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

2017 Research & Innovation Day Program

A one day showcase of applied research, social innovation, scholarship projects and activities.https://first.fanshawec.ca/cri_cripublications/1004/thumbnail.jp

IL17 Mediates Pelvic Pain in Experimental Autoimmune Prostatitis (EAP)

<div><p>Chronic pelvic pain syndrome (CPPS) is the most common form of prostatitis, accounting for 90–95% of all diagnoses. It is a complex multi-symptom syndrome with unknown etiology and limited effective treatments. Previous investigations highlight roles for inflammatory mediators in disease progression by correlating levels of cytokines and chemokines with patient reported symptom scores. It is hypothesized that alteration of adaptive immune mechanisms results in autoimmunity and subsequent development of pain. Mouse models of CPPS have been developed to delineate these immune mechanisms driving pain in humans. Using the experimental autoimmune prostatitis (EAP) in C57BL/6 mice model of CPPS we examined the role of CD4+T-cell subsets in the development and maintenance of prostate pain, by tactile allodynia behavioral testing and flow cytometry. In tandem with increased CD4+IL17A+ T-cells upon EAP induction, prophylactic treatment with an anti-IL17 antibody one-day prior to EAP induction prevented the onset of pelvic pain. Therapeutic blockade of IL17 did not reverse pain symptoms indicating that IL17 is essential for development but not maintenance of chronic pain in EAP. Furthermore we identified a cytokine, IL7, to be associated with increased symptom severity in CPPS patients and is increased in patient prostatic secretions and the prostates of EAP mice. IL7 is fundamental to development of IL17 producing cells and plays a role in maturation of auto-reactive T-cells, it is also associated with autoimmune disorders including multiple sclerosis and type-1 diabetes. More recently a growing body of research has pointed to IL17’s role in development of neuropathic and chronic pain. This report presents novel data on the role of CD4+IL17+ T-cells in development and maintenance of pain in EAP and CPPS.</p></div

EAP induces pain and expression of IL17 in C57BL/6 mice.

<p>A(i). EAP induces pain as assessed by tactile allodynia Von Frey testing in C57BL/6 mice. Pain is depicted as increased percentage response frequency above baseline every 7 days for 28 days. A(ii-iii), Responses frequencies to filaments on increased force showing increased response as early as day 7 post-EAP induction. B. Flow cytometry staining for T-cell extra and intracellular markers showing increases in CD4+ve IL17+ve cells in both EAP prostate, (i) and iliac lymph node, (iv) tissues compared to controls. Staining for CD4+ve IFNγ (ii, v) cells and CD4+ve CD25+ve FoxP3+ve (iii, vi) show no significant changes between EAP group and controls. Mean +/- SEM is shown, gating was performed as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125623#pone.0125623.s001" target="_blank">S1 Fig</a> Flow cytometry experiments are the average of at least two experiments with N = 4/5 mice per group. * = p<0.05, ** = p<0.01.</p

Increased IL7 in EPS is correlated with patient symptom scores.

<p>A (i-iv) Positive correlation between increasing patient scores and levels of IL7 in EPS. B Negative correlation between Total score and IL1RA. C (i-iii) Positive correlations between each patient score metric. * = p<0.05, ** = p<0.01.</p

IL7 expression is increased in a prostate specific manner in EAP mice.

<p>A (i-iv) QRTPCR of IL7 from murine splenic, bladder, prostate and iliac lymph tissues. B. Immuno-blotting and densitometry for IL7 using prostate tissue lysates. C. Immuno-histochemical staining for IL7, (i-ii) Isotype staining on EAP prostate (iii-iv) IL7 staining on control prostate and (v-vi) IL7 staining on EAP prostate, circle; epithelial, arrowhead; stromal cell staining. Immunoblot densitometry performed in ImageJ using area under the curve values followed by a T-test. Mean +/- SEM are shown, statistics performed in Prism using unpaired T-test, QRTPCR samples normalized to relevant controls with L19 as housekeeping gene and displayed as fold change, performed in technical duplicate and biological triplicate with N = 4/5 mice per group. * = p<0.05, ** = p<0.01.</p