Tumor Biology

, Volume 36, Issue 3, pp 1627–1642 | Cite as

p16 hypermethylation: A biomarker for increased esophageal cancer susceptibility in high incidence region of North East India

  • Mandakini Das
  • Bhaskar Jyoti Saikia
  • Santanu Kumar Sharma
  • Gaganpreet Singh Sekhon
  • Jagadish Mahanta
  • Rup Kumar Phukan
Research Article


Esophageal cancer is one of the most common cancers in North East India. The molecular mechanisms of esophageal cancer susceptibility in North East India have not been fully understood. There is a need for identification of biomarkers to identify people at risk of esophageal cancer. p16 is an essential G1 cell cycle regulatory gene whose loss of function is associated with carcinogenesis. Therefore, we conducted this study to determine the prevalence of p16 gene methylation in patients with esophageal cancer to assess the feasibility of using gene methylation as a biomarker. A total of 100 newly diagnosed esophageal cancer cases along with equal number of age, sex, and ethnicity-matched controls were included in this study. Methylation-specific PCR was used to determine the p16 methylation status. Aberrant promoter methylation of the p16 gene was detected in 81 of 100 (81 %) esophageal cancer cases. Hypermethylation of p16 gene was found to be influenced by lifestyle factors. Betel quid and tobacco chewing habit synergistically with p16 methylation elevated the risk for esophageal cancer development (adjusted odds ratio (OR) = 6.88, 95 % confidence interval (CI) = 1.64–28.81, p = 0.003 for betel quid chewing and adjusted OR = 7.02, 95 % CI = 1.87–26.38, p = 0.001 for tobacco chewing). Further, intake of green leafy vegetables and fruits lowered the risk of esophageal cancer (adjusted OR = 0.16, 95 % CI = 0.04–0.58, p = 0.05 for green leafy vegetables and adjusted OR = 0.15, 95 % CI = 0.04–0.64, p = 0.01 for fruits). Thus, p16 hypermethylation may aid as a biomarker in identifying habitués at greater risk for esophageal cancer susceptibility in high incidence region of North East India.


Esophageal cancer North East India p16 gene Methylation Biomarker 



This work was supported by the Indian Council of Medical Research (ICMR), Department of Health Research and Government of India under the extramural funding of Northeast Initiative. We would like to thank Dr. Nirmal Sahewala (Consultant Radiologist, Obsteristics and Gynecology) and Dr. Bimal Kumar Jalan (Chief Endoscopist), Aditya Hospital, Dibrugarh, Assam, India, for their support during sample collection. We would also like to thank the staff of Radiology Department, Assam Medical College and Hospital, Dibrugarh, Assam, India.

