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BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma
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1 Journal of Translational Medicine This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma Journal of Translational Medicine Sample (2015) 13:122 doi: /s Hakan Alakus Shawn E Yost Brian Woo Randall French Grace Y Lin Kristen Jepsen Kelly A Frazer Andrew M Lowy Olivier Harismendy Sample ISSN Article type Research Submission date 20 January 2015 Acceptance date 7 April 2015 Article URL For information about publishing your research in BioMed Central journals, go to Alakus et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.
2 BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma Hakan Alakus 1,3,4 Shawn E Yost 2 Brian Woo 3 Randall French 4 Grace Y Lin 5 Kristen Jepsen 6 Kelly A Frazer 2,3*,6 * Corresponding author Andrew M Lowy 3*,4 * Corresponding author Olivier Harismendy 3*,7 * Corresponding author 1 Department of General, Visceral and Cancer Surgery, University of Cologne, Köln, Germany 2 Division of Genome Information Sciences, Department of Pediatrics and Rady Children s Hospital, University of California San Diego, La Jolla, CA, USA 3 Moores UCSD Cancer Center, 3855 Health Science Drive, Maildrop 0820, La Jolla, USA 4 Division of Surgical Oncology, Department of Surgery, University of California San Diego, La Jolla, CA, USA 5 Department of Pathology, University of California San Diego, La Jolla, CA, USA
3 Abstract Background 6 Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA 7 Division of Biomedical Informatics, Department of Medicine, University of California San Diego, La Jolla, CA, USA Malignant mesothelioma (MM) arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces. The majority of MMs are pleural and have been associated with asbestos exposure. Previously, pleural MMs have been genetically characterized by the loss of BAP1 (40-60%) as well as loss of NF2 (75%) and CDKN2A (60%). The rare peritoneal form of MM occurs in ~10% cases. With only ~300 cases diagnosed in the US per year, its link to asbestos exposure is not clear and its mutational landscape unknown. Methods We analyzed the somatic mutational landscape of 12 peritoneal MM of epitheloid subtype using copy number analysis (N = 9), whole exome sequencing (N = 7) and targeted sequencing (N = 12). Results Peritoneal MM display few copy number alterations, with most samples having less than 10 Mbp total changes, mostly through deletions and no high copy number amplification. Chromosome band 3p21 encoding BAP1 is the most recurrently deleted region (5/9), while, in contrast to pleural MM, NF2 and CDKN2A are not affected. We further identified 87 nonsilent mutations across 7 sequenced tumors, with a median of 8 mutated genes per tumor, resulting in a very low mutation rate (median ). BAP1 was the only recurrently mutated gene (N = 3/7). In one additional case, loss of the entire chromosome 3 leaves a nonfunctional copy of BAP1 carrying a rare nonsense germline variant, thus suggesting a potential genetic predisposition in this patient. Finally, with targeted sequencing of BAP1 in 3 additional cases, we conclude that BAP1 is frequently altered through copy number losses (N = 3/12), mutations (N = 3/12) or both (N = 2/12) sometimes at a sub-clonal level. Conclusion Our findings suggest a major role for BAP1 in peritoneal MM susceptibility and oncogenesis and indicate important molecular differences to pleural MM as well as potential strategies for therapy and prevention. Keywords Genomics, Mutations, Tumor Suppressor, Peritoneal, Mesothelioma
4 Background Malignant mesothelioma (MM) is an aggressive tumor, which arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces [1]. The age-adjusted incidence of MM in the USA is about 1 per 100,000 [2]. The majority of MM cases are caused by asbestos or erionite exposure with a latency of typically years from exposure to diagnosis. MM is highly resistant to conventional cytotoxic therapies and no active molecularly targeted agents have been identified. As the disease is typically identified late in its course, most patients usually die within 2 years of diagnosis with a total of ~3,000 deaths in the US per year [3,4]. One of the largest studies to date screened 53 primary pleural MM for genomewide copy-number-aberrations (CNAs) and performed targeted sequencing of selected potential driver genes from the recurrent CNAs [5]. The majority of samples showed recurrent losses of 9p21, 22q and 3p21. BRCA1 associated protein-1 (BAP1) located at the epicenter of 3p21.1 was inactivated by somatic alterations in 42% of all tumors. This study also confirmed findings from previous reports, showing that CDKN2A (9p21) and NF2 (22q) are inactivated in ~60% and 75% of pleural MM respectively [6-10]. Finally other genes have been shown to be mutated in a smaller fraction of MM including LATS2 (10-30%) which is associated with activation of the YAP pathway [11]. While most studies have focused on pleural MM, about one in ten cases of MM arises from the peritoneum, which makes it an extremely rare condition (incidence ~1 per million in the U.S) and to our knowledge, the largest molecular study to date included only 6 cases [12]. In contrast to pleural MM, about 50% of peritoneal MMs do not have a clear history of asbestos exposure [13] and it is still unknown whether MMs from different sites of origin (pleural, peritoneal or pericardial) share genomic alterations or undergo similar oncogenic transformations [14-16]. Moreover, while whole exome sequencing has been applied to multiple common and rare tumors, it has never been used to determine the genome-wide mutational landscape of MM. Thus we sought to study the mutations present in peritoneal MM using a combination of whole exome sequencing (mutations), copy number arrays (CNA) or targeted sequencing. Examining 12 unique cases of epithelioid peritoneal mesothelioma, we identified the most recurrent somatic events present in this malignancy and compare the findings with what is known about pleural mesothelioma. Methods Samples and histology The acquisition and use of peritoneal MM samples was approved by the Institutional Review Board of the University of California, San Diego. Before enrolling in the study, patients gave informed consent. Blood samples for germline DNA extraction were collected before and tumor samples were collected during surgical tumor resection. The resected tumor samples were fixed in 10% formalin, embedded in paraffin and H&E-stained for evaluation by a surgical pathologist. Smaller parts of each tumor were put into 2x2x2 cm wells (Tissue Tek, Miles Scientific), covered with OCT and flash frozen. These samples were used for isolation of tumor DNA after cryosectioning, H&E-staining and evaluation for tumor cell content. Histologic examination of cases of mesothelioma may show many morphologic patterns and variable degrees of cytomorphologic atypia. The main histologic subtypes include epithelioid and sarcomatoid. Our study is limited to the epithelioid subtype. Genomic DNA was extracted from tumor samples with 70% tumor cell content using the AllPrep
5 DNA/RNA/miRNA kit (Qiagen ) and germline DNA was extracted from 100 μl buffy coats with the DNeasy Blood and Tissue kit (Qiagen ) according to the manufacturer s instructions. DNA concentration was determined by fluorometry (Qubit, Life Technologies). Exome capture and library preparation The sequencing libraries were prepared and captured using SureSelect Human All Exon V4 kit (Agilent Technologies) following the manufacturer s instructions. Briefly, 500 ng DNA was fragmented by Adaptive Focused Acoustics (E220 Focused Ultrasonicator, Covaris, Woburn, Massachusetts) to produce an average fragment size of ~175 base pairs. Fragmented DNA was purified using the Agencourt AMPure XP beads (Beckman Coulter, Fullerton, CA, USA). The quality of the fragmentation and purification was assessed with the Agilent 2100 Bioanalyzer. The fragment ends were repaired and adaptors were ligated to the fragments. The resulting DNA library was amplified by using manufacturer s recommended PCR conditions: 2 at 98 C followed by 6 cycles of (98 C 30 ; 65 C 30 ; 72 C 1 ) finished by 10 at 72 C. 500 ng of each library was captured by solution hybridization to biotinylated RNA library baits for 48 hrs at 65 C. Bound genomic DNA was purified with streptavidin coated magnetic Dynabeads (Invitrogen, Carlsbad, CA) and further amplified to add barcoding adapters using manufacturer s recommended PCR conditions: 2 at 98 C followed by 12 cycles of (98 C 30 ; 57 C 30 ; 72 C 1 ) finished by 10 at 72 C. Exome sequencing and analysis Sequencing was performed using the Illumina HiSeq 2000 system, generating 100 bp pairedend reads. All raw 100 bp paired-end reads were aligned to the human genome reference sequence (hg19) using BWA v0.5.9-r16 [17] with default parameters for paired-end reads except for seed length set to 35. Aligned reads were realigned using GATK s [18] IndelRealigner v g557da77 combining all reads from the same patients and subsequently splitting them. Duplicate reads were removed using Picard Tools v 1.65 MarkDuplicates. Finally the GATK s TableRecalibration tool was used to recalibrate the reads base quality scores. Additional file 1: Table S1 presents the summary statistics of the sequencing. The sequencing data is publically available via the NCBI Short Read Archive (SRA067608). We used VarScan2 v 2.3 [19] to compare the tumor to the normal sample and identify, for each patient, single nucleotide variants (SNVs) and small insertions and deletions (indels) that are: 1) inherited (germline variants); 2) acquired in the tumor (somatic mutations) as well as variants resulting from a loss of heterozygosity (LOH) or of unknown status. The required pileup files for VarScan2 were generated using SAMTools [20] mpileup v with default parameters except for q 5, Q 0, d 50000, and -B. We used the default parameters for filtering variants except changing the tumor, normal, and combined minimum coverage to 10X each, minimum mutant allele frequency of 0.1, and minimum average quality score to 17. We then applied additional filtering steps. 1) Low quality indels: somatic indels with <10X coverage depth or fewer than 3 supporting reads or with more than 5% frequency in the germline are removed. 2) VarScan default filters: 2a) Variant within 3 bp of an indel, 2b) clustering SNV: 3 SNVs located within 10 bp, 2c) less than 10% allelic frequency. 3) Low quality somatic variants: Somatic variants with Varscan Fisher p-value < 0.05 or with >5% alternate allele in the normal DNA (SNVs) or any alternate allele in the normal (indels) are filtered. 4) VarScan2 high quality filter: We finally applied VarScan2 s fpfilter script to both germline and somatic variants. This procedure filters variants based on their read position, strand bias, variant reads, variant frequency, distance to 3, homopolymer,
