Frequency and clinicopathologic profile of PIK3CA mutant GISTs: molecular genetic study of 529 cases.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors usually driven by the mutational activation of receptor tyrosine kinases, KIT, or PDGFRA. Oncogenic activation of phosphatidylinositide-3-kinase (PI3K), a downstream effector in the KIT signaling pathway, has been identified in different types of cancer, with the PI3K 110α subunit encoded by PIK3CA being a common mutational target. In this study, the mutational hotspot in the PIK3CA kinase domain encoded by exon 20 was evaluated in 529 imatinib-naive GISTs using PCR amplification and Sanger sequencing. Eight mutations (two co-existing in one tumor) were identified. Subsequently, The cobas PIK3CA Mutation Test was employed to evaluate mutational hotspots in exons 1, 4, 7, and 9 in 119 PIK3CA exon 20-wild type tumors. In two cases, mutations in exons 1 and 9 were identified. In one GIST, previously undetected by Sanger sequencing, the exon 20 mutation was discovered. Altogether, eight primary and two metastatic GISTs carried PIK3CA mutations. The size of primary PIK3CA-mutant GISTs was ≥14 cm (mean size 17 cm), and mitotic activity varied from 0 to 72 per 50HPF (mean 5/50HPF). Follow-up data showed short survival in 6 of 7 studied cases. Detection of PIK3CA mutations in large or metastatic KIT-mutant GISTs may suggest that PIK3CA-mutant clones have a proliferative advantage during disease progression. Tyrosine kinase inhibitors have been successfully used in GIST treatment. However, resistance frequently develops due to secondary KIT mutations or activation of downstream to KIT signaling pathways, such as the PI3K/AKT/mTOR pathway. PIK3CA mutations similar to the ones detected in GISTs have been shown to cause such activation. Therefore, genotyping of PIK3CA in GISTs might help to pinpoint primary and metastatic tumors with the potential to develop resistance to tyrosine kinase inhibitors and guide therapy with PI3K inhibitors.

No KRAS mutations found in gastrointestinal stromal tumors (GISTs): molecular genetic study of 514 cases.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. A great majority of GISTs is driven by pathological activation of KIT or platelet-derived growth factor receptor-α (PDGFRA), two closely related receptor tyrosine kinases. However, other genetic changes including gain-of-function BRAF mutations and loss of succinate dehydrogenase (SDH) complex activity have been identified in the subsets of KIT-, PDGFRA-wild type tumors. Genetic mutations affecting KIT, PDGFRA, BRAF and SDH complex functions are believed to be mutually exclusive events. Recently, KRAS codon 12 and 13 mutations were reported in a small subset of KIT or PDGFRA mutant GISTs. Moreover, in in vitro experiments, KIT mutants with concurrent KRAS mutation showed resistance to imatinib, a receptor tyrosine kinase inhibitor used in GIST treatment. The aim of this study was to evaluate a large cohort of GISTs to define frequency and clinical significance of KRAS mutations in this type of cancer. A well-characterized cohort of 514 GISTs was screened for KRAS mutations using Sanger sequencing (n=450) and pyrosequencing (n=64). In all, 350 gastric, 100 intestinal and 64 primary disseminated GISTs were analyzed. No KRAS mutations were found. In GIST, KRAS mutations are extremely rare if they exist (<0.2%). Thus, mutational activation of KRAS does not seem to play any significant role in the development and progression of this type of cancer.

Detection of the BRAF V600E mutation in colon carcinoma: critical evaluation of the imunohistochemical approach.

Recently BRAF V600E mutant-specific antibody (clone VE1) became available to immunohistochemically pinpoint the occurrence of these BRAF-mutant proteins in different tumors, such as colon carcinoma. Detection of BRAF mutations is important for the accurate application of targeted therapy against BRAF serine-threonine kinase activation. In this study, we evaluated 113 colon carcinomas including 95 primary and 27 metastatic tumors with the VE1 antibody using Leica Bond-Max automated immunohistochemistry. To ensure comprehensive BRAF V600E mutation detection, all cases were evaluated using 4 molecular methods (Sanger sequencing, the Cobas 4800 BRAF V600 Mutation Test, BRAF V600 allele-specific polymerase chain reaction, and BRAF V600 quantitative polymerase chain reaction) with nearly 100% concordance. Molecular and immunohistochemical studies were blinded. Furthermore, all cases were evaluated for KRAS and NRAS mutations as parameters mutually exclusive with BRAF mutations offering parallel evidence for BRAF mutation status. Strong to moderate VE1 positivity was seen in 34 tumors. Twelve colon carcinomas showed weak VE1 immunohistochemical staining, and 67 were entirely negative. An identical c.1799T>A single nucleotide substitution leading to the BRAF V600E mutation was identified in 27 of 113 (24%) colon carcinomas. A majority of BRAF-mutant tumors were located in the right side of the colon and had mismatch-repair deficiency. V600E mutation-negative carcinomas were more often sigmoid tumors and usually showed intact mismatch-repair proteins and KRAS or NRAS mutations. The sensitivity and specificity of positive results (strong to moderate staining) of VE1 immunohistochemistry were 85% and 68%, respectively. If any positivity would be considered, then the specificity declined to 51% with no significant improvement of sensitivity. Therefore, only strong positivity should be considered when using the VE1 antibody and Leica Bond-Max automated immunohistochemistry with these parameters. Although VE1 antibody can be useful in the screening of colon carcinomas for BRAF V600E-mutant proteins, molecular genetic confirmation is always necessary for mutation diagnosis.