We report for the first time to our knowledge that V600E and non-V600E BRAF mutations affect different patients with NSCLC. V600E mutations are significantly associated with female sex and represent a negative prognostic factor. In addition, we identified a number of other clinicopathologic parameters potentially useful for the selection of patients carrying BRAF mutations.

Systematic sequence profiling of the Glioblastoma Multiforme (GBM) genome has recently led to the identification of somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Interestingly, only the evolutionarily conserved residue R132 located in the substrate binding site of IDH1 was found mutated in GBM. At present, the occurrence and the relevance of p.R132 (IDH1 R132 ) variants in tumors other than GBMs is largely unknown. We searched for mutations at position R132 of the IDH1 gene in a panel of 672 tumor samples. These included high-grade glioma, gastrointestinal stromal tumors (GIST), melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, prostate, and thyroid carcinoma specimens. In addition, we assessed a panel of 84 cell lines from different tumor lineages. Somatic mutations affecting the IDH1 R132 residue were detected in 20% (23 of 113) high-grade glioma samples. In addition to the previously reported p.R132H and p.R132S alleles, we identified three novel somatic mutations (p.R132C, p.R132G, and p.R132L) affecting residue IDH1 R132 in GBM. Strikingly, no IDH1 mutations were detected in the other tumor types. These data indicate that cancer mutations affecting IDH1 R132 are tissue-specific, and suggest that it plays a unique role in the development of high-grade gliomas.

Introduction Lung cancer is formerly the highest cause of mortality among tumor pathologies worldwide. There are no validated techniques for an early detection of pulmonary cancer lesions other than low-dose helical CT-scan. Unfortunately, this method have some downside effects. Recent studies have laid the basis for development of exosomes-based techniques to screen/diagnose lung cancers. As the isolation of circulating exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostic applications. Methods We used a first set of 30 plasma samples from as many patients, including 10 patients affected by Lung Adenocarcinomas, 10 with Lung Granulomas and 10 healthy smokers matched for age and sex as negative controls. Wide range microRNAs analysis (742 microRNAs) was performed by quantitative RT-PCR. Data were compared by lesion characteristics using WEKA software for statistics and modeling. Subsequently, selected microRNAs were evaluated on an independent larger group of samples (105 specimens: 50 Lung Adenocarcinomas, 30 Lung Granulomas and 25 healthy smokers). Results This analysis led to the selection of 4 microRNAs to perform a screening test (miR-378a, miR-379, miR-139-5p and miR-200b-5p), useful to divide population into 2 groups: nodule (lung adenocarcinomas+carcinomas) and non-nodule (healthy former smokers). Six microRNAs (miR-151a-5p, miR-30a-3p, miR-200b-5p, miR-629, miR-100 and miR-154-3p) were selected for a second test on the “nodule” population to discriminate between lung adenocarcinoma and granuloma. Conclusions “Screening test” has shown 97.5% sensitivity, 72.0% specificity, AUC ROC of 90.8%. “Diagnostic test” had 96.0% sensitivity, 60.0% specificity, AUC ROC of 76.0%. Further evaluation is needed to confirm the predictive power of those models on higher cohorts of samples.

Purpose: KRAS mutations represent the main cause of resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (MoAbs) in metastatic colorectal cancer (mCRC). We evaluated whether highly sensitive methods for KRAS investigation improve the accuracy of predictions of anti-EGFR MoAbs efficacy.Experimental Design: We retrospectively evaluated objective tumor responses in mCRC patients treated with cetuximab or panitumumab. KRAS codons 12 and 13 were examined by direct sequencing, MALDI-TOF MS, mutant-enriched PCR, and engineered mutant-enriched PCR, which have a sensitivity of 20%, 10%, 0.1%, and 0.1%, respectively. In addition, we analyzed KRAS codon 61, BRAF, and PIK3CA by direct sequencing and PTEN expression by immunohistochemistry.Results: In total, 111 patients were considered. Direct sequencing revealed mutations in codons 12 and 13 of KRAS in 43/111 patients (39%) and BRAF mutations in 9/111 (8%), with almost all of these occurring in nonresponder patients. Using highly sensitive methods, we identified up to 13 additional KRAS mutations compared with direct sequencing, all occurring in nonresponders. By analyzing PIK3CA and PTEN, we found that of these 13 patients, 7 did not show any additional alteration in the PI3K pathway.Conclusions: The application of highly sensitive methods for the detection of KRAS mutations significantly improves the identification of mCRC patients resistant to anti-EGFR MoAbs.

IntroductionAssessment of EGFR mutation in non-small cell lung cancer (NSCLC) patients is mandatory for optimization of pharmacologic treatment. In this respect, mutation analysis of circulating tumor cells (CTCs) may be desirable since they may provide real-time information on patient's disease status.Experimental DesignBlood samples were collected from 37 patients enrolled in the TRIGGER study, a prospective phase II multi-center trial of erlotinib treatment in advanced NSCLC patients with activating EGFR mutations in tumor tissue. 10 CTC preparations from breast cancer patients without EGFR mutations in their primary tumors and 12 blood samples from healthy subjects were analyzed as negative controls. CTC preparations, obtained by the Veridex CellSearch System, were subjected to ultra-deep next generation sequencing (NGS) on the Roche 454 GS junior platform.ResultsCTCs fulfilling all Veridex criteria were present in 41% of the patients examined, ranging in number between 1 and 29. In addition to validated CTCs, potential neoplastic elements were seen in 33 cases. These included cells not fulfilling all Veridex criteria (also known as “suspicious objects”) found in 5 (13%) of 37 cases, and isolated or clustered large naked nuclei with irregular shape observed in 33 (89%) cases. EGFR mutations were identified by NGS in CTC preparations of 31 (84%) patients, corresponding to those present in matching tumor tissue. Twenty-five (96%) of 26 deletions at exon 19 and 6 (55%) of 11 mutations at exon 21 were detectable (P = 0.005). In 4 (13%) cases, multiple EGFR mutations, suggesting CTC heterogeneity, were documented. No mutations were found in control samples.ConclusionsWe report for the first time that the CellSearch System coupled with NGS is a very sensitive and specific diagnostic tool for EGFR mutation analysis in CTC preparations with potential clinical impact.