Conflicts of interest



  1. 1.
    Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al. GLOBOCAN 2012 v1.0, Cancer incidence and mortality worldwide: IARC Cancer Base No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer 2012. Accessed 11 Jul 2014.
  2. 2.
    NCRP. National cancer registry programme (NCRP) three year report of population based cancer registries : 2009–2011.Google Scholar
  3. 3.
    Phukan RK, Ali MS, Chetia CK, Mahanta J. Betel nut and tobacco chewing; potential risk factors of cancer of oesophagus in Assam, India. Br J Cancer. 2001;85(5):661–7. doi: 10.1054/bjoc.2001.1920.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Phukan RK, Chetia CK, Ali MS, Mahanta J. Role of dietary habits in the development of esophageal cancer in Assam, the north-eastern region of India. Nutr Cancer. 2001;39(2):204–9. doi: 10.1207/S15327914nc392_7.CrossRefPubMedGoogle Scholar
  5. 5.
    Phukan RK, Zomawia E, Narain K, Hazarika NC, Mahanta J. Tobacco use and stomach cancer in Mizoram, India. Cancer Epidemiol Biomarkers Prev. 2005;14:1892–6. doi: 10.1158/1055-9965.EPI-05-0074.CrossRefPubMedGoogle Scholar
  6. 6.
    Talukdar FR, Ghosh SK, Laskar RS, Mondal R. Epigenetic, genetic and environmental interactions in esophageal squamous cell carcinoma from northeast India. PLoS One. 2013;8(4):e60996. doi: 10.1371/journal.pone.0060996.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Adams L, Roth MJ, Abnet CC, Dawsey SP, Qiao YL, Wang GQ, et al. Promoter methylation in cytology specimens as an early detection marker for esophageal squamous dysplasia and early esophageal squamous cell carcinoma. Cancer Prev Res (Phila). 2008;1(5):357–61. doi: 10.1158/1940-6207.CAPR-08-0061.CrossRefPubMedCentralGoogle Scholar
  8. 8.
    Zhang XM, Guo MZ. The value of epigenetic markers in esophageal cancer. Front Med China. 2010;4(4):378–84. doi: 10.1007/s11684-010-0230-3.CrossRefPubMedGoogle Scholar
  9. 9.
    Agarwal A, Polineni R, Hussein Z, Vigoda I, Bhagat TD, Bhattacharyya S, et al. Role of epigenetic alterations in the pathogenesis of Barrett’s esophagus and esophageal adenocarcinoma. Int J Clin Exp Pathol. 2012;5(5):382–96.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Chik F, Szyf M, Rabbani SA. Role of epigenetics in cancer initiation and progression. Adv Exp Med Biol. 2011;720:91–104. doi: 10.1007/978-1-4614-0254-1_8.CrossRefPubMedGoogle Scholar
  11. 11.
    Xie GS, Hou AR, Li LY, Gao YN, Cheng SJ. Aberrant p16 promoter hypermethylation in bronchial mucosae as a biomarker for the early detection of lung cancer. Chin Med J. 2006;119(17):1469–72.PubMedGoogle Scholar
  12. 12.
    Bardhan K, Liu K. Epigenetics and colorectal cancer pathogenesis. Cancers. 2013;5(2):676–713. doi: 10.3390/cancers5020676.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhao SL, Zhu ST, Hao X, Li P, Zhang ST. Effects of DNA methyltransferase 1 inhibition on esophageal squamous cell carcinoma. Dis Esophagus. 2011;24(8):601–10. doi: 10.1111/j.1442-2050.2011.01199.x.CrossRefPubMedGoogle Scholar
  14. 14.
    Luczak MW, Roszak A, Pawlik P, Kedzia H, Kedzia W, Malkowska-Walczak B, et al. Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma. Int J Oncol. 2012;40(3):860–6. doi: 10.3892/ijo.2011.1183.PubMedGoogle Scholar
  15. 15.
    Morey Kinney SR, Smiraglia DJ, James SR, Moser MT, Foster BA, Karpf AR. Stage-specific alterations of DNA methyltransferase expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the transgenic adenocarcinoma of mouse prostate model. Mol Cancer Res. 2008;6(8):1365–74. doi: 10.1158/1541-7786.MCR-08-0040.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Li B, Wang B, Niu LJ, Jiang L, Qiu CC. Hypermethylation of multiple tumor-related genes associated with DNMT3b up-regulation served as a biomarker for early diagnosis of esophageal squamous cell carcinoma. Epigenetics. 2011;6(3):307–16.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Schildhaus HU, Krockel I, Lippert H, Malfertheiner P, Roessner A, Schneider-Stock R. Promoter hypermethylation of p16INK4a, E-cadherin, O6-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach. Int J Oncol. 2005;26(6):1493–500.PubMedGoogle Scholar
  18. 18.
    Wu J, Liang B, He J, Zhang H, Wang Z. Study on detection of aberrant promoter hypermethylation of p16 and DAP kinase in serum DNA from patients with non-small cell lung cancer. Zhong Fei Ai Za Zhi. 2002;5(3):188–90. doi: 10.3779/j.issn. 1009-3419.2002.03.09.Google Scholar
  19. 19.
    Ai L, Stephenson KK, Ling W, Zuo C, Mukunyadzi P, Suen JY, et al. The p16 (CDKN2a/INK4a) tumor-suppressor gene in head and neck squamous cell carcinoma: a promoter methylation and protein expression study in 100 cases. Mod Pathol. 2003;16(9):944–50. doi: 10.1097/01.MP.0000085760.74313.DD.CrossRefPubMedGoogle Scholar
  20. 20.
    Ito S, Ohga T, Saeki H, Watanabe M, Kakeji Y, Morita M, et al. Promoter hypermethylation and quantitative expression analysis of CDKN2A (p14ARF and p16INK4a) gene in esophageal squamous cell carcinoma. Anticancer Res. 2007;27(5A):3345–53.PubMedGoogle Scholar
  21. 21.
