The homeobox gene MEIS1 is methylated in BRAFp.V600E mutated colon tumors

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAFp.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAFp.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAFp.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF p.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAFp.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAFp.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAFp.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression. Copyright

Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients

Recurrent Coding Sequence Variation Explains only A Small Fraction of the Genetic Architecture of Colorectal Cancer

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10-7), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10-7); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10-

Germline variants in POLE are associated with early onset mismatch repair deficient colorectal cancer

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

COSORE: A community database for continuous soil respiration and other soil‐atmosphere greenhouse gas flux data

Globally, soils store two to three times as much carbon as currently resides in the atmosphere, and it is critical to understand how soil greenhouse gas (GHG) emissions and uptake will respond to ongoing climate change. In particular, the soil‐to‐atmosphere CO2 flux, commonly though imprecisely termed soil respiration (RS), is one of the largest carbon fluxes in the Earth system. An increasing number of high‐frequency RS measurements (typically, from an automated system with hourly sampling) have been made over the last two decades; an increasing number of methane measurements are being made with such systems as well. Such high frequency data are an invaluable resource for understanding GHG fluxes, but lack a central database or repository. Here we describe the lightweight, open‐source COSORE (COntinuous SOil REspiration) database and software, that focuses on automated, continuous and long‐term GHG flux datasets, and is intended to serve as a community resource for earth sciences, climate change syntheses and model evaluation. Contributed datasets are mapped to a single, consistent standard, with metadata on contributors, geographic location, measurement conditions and ancillary data. The design emphasizes the importance of reproducibility, scientific transparency and open access to data. While being oriented towards continuously measured RS, the database design accommodates other soil‐atmosphere measurements (e.g. ecosystem respiration, chamber‐measured net ecosystem exchange, methane fluxes) as well as experimental treatments (heterotrophic only, etc.). We give brief examples of the types of analyses possible using this new community resource and describe its accompanying R software package

Gene expression of <i>MEIS1</i> and <i>MEIS1D</i>_<i>27</i> expression in human colorectal samples and CRC cell lines.

<p><i>MEIS1</i> gene expression as measured by RT-qPCR corrected for the geometric mean of the housekeeping genes <i>CPSF6</i> and <i>HNRNPM</i>. (<b>A</b>) <i>BRAF</i>^p.V600E tumors (black bars) that were all <i>MEIS1</i> methylated showed lower expression of the full length <i>MEIS1</i> gene, when compared to the paired normal tissues (white bars). (<b>B</b>) Colorectal cancer cell lines that were methylated for the <i>MEIS1</i> gene promoter were devoid of <i>MEIS1</i> gene expression. HT29, LS411N and RKO colon cancer cell lines carried the <i>BRAF</i>^p.V600E mutation, whereas the remaining cell lines were wild type for <i>BRAF</i>. (<b>C</b>) Gene expression of the truncated <i>MEIS1D</i>_<i>27</i> transcript in <i>BRAF</i>^p.V600E tumors (black bars) was low to absent when compared to paired normal tissue (white bars). Primer sets used, uniquely detect the truncated transcript. (<b>D</b>) <i>MEIS1</i> methylated colon cancer cells were devoid of <i>MEIS</i>_<i>D27</i> (i.e. exon 8 skipped <i>MEIS1</i>). Gene expression was expressed relative to SW837.</p

<i>MEIS1</i> is methylated in <i>BRAF</i>^p.V600E relative to <i>BRAF</i> wild type tumors.

<p>(<b>A</b>) The <i>MEIS1</i> promoter is hypermethylated in colorectal tumors with a <i>BRAF</i>^p.V600E mutation (black dots) when compared to wild type <i>BRAF</i> (white dots). The Y-axis represents the tumor vs. normal log₂ ratio for the median probe per CpG fragment. The horizontal dotted line at log₂ ratio 0 indicates an equal extent of <i>MEIS1</i> methylation in tumor and normal samples. (<b>B</b>) Overview of the analyzed <i>MEIS1</i> promoter, CpG islands within the promoter and the locus analyzed by MSP primers. Locations were based on the human genome browser (UCSC assembly March 2006, hg18). (<b>C</b>) <i>MEIS1</i>-MSP data showing hypermethylation in <i>BRAF</i>^p.V600E colorectal tumors when compared to <i>BRAF</i> wild types. T: tumor; N: normal tissue; M: methylated <i>MEIS1</i> promoter (168 bp); Um: Unmethylated <i>MEIS1</i> promoter (176 bp).</p

Colorectal cancer risk variants on 11q23 and 15q13 are associated with unexplained adenomatous polyposis

Background Colorectal adenomatous polyposis is associated with a high risk of colorectal cancer (CRC) and is frequently caused by germline mutations in APC or MUTYH. However, in about 20–30% of patients no underlying gene defect can be identified. In this study, we tested if recently identified CRC risk variants play a role in patients with &gt;10 adenomas. Methods We analysed a total of 16 SNPs with a reported association with CRC in a cohort of 252 genetically unexplained index patients with &gt;10 colorectal adenomas and 745 controls. In addition, we collected detailed clinical information from index patients and their first-degree relatives (FDRs). Results We found a statistically significant association with two of the variants tested: rs3802842 (at chromosome 11q23, OR=1.60, 95% CI 1.3 to 2.0) and rs4779584 (at chromosome 15q13, OR=1.50, 95% CI 1.2 to 1.9). The majority of index patients (84%) had between 10 and 100 adenomas and 15% had &gt;100 adenomas. Only two index patients (1%), both with &gt;100 adenomas, had FDRs with polyposis. Forty-one per cent of the index patients had one or more FDRs with CRC. Conclusions These SNPs are the first common, low-penetrant variants reported to be associated with adenomatous polyposis not caused by a defect in the APC, MUTYH, POLD1 and POLE genes. Even though familial occurrence of polyposis was very rare, CRC was over-represented in FDRs of polyposis patients and, if confirmed, these relatives will therefore benefit from surveillance