Skip to main content
SpringerLink
Log in

Pyrosequencing Assay to Measure LINE-1 Methylation Level in Esophageal Squamous Cell Carcinoma

  • Translational Research and Biomarkers
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element 1 L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. LINE-1 methylation is a useful marker for predicting cancer prognosis and monitoring efficacy of adjuvant therapy. Nonetheless, no study has examined LINE-1 methylation in esophageal squamous cell carcinoma (ESCC). The aim of this study is to assess the precision of sodium bisulfite conversion and polymerase chain reaction (PCR) pyrosequencing assay for evaluating LINE-1 methylation in ESCC.

Methods

To measure assay precision, we performed bisulfite conversion on 5 different DNA specimen aliquots (bisulfite-to-bisulfite) and repeated PCR pyrosequencing five times (run to run). Second, to assess heterogeneity of LINE-1 methylation levels within tumor, we made 5 different tissue sections from one tumor and examined LINE-1 methylation level of each section (section to section). Third, to evaluate LINE-1 methylation status in ESCC, we applied this assay to 30 ESCCs and 30 matched normal esophageal mucosa.

Results

Bisulfite-to-bisulfite standard deviation (SD) ranged from 1.44 to 2.90 (median 2.32) in ESCCs; and 0.57 to 4.02 (median 1.23) in normal esophagus. Run-to-run SD ranged from 0.63 to 3.25 (median 1.54) in ESCCs. Section-to-section SD ranged from 1.37 to 3.31 (median 1.94). ESCC tissues showed significantly lower levels of LINE-1 methylation than matched normal mucosa (P < .0001; n = 30). There was no significant relationship between LINE-1 methylation level and tumor stage (P = 0.14).

Conclusions

Bisulfite conversion and PCR pyrosequencing assay can measure LINE-1 methylation in ESCC, and may be useful in clinical and research settings.

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. Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med. 2003;349:2241–52.

    Article  PubMed  CAS  Google Scholar 

  2. Kleinberg L, Forastiere AA. Chemoradiation in the management of esophageal cancer. J Clin Oncol. 2007;25:4110–7.

    Article  PubMed  CAS  Google Scholar 

  3. Wouters MW, Karim-Kos HE, le Cessie S, et al. Centralization of esophageal cancer surgery: does it improve clinical outcome? Ann Surg Oncol. 2009;16:1789–98.

    Article  PubMed  CAS  Google Scholar 

  4. Brucher BL, Swisher SG, Konigsrainer A, et al. Response to preoperative therapy in upper gastrointestinal cancers. Ann Surg Oncol. 2009;16:878–86.

    Article  PubMed  Google Scholar 

  5. Rodriguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med. 2011;17:330–9.

    Article  PubMed  CAS  Google Scholar 

  6. Taby R, Issa JP. Cancer epigenetics. CA Cancer J Clin. 2011;60:376–92.

    Article  Google Scholar 

  7. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–92.

    Article  PubMed  CAS  Google Scholar 

  8. Gaudet F, Hodgson JG, Eden A, et al. Induction of tumors in mice by genomic hypomethylation. Science. 2003;300:489–92.

    Article  PubMed  CAS  Google Scholar 

  9. Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout WM 3rd, Jaenisch R. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell. 2005;8:275–85.

    Article  PubMed  CAS  Google Scholar 

  10. Suzuki K, Suzuki I, Leodolter A, et al. Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell. 2006;9:199–207.

    Article  PubMed  CAS  Google Scholar 

  11. Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10:691–703.

    Article  PubMed  CAS  Google Scholar 

  12. Ogino S, Nosho K, Kirkner GJ, et al. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst. 2008;100:1734–8.

    Article  PubMed  CAS  Google Scholar 

  13. Saito K, Kawakami K, Matsumoto I, Oda M, Watanabe G, Minamoto T. Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non–small cell lung cancer. Clin Cancer Res. 2010;16:2418–26.

    Article  PubMed  CAS  Google Scholar 

  14. Sigalotti L, Fratta E, Bidoli E, et al. Methylation levels of the “long interspersed nucleotide element-1” repetitive sequences predict survival of melanoma patients. J Transl Med. 2011;9:78.

    Google Scholar 

  15. Zhang C, Xu Y, Zhao J, et al. Elevated expression of the stem cell marker CD133 associated with line-1 demethylation in hepatocellular carcinoma. Ann Surg Oncol. 2011;18:2373–80.

    Article  PubMed  Google Scholar 

  16. Nosho K, Kure S, Irahara N, et al. A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology. 2009;137:1609–20.

    Google Scholar 

  17. Maesawa C, Tamura G, Nishizuka S, et al. Inactivation of the CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinoma. Cancer Res. 1996;56:3875–8.

    PubMed  CAS  Google Scholar 

  18. Kuroki T, Trapasso F, Yendamuri S, et al. Allele loss and promoter hypermethylation of VHL, RAR-beta, RASSF1A, and FHIT tumor suppressor genes on chromosome 3p in esophageal squamous cell carcinoma. Cancer Res. 2003;63:3724–8.

    PubMed  CAS  Google Scholar 

  19. Wang J, Sasco AJ, Fu C, 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 Biomarkers Prev. 2008;17:118–25.

    Article  PubMed  CAS  Google Scholar 

  20. Rice TW, Blackstone EH, Rusch VW. 7th edition of the AJCC Cancer Staging Manual: esophagus and esophagogastric junction. Ann Surg Oncol. 2010;17:1721–4.