6 mapping quality difference, read length difference, and mismatch quality sum difference. Variants were queried against dbsnp135 to determine novel or known variants. Next we used snpeff [21] v or ANNOVAR [version ] [22] to identify the different the functional and impact on coding genes. Illumina exome array and copy number analysis The tumor DNA from 9 cases was analyzed on the Illumina CoreExome Array. For our analysis, we utilized the Genotyping module (1.9.4) within Illumina s GenomeStudio (GS) V Data was normalized using default parameters and Log R Ratios and B Allele Frequencies were exported for further analysis in R. Because the platform is designed to detect additional rare variants that may interfere with CNA analysis, we used only the probes found on Illumina s HumanCore BeadChip, which is dedicated to Copy Number Analysis, and excluded the rare variants designed from the human exome. We removed probes with missing values for any of the samples. To perform segmentation analysis, we used the copynumber package from Bioconductor using default parameters. For analysis purposes, we assigned a segment with a LogR ratio greater than 0.25 as being an amplified region and a LogR ratio less than 0.25 as being a deleted region. Due to the normalization procedure, chromosome 3 aneuploidy in sample AA2463T was only identified by manual review and added to the segmentation results. A cytoband was called deleted (respectively amplified) when more than 75% of its length belonged to a deleted (resp. amplified) segment. BAP1 targeted sequencing and PCR amplification We used Illumina TruSeq custom amplicons panel. We used DesignStudio (Illumina, Inc San Diego CA) to design custom primers to amplify all exons in BAP1. Following the manufacturer s recommendation we used the DNA from 12 fresh frozen tumor specimens and matched blood DNA from 2 patients to amplify the targets using 14 multiplexing primers. After purification and quantification, we combined the 14 libraries in equimolar amounts and sequenced them using Illumina MiSeq sequencer for 2x150 bp reads. The sequencing reads were then aligned and mutation called using the Illumina BaseSpace cloud with TruSeq DNA amplicon application or using Mutascope locally [23]. To increase the detection sensitivity the reads spanning the expected 42 nt deletion in AA2476T tumor were aligned independently using BLAT [24]. To analyze the large deletion in sample AA2476T we performed the PCR amplification using the following primers: BAP142delF: AGCCAGCATGGAGATAAAGG and BAP142delR TGCCTCAAGGAGGAGGTAGA. The results of the analysis of the BAP1 mutation in AA2476T are presented in Additional file 2: Figure S1. Results and discussion To date, most genome-wide analysis of MM have used copy number (or CGH) arrays to identify potentially recurrent chromosomal alterations in the tumors. We analyzed 9 tumors using the Illumina CoreExome arrays, identifying high confidence copy number segments. We observed that 3/9 tumors had no large copy number events, while 3/9 had between 1 and 6 large copy number events, and the remaining 3 had between 14 and 95 segments with a maximum of 290 Mb net copy number changes, including the loss of chromosome 3 in one tumor (Figure 1A Additional file 1: Table S2). About 14/82 deleted segments also show evidence of a loss of heterozygosity (B Allele Frequency > 0.55), increasing our confidence
7 in these calls. There were no detectable copy number gains recurring in more than 2 samples across the 9 tumors analyzed and none of them were high-level amplifications (max LogR = 0.55). We identified five cytobands where copy number losses or strong loss of heterozygosity are present in 3 or more samples (Additional file 1: Table S3): 12q24.13 (N = 4), 3p21 (N = 3) and 3p14 (N = 3), 15q15.3 (N = 3) and 15q21.1 (N = 3). An analysis of the 369 genes located in these regions reveals that 3p21 harbors the most significantly affected genes, with the lowest net loss (sum of log ratio across all samples) and highest net LOH (Figure 1B). Among those, BAP1 and PBRM1, located 135 kb apart, are known tumor suppressors in pleural MM [5] and renal clear cell carcinoma [25] respectively. At the resolution permitted by our analysis, we did not identify other significant copy number alterations. In particular NF2 (22q) and CDKN2A (9p21), known to be frequently lost in pleural MM do not exhibit any copy number alterations in peritoneal MM (Additional file 2: Figures S3-S4). Overall these results suggest that peritoneal MM exhibits few copy number alterations, mostly losses, and that BAP1 and its neighboring genes on 3p21 are lost in 5/9 tumors (Additional file 2: Figure S2). Figure 1 Copy number alterations in 9 peritoneal MM tumors. (A) The cumulative number of base pairs affected by losses (blue) or gains (red) in large segments (>1 Mb) is indicated for each tumor. The corresponding number of segments (Lost/Gained) is mentioned next to the sample ID. (B) Net Log Ratio (x axis) and B allele frequency (y axis) calculated across all 9 tumors for 369 genes located in the 5 recurrently lost cytobands. 3p21 (red genes) shows the strongest net loss (x axis) and net LoH (y axis). The data points corresponding to PBRM1 and BAP1 genes are labeled. (*) the BAP1 data point overlaps with other genes located in the same segment. We next performed whole exome sequencing on 7 out of the 9 tumors for which matched normal DNA was available and called somatic mutations in all 7 tumors (Additional file 1: Table S4). We were able to identify between 47 and 133 mutations per tumor, of which 2 to 33 are non-silent (Figure 2). With a median of 1.3 mutations per million base pairs, peritoneal MM has a much lower mutation rate than other adult solid tumors, and comparable to pediatric cancers, leukemias or endocrine cancers [26]. Across all seven cases, 87 somatic mutations were affecting the coding region of 83 genes (Additional file 1: Table S5). BAP1 was the only recurrently mutated gene, affecting 3 tumors through 2 nonsense mutations and one 42 nt frameshift. The BAP1-K453* mutation has an allelic fraction of 48% in a tumor without BAP1 loss (AA2273T), indicating that one WT copy remains. Similarly, BAP1- Q393* is identified in 12% of the sequencing reads consistent with the genetic heterogeneity of this tumor where a subclonal loss of 3p21 was observed (AA1844T - Additional file 2: Figure S2). Finally BAP1-I71fs was identified in only 14% of the sequencing reads in a tumor with BAP1 LOH (AA2476T). The unusual length of the deletion (42 nt) may have prevented additional reads from aligning to the reference genome resulting in an underestimation of the allelic fraction. Alternatively, both loss and mutations in this tumor may occur at a sub-clonal level. Overall, our results suggest that BAP1 is affected by concurrent copy number loss and mutations (2/7), mutation only (1/7) or loss only (3/7). Figure 2 Somatic mutations in 7 peritoneal MM cases. The total number of somatic mutations as well as their predicted effect is represented. With the paired sequencing of tumor and normal DNA, we are able to distinguish with confidence germline variants from somatic mutations. In particular, we identified a loss of function variant in BAP1 (BAP1-Y44*) in the germline DNA of one patient (AA2463T). This
