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Annals of Surgical Oncology
Published in: Annals of Surgical Oncology 8/2012

01-08-2012 | Translational Research and Biomarkers

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

Authors: 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

Published in: Annals of Surgical Oncology | Issue 8/2012

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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.
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Metadata
Title
Pyrosequencing Assay to Measure LINE-1 Methylation Level in Esophageal Squamous Cell Carcinoma
Authors
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
Publication date
01-08-2012
Publisher
Springer-Verlag
Published in
Annals of Surgical Oncology / Issue 8/2012
Print ISSN: 1068-9265
Electronic ISSN: 1534-4681
DOI
https://doi.org/10.1245/s10434-011-2176-3

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