Epigenetics: DNA Methylation Analysis in Esophageal Adenocarcinoma

  • Farhadul Islam
  • Johnny C. Tang
  • Vinod Gopalan
  • Alfred K. Lam
Part of the Methods in Molecular Biology book series (MIMB, volume 1756)


The aberrant DNA methylation has been noted to occur at promoter of tumor suppressor, cell adhesion, DNA repair, and other growth regulating genes during the progression of nonneoplastic esophageal mucosa to Barrett esophagus to esophageal adenocarcinoma. Methylation-mediated silencing of individual gene or concurrent loss of a number of genes plays crucial roles in dysplasia-metaplasia-neoplasia sequence of esophageal adenocarcinoma. In addition, promoter methylation of genes had shown significant prognostic potential in patients with esophageal adenocarcinoma. Thus, determination of methylation status of genes of interest can be used as a molecular marker for risk stratification and/or better prognosis of patients with esophageal adenocarcinoma. There are a number of methods including bead array, PCR and sequencing, pyrosequencing, methylation-specific PCR, and PCR with high-resolution melt curve available to determine the methylation status of particular gene of interest. Herein, we describe the polymerase chain reaction followed by sequencing-based protocol for identifying DNA methylation status in esophageal adenocarcinoma.

Key words

DNA methylation Bisulfite conversion PCR Esophageal adenocarcinoma Barrett esophagus 



Farhadul Islam and Johnny C. Tang contributed equally to this work as first authors.


