPubMedGoogle Scholar
  6. 6.
    Toyota M, Ohe-Toyota M, Ahuja N, et al. Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype. Poc Natl Acad Sci USA. 2000;97:710–5.CrossRefGoogle Scholar
  7. 7.
    Dahlin AM, Palmqvist R, Henriksson ML, et al. The role of the CpG island methylator phenotype in colorectal cancer prognosis depends on microsatellite instability screening status. Clin Cancer Res. 2010;16:1845–55.CrossRefPubMedGoogle Scholar
  8. 8.
    Ogino S, Meyerhardt JA, Kawasaki T. CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma. Virchows Arch. 2007;450:529–37.CrossRefPubMedGoogle Scholar
  9. 9.
    Kim JH, Shin SH, Kwon HJ, et al. Prognostic implications of CpG island hypermethylator phenotype in colorectal cancers. Virchows Arch. 2009;455:485–94.CrossRefPubMedGoogle Scholar
  10. 10.
    Li X, Hu F, Wang Y, Yao X, et al. CpG island methylator phenotype and prognosis of colorectal cancer in Northeast China. Biomed Res Int. 2014;2014:236361.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Brim H, Abu-Asab MS, Nouraie M, et al. An integrative CGH, MSI and candidate genes methylation analysis of colorectal tumors. PLoS One. 2014;9(1):e82185.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Han SW, Lee HJ, Bae JM, et al. Methylation and microsatellite status and recurrence following adjuvant FOLFOX in colorectal cancer. Int J Cancer. 2013;132(9):2209–16.CrossRefPubMedGoogle Scholar
  13. 13.
    Bae JM, Kim JH, Cho NY, et al. Prognostic implication of the CpG island methylator phenotype in colorectal cancers depends on tumour location. Br J Cancer. 2013;109(4):1004–12.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kim JH, Rhee YY, Bae JM, et al. Subsets of microsatellite-unstable colorectal cancers exhibit discordance between the CpG island methylator phenotype and MLH1 methylation status. Mod Pathol. 2013;26(7):1013–22.CrossRefPubMedGoogle Scholar
  15. 15.
    Yamauchi M, Morikawa T, Kuchiba A, et al. Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum. Gut. 2012;61(6):847–54.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Liggett WH, Sidransky D. Role of the p16 tumor suppressor gene in cancer. J Clin Oncol. 1998;16:1197–206.CrossRefPubMedGoogle Scholar
  17. 17.
    Burri N, Shaw P, Bouzourene H, et al. Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer. Lab Invest. 2001;81:217–29.CrossRefPubMedGoogle Scholar
  18. 18.
    Tommasi S, Pinto R, Petriella D, et al. Oncosuppressor methylation: a possible key role in colon metastatic progression. J Cell Physiol. 2011;226:1934–39.CrossRefPubMedGoogle Scholar
  19. 19.
    Sanz-Casla MT, Maestro ML, Vidaurreta M, et al. p16 gene methylation in colorectal tumors: correlation with clinicopathological features and prognostic value. Dig Dis. 2005;23:151–5.CrossRefPubMedGoogle Scholar
  20. 20.
    Kanai Y, Ushijima S, Kondo Y, et al. DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancer. In J Cancer. 2001;91:205–12.CrossRefGoogle Scholar
  21. 21.
    Ogino S, Odze RD, Kawasaki T, et al. Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma. Am J Surg Pathol. 2006;30:1175–83.CrossRefPubMedGoogle Scholar
  22. 22.
    Iacopetta B, Grieu F, Li W, et al. APC gene methylation is inversely correlated with features of the CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2006;119:2272–8.CrossRefPubMedGoogle Scholar
  23. 23.
    Psofaki V, Kalogera C, Tzambouras N, et al. Promoter methylation status of HMLH1, MGMT, and CDKN2A/p16 in colorectal adenomas. World J Gastroenterol. 2010;16:3553–60.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Mitomi H, Fukui N, Tanaka N, et al. Aberrant p16(INK4a) methylation is a frequent event in colorectal cancers: prognostic value and relation to mRNA expression and immunoreactivity. J Cancer Res Clin Oncol. 2010;136:323–31.CrossRefPubMedGoogle Scholar
  25. 25.
