Modern Pathology (2017) 30, 1133–1143 © 2017 USCAP, Inc All rights reserved 0893-3952/17 $32.00 1133 Next-generation sequencing-based molecular characterization of primary urinary bladder adenocarcinoma Somak Roy1, Dinesh Pradhan1, Wayne L Ernst1, Stephanie Mercurio1, Yana Najjar2, Rahul Parikh2, Anil V Parwani3, Reetesh K Pai1, Rajiv Dhir1 and Marina N Nikiforova1 1Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 2Department of Medical Oncology and Hematology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA and 3Department of Pathology, Ohio State University, Columbus, OH, USA Primary bladder adenocarcinoma is a rare and aggressive tumor with poor clinical outcomes and no standard of care therapy. Molecular biology of this tumor is unknown due to the lack of comprehensive molecular profiling studies. The study aimed to identify genomic alterations of clinical and therapeutic significance using next- generation sequencing and compare genomic profile of primary bladder adenocarcinoma with that of high-grade urothelial carcinoma and colorectal adenocarcinoma. A cohort of 15 well-characterized primary bladder adenocarcinoma was subjected to targeted next-generation sequencing for the identification of mutations and copy-number changes in 51 cancer-related genes. Genomic profiles of 25 HGUCs and 25 colorectal adenocarcinomas using next-generation sequencing of 50 genes were compared with primary bladder adenocarcinoma. Genomic profiles were visualized using JavaScript library D3.js. A striking finding was the distinct lack of genomic alterations across the 51 genes assessed in mucinous subtype of primary bladder adenocarcinoma. Eleven of 15 primary bladder adenocarcinoma harbored at least one genomic alteration in TP53, KRAS, PIK3CA, CTNNB1, APC, TERT, FBXW7, IDH2 and RB1, many of which are novel findings and of potential therapeutic significance. CTNNB1 and APC mutations were restricted to enteric subtype only. While genomic alterations of primary bladder adenocarcinoma showed substantial overlap with colorectal adenocarcinoma, FGFR3 and HRAS mutations and APC, CTNNB1 and IDH2 alterations were mutually exclusive between primary bladder adenocarcinoma and high-grade urothelial carcinoma. These alterations affecting the MAP kinase, PI3K/Akt, Wnt, IDH (metabolic) and Tp53/Rb1 signaling pathways may provide the opportunity for defining targeted therapeutic approaches. Modern Pathology (2017) 30, 1133–1143; doi:10.1038/modpathol.2017.33; published online 26 May 2017 Primary bladder adenocarcinoma is a rare tumor, clinical outcomes unless detected at a very early accounting for 0.5–2% of all primary bladder stage.3,4 Unfortunately there exists no effective malignancies. It usually affects middle age to elderly conventional or targeted therapy for primary bladder individuals, with a male preponderance. Enteric, adenocarcinoma. Confirming urinary bladder origin mucinous, signet ring cell and mixed subtypes of primary bladder adenocarcinoma is a significant represent the morphologic heterogeneity in primary diagnostic challenge because of morphologic simi- bladder adenocarcinoma.1,2 These are clinically larity with primary adenocarcinoma of other sites, aggressive tumors that usually present with such as colorectal and female genital tract. Morpho- advanced stage disease at diagnosis with nodal logic and immunophenotypic studies of primary bladder adenocarcinoma have not identified a involvement in 30–40% of the cases and poor specific marker. While nuclear expression of beta- catenin may suggest a colonic origin, rare cases of Correspondence: Dr S Roy, MD, Director of Genetics Services and primary bladder adenocarcinoma may demonstrate Molecular Informatics, Molecular and Genomic Pathology Labora- nuclear expression.1,2,5 Clinical exclusion of extra- tory, Department of Pathology, UPMC, 3477 Euler Way, Room# vesical primary adenocarcinoma is necessary to 7028, Clinical Laboratory Building, Pittsburgh, PA 15213, USA. E-mail: [email protected] establish the diagnosis. There is limited data on Received 25 September 2016; revised 13 March 2017; accepted 13 molecular biology of primary bladder adenocarci- March 2017; published online 26 May 2017 noma and little is known about its genomic profile. www.modernpathology.org Genomics of bladder adenocarcinoma 1134 S Roy et al A few studies have investigated molecular altera- was isolated from each target with the DNeasy Blood tions using single gene assay in primary bladder & Tissue Kit on the automated QIAcube (Qiagen, adenocarcinoma to reveal low frequency of KRAS CA, USA) instrument according to the manufac- and TERT promoter mutations in 11.5% (3/26) and turer's instructions and amplified using the Ion 28.5% (4/14) of primary bladder adenocarcinoma, AmpliSeq