Gastroenterology

Gastroenterology

Volume 147, Issue 2, August 2014, Pages 418-429.e8
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Differences in DNA Methylation Signatures Reveal Multiple Pathways of Progression From Adenoma to Colorectal Cancer

https://doi.org/10.1053/j.gastro.2014.04.039Get rights and content

Background & Aims

Genetic and epigenetic alterations contribute to the pathogenesis of colorectal cancer (CRC). There is considerable molecular heterogeneity among colorectal tumors, which appears to arise as polyps progress to cancer. This heterogeneity results in different pathways to tumorigenesis. Although epigenetic and genetic alterations have been detected in conventional tubular adenomas, little is known about how these affect progression to CRC. We compared methylomes of normal colon mucosa, tubular adenomas, and colorectal cancers to determine how epigenetic alterations might contribute to cancer formation.

Methods

We conducted genome-wide array-based studies and comprehensive data analyses of aberrantly methylated loci in 41 normal colon tissue, 42 colon adenomas, and 64 cancers using HumanMethylation450 arrays.

Results

We found genome-wide alterations in DNA methylation in the nontumor colon mucosa and cancers. Three classes of cancers and 2 classes of adenomas were identified based on their DNA methylation patterns. The adenomas separated into classes of high-frequency methylation and low-frequency methylation. Within the high-frequency methylation adenoma class a subset of adenomas had mutant KRAS. Additionally, the high-frequency methylation adenoma class had DNA methylation signatures similar to those of cancers with low or intermediate levels of methylation, and the low-frequency methylation adenoma class had methylation signatures similar to that of nontumor colon tissue. The CpG sites that were differentially methylated in these signatures are located in intragenic and intergenic regions.

Conclusions

Genome-wide alterations in DNA methylation occur during early stages of progression of tubular adenomas to cancer. These findings reveal heterogeneity in the pathogenesis of colorectal cancer, even at the adenoma step of the process.

Section snippets

Primary Human Tissue Samples

DNA extracted from snap-frozen tissues was used for the studies using the HumanMethylation450 arrays. A detailed description of the samples used is available in the Supplementary Methods.

DNA Isolation and Bisulfite Conversion

Genomic DNA was extracted and bisulfite modified as described previously.24

Molecular Characterization

The CIMP status and MSI status of the CRCs were assessed using methods as described previously.24, 25 Gene mutation status of KRAS, BRAFV600E, APC, TP53, and PIK3CA was determined using the qBiomarker Somatic Mutation PCR System

Identification and Validation of Methylated Probes on the Human Methylation450 Arrays That Are Differentially Methylated Between Normal Colon, Tubular Adenomas, and Colorectal Cancer

We and others have reported previously that results from the HM450 BeadChips are technically robust, but that there is a measurable false discovery rate.26, 27 Therefore, we initially conducted technical validation studies and biological validation studies of a subset of differentially methylated CpGs (n = 4) found on the HM450 arrays (described in detail in the Supplementary Methods and Results). All of the CpG probes (n = 4) that were either aberrantly methylated in cancers or adenomas were

Discussion

In these studies we have found considerable genetic and epigenetic heterogeneity among not only CRCs but also among adenomas.6, 38, 39 The existence of different classes of CRC that differ based on DNA methylation patterns was first proposed by Issa and colleagues in 1999 when they identified a CIMP class of CRCs.8 Our studies provide additional insight into CIMP CRCs by showing that in the high methylation pattern CRCs there is a low frequency of APC mutations, consistent with findings from

Acknowledgments

The authors would like to acknowledge the outstanding service provided by the Genomics Shared Resources (FHCRC) and the Cooperative Human Tissue Network for the tissues they provided. We also thank the ColoCare team (Chris Velicer, Rebecca Holmes, Stephanie Zschäbitz, Kathy Vickers, Rachel Wilbur, Shannon Rush, and Sara Bates and others) for their assistance on these studies. In addition, we would like to thank Toshinori Hinoue and Peter W. Laird for kindly sharing their data. Finally, we would

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    Funding This research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award number RO1CA115513, P30CA15704, UO1CA152756, U54CA143862, and P01CA077852 (WMG), P50CA95103 (ZW), R01CA121060, P30CA68485, K07CA122451 (MJS). The content is solely the responsibility of the authors, and does not necessarily represent the official views of the National Institutes of Health. Support for these studies was also provided by a Burroughs Wellcome Fund Translational Research Award for Clinician Scientist (WMG), Program of Introducing Talents of Discipline to Universities of China (B12003, JW) and International Science & Technology Cooperation Program of China (2011DFA32570, JW), National Natural Science Foundation of China (81201920, YL); and 5P50CA150964 (SDM).

    Author names in bold designate shared co-first authorship.

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