Mechanisms of nuclear content loading to exosomes.

Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development

Gene Expression Analysis Identifies Novel Targets for Cervical Cancer Therapy

Although there has been significant progress in prevention and treatment of cervical cancer, this malignancy is still a leading cause of cancer death for women. Anti-angiogenesis and immunotherapy approaches have been providing survival benefits, however, response rates and durability of response need to be improved. There is a clear need for combination therapies that increase effectiveness of these agents and further improve patient outcome. Previous studies have largely focused on gene expression and molecular pathways in untreated cervix cancer. The goal of this study was to evaluate cancer-specific molecular pathways and their correlation with tumor immune profile in recurrent cervical cancer. Tumor and adjacent normal tissues were used to identify potential combination therapy targets. We found that DNA damage repair pathway genes were significantly overexpressed in the tumor. Based on our results and other recent investigations, we suggest that combination immune checkpoint and PARP inhibitor therapy is a high priority consideration for patients with recurrent, previously treated cervical cancer. We also show that multiple epithelial-mesenchymal transition-related genes, including MAP2K4, ID2, JAK1, FGF2, PIK3R1, AKT3, FGF13, and STAT3 may be potential targets. Interestingly, high-throughput analysis of Cancer Genome Atlas data identified distinct targets, including Fatty acid synthase FASN and Matrix Metallopeptidase 1 MMP1 as novel, promising combination therapy partners

Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan–Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R(2) = 0.73 and R(2) = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-016-0705-4) contains supplementary material, which is available to authorized users

Beyond BRAFV600: Clinical Mutation Panel Testing by Next-Generation Sequencing in Advanced Melanoma

The management of melanoma has evolved owing to improved understanding of its molecular drivers. To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next-generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed. Mutations were identified in 43 of the 46 genes on the panel. The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%). Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype. Concurrent TP53 mutations were the most frequent events in tumors with BRAFV600and NRAS mutations. Melanomas with BRAFNon-V600mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%). The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location. Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600and NRAS mutations. These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma

A Pan-cancer analysis reveals high-frequency genetic alterations in mediators of signaling by the tgf-β superfamily

We present an integromic analysis of gene alterations that modulate transforming growth factor β (TGF-β)-Smad-mediated signaling in 9,125 tumor samples across 33 cancer types in The Cancer Genome Atlas (TCGA). Focusing on genes that encode mediators and regulators of TGF-β signaling, we found at least one genomic alteration (mutation, homozygous deletion, or amplification) in 39% of samples, with highest frequencies in gastrointestinal cancers. We identified mutation hotspots in genes that encode TGF-β ligands (BMP5), receptors (TGFBR2, AVCR2A, and BMPR2), and Smads (SMAD2 and SMAD4). Alterations in the TGF-β superfamily correlated positively with expression of metastasis-associated genes and with decreased survival. Correlation analyses showed the contributions of mutation, amplification, deletion, DNA methylation, and miRNA expression to transcriptional activity of TGF-β signaling in each cancer type. This study provides a broad molecular perspective relevant for future functional and therapeutic studies of the diverse cancer pathways mediated by the TGF-β superfamily

The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter

Comprehensive and Integrated Genomic Characterization of Adult Soft Tissue Sarcomas

Summary Sarcomas are a broad family of mesenchymal malignancies exhibiting remarkable histologic diversity. We describe the multi-platform molecular landscape of 206 adult soft tissue sarcomas representing 6 major types. Along with novel insights into the biology of individual sarcoma types, we report three overarching findings: (1) unlike most epithelial malignancies, these sarcomas (excepting synovial sarcoma) are characterized predominantly by copy-number changes, with low mutational loads and only a few genes (TP53, ATRX, RB1) highly recurrently mutated across sarcoma types; (2) within sarcoma types, genomic and regulomic diversity of driver pathways defines molecular subtypes associated with patient outcome; and (3) the immune microenvironment, inferred from DNA methylation and mRNA profiles, associates with outcome and may inform clinical trials of immune checkpoint inhibitors. Overall, this large-scale analysis reveals previously unappreciated sarcoma-type-specific changes in copy number, methylation, RNA, and protein, providing insights into refining sarcoma therapy and relationships to other cancer types