Sporadic hemangioblastomas are characterized by cryptic VHL inactivation

Abstract Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.http://deepblue.lib.umich.edu/bitstream/2027.42/110224/1/40478_2014_Article_167.pd

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation

Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas

The Public Repository of Xenografts enables discovery and randomized phase II-like trials in mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease

Evaluation of three methods for the detection of DNA single-strand breaks in human lymphocytes: alkaline elution, nick translation, and single-cell gel electrophoresis.

The aim of this study is to assess the ability of three methods, alkaline elution (AE), nick translation (NT), and single-cell gel electrophoresis (SCGE), to detect DNA single-strand breaks (ssb) in human peripheral blood lymphocytes (HPBL) exposed in vitro to three genotoxic agents; gamma-rays, ethyl methanesulfonate (EMS) and benzo[a]pyrene diol epoxide (BPDE). The ultimate objective is to select the most feasible, sensitive, and reproducible method for the monitoring of populations exposed to genotoxic agents. AE and NT do not seem suitable assays. AE is able to detect DNA lesions induced by the three compounds, but only at relatively high doses (2 Gy, 5 mM EMS and 20 microM BPDE). With NT, DNA alterations induced by gamma-rays are not detected and ssb are only evidenced after exposure to EMS (80 mM), which already alters the viability of the lymphocytes. Nick translation is able to detect ssb induced by 10 microM BPDE. Compared to the other assays, the sensitivity of the SCGE assay is significantly higher since statistically significant changes were detected after incubation with 0.5 mM EMS and 1.25 microM BDPE. SCGE is a relatively simple method, not time-consuming and applicable to a large number of samples per working day. In conclusion, on the basis of the results of this in vitro comparison, SCGE seems a promising method for the monitoring of populations exposed to genotoxic chemicals

Somatic Copy Number Abnormalities and Mutations in PI3K/AKT/mTOR Pathway Have Prognostic Significance for Overall Survival in Platinum Treated Locally Advanced or Metastatic Urothelial Tumors

Background: An integrative analysis was conducted to identify genomic alterations at a pathway level that could predict overall survival (OS) in patients with advanced urothelial carcinoma (UC) treated with platinum-based chemotherapy. Patients and Methods DNA and RNA were extracted from 103 formalin-fixed paraffin embedded (FFPE) invasive high-grade UC samples and were screened for mutations, copy number variation (CNV) and gene expression analysis. Clinical data were available from 85 cases. Mutations were analyzed by mass-spectrometry based on genotyping platform (Oncomap 3) and genomic imbalances were detected by comparative genomic hybridization (CGH) analysis. Regions with threshold of log2 ratio ≥0.4, or ≤0.6 were defined as either having copy number gain or loss and significantly recurrent CNV across the set of samples were determined using a GISTIC analysis. Expression analysis on selected relevant UC genes was conducted using Nanostring. To define the co-occurrence pattern of mutations and CNV, we grouped genomic events into 5 core signal transduction pathways: 1) TP53 pathway, 2) RTK/RAS/RAF pathway, 3) PI3K/AKT/mTOR pathway, 4) WNT/CTNNB1, 5) RB1 pathway. Cox regression was used to assess pathways abnormalities with survival outcomes. Results: 35 samples (41%) harbored mutations on at least one gene: TP53 (16%), PIK3CA (9%), FGFR3 (2%), HRAS/KRAS (5%), and CTNNB1 (1%). 66% of patients had some sort of CNV. PIK3CA/AKT/mTOR pathway alteration (mutations+CNV) had the greatest impact on OS (p=0.055). At a gene level, overexpression of CTNNB1 (p=0.0008) and PIK3CA (p=0.02) were associated with shorter OS. Mutational status on PIK3CA was not associated with survival. Among other individually found genomic alterations, TP53 mutations (p=0.07), mTOR gain (p=0.07) and PTEN overexpression (p=0.08) have a marginally significant negative impact on OS. Conclusions: Our study suggests that targeted therapies focusing on the PIK3CA/AKT/mTOR pathway genomic alterations can generate the greatest impact in the overall patient population of high-grade advanced UC

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings

Prediction of lymph node involvement in breast cancer from primary tumor tissue using gene expression profiling and miRNAs.

The aim of this study was to investigate whether lymph node involvement in breast cancer is influenced by gene or miRNA expression of the primary tumor. For this purpose, we selected a very homogeneous patient population to minimize heterogeneity in other tumor and patient characteristics. First, we compared gene expression profiles of primary tumor tissue from a group of 96 breast cancer patients balanced for lymph node involvement using Affymetrix Human U133 Plus 2.0 microarray chip. A model was built by weighted Least-Squares Support Vector Machines and validated on an internal and external dataset. Next, miRNA profiling was performed on a subset of 82 tumors using Human MiRNA-microarray chips (Illumina). Finally, for each miRNA the number of significant inverse correlated targets was determined and compared with 1000 sets of randomly chosen targets. A model based on 241 genes was built (AUC 0.66). The AUC for the internal dataset was 0.646 and 0. 651 for the external datasets. The model includes multiple kinases, apoptosis-related, and zinc ion-binding genes. Integration of the microarray and miRNA data reveals ten miRNAs suppressing lymph node invasion and one miRNA promoting lymph node invasion. Our results provide evidence that measurable differences in gene and miRNA expression exist between node negative and node positive patients and thus that lymph node involvement is not a genetically random process. Moreover, our data suggest a general deregulation of the miRNA machinery that is potentially responsible for lymph node invasion.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Tumor mutational analysis of GOG248, a phase II study of temsirolimus or temsirolimus and alternating megestrol acetate and tamoxifen for advanced endometrial cancer (EC): An NRG Oncology/Gynecologic Oncology Group study

OBJECTIVE: Rapamycin analogs have reproducible but modest efficacy in endometrial cancer (EC). Identification of molecular biomarkers that predict benefit could guide clinical development. METHODS: Fixed primary tissue and whole blood were collected prospectively from patients enrolled on GOG 248. DNA was isolated from macro-dissected tumors and blood; next-generation sequence analysis was performed on a panel of cancer related genes. Associations between clinical outcomes [response rate (RR) 20%; progression-free survival (PFS) median 4.9months] and mutations (PTEN, PIK3CA, PIK3R1, KRAS, CTNNB1, AKT1, TSC1, TSC2, NF1, FBXW7) were explored. RESULTS: Sequencing data was obtained from tumors of 55 of the 73 enrolled pts. Mutation rates were consistent with published reports: mutations in PTEN (45%), PIK3CA (29%), PIK3R1 (24%), K-RAS (16%), CTNNB1 (18%) were common and mutations in AKT1 (4%), TSC1 (2%), TSC2 (2%), NF1 (9%) and FBXW7 (4%) were less common. Increased PFS (HR 0.16; 95% CI 0.01-0.78) and RR (response difference 0.83; 95% CI 0.03-0.99) were noted for AKT1 mutation. An increase in PFS (HR 0.46; 95% CI 0.20-0.97) but not RR (response difference 0.00, 95% CI -0.34-0.34) was identified for CTNNB1 mutation. Both patients with TSC mutations had an objective response. There were no statistically significant associations between mutations in PIK3CA, PTEN, PIK3R1, or KRAS and PFS or RR. CONCLUSIONS: Mutations in AKT1, TSC1 and TSC2 are rare, but may predict clinical benefit from temsirolimus. CTNNB1 mutations were associated with longer PFS on temsirolimus