    Shen L, Catalano PJ, Benson III AB, et al. Association between DNA methylation and shortened survival in patients with advanced colorectal cancer treated with 5-Xuorouracil-based chemotherapy. Clin Cancer Res. 2007;13:6093–8.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Ogino S, Cantor M, Kawasaki T, et al. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative ADN methylation analysis and prospective cohort studies. Gut. 2006;55:1000–6.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Tanaka N, Huttenhower C, Nosho K, et al. Novel application of structural equation modeling to correlation structure analysis of CpG island methylation in colorectal cancer. Am J Pathol. 2010;177:2731–40.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Shima K, Nosho K, Baba Y, et al. Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: cohort study and literature review. Int J Cancer. 2011;128:1080–94.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Ahn JB, Chung WB, Maeda O, et al. DNA methylation predicts recurrence from resected stage III proximal colon cancer. Cancer. 2011;117:1847–54.CrossRefPubMedGoogle Scholar
  30. 30.
    Veigl ML, Kasturi L, Olechnowicz J, et al. Biallelic inactivation of HMLH1 by epigenetic gene silencien, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A. 1998;95:8698–702.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of HMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A. 1998;95:6870–75.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Li X, Yao X, Wang Y, et al. MLH1 promoter methylation frequency in colorectal cancer patients and related clinicopathological and molecular features. PLoS One. 2013;8(3):e59064.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kumar K, Brim H, Giardiello F, et al. Distinct BRAF (V600E) and KRAS mutations in high microsatellite instability sporadic colorectal cancer in African Americans. Clin Cancer Res. 2009;15(4):1155–61.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Koinuma K, Shitoh K, Miyakura Y, et al. Mutations of BRAF are associated with extensive HMLH1 promoter methylation in sporadic colorectal carcinomas. Int J Cancer. 2004;108:237–42.CrossRefPubMedGoogle Scholar
  35. 35.
    Rajagopalan H, Bardelli A, Lengauer C, et al. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature. 2002;418:934.CrossRefPubMedGoogle Scholar
  36. 36.
    Vidaurreta M, Sanz-Casla MT, Maestro ML, et al. Microsatellite instability predicts better outcome in colorectal cancer patients. Med Clin (Barc). 2005;124(4):121–5.CrossRefGoogle Scholar
  37. 37.
    Maestro ML, Vidaurreta M, Sanz-Casla MT, et al. Role of the BRAF mutations in the microsatellite instability genetic pathway in sporadic colorectal cancer. Ann Surg Oncol. 2007;14(3):1229–36.CrossRefPubMedGoogle Scholar
  38. 38.
    Suehiro Y, Wong CW, Chirieac LR, et al. Epigenetic-genetic interactions in the APC/WNT, RAS/RAF, and P53 pathways in colorectal carcinoma. Clin Cancer Res. 2008;14(9):2560–9.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Bihl MP, Foerster A, Lugli A, et al. Characterization of CDKN2A(p16) methylation and impact in colorectal cancer: systematic analysis using pyrosequencing. J Transl Med. 2012;10:173.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kim JH, Kang GH. Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer. World J Gastroenterol. 2014;20:4230–43.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998;58:5248–57.PubMedGoogle Scholar
  42. 42.
    Veganzones S, Rafael S, Vidaurreta M, et al. p16 gene methylation in colorectal cancer patients with long-term follow-up. Rev Esp Enferm Dig. 2012;104:111–7.CrossRefGoogle Scholar
  43. 43.
    Muzny DM, Bainbridge MN, Chang K, et al. The cancer genome atlas network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–7.CrossRefGoogle Scholar
  44. 44.
    Yamashita K, Dai T, Dai Y, et al. Genetics supersedes epigenetics in colon cancer phenotype. Cancer Cell. 2003;4:121–31.CrossRefPubMedGoogle Scholar
  45. 45.
    Perucho M. Cancer of the microsatellite mutator phenotype. Biol Chem. 1996;377:675–84.PubMedGoogle Scholar
  46. 46.