Library Kit 2.0 and Ion AmpliSeq Cancer respectively.6,7 Although EGFR mutation or amplifi- Hotspot Panel v2 primer pool (Life Technologies, cation and ALK rearrangement was not identified in Carlsbad, CA, USA) covering hotspots across 50 any primary bladder adenocarcinoma, 36% of the cancer-related genes (Supplementary Table 1). Sub- tumors demonstrated EGFR polysomy.8 In this study sequently, the multiplexed, barcoded libraries were we aimed to genomically profile a cohort of primary enriched by clonal amplification and massively bladder adenocarcinoma using targeted next- parallel sequencing was carried out on an Ion Proton generation sequencing of 51 cancer-related genes to sequencer (Life Technologies). After a successful identify potential therapeutically useful molecular sequencing reaction, the raw signal data were markers. In addition, we compare the targeted analyzed using Torrent Suite v4.2.1 (Life Technolo- molecular profile of primary bladder adenocarcino- gies). The short sequence reads were aligned to the mas with colorectal adenocarcinoma and high-grade human genome reference sequence (GRCh37 patch urothelial carcinoma. 13, GCF_000001405.25). Variant calling was per- formed using Variant Caller v4.4.3.3 plugin (inte- grated with Torrent Suite) that generated a list of Materials and methods identified sequence variations (single-nucleotide variants (SNV) and insertions or deletions (indels)) in Sample Cohort a variant calling file (VCF v4.2; https://samtools.github. After approval form the Institutional Review Board io/hts-specs/VCFv4.2.pdf). Copy-number analysis from next-generation sequencing data was performed at The University of Pittsburgh, the institutional 9 archive of the Department of Pathology was retro- as previously described. The variant calls were spectively reviewed from 1999 to 2016. We used the annotated using inhouse developed custom World Health Organization (WHO) 2016/Interna- software. Subsequently, HGVS variant nomenclature was normalized and variants prioritized using tional Society of Urologic Pathology definition of 10 primary bladder adenocarcinoma as inclusion cri- SeqReporter. An in-house knowledge base and teria. The cases of urothelial carcinoma with focal or several publically available databases were used extensive glandular differentiation as well as those (Supplementary Methods) for variant annotation. with clinical evidence of metastasis from other sites, Sequence variants with at least 5% allelic fraction such as colorectal or female genital tract and urachal with at least 300 × depth of coverage or 3% allelic adenocarcinomas were excluded. This yielded a fraction with at least 1000 × depth of coverage were total of 15 cases of primary bladder adenocarcinoma, included for analysis. Integrated Genomics Viewer which had adequate and viable tumor content for (IGV; Broad Institute, Cambridge, MA, USA) was molecular analysis. For comparative analysis, geno- used for manual review of the sequence read pileups. mic data from consecutive 25 cases of high-grade Visualization plots were created using javascript urothelial carcinoma without adenocarcinoma com- library D3.js (https://d3js.org/; last accessed 29 May ponent and consecutive 25 cases of colorectal 2016). The TERT promoter was amplified by PCR on extracted DNA using and interrogated for the hotspot adenocarcinoma that were received for routine 4 4 clinical molecular testing, were retrospectively mutation loci for g.1295228C T(c.-124C T; C228T) 4 4 retrieved from our laboratory information system and g.1295250C T (c.-146C T; C250T) using (LIS). Data on TERT promoter sequencing was not bi-directional Sanger sequencing (supplementary available for these 50 clinical cases. All histologic Material). slides including Hematoxylin and Eosin (H&E) and Immunohistochemistry were reviewed and a specific formalin-fixed paraffin embedded tissue block was Immunohistochemistry selected for molecular analysis. Paired normal tissue Immunohistochemistry for β-catenin, MLH1, PMS2, was not used sequencing analysis. MSH2 and MSH4 were performed on all cases of primary bladder adenocarcinoma (Supplementary Targeted Next-Generation Sequencing Table 2). Briefly, formalin-fixed paraffin embedded sections were cut at 4μ, deparaffinized and hydrated. Next-generation sequencing analysis was performed Subsequent to heat induced epitope retrieval and in a Clinical Laboratory Improvements and Amend- endogenous peroxidase quenching, slides were ments (CLIA)-certified molecular laboratory. Tumor- incubated with primary antibody for 16–30 min rich areas (450% neoplastic cells), targeted using depending on the antibody, followed by secondary Hematoxylin and Eosin (H&E) stain guidance by a antibody (Mach 2 Mouse HRP, Biocare,