    Article  PubMed  Google Scholar 

  21. Ogino S, Brahmandam M, Cantor M, et al. Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component. Mod Pathol. 2006;19:59–68.

    Article  PubMed  CAS  Google Scholar 

  22. Ogino S, Kawasaki T, Brahmandam M, et al. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn. 2006;8:209–17.

    Article  PubMed  CAS  Google Scholar 

  23. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32:e38.

    Article  PubMed  Google Scholar 

  24. Estecio MR, Gharibyan V, Shen L, et al. LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS One. 2007;2:e399.

    Google Scholar 

  25. Ogino S, Kawasaki T, Nosho K, et al. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2008;122:2767–73.

    Article  PubMed  CAS  Google Scholar 

  26. Irahara N, Nosho K, Baba Y, et al. Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J Mol Diagn. 2010;12:177–83.

    Article  PubMed  CAS  Google Scholar 

  27. Baba Y, Huttenhower C, Nosho K, et al. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer. 2010;9:125.

    Google Scholar 

  28. Kawakami K, Matsunoki A, Kaneko M, Saito K, Watanabe G, Minamoto T. Long interspersed nuclear element-1 hypomethylation is a potential biomarker for the prediction of response to oral fluoropyrimidines in microsatellite stable and CpG island methylator phenotype-negative colorectal cancer. Cancer Sci. 2011;102:166–74.

    Article  PubMed  CAS  Google Scholar 

  29. Aparicio A, North B, Barske L, et al. LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors. Epigenetics. 2009;4:176–84.

    Article  PubMed  CAS  Google Scholar 

  30. Fakhrai-Rad H, Pourmand N, Ronaghi M. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum Mutat. 2002;19:479–85.

    Article  PubMed  CAS  Google Scholar 

  31. Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res. 2001;11:3–11.

    Article  PubMed  CAS  Google Scholar 

  32. Ogino S, Chan AT, Fuchs CS, Giovannucci E. Molecular pathological epidemiology of colorectal neoplasia: an emerging transdisciplinary and interdisciplinary field. Gut. 2011;60:397–411.

    Article  PubMed  Google Scholar 

  33. Schernhammer ES, Giovannucci E, Kawasaki T, Rosner B, Fuchs CS, Ogino S. Dietary folate, alcohol and B vitamins in relation to LINE-1 hypomethylation in colon cancer. Gut. 2010;59:794–9.

    Article  PubMed  CAS  Google Scholar 

  34. Sepulveda AR, Jones D, Ogino S, et al. CpG methylation analysis—current status of clinical assays and potential applications in molecular diagnostics: a report of the Association for Molecular Pathology. J Mol Diagn. 2009;11:266–78.

    Article  PubMed  CAS  Google Scholar 

  35. Speek M. Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes. Mol Cell Biol. 2001;21:1973–85.

    Article  PubMed  CAS  Google Scholar 

  36. Peaston AE, Evsikov AV, Graber JH, et al. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev Cell. 2004;7:597–606.

    Article  PubMed  CAS  Google Scholar 

  37. Faulkner GJ, Kimura Y, Daub CO, et al. The regulated retrotransposon transcriptome of mammalian cells. Nat Genet. 2009;41:563–71.

    Article  PubMed  CAS  Google Scholar 

  38. Han JS, Szak ST, Boeke JD. Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature. 2004;429:268–74.

    Article  PubMed  CAS  Google Scholar 

  39. Yamada Y, Jackson-Grusby L, Linhart H, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci USA. 2005;102:13580–5.

    Article  PubMed  CAS  Google Scholar 

  40. Howard G, Eiges R, Gaudet F, Jaenisch R, Eden A. Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Oncogene. 2008;27:404–8.

    Article  PubMed  CAS  Google Scholar 

  41. Chalitchagorn K, Shuangshoti S, Hourpai N, et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene. 2004;23:8841–6.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

Supported in part by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (grant 23689061) and the Kobayashi Foundation for Cancer Research.

Author information

Authors and Affiliations

  1. Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan

    Shiro Iwagami MD, Yoshifumi Baba MD, PhD, Masayuki Watanabe MD, PhD, Hironobu Shigaki MD, Keisuke Miyake MSc, Satoshi Ida MD, PhD, Yohei Nagai MD, Takatsugu Ishimoto MD, PhD, Masaaki Iwatsuki MD, PhD, Yasuo Sakamoto MD, PhD, Yuji Miyamoto MD, PhD & Hideo Baba MD, PhD

Authors
  1. Shiro Iwagami MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yoshifumi Baba MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Masayuki Watanabe MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hironobu Shigaki MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Keisuke Miyake MSc
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Satoshi Ida MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yohei Nagai MD
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takatsugu Ishimoto MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Masaaki Iwatsuki MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yasuo Sakamoto MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yuji Miyamoto MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hideo Baba MD, PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hideo Baba MD, PhD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Iwagami, S., Baba, Y., Watanabe, M. et al. Pyrosequencing Assay to Measure LINE-1 Methylation Level in Esophageal Squamous Cell Carcinoma. Ann Surg Oncol 19, 2726–2732 (2012). https://doi.org/10.1245/s10434-011-2176-3

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1245/s10434-011-2176-3

Keywords

Search

Navigation