8 patient s tumor was also characterized by the loss of chromosome 3. The Y44* variant has not been identified in the NHLBI Exome Sequencing Project [27] nor by the Exome Aggregation Consortium [28] and may thus have a minor allele frequency of less than 10 5, or be a de novo acquired variant in this patient. Inherited loss of function variants in BAP1 are known to increase susceptibility to melanoma, renal cell carcinomas and malignant mesothelioma [29-31]. A more detailed evaluation of this patient s family history would however be necessary to establish the true genetic risk associated with this nonsense variant. To further establish the prevalence of somatic mutations in peritoneal MM, we performed targeted sequencing of BAP1 exons in 12 tumors, including 3 cases not evaluated by exome or copy number analysis. This analysis allows us to confidently call mutations at high coverage depth. Across all exons and samples, greater than 91% of BAP1 coding base pairs were covered by 100 reads or more. We were able to confirm in three samples the presence of BAP1-Q393*, K453* and I71fs at an allelic fraction of 20%, 51% and 20% respectively, consistent with the whole exome sequencing findings (Additional file 1: Table S6). We find additional BAP1 somatic mutations in two samples: at Q684* (50% allelic fraction) and A95fs (39% allelic fraction). In summary, BAP1 is the most altered gene in peritoneal MM (Table 1), affecting 66% (8/12) of the studied cases through somatic mutations (N = 3), allelic copy number loss (N = 3), or both (N = 2). Table 1 BAP1 genetic status (germline or somatic) across all 12 cases Sample Assay 1 BAP1 Germline LOH Somatic (Allelic Fraction) AA1844T A + E + T subclonal Q393* (E:12%, T:20%) AA1968T A + E + T AA2273T A + E + T K453* (E:48%, T:51%) AA2463T A + E + T Y44* chr3 AA2476T A + E + T 15 Mb I71fs (E:14%, T:20%) AA2528T A + E + T AA2253T A + E + T 0.34 Mb AA2489T A + T 54 Mb AA2617T A + T NA A95fs (39%) AA2830T T NA NA AA2627T T NA NA Q684* (50%) AA2819T T NA NA 1: A: copy number array; E: whole exome sequencing; T: targeted sequencing. Peritoneal MM is an extremely rare malignancy and, in contrast to pleural MM, has never been analyzed on a genome-wide scale before. Early cytogenetic and loss of heterozygosity (LOH) analyses of pleural MMs described deletions as the most common cytogenetic aberration, suggesting that the inactivation of tumor suppressor genes residing in these deleted chromosomal regions may be responsible for neoplastic transformation [32]. Losses of 3p21 were described as a common alteration (13/23, 57%) in pleural MM twenty years ago [33] and in a recent study, the BAP1 tumor suppressor gene at 3p21 was frequently affected by loss, mutations or both (22/53; 42%). It has therefore been concluded that BAP1 loss drives the selection for 3p21 deletions in pleural MM [4]. Furthermore, germline mutations of BAP1 in two families have also been shown to predispose to pleural MM [29] and heterozygous BAP1 /+ mice are more susceptible to mesothelioma than their wild type littermate [34] thus strongly implicating BAP1 as a central player in MM tumorigenesis,.
9 Here we report that somatic alterations in 3p21 are also present in the majority of peritoneal MM. Five samples revealed deletions at 3p21 resulting in loss of one or two cancer genes (BAP1 and/or PBRM1) per sample. Further investigation by whole exome and targeted sequencing suggests that BAP1 is the most significant gene in this region as it is recurrently altered by somatic loss of function mutations in 5 tumors. While BAP1 is a natural candidate driver in 3p21 given its somatic alterations in pleural MM, PBRM1 is another potential tumor suppressor in the region. Frequent inactivation PBRM1 by somatic alterations have been described for kidney clear cell carcinoma (92/227, 42%) based on exome sequencing of 7 and targeted sequencing of 257 tumor samples [35]. However in our study PBRM1 is recurrently lost in 6 peritoneal MM cases but we did not identify any somatic mutations in the 7 samples sequenced. Thus, PBRM1 is less likely to be the most relevant gene in 3p21, except perhaps in tumor AA1968T where the 3p21 deletion specifically affects PBRM1 and not BAP1. Beyond BAP1, it is important to determine which other genes or pathways may be involved in peritoneal MM. Surprisingly, while somatic alterations of NF2 (39/53, 74%) and CDKN2A (31/53, 58%) are common in pleural MM [5], they are not observed in any of the 12 peritoneal MM cases we evaluated, despite adequate coverage depth (Additional file 1: Table S7). This suggests an important molecular difference between pleural and peritoneal MM. A recent study demonstrated that, in contrast to wild-type animals, CDKN2A loss is not required for tumorigenesis in BAP1 +/ mice, They further demonstrated that BAP1 mediates Rb1 expression loss via epigenetic down-regulation independent of CDKN2A status [34]. This observation may imply differences in the pathogenesis of pleural vs peritoneal MM in which the prevalence of CDKN2A loss differs. In our study, only one case of peritoneal MM had a clear loss of function of both BAP1 alleles, harboring both a chromosome 3 deletion