Skip to main content
SpringerLink
Log in

Promoter methylation and immunohistochemical expression of hMLH1 and hMSH2 in sporadic colorectal cancer: a study from India

Tumor Biology

Cite this article

Abstract

To determine the etiological factors of human colorectal cancer (CRC) we assessed the frequency and prognostic significance of hMLH1 and hMSH2 genes in conjunction with hMLH1 and hMSH2 protein expression in 30 Indian CRC patients. The protein expression and promoter methylation of hMLH1 and hMSH2; Mismatch Repair genes (MMR) were analyzed by immunohistochemistry and methylation-specific PCR (MSP), respectively. A loss of hMLH1 expression was recognized in 4(13.3 %) and loss of hMSH2 expression was recognized in 2(6.6 %) of 30 CRC cases whereas 50 % tumors showed reduced expression of hMLH1 and 33.3 % showed reduced expression of hMSH2 protein. One tumor showed a loss of both hMLH1 and hMSH2 expression. Normal nuclear staining pattern of hMLH1 and hMSH2 was observed in almost all the adjoining and normal mucosa. Promoter hypermethylation of the hMLH1 gene was detected in 15 of 30 CRC cases (50 %) and of hMSH2 gene was only in 3 of 30 CRC cases (10 %). No promoter methylation of hMLH1 and hMSH2 genes was observed in adjoining and normal mucosa. Combination of methylation of hMLH1 and hMSH2 gene was observed in two tumors (6.6 %). A significant correlation between histological grade of the tumor, methylation and expression of hMLH1 gene (p < 0.05) was observed. Normal expression of hMLH1 and hMSH2 was seen in all of the unmethylated tumors (100 %). Nuclear staining and promoter methylation of hMLH1 and hMSH2 did not significantly influence survival. hMLH1 methylation was common and was significantly correlated with loss of hMLH1 protein expression. In contrast, hMSH2 methylation was infrequent. These findings suggest that the inactivation of MMR gene expression probably via hypermethylation may lead to inactivation of their functions which finally leads to tumor aggressiveness and the immunostaining of hMLH1 protein can be used as a prognostic factor for determining the grade of the tumor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55(2):74–108.

    Article  PubMed  Google Scholar 

  2. Sung JJ, Lau JY, Goh KL, Leung WK, Asia Pacific Working Group on Colorectal Cancer. Increasing incidence of colorectal cancer in Asia: implications for screening. Lancet Oncol. 2005;6:871–6. doi:10.1016/S1470-2045(05)70422-8.

    Article  PubMed  Google Scholar 

  3. Bacani J, Zwingerman R, Di Nicola N, Spencer S, Wegrynowski T, Mitchell K, et al. Tumor microsatellite instability in early onset gastric cancer. J Mol Diagn. 2005;7(4):465–77.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Gologan A, Sepulveda AR. Microsatellite instability and DNA mismatch repair deficiency testing in hereditary and sporadic gastrointestinal cancers. Clin Lab Med. 2005;25(1):179–96.

    Article  PubMed  Google Scholar 

  5. Aaltonen LA, Salovaara R, Kristo P, Canzian F, Hemminki A, Peltomaki P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med. 1998;338:1481–7.

    Article  CAS  PubMed  Google Scholar 

  6. Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, et al. Science. 1993;260:812–6.

    Article  CAS  PubMed  Google Scholar 

  7. Thibodeau SN, Bren G, Schaid D. Science. 1993;260:816–9.

    Article  CAS  PubMed  Google Scholar 

  8. Peltoma¨ki P, Vasen HF. Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology. 1997;113:1146–58.

    Article  Google Scholar 

  9. Borresen AL, Lothe RA, Meling GI, Lystad S, Morrison P, Lipford J, et al. Somatic mutations in the hMSH2 gene in microsatellite unstable colorectal carcinomas. Hum Mol Genet. 1995;4:2065–72.

    Article  CAS  PubMed  Google Scholar 

  10. Liu B, Nicolaides NC, Markowitz S, Willson JK, Parsons RE, Jen J, et al. Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet. 1995;9:48–55.

    Article  CAS  PubMed  Google Scholar 

  11. Moslein G, Tester DJ, Lindor NM, Honchel R, Cunningham JM, French A, et al. Microsatellite instability and mutation analysis of hMSH2 and hMLH1 in patients with sporadic, familial and hereditary colorectal cancer. Hum Mol Genet. 1996;5:1245–52.

    Article  CAS  PubMed  Google Scholar 

  12. Bubb VJ, Curtis LJ, Cunningham C, Dunlop MG, Carothers AD, Morris R, et al. Microsatellite instability and the role of hMSH2 in sporadic colorectal cancer. Oncogene. 1996;12:2641–9.

    CAS  PubMed  Google Scholar 

  13. Thibodeau SN, French AJ, Roche PC, Cunningham JM, Tester DJ, Lindor NM, et al. Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res. 1996;56:4836–40.