    Issa JP. Colon cancer: it’s CIN or CIMP. Clin Cancer Res. 2008;14:5939–40.CrossRefPubMedGoogle Scholar
  47. 47.
    Ahuja N, Mohan AL, Li Q, et al. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res. 1997;57:3370–4.PubMedGoogle Scholar
  48. 48.
    Norrie MW, Hawkins NJ, Todd AV, et al. Inactivation of p16INK4a by CpG hypermethylation is not a frequent event in colorectal cancer. J Surg Oncol. 2003;84:143–50.CrossRefPubMedGoogle Scholar
  49. 49.
    Barault L, Charon-Barra C, Jooste V, et al. Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases. Cancer Res. 2008;68:8541–6.CrossRefPubMedGoogle Scholar
  50. 50.
    Samadder NJ, Vierkant RA, Tillmans LS, et al. Associations between colorectal cancer molecular markers and pathways with clinicopathologic features in older women. Gastroenterology. 2013;145:348–56.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Ward RL, Cheong K, Ku SL, et al. Adverse prognostic effect of methylation in colorectal cancer is reversed by microsatellite instability. J Clin Oncol. 2003;21:3729–36.CrossRefPubMedGoogle Scholar
  52. 52.
    Samowitz WS, Sweeney C, Herrick J, et al. Cancer Res. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res. 2005;65:6063–9.CrossRefPubMedGoogle Scholar
  53. 53.
    Lee S, Cho NY, Yoo EJ, et al. CpG island methylator phenotype in colorectal cancers: comparison of the new and classic CpG island methylator phenotype marker panels. Arch Pathol Lab Med. 2008;132:1657–65.PubMedGoogle Scholar
  54. 54.
    Donada M, Bonin S, Barbazza R, et al. Management of stage II colon cancer—the use of molecular biomarkers for adjuvant therapy decision. BMC Gastroenterology. 2013;13:36.CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Ogino S, Shima K, Meyerhardt JA, McCleary NJ, Ng K, Hollis D, et al. Predictive and prognostic roles of BRAF mutation in stage III colon cancer: results from intergroup trial CALGB 89803. Clin Cancer Res. 2012;18:890–900.CrossRefPubMedGoogle Scholar
  56. 56.
    Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol. 2010;28:3219–26.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Van Rijnsoever M, Elsaleh H, Joseph D, et al. CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer. Clin Cancer Res. 2003;9:2898–903.PubMedGoogle Scholar
  58. 58.
    Jover R, Nguyen TP, Pérez-Carbonell L, et al. 5-Fluorouracil adjuvant chemotherapy does not increase survival in patients with CpG island methylator phenotype colorectal cancer. Gastroenterology. 2011;140:1174–81.CrossRefPubMedGoogle Scholar
  59. 59.
    An B, Kondo Y, Okamoto Y, et al. Characteristic methylation profile in CpG island methylator phenotype-negative distal colorectal cancers. Int J Cancer. 2010;127:2095–105.CrossRefPubMedGoogle Scholar
  60. 60.
    Arends MJ. Pathways of colorectal carcinogenesis. Appl Immunohistochem Mol Morphol. 2013;21(2):97–102.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • S. Veganzones
    • 1
  • M. L. Maestro
    • 1
  • S. Rafael
    • 1
  • V. de la Orden
    • 1
  • M. Vidaurreta
    • 1
  • B. Mediero
    • 1
  • M. Espantaleón
    • 2
  • J. Cerdán
    • 3
  • E. Díaz-Rubio
    • 4
    • 5
  1. 1.Department of Clinical Analysis, Genomic SectionHospital Clínico San CarlosMadridSpain
  2. 2.Department of DocumentationHospital Clínico San CarlosMadridSpain
  3. 3.Department of General SurgeryHospital Clínico San CarlosMadridSpain
  4. 4.Department of Medical Oncology, Instituto Investigación BiomédicaHospital Clínico San CarlosMadridSpain
  5. 5.Department of Medicine, Facultad Medicina Universidad Complutense, Center Affiliated to the Red Tematica de Investigacion Cooperativa (RD/12/0036/006), Instituto Carlos IIISpanish Ministery of Economy and CompetitivyMadridSpain

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