and a germline nonsense variant. In all other cases, one wild type copy of BAP1 likely remains or the somatic alterations are only present at a sub-clonal level. Thus, while the mouse model may recapitulate a typical tumor suppressor loss pattern, with one inherited variant and one subsequent somatic loss, it appears that alteration or loss of the second allele may not be required in the majority of sporadic peritoneal MMs. Rather, the presence of a wild type allele in most studied cases, suggests that BAP1 haplo-insufficiency may lead to peritoneal MM. The analysis of more samples would be required to correlate somatic BAP1 ploidy, somatic status, Rb1 expression and epigenetic landscape with known clinical and environmental features. BAP1 is ubiquitously expressed, and involved in multiple processes such as transcriptional regulation, chromatin remodeling or BRCA1 mediated mismatch repair [36]. It may be feasible to exploit BAP1 loss of function in MM using synthetic lethal approaches with PARP inhibitors or HDAC inhibitors for example to leverage a potential defect in DNA repair or chromatin remodeling, respectively. This is a hypothesis that remains untested at this time. However a more systematic investigation of BAP1 partners and targets will likely be required to reveal effective ways to target BAP1 loss in MM. Conclusion In conclusion, our report suggests that the loss of BAP1 is a molecular alteration characteristic of peritoneal MM, occurring in the absence of any other strong recognized oncogenic drivers. Inactivation of BAP1 was previously identified as a common event in pleural MM but had never been described in peritoneal MM before. The lack of alterations in NF2 and CDKN2A supports the idea of distinct genomic features between pleural and
10 peritoneal MM. Further studies with more samples will be required to determine the molecular consequences and potential association with clinical and environmental features. Abbreviations MM, Malignant Mesothelioma; CNA, Copy Number Aberration. Competing interests The authors declare that they have no competing interests. Authors contributions SEY, BW, OH analyzed the data. HA, KJ, and RF prepared the samples and generated the data. KAF, AL and OH designed the study. AML, HA, KAF and OH prepared the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by UCSD Academic Senate Health Sciences Research Grant to KAF and AL, by the Brian Deily Living Trust, by the NIH/NCI Moores Cancer Center Support (P30CA023100, PI: Scott Lippman) by idash (U54HL108460, PI: Lucila Ohno- Machado), by the UCSD Clinical and Translational Research Institute (TL1TR000098, PI: Gary Firestein M.D) and by NIH/NCI R21CA to OH. HA is supported by a grant from the German Cancer Aid (#109790). References 1. Rudd RM. Malignant mesothelioma. Br Med Bull. 2010;93: Bridda A, Padoan I, Mencarelli R, Frego M. Peritoneal mesothelioma: a review. MedGenMed. 2007;9: Bueno R, De Rienzo A, Dong L, Gordon GJ, Hercus CF, Richards WG, et al. Second generation sequencing of the mesothelioma tumor genome. PLoS One. 2010;5:e Sugarbaker DJ, Richards WG, Gordon GJ, Dong L, De Rienzo A, Maulik G, et al. Transcriptome sequencing of malignant pleural mesothelioma tumors. Proc Natl Acad Sci USA. 2008;105: Bott M, Brevet M, Taylor BS, Shimizu S, Ito T, Wang L, et al. The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma. Nat Genet. 2011;43: Xio S, Li D, Vijg J, Sugarbaker DJ, Corson JM, Fletcher JA. Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene. 1995;11:511 5.
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16 Additional files provided with this submission: Additional file 1. Table S1 Sequencing Statistics. Table S2 Number and total size of the large (>1 Mbp) copy number aberrations identified in all 9 samples. Table S3 List of cytobands with significant losses or gains. Table S4 Whole exome sequencing variants and mutations statistics. Table S5 List of non-silent somatic mutations. Table S6 BAP1 somatic variants identified by targeted sequencing. Table S7 Fraction of coding base pairs covered at 20 or more in BAP1, CDKN2A and NF2 genes in whole exome sequencing data (36kb) Additional file 2. Figure S1 BAP1 42 nt frameshift deletion in AA2476T. Figure S2 BAP1 Copy Number Analysis. Figure S3 CDKN2A Copy Number Analysis. Figure S4 NF2 Copy Number Analysis (1678kb)
Data Analysis for Ion Torrent Sequencing
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You just can't make this stuff up...
A Buffalo, N.Y. community activist who is well known locally for pushing for a highly restrictive 2013 gun control law has been arrested for — wait for it — carrying a gun illegally at a public elementary school.Now, just to make sure we're clear on this, there's no hypocrisy inherent in legally carrying and insisting on a given law, rather the slap in the face comes primarily in his response to this issue.
The arrested gun-control advocate, Dwayne Ferguson, caused quite a scene at Harvey Austin Elementary School, reports local CBS affiliate WIVB.
The community activist has claimed that he forgot he was carrying his gun in a felony gun-free zone he helped create.
Rev. James E. Giles, Ferguson's friend and the president of Buffalo's Back to Basics Outreach Ministries, vouched for this claim.
"I'm sure Dwayne went into the school not thinking he had the gun on him," Giles told The Buffalo News.
Giles said Ferguson even asked police on the scene what was going on.
"Dwayne's reaction was to get his kids — he had about 50 of them — and make sure they were safe," Giles explained.
Now Dwayne should be advocating that he receive the maximum sentence possible for the heinous offense of exposing these children to the mere possibility that the gun just might inflict thought-hurt on them. Look at the massive trauma that those poor police and children had to go through just to ensure their safety!
