Background: A small subset (10-15%) of gastrointestinal stromal tumours (GISTs) lack mutations in KIT and PDGFRA (wild-type GIST). Recently, a novel BRAF exon 15 mutation (V600E) was detected in imatinib-naive wildtype high-risk intestinal GISTs (4%). However, the frequency and distribution of BRAF mutations within the spectrum of GISTs, and whether they might represent secondary events acquired during tumour progression, remain unknown. Methods: 69 GISTs (39 KIT mutants, 2 PDGFRA mutants and 28 wild-type) were analysed for mutations in BRAF exon 15 and KRAS exon 2. To assess the stage at which these mutations might occur in GIST, a considerable number of incidental gastric (n = 23) and intestinal (n = 2) tumours were included. Results: BRAF mutations (V600E) were detected in 2 of 28 wild-type GISTs (7%), but in none of the 41 KIT/ PDGFRA mutants. No KRAS mutation was detected. The two BRAF-mutated GISTs measured 4 mm in diameter and originated in the gastric body and the jejunum in two men (mean age, 76 years). Both tumours were mitotically inactive KIT-positive spindle-cell GISTs that were indistinguishable histologically from their more common KITmutated counterparts. Conclusion: BRAF mutations represent an alternative molecular pathway in the early tumorigenesis of a subset of KIT/PDGFRA wild-type GISTs and are per se not associated with a high risk of malignancy. Mutations in KIT, PDGFRA and BRAF were mutually exclusive in this study. Results from this and a previous study indicate that BRAF-mutated GISTs show a predilection for the small bowel (four of five tumours), but this needs further evaluation in larger studies.

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAF V600E have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAF V600E and specific KRAS mutation (Gly fi Asp; G12D, Gly fi Asp, G13D; Gly fi Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAF V600E was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild-type cancers (p 5 0.038). With MSI, specific KRAS and BRAF V600E mutations, 3 distinct prognostic subgroups were observed in univariate (p 5 0.006) and multivariable (p 5 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAF V600E mutation, (ii) KRAS/BRAF V600E wild-type or KRAS G13D mutations in MSS/MSI-L and (iii) MSI-H and KRAS G13D mutations. Moreover, none of the sporadic MSI-H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAF V600E and MSI may identify sporadic CRC patients with poor clinical outcome.Sporadic colorectal cancer (CRC) is a heterogeneous disease arising from at least 2 different and seemingly independent pathways, namely the chromosomal instability (CIN, or microsatellite stable, MSS) and microsatellite instability (MSI) pathways. 1 CRCs from the MSS pathway are thought to originate from a relatively uniform and linear accumulation of genetic changes in APC, KRAS and TP53. 2 However, in practice, only 10% of these tumors are characterized by this classic genotype. 3,4 Sporadic MSI-high (MSI-H) cancers occur through promoter hypermethylation of the MLH1 gene, more frequently found in females, and tend to be poorly differentiated tumors, of mucinous subtype, associated with a Crohn's like reaction at the invasive tumor front and often harboring somatic mutations in BRAF V600E . 5-7 MSI-H CRCs can also arise in the context of Lynch syndrome/hereditary nonpolyposis CRC (HNPCC), an autosomal dominant disorder, accounting for 3% of all CRC cases, characterized by germline mutations of mismatch-repair genes. 8 Both sporadic and Lynch syndrome-associated MSI-H tumors are characterized by dense tumor-infiltrating lymphocyte populations and less frequent metastases suggesting enhanced immunogenicity irrespective of the mechanism of DNA mismatch repair inactivation. 9,10 Recently, MSI status, BRAF V600E and KRAS mutation status have attracted significant attention due to their potential prognostic and predictive role in CRC. 11 On one hand, patients with MSI-H cancers are specifically thought to have a favorable outcome; yet on the other hand, they are generally nonresponsive to certain chemotherapeutic agents such as 5-flurouracil (5-FU). 6,12 The prognostic effect of somatic KRAS mutations, described in 30...

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ≥ 4/5 methylated genes), MSI (MSI-H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

Our comprehensive data would be compatible with a two-step process leading to loss of p16 expression in NSCLC. Hypermethylation of the promoter region may represent an early event, followed by heterozygous deletion of the p16 locus. Because of the possibility of detection of hypermethylated gene regions, these data may lead to the identification of specific patient subgroups more likely to benefit from upcoming demethylating treatment strategies.

These findings underline that MSI, rather than CIMP, plays a role in conferring a tumour budding phenotype. Budding retains its unfavourable prognostic effect independently of these five molecular features. Continued efforts to standardize the assessment of tumour budding are necessary to integrate this feature into daily diagnostic routine.