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. 2010 Dec;139(6):1855-64.
doi: 10.1053/j.gastro.2010.07.050. Epub 2010 Aug 2.

Hypomethylation of the IGF2 DMR in colorectal tumors, detected by bisulfite pyrosequencing, is associated with poor prognosis

Yoshifumi Baba  1 , Katsuhiko Nosho, Kaori Shima, Curtis Huttenhower, Noriko Tanaka, Aditi Hazra, Edward L Giovannucci, Charles S Fuchs, Shuji Ogino
Affiliations

Hypomethylation of the IGF2 DMR in colorectal tumors, detected by bisulfite pyrosequencing, is associated with poor prognosis

Yoshifumi Baba et al. Gastroenterology. 2010 Dec.

Abstract

Background & aims: The insulin-like growth factor 2 (IGF2) gene is normally imprinted. Constitutive loss of imprinting (LOI) of IGF2 has been associated with increased risks of colon cancer and adenoma, indicating its role in carcinogenesis. The conventional LOI assay relies on a germline polymorphism to distinguish between 2 allelic expression patterns but results in many uninformative cases. IGF2 LOI correlates with hypomethylation at the differentially methylated region (DMR)-0. An assay for methylation of the DMR0 could overcome the limitations of the conventional IGF2 LOI assay.

Methods: We measured methylation at the IGF2 DMR0 using a bisulfite-pyrosequencing assay with 1178 paraffin-embedded colorectal cancer tissue samples from 2 prospective cohort studies. A Cox proportional hazard model was used to calculate mortality hazard ratio (HR); calculations were adjusted for microsatellite instability; the CpG island methylator phenotype; LINE-1 methylation; and KRAS, BRAF, and PIK3CA mutations.

Results: Methylation at the IGF2 DMR0 was successfully measured in 1105 (94%) of 1178 samples. Colorectal tumors had significantly less methylation at the DMR0 compared with matched, normal colonic mucosa (P < .0001; N = 51). Among 1033 patients eligible for survival analysis, hypomethylation of the IGF2 DMR0 was significantly associated with higher overall mortality (log-rank P = .0006; univariate HR, 1.41; 95% confidence interval, 1.16-1.71; P = .0006; multivariate HR, 1.33; 95% confidence interval, 1.08-1.63; P = .0066).

Conclusions: A bisulfite-pyrosequencing assay to measure methylation of the IGF2 DMR0 is robust and applicable to paraffin-embedded tissue. IGF2 DMR0 hypomethylation in colorectal tumor samples is associated with shorter survival time, so it might be developed as a prognostic biomarker.

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Figures

Figure 1
Figure 1. IGF2 DMR0 methylation levels in colorectal cancer and normal mucosa
A. Representative pyrograms in IGF2 DMR0 methylation assay. The top panel shows a tumor with high methylation level (47.5%) and the bottom panel shows a tumor with low methylation level (13.3%). The % numbers (in blue shade) are proportions of C and T at each CpG site after bisulfite conversion, and the proportion of C indicates the methylation level at each CpG site. The second and third CpG sites follow stretches of Ts leading to apparently high T peaks relative to C peaks. B. IGF2 DMR0 methylation levels in 51 colorectal cancers and matched normal mucosa. Cancer tissues showed significantly lower level of methylation (mean 31.9; median 29.7) than matched normal mucosa (mean 50.6; median 51.4) (p<0.0001 by the paired t-test).
Figure 2
Figure 2. IGF2 DMR0 methylation levels in colorectal cancer
A. Distribution of IGF2 DMR0 methylation levels in 1105 colorectal cancers. B. Significant correlation between IGF2 DMR0 methylation level and LINE-1 methylation level in colorectal cancer (r=0.29, p<0.0001),
Figure 3
Figure 3
Kaplan-Meier curves for colorectal cancer-specific survival (top panels) and overall survival (bottom panels) according to quartiles (Q1-4) of IGF2 DMR0 methylation level in colorectal cancer. On the right panels, Q4 represents the “hypomethylated group” and Q1, Q2 and Q3 represent the “hypermethylated group”.
See this image and copyright information in PMC

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