At about 4:15 p.m. on Thursday, police acted on a pair of anonymous 911 tips. A battalion of cops quickly swarmed the school. The brigade included over a dozen squad cars, the SWAT team and K9 units. The Erie County Sheriff's Air One helicopter and what appears to be an armored vehicle also turned up.I am certain that responsible administrators should mandate that counseling immediately be offered to these 60 students with a distinct emphasis placed on how they survived through the pain that was 'Buffalo Lockdown 2014'. Why, just think, they too could have been hostages! If only we could sell some purple ribbons to raise awareness for these unfortunate souls.
The school was immediately placed on lockdown. Parts of two streets were closed.
About 60 students who were still on campus participating in after-school activities were funneled to the cafeteria.
Cops searched the school room by room and would not let parents on campus until they were satisfied that no shooting threat existed.
In all seriousness, this illuminates the absolute absurdity placed on gun owners in America today. When just the mere sight of a firearm creates an obviously insane escalation that has real costs in terms of police manpower, lost services due to aforementioned police manpower, educational instruction time, administrative overhead, publicity, court time, not to mention the 'trauma' inflicted on all of the students and staff at the hands of the police. This bozo shouldn't get a pass in any way in his sentencing, he should do 50 years for merely advocating this stupidity.

subset: commoncrawl


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For centuries, spies could only listen to enemy communications. In this thoughtful, opinionated history, a Pulitzer Prize–winning journalist warns that in today's cyberage, "once they hacked a computer, they could prowl the entire network…they could not only read and download scads of information, they could change its contents—disrupt, corrupt, or erase it—and mislead or disorient the officials who relied on it."
In the 1983 movie, WarGames, a teenager unwittingly hacks into the United States' defense system, nearly causing World War III. One viewer, an alarmed President Ronald Reagan, commissioned a groundbreaking 1984 directive giving our largest intelligence agency, the National Security Agency, responsibility for securing computer networks. Then the issue basically vanished for a decade. Soviet technology was far behind America's. In the mid-1990s, teenage hackers broke into American military computers, and a Russian intelligence agency did the same, so the issue was revived. Experts agreed that attack is the best defense, and Slate "War Stories" columnist Kaplan (The Insurgents: David Petraeus and the Plot to Change the American Way of War, 2013, etc.) delivers an eye-opening account of the dawn of cyberwar in 1995, when the air war in Serbia was won through crippling of its air defenses by information warfare. A decade later, the Stuxnet computer worm wreaked havoc on Iran's nuclear centrifuges, a triumph of digital skulduggery but perhaps an act of war. Though enthusiasts ignore the implications, Kaplan does not. Iranian hackers are inflicting expensive revenge, and a Chinese government agency is devoted to extracting useful information from American computers. Readers may take comfort knowing that we have the capacity to do the same.
An important, disturbing, and gripping history arguing convincingly that, as of 2015, no defense exists against a resourceful cyberattack.


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TRENTON – Gov. Jon S. Corzine signed legislation to establish a Veterans' Oral History Foundation on Sunday. The foundation will be devoted to sponsoring activities and raising money for the support and promotion of a veterans' oral history program
"This will keep the memories and stories of New Jersey's veterans alive for generations to come," said Assemblyman John S. Wisniewski (D-Middlesex). "Each generation has a different history to tell, and who better for to hear it from than the people that created that very history. The foundation will ensure the heroic efforts of veterans will never be forgotten."
"We owe our freedoms to those who have served this nation over the many years, and their histories must never be forgotten," said Assemblywoman Nilsa Cruz-Perez (D-Camden). "With this new law, quite simply, we'll be keeping their stories alive, and providing vivid history lessons for our children on what it means to be an American and to sacrifice to protect our liberty."
The foundation may incorporate, organize and operate in such manner as to be eligible under applicable federal law for tax-exempt status and for the receipt of tax-deductible contributions. All funds received by the foundation, other than those necessary to pay the expenses of the foundation, will be used exclusively for the support and promotion of a veterans' oral history program.
The foundation will be established in the Department of Military and Veterans' Affairs and will consist of nine members, as a board of directors, as follows: the Adjutant General of the Department of Military and Veterans' Affairs, or his designee; six members from recognized veterans groups in the state, to be appointed by the Adjutant General; and two representatives from the New Jersey Militia Museum, to be appointed by the Adjutant General.
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I was meeting with doctors; a 72 year old woman driving in a strange & busy city like Queretaro is rather difficult so I was delighted with the shuttle service that was provided!
Nothing!!!! My experience was excellent!
