Identification of Nine Genomic Regions of Amplification in Urothelial Carcinoma, Correlation with Stage, and Potential Prognostic and Therapeutic Value

We performed a genome wide analysis of 164 urothelial carcinoma samples and 27 bladder cancer cell lines to identify copy number changes associated with disease characteristics, and examined the association of amplification events with stage and grade of disease. Multiplex inversion probe (MIP) analysis, a recently developed genomic technique, was used to study 80 urothelial carcinomas to identify mutations and copy number changes. Selected amplification events were then analyzed in a validation cohort of 84 bladder cancers by multiplex ligation-dependent probe assay (MLPA). In the MIP analysis, 44 regions of significant copy number change were identified using GISTIC. Nine gene-containing regions of amplification were selected for validation in the second cohort by MLPA. Amplification events at these 9 genomic regions were found to correlate strongly with stage, being seen in only 2 of 23 (9%) Ta grade 1 or 1–2 cancers, in contrast to 31 of 61 (51%) Ta grade 3 and T2 grade 2 cancers, p<0.001. These observations suggest that analysis of genomic amplification of these 9 regions might help distinguish non-invasive from invasive urothelial carcinoma, although further study is required. Both MIP and MLPA methods perform well on formalin-fixed paraffin-embedded DNA, enhancing their potential clinical use. Furthermore several of the amplified genes identified here (ERBB2, MDM2, CCND1) are potential therapeutic targets

Bladder Cancer Genomics: Mutations, Deletions, and Amplifications

Bladder cancer is a complex disease caused by multiple environmental and genetic factors, and is associated with advanced age that causes 15,000 deaths annually in the US population. Bladder cancer arises from transitional cells of the mucosal urothelium with two distinct morphological forms: papillary, a low-grade intraurothelial neoplasia which make up 80% of bladder transitional cell carcinoma (TCC); and non-papillary tumors which make up the remaining 20% and are typically invasive into the muscle layer or beyond. Multiple recurrent mutations in both dominantly acting oncogenes, and tumor suppressor genes have been identified in bladder TCC, as well as genes identified as targets for genomic loss or amplification. We applied a novel technology for the analysis of both mutations and copy number changes in a set of 80 bladder cancers samples. This technology, multiplex inversion probe (MIP) assay by Affymetrix, enabled us to analyze 330,000 single nucleotide polymorphisms (SNPs) for allele ratios and copy number, and to analyze 412 common cancer mutations. Mutation findings were validated by Sanger sequencing in 20 of 33 cases in which the MIP score was greater than or equal to 9.0. Nexus v6.0 was used to visualize and analyze MIP data on copy number and allele frequency. 73 of the 80 TCC samples that met quality control standards were analyzed using Genomic Identification of Significant Targets in Cancer (GISTIC) that identified 44 genomic regions having significant gains or loss (q \u3c 0.1). Subsequent manual review was used to assess reliability and to refine the extent of the regions and the genes contained therein. I then chose to focus on regions of amplification because they usually act in a dominant manner, and are more targetable in general than tumor suppressor genes lost in deletions. My plan is to validate these results using Multiplex Ligation-dependent Probe Amplification (MLPA) on bladder cancer cell lines, and additional clinical samples from bladder cancer patients. Future studies will examine the importance of these amplified genes in bladder cancer development and growth, and will hopefully lead to novel targeted therapy

BAP1 missense mutation c.2054 A>T (p.E685V) completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line.

BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val), identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1) a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU), which resulted in the deletion of 4 base pairs and presumably protein truncation; 2) a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3) partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG) in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V) variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing

Comparative sequence analysis reveals the <i>BAP1 c</i>.<i>2054A</i> (p.E685) is highly conserved.

<p>A. Multi-species comparative genomic analysis of <i>BAP1 c</i>.<i>2054A</i> in ten distantly related species: human, chimpanzee, mouse, rat, dog, cat, rabbit, chicken, xenopus and zebrafish. B. Multiple sequence alignment indicates BAP1 p.E685 is well conserved across the same ten distantly related species.</p

Summary of splicing variants cloned from RT-PCR products of HMeso01A harboring the c.2054 A>T (p.Glu685Val) mutation.

<p>A. Retention of introns 14, 15, 16; B. Retention of intron 15, 16; C. Retention of intron 14 and 4 bp deletion (GAAG) at the end of exon 16; D. Retention of intron 16; E. Partial retention of 3’ 69 bp in intron 14 and 4 bp deletion (GAAG) at the end of exon 16; F. Partial retention of 3’ 18 bp in intron 15 and 4 bp deletion (GAAG) at the end of exon 16; G. 4 bp deletion (GAAG) at then end of exon 16 (Δ4). //: partial exon.</p

Detection of aberrant splicing products in HMeso01A harboring the <i>BAP1 c</i>.<i>2054 A>T</i> (p.E685V) mutation.

<p>PCR fragments generated from amplification of <i>BAP1</i> exons 14–17 on cDNAs from HMeso01A and 4 controls were separated by agarose gel electrophoresis. Marker: DNA marker PhiX 174-HaeIII digest ladder.</p

Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to germline mutations in either TSC1 or TSC2. 10-15% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency < 5%, five at an allele frequency < 1%, and two were identified in skin tumor biopsies only, and were not seen at appreciable frequency in blood or saliva DNA. These findings illuminate the extent of mosaicism in TSC, indicate the importance of full gene coverage and next generation sequencing for mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counseling to the substantial population of TSC individuals who are currently NMI

Chromosomal regions and genes analyzed by MLPA.

<p>Chromosomal regions and genes analyzed by MLPA.</p

Summary of MLPA findings in FFPE bladder cancer samples according to stage.

<p>Groups 1, 2, and 3 have a statistically significant difference in the frequency of any amplification event, with p = 0.0020 comparing groups 1 and 2, and p = 0.0011 comparing groups 1 and 3. P is not significant comparing group 2 with group 3. Fisher exact test.</p