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The objective of this study was to investigate the mutational profiles of cancers arising in different colon segments. To this aim, we have analyzed 37 colon cancer samples by use of the Ion AmpliSeq™ Comprehensive Cancer Panel. Overall, we have found 307 mutated genes, most of which already implicated in the development of colon cancer. Among these, 15 genes were mutated in tumors originating in all six colon segments and were defined "common genes" (i.e. APC, PIK3CA, TP53) whereas 13 genes were preferentially mutated in tumors originating only in specific colon segments and were defined "site-associated genes" (i.e. BLNK, PTPRD). In addition, the presence of mutations in 10 of the 307 identified mutated genes (NBN, SMUG1, ERBB2, PTPRT, EPHB1, ALK, PTPRD, AURKB, KDR and GPR124) were found to be of clinical relevance. Among clinically relevant genes, NBN and SMUG1 were identified as independent prognostic factors that predicted poor survival in colon cancer patients. In conclusion, the findings reported here indicate that tumors arising in different colon segments present differences in the type and/or frequency of genetic variants, with two of them being independent prognostic factors that predict poor survival in colon cancer patients., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

In this study, we have set-up a routine pipeline to evaluate the clinical application of Oncomine™ Focus Assay, a panel that allows the simultaneous detection of single nucleotide hotspot mutations in 35 genes, copy number alterations (CNAs) in 19 genes and gene fusions involving 23 genes in cancer samples. For this study we retrospectively selected 106 patients that were submitted to surgical resection for lung, gastric, colon or rectal cancer. We found that 56 patients out of 106 showed at least one alteration (53%), with 47 patients carrying at least one relevant nucleotide variant, 10 patients carrying at least one CNA and 3 patients carrying one gene fusion. On the basis of the mutational profiles obtained, we have identified 22 patients (20.7%) that were potentially eligible for targeted therapy. The most frequently mutated genes across all tumor types included KRAS (30 patients), PIK3CA (16 patients), BRAF (6 patients), EGFR (5 patients), NRAS (4 patients) and ERBB2 (3 patients) whereas CCND1, ERBB2, EGFR and MYC were the genes most frequently subjected to copy number gain. Finally, gene fusions were identified only in lung cancer patients and involved MET [MET(13)-MET(15) fusion] and FGFR3 [FGFR3(chr 17)-TACC3(chr 11)]. In conclusion, we demonstrate that the analysis with a multi-biomarker panel of cancer patients after surgery, may present several potential advantages in clinical daily practice, including the simultaneous detection of different potentially druggable alterations, reasonable costs, short time of testing and automated interpretation of results., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

The objective of this study was to determine the feasibility to detect copy number alterations in colon cancer samples using Next Generation Sequencing data and to elucidate the association between copy number alterations in specific genes and the development of cancer in different colon segments. We report the successful detection of somatic changes in gene copy number in 37 colon cancer patients by analysis of sequencing data through Amplicon CNA Algorithm. Overall, we have found a total of 748 significant copy number alterations in 230 significant genes, of which 143 showed CN losses and 87 showed CN gains. Validation of results was performed on 20 representative genes by quantitative qPCR and/or immunostaining. By this analysis, we have identified 4 genes that were subjected to copy number alterations in tumors arising in all colon segments (defined "common genes") and the presence of copy number alterations in 14 genes that were significantly associated to one specific site (defined "site-associated genes"). Finally, copy number alterations in ASXL1, TSC1 and IL7R turned out to be clinically relevant since the loss of TSC1 and IL7R was associated with advanced stages and/or reduced survival whereas copy number gain of ASXL1 was associated with good prognosis., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Hyperactivation of the PI3K/AKT pathway is observed in most human cancer including lung carcinomas. Here we have investigated the role of miRNAs as downstream targets of activated PI3K/AKT signaling in Non Small Cell Lung Cancer (NSCLC). To this aim, miRNA profiling was performed in human lung epithelial cells (BEAS-2B) expressing active AKT1 (BEAS-AKT1-E17K), active PI3KCA (BEAS-PIK3CA-E545K) or with silenced PTEN (BEAS-shPTEN).Twenty-four differentially expressed miRNAs common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells were identified through this analysis, with miR-196a being the most consistently up-regulated miRNA. Interestingly, miR-196a was significantly overexpressed also in human NSCLC-derived cell lines (n=11) and primary lung cancer samples (n=28).By manipulating the expression of miR-196a in BEAS-2B and NCI-H460 cells, we obtained compelling evidence that this miRNA acts downstream the PI3K/AKT pathway, mediating some of the proliferative, pro-migratory and tumorigenic activity that this pathway exerts in lung epithelial cells, possibly through the regulation of FoxO1, CDKN1B (hereafter p27) and HOXA9.

Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC.

The hotspot E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. In this manuscript, we sought to determine whether this AKT1 variant is a bona-fide activating mutation and plays a role in the development of lung cancer. Here we report that in immortalized human bronchial epithelial cells (BEAS-2B cells) mutant AKT1-E17K promotes anchorage-dependent and -independent proliferation, increases the ability to migrate, invade as well as to survive and duplicate in stressful conditions, leading to the emergency of cells endowed with the capability to form aggressive tumours at high efficiency. We provide also evidence that the molecular mechanism whereby AKT1-E17K is oncogenic in lung epithelial cells involves phosphorylation and consequent cytoplasmic delocalization of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these results, cytoplasmic p27 is preferentially observed in primary NSCLCs with activated AKT and predicts poor survival.