If you are looking for lodging in a hotel that has the support of an unstoppable brand with irresistible personality, a 100% satisfaction guarantee!, a warm and friendly staff eager to offer consistency, value and great service; situated in the middle of a fascinating, cultural, industrial, diverse city, which by the way… is home to a World Heritage Site, then look nowhere else because you have come to the right place! At Hampton Inn By Hilton Querétaro Tecnológico, you'll find exactly what you need. Whether you're traveling for business or pleasure, in our spacious rooms and studio king suites you'll enjoy all the extras our hotel has to offer. Along with earning Hilton HHonors points and miles, you will enjoy free hot breakfast everyday including waffles (now served on China), on the run breakfast bags, free stay for children under 18 accompanied by parents, exercise facilities, outdoor pool, laundry and dry cleaning service, a welcoming reception Tuesdays through Thursdays (please check schedule at Front Desk), meeting rooms with audio and video equipment available and free local transportation. You can start your weekday morning with a free local newspaper in our lobby while relaxing with a free cup of coffee or tea, offered 24 hours a day; or you can end your days in our Bar (part of the new Latin American Design) talking with your "friends in town" (that is how we like to think of ourselves). Each room features mahogany furniture with a 32" flat panel LCD TV. Rooms include 2 phones with data port access, free high speed internet access (wired and wireless), clock radios, free local and 800 telephone calls, no surcharge for using a calling card, a coffee maker, an iron and ironing board, curved shower rod, WaterPik Shower head, in-room movie channel and the Cloud 9 bed for The Hampton Experience. If you like to think of your room as a personal office, you´ll find convenient a built in desk, a desk phone with voice mail, and a lapdesk for a comfortable working experience. Hampton Inn By Hilton Querétaro Tecnológico is conveniently situated in the heart of the financial District in Queretaro on Bernardo Quintana Boulevard; just 1 mile away from all major companies, 2 miles away from most important attractions and within walking distance to great local restaurants. You will notice that Querétaro is a quite calm place, with a high standard of living. International Queretaro Airport is just 46 minutes away. Despite its small size, the State of Querétaro offers many interesting cultural and natural attractions, thanks to its diverse geography and to its crucial role in Mexican history. Three World Heritage Sites, a Pueblo Mágico (Bernal), the first vineyards in America (Tequisquiapan and surroundings), beautiful haciendas as well as lush natural scenery are only part of what visitors enjoy. The state is really accessible to any traveler; it is the crossroads of Mexico, since the highways that connect Northeastern, Western and Central Mexico join here. Highway 57 leaves Mexico City and reaches Laredo, Texas in the border with the United States, and is also called the Pan-American Highway. The municipalities located along its route are referred to as the "industrial corridor" and are the most developed ones in the state. Highway 45 connects Querétaro with the state of Guanajuato. Due to its strategic importance, Federal roads in the state receive a high level of investment. You can visit Querétaro any time of the year, its great weather makes your stay a pleasant one, and its numerous activities make it unforgettable. In short, it's a warm and friendly place, a destination that offers through its festivities, history, culture and gastronomy, a good life. Dare to re-live the passage of time and history walking along its streets full of art, tradition and charm. We love having you here!
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Artist: Blunt Chunks
Song: [second piece]
Recorded at The Imperial Pub's back room, November 15, 2015.Blunt Chunks - [second piece]
Alongside Karen Ng from Somewhere There, I had the opportunity to help put together a night for some new out-of-town pals. Caitlin Woelfle O'Brien was the first to pop into my mind for local content on the bill — she refers to her improvisational work as "noise", but while that might lead someone to expect something harsh or discordant, the result is generally quite ethereally beautiful, employing layers of ascendant vocal loops.
[Blunt Chunks will be playing at the next Long Winter on Saturday, December 12th.]


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The Marisa Tufaro Foundation commemorates what would have been Marisa's 14th birthday on July 30, 2017 by unveiling its strategic plan and outlining its first wave of initiatives to help children in need throughout the greater Middlesex County area.
Our mission statement is broad-based by design. The foundation wants to extend assistance to children and their families beyond those in medical crisis, although such individuals will forever remain a primary focus. The foundation will also make community service an integral part of its mission.
Through the generosity of others, who have made private donations or participated in fundraising events over the past six months, our nonprofit corporation, with 501 (c)(3) status pending, has a sound financial base from which to start turning its vision into reality.
As the foundation grows in the ensuing year, so will its outreach. We ask that you please revisit this website with frequency to keep abreast of future endeavors.
We encourage you to read in detail about our first wave of initiatives – each of which in some way reflects Marisa's inspirational life – by clicking on the links provided below:
- The foundation is committed to being a source of comfort to hospitalized children and their families
- The foundation will partner with Middlesex county schools, whose students will become "Marisa's Missionaries," to fulfill a specific "Marisa's Mission" that is designed to help children in need
- The foundation will provide scholarship opportunities
- The foundation will partner with other organizations who share our mission to help children in need
- The foundation will help provide hippotherapy to children whose families do not have health insurance or whose family's health insurer will not cover the cost of such physical therapy
- Old Bridge High School football partners with The Marisa Tufaro Foundation to host "Holiday Knight" Toy Drive during Childhood Cancer Awareness Month
- The foundation will be a resource for the Ronald McDonald House of Central Jersey
- The foundation is making itself available as a resource, through funding and/or providing volunteers, to help make the innovative ideas of those wanting to assist children in need come to fruition
The foundation is a fitting way to honor Marisa and keep alive her spirit. It allows her parents, Cyndi and Greg, to give back to the residents of a community that has forever provided them and Marisa with overwhelming love and support.
To those who have donated or are considering donating to the foundation, we can never find the perfect way to thank you for your kindness and generosity.
We can only believe the actions of our foundation will speak louder than words.


text: Seth Myers has only been at the helm of Late Night for a few days and he's already doing important work. Last night he used his new platform to point the spotlight at Florida, the Jewel of the Union. The Sunshine State has been ground zero for some of the most bizarre, inane, baffling, insane, perplexing and infuriating news stories for years and it's about time somebody did something about it. Do you think this is a game? It is.


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By: Susane Colasanti
Pub date: May 3rd, 2011
Scott Abrams is the love of Brooke's life. He just doesn't know it yet. So when his family moves to New York City the summer before senior year, Brooke has no choice but to follow Scott. It's her last chance to prove to him that they're meant to be together.
