We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score ≥ 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (∼2%; 4/216) and TRAF7 (∼2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK(1) cells even in the absence of hormonal stimulation. Incubating cells at 20 degrees C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H(+)-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20 degrees C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (p<0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that non-epitheliod histology (p=0.037), whereas CDKN2A deletions occurred more frequently in non-epithelioid subtypes among men (p=0.021) and were correlated with shorter overall survival for the entire cohort (p=0.002) and for men (p=0.012). Furthermore, women were more likely to harbor TP53 mutations (p=0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in non-epithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive disease related to asbestos exposure, with no effective therapeutic options. Methods: We undertook unsupervised analyses of RNA-sequencing data of 284 MPMs, with no assumption of discreteness. Using immunohistochemistry, we performed an orthogonal validation on a subset of 103 samples and a biological replication in an independent series of 77 samples. Findings: A continuum of molecular profiles explained the prognosis of the disease better than any discrete model. The immune and vascular pathways were the major sources of molecular variation, with strong differences in the expression of immune checkpoints and pro-angiogenic genes; the extrema of this continuum had specific molecular profiles: a "hot" bad-prognosis profile, with high lymphocyte infiltration and high expression of immune checkpoints and pro-angiogenic genes; a "cold" badprognosis profile, with low lymphocyte infiltration and high expression of pro-angiogenic genes; and a "VEGFR2+/VISTA+" better-prognosis profile, with high expression of immune checkpoint VISTA and proangiogenic gene VEGFR2. We validated the gene expression levels at the protein level for a subset of five selected genes belonging to the immune and vascular pathways (CD8A, PDL1, VEGFR3, VEGFR2, and VISTA), in the validation series, and replicated the molecular profiles as well as their prognostic value in the replication series. Interpretation: The prognosis of MPM is best explained by a continuous model, which extremes show specific expression patterns of genes involved in angiogenesis and immune response.

Introduction:The aim of this study was to validate a molecular expression signature [cell cycle progression (CCP) score] that identifies patients with a higher risk of cancer-related death after surgical resection of early stage (I-II) lung adenocarcinoma in a large patient cohort and evaluate the effectiveness of combining CCP score and pathological stage for predicting lung cancer mortality.Methods:Formalin-fixed paraffin-embedded surgical tumor samples from 650 patients diagnosed with stage I and II adenocarcinoma who underwent definitive surgical treatment without adjuvant chemotherapy were analyzed for 31 proliferation genes by quantitative real-time polymerase chain reaction. The prognostic discrimination of the expression score was assessed by Cox proportional hazards analysis using 5-year lung cancer-specific death as primary outcome.Results:The CCP score was a significant predictor of lung cancer-specific mortality above clinical covariates [hazard ratio (HR) = 1.46 per interquartile range (95% confidence interval = 1.12–1.90; p = 0.0050)]. The prognostic score, a combination of CCP score and pathological stage, was a more significant indicator of lung cancer mortality risk than pathological stage in the full cohort (HR = 2.01; p = 2.8 × 10−11) and in stage I patients (HR = 1.67; p = 0.00027). Using the 85th percentile of the prognostic score as a threshold, there was a significant difference in lung cancer survival between low-risk and high-risk patient groups (p = 3.8 × 10−7).Conclusions:This study validates the CCP score and the prognostic score as independent predictors of lung cancer death in patients with early stage lung adenocarcinoma treated with surgery alone. Patients with resected stage I lung adenocarcinoma and a high prognostic score may be candidates for adjuvant therapy to reduce cancer-related mortality.

Genomic instability, as demonstrated by the presence of additional alleles at short tandemly repeated (STR) loci, has recently been observed in colorectal tumours from individuals with hereditary nonpolyposis colorectal cancer (HNPCC), and in some sporadic tumours. These neoplasms have been called replication error positive (RER+). In this study, we confirm the presence of genomic instability in a proportion of unselected colorectal carcinomas but find no evidence of instability in adenomas. We further report replication errors in a tetranucleotide sequence, and in STRs within two tumour suppressor genes. 108 colorectal adenocarcinomas and 46 adenomas were analysed for the presence of variant bands at 4-15 microsatellite markers. Seven (6.5%) of carcinomas were RER+, four of which originated from the proximal colon. Analysis of the adenomas and of matched adenoma-carcinoma and carcinoma-metastatic samples from four patients suggests that the replication errors may occur during the development of carcinomas but are rare in adenomas.

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