BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status

BACKGROUND: BRAF is a member of RAF family of serine/threonine kinases and mediates cellular responses to growth signals through the RAS-RAF-MAP kinase pathway. Activating mutations in BRAF have recently been found in about 10% of colorectal cancers, with the vast majority being a V600E hotspot mutation. The aim of the present study was to evaluate the clinical, pathological and molecular phenotype of colorectal tumors with BRAF mutations. RESULTS: Mutations in BRAF were identified in 8% (23/275) of colorectal cancers. They were 5–10-fold more frequent in tumors with infiltrating lymphocytes, location in the proximal colon, poor histological grade and mucinous appearance (P < 0.002 for each). Tumors with BRAF mutation were also 10-fold more likely to show microsatellite instability and frequent DNA methylation (P < 0.0001) compared to tumors without this mutation. The characteristic morphological features of tumors with BRAF mutation (infiltrating lymphocytes, poor grade, mucinous) remained after stratification according to microsatellite instability and methylator phenotypes. Mutations in BRAF were mutually exclusive with mutations in KRAS but showed no clear association with the presence of TP53 mutation. CONCLUSION: BRAF mutation identifies a colorectal cancer subgroup with distinctive phenotypic properties independent of microsatellite instability status and thus could be a valuable marker for studies into the clinical properties of these tumors

K-RAS testing as part of routine care for colorectal cancer patients

Recent clinical evidence strongly suggests that only those colorectal cancer patients with wild type K-ras respond to the anti-EGFR monoclonal antibody, Cetuximab. As such K-ras mutation testing is now a pre-requisite for inclusion in forthcoming CRC clinical trials using Cetuximab and is a requirement for access through the Interim Access Program (IAP) operated by Merck. SJOG Pathology in West Australia has recently implemented K-ras mutation testing and performed 300 such tests on DNA extracted from fresh frozen material as part of creating a colorectal tissue bank within routine patient management. Approximately 32 % of cases were K-ras mutant. The results have been included in the routine histopathology report. The test is cheap and easy to perform. At the same time we have established a service for K-ras testing from archival cases using paraffin embedded samples. In contrast to the prospective cases this service has proven difficult to establish due to several reasons, the most important being that accessing archival material is relatively difficult. In addition, jurisdiction over tissue blocks and the time lapsed between the initial surgery and when the test is requested (often some years) creates significant logistical issues for sample retrieval and testing. In addition, a survey we conducted demonstrated a wide range of knowledge about the testing by oncologists and this contributed to delays in obtaining the test results. Given the increased likelihood of similar tests for other molecular markers (eg. Braf, MSI) and with an ever increasing array of targeted therapies in the pipeline we suggest that routine diagnostic pathology labs should harvest DNA from primary tumours as part of their routine practice. In this manner such tests may be optimally performed whilst at the same time avoiding double handling of samples and so reducing both the cost of and time taken to perform molecular tests

Low expression of γ-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype

金沢大学がん研究所分子標的がん医療研究開発センターThe CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of γ-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin. © 2008 Cancer Research UK

Comprehensive profiling of DNA methylation in colorectal cancer reveals subgroups with distinct clinicopathological and molecular features

<p>Abstract</p> <p>Background</p> <p>Most previous studies of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been conducted on a relatively small numbers of CpG sites. In the present study we performed comprehensive DNA methylation profiling of CRC with the aim of characterizing CIMP subgroups.</p> <p>Methods</p> <p>DNA methylation at 1,505 CpG sites in 807 cancer-related genes was evaluated using the Illumina GoldenGate^®methylation array in 28 normal colonic mucosa and 91 consecutive CRC samples. Methylation data was analyzed using unsupervised hierarchical clustering. CIMP subgroups were compared for various clinicopathological and molecular features including patient age, tumor site, microsatellite instability (MSI), methylation at a consensus panel of CpG islands and mutations in <it>BRAF </it>and <it>KRAS</it>.</p> <p>Results</p> <p>A total of 202 CpG sites were differentially methylated between tumor and normal tissue. Unsupervised hierarchical clustering of methylation data from these sites revealed the existence of three CRC subgroups referred to as CIMP-low (CIMP-L, 21% of cases), CIMP-mid (CIMP-M, 14%) and CIMP-high (CIMP-H, 65%). In comparison to CIMP-L tumors, CIMP-H tumors were more often located in the proximal colon and showed more frequent mutation of <it>KRAS </it>and <it>BRAF </it>(<it>P </it>< 0.001).</p> <p>Conclusions</p> <p>Comprehensive DNA methylation profiling identified three CRC subgroups with distinctive clinicopathological and molecular features. This study suggests that both <it>KRAS </it>and <it>BRAF </it>mutations are involved with the CIMP-H pathway of CRC rather than with distinct CIMP subgroups.</p

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: A comprehensive profiling approach

10.1186/1471-230X-14-55BMC Gastroenterology141-BGMA

Genetic factors associated with intestinal metaplasia in a high risk Singapore-Chinese population: a cohort study

BMC Gastroenterology97

Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features

Activation of RAS signalling induced by K-ras/BRAF mutations is a hallmark of colorectal tumours. In addition, Ras association domain families 1 and 2 (RASSF1 and RASSF2), the negative regulators of K-ras, are often inactivated by methylation of the promoter region in those tumours. However, reports showing differences in the occurrence of these alterations on the basis of tumour characteristics have been scarce. We analysed K-ras/BRAF mutations and the methylation status of RASSF1 and RASSF2 promoter regions in 120 colorectal adenomas with respect to their clinicopathological features. K-ras/BRAF mutations and RASSF2 methylation were observed in 49 (41%) and 30 (25%) of the samples, respectively, while RASSF1 methylation was observed in only 3 (2.5%). Adenomas with RASSF2 methylation often carried K-ras/BRAF mutations simultaneously (22 out of 30, P<0.01). Multivariate analysis revealed that the concomitance of these alterations was frequently observed in serrated adenomas (odds ratio (OR) 11.11; 95% confidence interval (CI) 1.96–63.00), but rarely in adenomas located in the sigmoid or descending colon (OR 0.13; 95% CI 0.03–0.58). A comparison between adenomas and cancers showed a significantly higher prevalence of these alterations in cancers than in adenomas in the proximal colon (58 vs 27%, P=0.02). Frequency and the time point of the occurrence of Ras signalling disorders differ according to colorectal neoplasia's characteristics, particularly the location