    CAS  PubMed  Google Scholar 

  14. Wu Y, Nystrom Lahti M, Osinga J, Looman MW, Peltomaki P, Aaltonen L, et al. MSH2 and MLH1 mutations in sporadic replication error-positive colorectal carcinoma as assessed by two-dimensional DNA electrophoresis. Gene Chromosome Canc. 1997;18:269–78.

    Article  CAS  Google Scholar 

  15. Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet. 1999;21:163–7.

    Article  CAS  PubMed  Google Scholar 

  16. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, et al. 59 CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med. 1995;1:686–92.

    Article  CAS  PubMed  Google Scholar 

  17. Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998;72:141–96.

    Article  CAS  PubMed  Google Scholar 

  18. Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A. 1994;91:9700–4.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A. 1998;95:6870–5.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 1997;57:808–11.

    CAS  PubMed  Google Scholar 

  21. Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res. 1998;58:3455–60.

    CAS  PubMed  Google Scholar 

  22. Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S, et al. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A. 1998;95:8698–702.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Thibodeau SN, French AJ, Roche PC, Cunningham JM, Tester DJ, Lindor NM, et al. Cancer Res. 1996;56:4836–40.

    CAS  PubMed  Google Scholar 

  24. Shin KH, Shin JH, Kim JH. Park JG Mutational analysis of promoters of mismatch repair genes hMSH2 and hMLH1 in hereditary nonpolyposis colorectal cancer and early onset colorectal cancer patients: identification of three novel germ-line mutations in promoter of the hMSH2 gene. Cancer Res. 2002;62(1):38–42.

    PubMed  Google Scholar 

  25. Yamada K, Zhong X, Kanazawa S, Koike J, Tsujita K, Hemmi H. Oncogenic pathway of sporadic colorectal cancer with novel germline missense mutations in the hMSH2 gene. Oncol Rep. 2003;10(4):859–66.

    CAS  PubMed  Google Scholar 

  26. Jeong SY, Shin KH, Shin JH, Ku JL, Shin YK, Park SY, et al. Microsatellite instability and mutations in DNA mismatch repair genes in sporadic colorectal cancers. Dis Colon Rectum. 2003;46(8):1069–77.

    Article  PubMed  Google Scholar 

  27. Wu Y, Nystrom-Lahti M, Osinga J, Looman MW, Peltomaki P, Aaltonen LA, et al. Gene Chromosome Canc. 1997;18:269–78.

    Article  CAS  Google Scholar 

  28. Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res. 1998;58(15):3455–60.

    CAS  PubMed  Google Scholar 

  29. Kim HC, Kim CN, Yu CS, Roh SA, Kim JC. Methylation of the hMLH1 and hMSH2 promoter in early-onset sporadic colorectal carcinomas with microsatellite instability. Int J Colorectal Dis. 2003;18(3):196–202.

    PubMed  Google Scholar 

  30. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A. 1998;95(12):6870–5.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Miyakura Y, Sugano K, Konishi F, Ichikawa A, Maekawa M, Shitoh K, et al. Extensive methylation of hMLH1 promoter region predominates in proximal colon cancer with microsatellite instability. Gastroenterol. 2001;121(6):1300–9.

    Article  CAS  Google Scholar 

  32. Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. p. 6.4–6.12.

    Google Scholar 

  33. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:9821–6. doi:10.1073/pnas.93.18.9821].

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Lanza G, Gafa R, Santini A, Maestri I, Guerzoni L, Cavazzini L. Immunohistochemical test for MLH1 and MSH2 expression predicts clinical outcome in stage II and III colorectal cancer patients. J Clin Oncol. 2006;24(5):2359–67.

    Article  CAS  PubMed  Google Scholar 

  35. Hameed F, Goldberg PA, Hall P, Algar U, van Wijk R, Ramesar R. Immunohistochemistry detects mismatch repair gene defects in colorectal cancer. Colorectal Dis. 2006;8(5):411–7.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We acknowledge Dr J. D. Wig and the staff of OT and the Department of Exp. Med. and Biotech for helping in the collection of samples.

Author information

Authors and Affiliations

  1. Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Pooja Malhotra & Rakesh Kochhar

  2. Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Mumtaz Anwar, Shabeer Ahmad & Safrun Mahmood

  3. Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, 160012, India

    Kim Vaiphei

Authors
  1. Pooja Malhotra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mumtaz Anwar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rakesh Kochhar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shabeer Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kim Vaiphei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Safrun Mahmood
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Safrun Mahmood.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Malhotra, P., Anwar, M., Kochhar, R. et al. Promoter methylation and immunohistochemical expression of hMLH1 and hMSH2 in sporadic colorectal cancer: a study from India. Tumor Biol. 35, 3679–3687 (2014). https://doi.org/10.1007/s13277-013-1487-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-013-1487-3

Keywords

search

Navigation