A panel of genes methylated with high frequency in colorectal cancer.

Susan M Mitchell ,Iain Beetson,Aidan Mcevoy,Thu Ho,Horace R Drew ,Jason P Ross ,Susanne K Pedersen ,Lawrence C Lapointe ,Michael Buckley ,Neil F W Saunders ,David Wattchow ,Trevor Lockett ,Glenn Brown ,Keith N Rand ,Robert Dunne ,Rohan T Baker ,Konsta Duesing ,Melissa L Thomas ,Graeme P Young ,Peter L Molloy

doi:10.1186/1471-2407-14-54

Abstract

BackgroundThe development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.MethodsCombined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).ResultsCombined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.ConclusionsThis study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Highlights

The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation of gene promoter regions
We had previously identified [32] in a large set of colorectal tissues a panel of 429 genes that were down-regulated in both colorectal cancers and adenomas relative to normal tissue
We had developed two novel methods of genome-wide DNA methylation analysis, BisulfiteTag and Streptavidin bisulfite ligand methylation enrichment (SuBLiME), that interrogated different but overlapping portions of the methylome (Figure 1); these were applied to clinical specimens and/or CRC cell lines and wbcDNA respectively

Summary

Introduction

The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation of gene promoter regions. It is well established that widespread epigenetic changes, including of DNA methylation profiles, relative to non-neoplastic tissue are a characteristic of many cancer types [1,2] These changes typically involve the hypermethylation of promoter regions, characterised by CpG islands, of many genes as well as reduced methylation of repeated DNA sequences and some individual genes [2,3,4]. While many genes are relevant to CRC subtypes, some genes such as SEPT9 [12] and VIM [13] become methylated in a high fraction of cancers and are being commercialised as diagnostic markers Despite their promise, there is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests

Methods

Results

Discussion

Conclusion