Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile

Background: Sessile serrated polyps (SSPs) have emerged as important precursors for a large number of sporadic colorectal cancers. They are difficult to detect during colonoscopy due to their flat shape and the excessive amounts of secreted mucin that cover the polyps. The underlying genetic and epigenetic basis for the emergence of SSPs is largely unknown with existing genetic studies confined to a limited number of oncogenes and tumor suppressors. A full characterization of the genetic and epigenetic landscape of SSPs would provide insight into their origin and potentially offer new biomarkers useful for detection of SSPs in stool samples. Methods: We used a combination of genome-wide mutation detection, exome sequencing and DNA methylation profiling (via methyl-array and whole-genome bisulfite sequencing) to analyze multiple samples of sessile serrated polyps and compared these to familial adenomatous polyps. Results: Our analysis revealed BRAF-V600E as the sole recurring somatic mutation in SSPs with no additional major genetic mutations detected. The occurrence of BRAF-V600E was coincident with a unique DNA methylation pattern revealing a set of DNA methylation markers showing significant (~3 to 30 fold) increase in their methylation levels, exclusively in SSP samples. These methylation patterns effectively distinguished sessile serrated polys from adenomatous polyps and did so more effectively than parallel gene expression profiles. Conclusions: This study provides an important example of a single oncogenic mutation leading to reproducible global DNA methylation changes. These methylated markers are specific to SSPs and could be of important clinical relevance for the early diagnosis of SSPs using non-invasive approaches such as fecal DNA testing

Activating mutation in MET oncogene in familial colorectal cancer

<p>Abstract</p> <p>Background</p> <p>In developed countries, the lifetime risk of developing colorectal cancer (CRC) is 5%, and it is the second leading cause of death from cancer. The presence of family history is a well established risk factor with 25-35% of CRCs attributable to inherited and/or familial factors. The highly penetrant inherited colon cancer syndromes account for approximately 5%, leaving greater than 20% without clear genetic definition. Familial colorectal cancer has been linked to chromosome 7q31 by multiple affected relative pair studies. The <it>MET </it>proto-oncogene which resides in this chromosomal region is considered a candidate for genetic susceptibility.</p> <p>Methods</p> <p><it>MET </it>exons were amplified by PCR from germline DNA of 148 affected sibling pairs with colorectal cancer. Amplicons with altered sequence were detected with high-resolution melt-curve analysis using a LightScanner (Idaho Technologies). Samples demonstrating alternative melt curves were sequenced. A TaqMan assay for the specific c.2975C <b>></b>T change was used to confirm this mutation in a cohort of 299 colorectal cancer cases and to look for allelic amplification in tumors.</p> <p>Results</p> <p>Here we report a germline non-synonymous change in the <it>MET </it>proto-oncogene at amino acid position T992I (also reported as <it>MET </it>p.T1010I) in 5.2% of a cohort of sibling pairs affected with CRC. This genetic variant was then confirmed in a second cohort of individuals diagnosed with CRC and having a first degree relative with CRC at prevalence of 4.1%. This mutation has been reported in cancer cells of multiple origins, including 2.5% of colon cancers, and in <1% in the general population. The threonine at amino acid position 992 lies in the tyrosine kinase domain of MET and a change to isoleucine at this position has been shown to promote metastatic behavior in cell-based models. The average age of CRC diagnosis in patients in this study is 63 years in mutation carriers, which is 8 years earlier than the general population average for CRC.</p> <p>Conclusions</p> <p>Although the <it>MET </it>p.T992I genetic mutation is commonly found in somatic colorectal cancer tissues, this is the first report also implicating this <it>MET </it>genetic mutation as a germline inherited risk factor for familial colorectal cancer. Future studies on the cancer risks associated with this mutation and the prevalence in different at-risk populations will be an important extension of this work to define the clinical significance.</p

The biopsy samples.

<p>Mutations known for each sample are listed. SSP-1 to SSP-8 are exome sequenced. Mutations in other samples are tested by OncoCarta3. BRAF-V600E mutation and KRAS codon 12 and 13 mutations are tested by PCR-sequencing in all samples.</p

Gene expression profiling of colon samples.

<p>Hierarchical clustering and sample-to-sample distance heatmap of the expression of protein coding genes. SSP samples are clustered together, FAP samples also clustered together. Gene expression in TSA-2 that has both APC and BRAF mutation is more similar to the pattern of SSP expression cluster. The gene expression pattern of FAP show about 50% similarity to the gene expression of SSP, although the DNA methylation patterns of these two polyp types differ significantly. Therefore studying the DNA methylation marker can lead to detect SSP more specifically than studying the gene expression.</p

Hierarchical clustering of all CpGs in 450K methyl-array on colon samples.

<p>The SSP samples are clustered together based on DNA methylation. The TSA-2 sample that is mutant for BRAF gene also clustered with SSPs.</p

Global methylation changes in SSP and FAP.

<p><b>(a)</b> Comparison of FAP with normal samples shows only few DNA methylation changes. From 395,899 CpGs tested on the Infinium array, only 52 CpGs show more than 2 fold increase in methylation in FAP compared to normal colon tissue and 6 CpGs show more than 2 fold reduction in methylation (p-value <0.05). <b>(b)</b> In contrast comparison of SSP with normal samples shows significant DNA methylation changes. From 395,899 CpGs tested on the Infinium array, 42,965 (10.85%) show more than 2 fold increase in methylation in SSP compared to normal colon tissue and 19,019 (4.80%) show more than 2 fold reduction in methylation (p-value <0.05).</p

Global loss of methylation and partial methylation in SSP.

<p><b>(A)</b> Global level of cytosine methylation in SSP, GU and blood. <b>(B)</b> Global level of CpG methylation in SSP, GU and blood. Histogram of fraction methylation in colon SSP, GU and blood <b>(C-E). (C)</b> In normal tissues most of the CpGs are either unmethylated or highly methylated (75–95% methylated). In SSP the very high level of methylation is lost and methylation level shows a more disperse distribution (35–80% methylated). <b>(D)</b> The EMRs show mostly 5–15% methylation in uninvolved colon tissue, while they are 15–45% methylated in SSP. <b>(E)</b> The RMRs in SSP were originally 65–90% methylated in uninvolved colon but this level is reduced to 15–40% in SSP.</p