  1. 1.
    Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9:465–476CrossRefGoogle Scholar
  2. 2.
    Sharma S, Kelly TK, Jones PA (2010) Epigenetics in cancer. Carcinogenesis 31:27–36CrossRefGoogle Scholar
  3. 3.
    Feinberg AP, Tycko B (2004) The history of cancer epigenetics. Nat Rev Cancer 4:143–153CrossRefGoogle Scholar
  4. 4.
    Kailasam A, Mittal SK, Agrawal DK (2015) Epigenetics in the pathogenesis of esophageal adenocarcinoma. Clin Transl Sci 8:394–402CrossRefGoogle Scholar
  5. 5.
    Agarwal A, Polineni R, Hussein Z, Vigoda I, Bhagat TD, Bhattacharyya S, Maitra A, Verma A (2012) Role of epigenetic alterations in the pathogenesis of Barrett's esophagus and esophageal adenocarcinoma. Int J Clin Exp Pathol 5:382–396PubMedCentralPubMedGoogle Scholar
  6. 6.
    Peng D, Hu TL, Jiang A, Washington MK, Moskaluk CA, Schneider-Stock R, El-Rifai W (2011) Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas. PLoS One 6:e22009CrossRefPubMedGoogle Scholar
  7. 7.
    Kaz AM, Grady WM (2014) Epigenetic biomarkers in esophageal cancer. Cancer Lett 342:193–199CrossRefGoogle Scholar
  8. 8.
    Wong DJ, Barrett MT, Stoger R et al (1997) p16INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas. Cancer Res 57:2619–2622Google Scholar
  9. 9.
    Klump B, Hsieh CJ, Holzmann K, Gregor M, Porschen R (1998) Hypermethylation of the CDKN2/p16 promoter during neoplastic progression in Barrett’s esophagus. Gastroenterology 115:1381–1386CrossRefGoogle Scholar
  10. 10.
    Eads CA, Lord RV, Wickramasinghe K, Long TI, Kurumboor SK, Bernstein L, Peters JH, DeMeester SR, DeMeester TR, Skinner KA, Laird PW (2001) Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 61:3410–3418Google Scholar
  11. 11.
    Wild Christopher P, Hardie LJ (2003) Molecular events associated with the development of Barrett’s esophagus and esophageal adenocarcinoma. Nat Rev Cancer 3:676–684CrossRefGoogle Scholar
  12. 12.
    Xu E, Gu J, Hawk ET, Wang KK, Lai M, Huang M, Ajani J, Wu X (2013) Genome-wide methylation analysis shows similar patterns in Barrett's esophagus and esophageal adenocarcinoma. Carcinogenesis 34:2750–2756CrossRefPubMedGoogle Scholar
  13. 13.
    Corn PG, Heath EI, Heitmiller R, Fogt F, Forastiere AA, Herman JG, Wu TT (2001) Frequent hypermethylation of the 5′ CpG island of E-cadherin in esophageal adenocarcinoma. Clin Cancer Res 7:2765–2769Google Scholar
  14. 14.
    Kuester D, El-Rifai W, Peng D, Ruemmele P, Kroeckel I, Peters B, Moskaluk CA, Stolte M, Mönkemüller K, Meyer F, Schulz HU, Hartmann A, Roessner A, Schneider-Stock R (2009) Silencing of MGMT expression by promoter hypermethylation in the metaplasia-dysplasia-carcinoma sequence of Barrett’s esophagus. Cancer Lett 275:117–126CrossRefGoogle Scholar
  15. 15.
    Smith E, Ruszkiewicz AR, Jamieson GG, Drew PA (2014) IGFBP7 is associated with poor prognosis in esophageal adenocarcinoma and is regulated by promoter DNA methylation. Br J Cancer 110:775–782CrossRefGoogle Scholar
  16. 16.
    Brock MV, Gou M, Akiyama Y, Muller A, Wu TT, Montgomery E, Deasel M, Germonpré P, Rubinson L, Heitmiller RF, Yang SC, Forastiere AA, Baylin SB, Herman JG (2003) Prognostic importance of promoter hypermethylation of multiple genes in esophageal adenocarcinoma. Clin Cancer Res 9:2912–2919Google Scholar
  17. 17.
    Wang JS, Guo M, Montgomery EA, Thompson RE, Cosby H, Hicks L, Wang S, Herman JG, Canto MI (2009) DNA promoter hypermethylation of p16 and APC predicts neoplastic progression in Barrett’s esophagus. Am J Gastroenterol 104:2153–2160CrossRefPubMedGoogle Scholar
  18. 18.
    Kuester D, Dar AA, Moskaluk CC, Krueger S, Meyer F, Hartig R, Stolte M, Malfertheiner P, Lippert H, Roessner A, El-Rifai W, Schneider-Stock R (2007) Early involvement of death-associated protein kinase promoter hypermethylation in the carcinogenesis of Barrett’s esophageal adenocarcinoma and its association with clinical progression. Neoplasia 9:236–245CrossRefPubMedGoogle Scholar
  19. 19.
    Jin Z, Cheng Y, Olaru A, Kan T, Yang J, Paun B, Ito T, Hamilton JP, David S, Agarwal R, Selaru FM, Sato F, Abraham JM, Beer DG, Mori Y, Shimada Y, Meltzer SJ (2008) Promoter hypermethylation of CDH13 is a common, early event in human esophageal adenocarcinogenesis and correlates with clinical risk factors. Int J Cancer 123:2331–2336CrossRefGoogle Scholar
  20. 20.
    Jin Z, Mori Y, Yang J, Sato F, Ito T, Cheng Y, Paun B, Hamilton JP, Kan T, Olaru A, David S, Agarwal R, Abraham JM, Beer D, Montgomery E, Meltzer SJ (2007) Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma. Oncogene 26:6332–6340CrossRefGoogle Scholar
  21. 21.
    Tischoff I, Hengge UR, Vieth M, Ell C, Stolte M, Weber A, Schmidt WE, Tannapfel A (2007) Methylation of SOCS-3 and SOCS-1 in the carcinogenesis of Barrett’s adenocarcinoma. Gut 56:1047–1053CrossRefPubMedGoogle Scholar
  22. 22.
    Jin Z, Olaru A, Yang J, Sato F, Cheng Y, Kan T, Mori Y, Mantzur C, Paun B, Hamilton JP, Ito T, Wang S, David S, Agarwal R, Beer DG, Abraham JM, Meltzer SJ (2007) Hypermethylation of tachykinin-1 is a potential biomarker in human esophageal cancer. Clin Cancer Res 13:6293–6300CrossRefGoogle Scholar
  23. 23.
    Clément G, Guilleret I, He B, Yagui-Beltrán A, Lin YC, You L, Xu Z, Shi Y, Okamoto J, Benhattar J, Jablons D (2008) Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett’s esophagus. Cancer Sci 99:46–53Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Farhadul Islam
    • 1
    • 2
  • Johnny C. Tang
    • 3
  • Vinod Gopalan
    • 1
  • Alfred K. Lam
    • 1
  1. 1.Cancer Molecular Pathology of School of MedicineGriffith UniversityGold CoastAustralia
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of RajshahiRajshahiBangladesh
  3. 3.Department of Applied Biology and Chemical TechnologyThe Hong Kong Polytechnic UniversityHong KongHong Kong SAR

Personalised recommendations