[946] Regional Chromosome 9 Targeted Capture and Sequencing Detects Papillary 2 Renal Cell Carcinoma

William LaFramboise, Patti Petrosko, Maureen Lyons, Michael Belsky, Michael Becich, Christin Sciulli, Melissa Bastacky, Gavin Meredith, Sheldon Bastacky, Anil Parwani, Rajiv Dhir. University of Pittsburgh, Pittsburgh, PA; Life Technologies, Inc., Carlsbad, CA

Background: Early detection and classification of renal tumors by fine needle biopsy could prove beneficial but efficacious molecular markers remain to be identified. Our SNP analysis (Krill-Burger et al, AJP, 2012) identified shared, tumor type-specific copy number (CN) changes in renal cancers including 2 CN losses in chromosomes 9 and 14 specific to Papillary 2 carcinoma (PRCC2). The goal of this study was to use precisely focused, deep sequencing of the exons spanning the PRCC2 CN "hot spot" in chromosome 9 to identify tumor specific changes within this small molecular domain as a potential diagnostic biomarker.
Design: We purified DNA from 7 macrodissected PRCC2 tumors with matched blood (n=3) or normal adjacent tissue (n=4). DNA was sheared, barcoded, adaptors ligated, selected to 200bp and PCR amplified (12 cycles). Tumor-normal pairs were pooled in equimolar amounts and hybridized with capture "baits" for 457 exons in 40 genes on chromosome 9 (p24.3 to p22.3) followed by emulsion PCR and Ionsphere enrichment. Libraries were deposited on Ion 316 chips and sequenced as matched pairs on the Ion Torrent PGM. Raw depth of coverage comparisons were used to calculate CN of tumor vs. normal paired samples (mean read depth= 833 +/- 396 templates per sample) and single nucleotide variants were called using Gene Spring NGS Analysis Software (p<0.00001, >30 bases calls per variant per sample).
Results: Twenty-two genes contained individual exons with significant CN alterations (P<.05) comprising predominantly deletions or CN reductions. Three genes (CDC37L1, RCL1, and SCL1A1) exhibited significant losses in all seven tumor samples. Although no genes exhibited single nucleotide variants across all samples, CDC37L1 and INSL4 contained variants common to five of seven patients at the somatic and germline level, respectively, after filtering known SNPs.
Conclusions: We identified exon CN losses in 3 genes in all 7 PRCC2 specimens tested (100% Sensitivity). Changes occurred in one gene prominent in apoptosis (CDC37L1), suggesting that these alterations play a role in mechanisms underlying tumorigenesis. If targeted sequencing specificity holds true to our SNP studies, utilization of these results as a Papillary 2 specific biomarker remains promising inasmuch as CN variants affecting these 3 genes were not detected in 5 Chromophobe and 5 Oncocytomas and were in only 1 of 5 Clear Cell and 1 of 5 PRCC1 tumors. This approach provides a prototype for development of a panel of exclusionary diagnostic biomarkers for renal cell carcinomas.
Category: Genitourinary (including renal tumors)

Monday, March 4, 2013 9:15 AM

Proffered Papers: Section A, Monday Morning


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