But the city is full of surprises that Brooke never expected. Ever since a painful family trauma, she's been closed off to her parents, to her friends, and even to herself. Now, inspired by the thrilling energy of the bustling and creative city around her, Brooke starts to explore her talents and become someone she never dreamed she'd be.
So what will she do when her greatest dream of love finally comes true . . . and instead, she wants something more?
Chapter One
Today I'm telling Scott Abrams that I'm in love with him.
Sometimes I think that if he knew, he would admit he feels the same way. Other times I think he would laugh in my face so hard I would never get over it.
It could be so easy. Just go up to him, tell him, and see what happens. Put it all out there. Finally know how he feels about me.
It would probably be easier if he knew I existed.
The hope that Scott Abrams could like me back has kept me going for two years. It's like this energy I live on. The idea of being with him is almost more exciting than being with him for real. But of course I want to turn this fantasy into reality.
The thing is, he's never noticed me. Saying sorry because he accidentally bumped into me in the hall last year doesn't count. So telling him that I know we belong together is probably a crazy thing to do.
I guess I'm crazy then. Because I'm doing it anyway...
To read the remainder of this excerpt you can do so here. For more on Susane and her other novels you can visit her official website and or blog!
Have a wonderful weekend everyone! :)
Have a wonderful weekend everyone! :)


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Chatting web cam porn germany live. She closed her eyes and let her mind drift back to the memory.
He had opened her legs, but kept them in the air above her and looked down at her slit as he watched the pearly moisture seep from her perfectly shaved hole. He layed on the bed, his head hovering above her pussy, and she watched as his tongue extended and teasingly licked her dripping pussy, her head fell back and her eyes closed as she moaned, her feet resting either side of his head, on his manly shoulders. Chatting web cam porn germany live.


text: well, my brother and myself both have the new htc thunderbolt. we have had them for 11 days now, and both have had constant issues. they both keep going fron 3G down to 1X, and just while sitting here at home they go from 3 or 4 bars to NONE at all and saying no service. (we do not have 4G LTE anywhere near here at all) so should i try to return it and get a different phone, like a fascinate or droid x? on mine, when ever i am on the phone, people are constantly saying they cant hear me talking hardly at all. one issue i see, and this is purely cosmetic, but where the charger plugs in, the little "prongs" that stick up to hold the charger have started scratching the plastic when removing the plug in. thats a poor design there. that should be all metal in there, but this is all cosmetic, nothing more nothing less. I am mainly concerned about the issues above.


text: So I finally got to remove the bandage that the hospital put on my finger and I find that some of it has hardened and is stuck to my stitches. I think it look hot. I really do. Now that I see it I really should have just duct tapped it shut and called it a day. Duct tape... the poor mans cure for open wounds.


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- Description
MSR Tyker Kids snowshoes enable young adventurers to join Mom and Dad on winter hikes. Based on the same technology utilized in MSR's adult offerings, the snowshoes provide the fit and traction needed for snow and ice.
Taking the Tyker Kids snowshoes on and off is quick and easy. The 3-strap binding is simple to adjust even for small, mitten-clad hands. The bindings will fit kids' shoe sizes ranging from 7.5-13.5 and even fits adult sizes 1-4.5.
Molded traction bars and steel crampons affixed to the frame of the Tyker Kids snowshoes provide optimal traction in winter conditions. The durable construction of the snowshoes holds up well to wear and tear.
Sold in pairs, MSR Tyker Kids snowshoes are suitable for a total weight (user and gear) of up to 80 pounds.
California residents: view proposition 65 WARNING
- Sold in pairs
- Secure bindings designed for smaller,mitten-clad hands and can be used on most any footwear
- Molded traction bars add safety without compromising traction
- Injection-molded decks stand up to abuse
- Fits kids' shoe sizes 7.5-13.5 and adult sizes 1-4.5 US
- Specs
Best Use Snow Sports Warranty Lifetime Type Snowshoes (Pair) Item Weight 1 lb 15 oz Dimensions 6.5" x 17" Capacity up to 80 lbs
- Reviews
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Framed Photo Bridal Shower Dark Grey Wedding Invite
Item# 23505
These bridal shower invites feature a rich milk chocolate background with the silhouette of an eager bride in the bottom left. They also have a pink frame with rose details to show a photo of the new bride-to-be. Upload your photo and let us print it for you! Lots of room for shower details in a matching cream with a fun pink script for the guest of honor.
Card Size: 5.00" x 7.00" (as shown image)
Paper thickness: 110 lb. premium quality 100% post-consumer recycled paper matte feel white finish card stock. FSC Certified eco-friendly paper, made in USA.
You have the chance to review digital E-Proof image of your text, in your choosen font, laid out on your selected card for your approval prior to submitting your order.
Digital printing press delivers high resolution digital images that rivals the 4 color offset printing press that is used on traditional mass-produced stationery.
Digital print output quality is outstanding, with an emphasis on rich, vibrant color and unsurpassed detail with crisp, clean text.
This means you get the best print quality for graphics, text and photographs. With this exclusive design, we offer premium quality print output since this order is always printed on-demand.
The use of a laser printer is not recommended due to the heat process involved, which tends to interfere and distort the image of your customized finish digital print-out.| Inkjet Printable: No (?)
Traditional inkjet printer may have difficulties to properly self-feed and tends to smear the ink due to heavy card stock involved.


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Print, scan and copy everyday documents with the compact, reliable HP Deskjet 1056 Inkjet Multifunction Printer/Copier/Scanner. Designed to fit your taste, space and budget, it is easy to use and maintain, helps you conserve resources and is backed by HP's award-winning service and support.
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