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

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

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

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

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

DNA methylation markers for SSP characterization and early diagnosis.

<p>DNA methylation markers for SSP characterization and early diagnosis.</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