    Roth MJ, Abnet CC, Hu N, Wang QH, Wei WQ, Green L, et al. p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in esophageal squamous cell carcinoma and its precursor lesions. Oncol Rep. 2006;15(6):1591–7.PubMedGoogle Scholar
  22. 22.
    Sibin MK, Bhat DI, Lavanya C, Manoj MJ, Aakershita S, Chetan GK. CDKN2A exon-wise deletion status and novel somatic mutations in Indian glioma patients. Tumour Biol. 2014;35(2):1467–72. doi: 10.1007/s13277-013-1201-5.CrossRefPubMedGoogle Scholar
  23. 23.
    Wang J, Lee JJ, Wang L, Liu DD, Lu C, Fan YH, et al. Value of p16INK4a and RASSF1A promoter hypermethylation in prognosis of patients with resectable non-small cell lung cancer. Clin Cancer Res. 2004;10(18 Pt 1):6119–25. doi: 10.1158/1078-0432.CCR-04-0652.CrossRefPubMedGoogle Scholar
  24. 24.
    Sharma G, Mirza S, Prasad CP, Srivastava A, Gupta SD, Ralhan R. Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients. Life Sci. 2007;80(20):1873–81. doi: 10.1016/j.lfs.2007.02.026.CrossRefPubMedGoogle Scholar
  25. 25.
    Wang CC, Mao WM, Ling ZQ. DNA methylation status of RARbeta2 and p16(INK4alpha) in peripheral blood and tumor tissue in patients with esophageal squamous cell carcinoma. Zhonghua Zhong Liu Za Zhi. 2012;34(6):441–5. doi: 10.3760/cma.j.issn.0253-3766.2012.06.009.PubMedGoogle Scholar
  26. 26.
    Wang X, Zhu YB, Cui HP, Yu TT. Aberrant promoter methylation of p15 and p16 genes may contribute to the pathogenesis of multiple myeloma: a meta-analysis. Tumour Biol. 2014. doi: 10.1007/s13277-014-2054-2.Google Scholar
  27. 27.
    do Nascimento Borges B, Burbano RM, Harada ML. Analysis of the methylation patterns of the p16 INK4A, p15 INK4B, and APC genes in gastric adenocarcinoma patients from a Brazilian population. Tumour Biol. 2013;34(4):2127–33. doi: 10.1007/s13277-013-0742-y.CrossRefPubMedGoogle Scholar
  28. 28.
    Bhagat R, Kumar SS, Vaderhobli S, Premalata CS, Pallavi VR, Ramesh G, et al. Epigenetic alteration of p16 and retinoic acid receptor beta genes in the development of epithelial ovarian carcinoma. Tumour Biol. 2014. doi: 10.1007/s13277-014-2136-1.Google Scholar
  29. 29.
    Yang W, Wang X, Li X, Wang M, Chen X, Wu X, et al. The specific methylation characteristics of cancer related genes in Chinese colorectal cancer patients. Tumour Biol. 2014. doi: 10.1007/s13277-014-2100-0.Google Scholar
  30. 30.
    Zhang JC, Gao B, Yu ZT, Liu XB, Lu J, Xie F, et al. Promoter hypermethylation of p14 (ARF), RB, and INK4 gene family in hepatocellular carcinoma with hepatitis B virus infection. Tumour Biol. 2014;35(3):2795–802. doi: 10.1007/s13277-013-1372-0.CrossRefPubMedGoogle Scholar
  31. 31.
    Yi JM, Dhir M, Guzzetta AA, Iacobuzio-Donahue CA, Heo K, Yang KM, et al. DNA methylation biomarker candidates for early detection of colon cancer. Tumour Biol. 2012;33(2):363–72. doi: 10.1007/s13277-011-0302-2.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Wang J, Sasco AJ, Fu C, Xue H, Guo G, Hua Z, et al. Aberrant DNA methylation of P16, MGMT, and hMLH1 genes in combination with MTHFR C677T genetic polymorphism in esophageal squamous cell carcinoma. Cancer Epidemiol Biomark Prev. 2008;17(1):118–25. doi: 10.1158/1055-9965.EPI-07-0733.CrossRefGoogle Scholar
  33. 33.
    NCRP. National cancer registry programme (NCRP) three year report of population based cancer registries: 2006–2008.Google Scholar
  34. 34.
    Xu R, Wang F, Wu L, Wang J, Lu C. A systematic review of hypermethylation of p16 gene in esophageal cancer. Cancer Biomark. 2013;13(4):215–26. doi: 10.3233/CBM-130355.CrossRefPubMedGoogle Scholar
  35. 35.
    Lin RK, Hsieh YS, Lin P, Hsu HS, Chen CY, Tang YA, et al. The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients. J Clin Invest. 2010;120(2):521–32. doi: 10.1172/JCI40706.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Zhao P, Lin F, Li Z, Lin B, Lin J, Luo R. Folate intake, methylenetetrahydrofolate reductase polymorphisms, and risk of esophageal cancer. Asian Pac J Cancer Prev. 2011;12(8):2019–23.PubMedGoogle Scholar
  37. 37.
    Aune D, Deneo-Pellegrini H, Ronco AL, Boffetta P, Acosta G, Mendilaharsu M, et al. Dietary folate intake and the risk of 11 types of cancer: a case–control study in Uruguay. Ann Oncol. 2011;22(2):444–51. doi: 10.1093/annonc/mdq356.CrossRefPubMedGoogle Scholar
  38. 38.
    Stidley CA, Picchi MA, Leng S, Willink R, Crowell RE, Flores KG, et al. Multivitamins, folate, and green vegetables protect against gene promoter methylation in the aerodigestive tract of smokers. Cancer Res. 2010;70(2):568–74. doi: 10.1158/0008-5472.CAN-09-3410.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Barrera LN, Cassidy A, Johnson IT, Bao Y, Belshaw NJ. Epigenetic and antioxidant effects of dietary isothiocyanates and selenium: potential implications for cancer chemoprevention. Proc Nutr Soc. 2012;71(2):237–45. doi: 10.1017/S002966511200016X.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Mandakini Das
    • 1
  • Bhaskar Jyoti Saikia
    • 1
  • Santanu Kumar Sharma
    • 1
  • Gaganpreet Singh Sekhon
    • 1
  • Jagadish Mahanta
    • 1
  • Rup Kumar Phukan
    • 1
  1. 1.Regional Medical Research Centre, N.E. Region (ICMR)DibrugarhIndia

Personalised recommendations