Whole Exome Sequence Analysis of Merkel Cell Carcinoma
Patrick J Cimino, Jr., Eric J Duncavage. Washington University School of Medicine, St. Louis, MO
Background: Merkel cell carcinoma (MCC) is a rare neuroendocrine tumor with an aggressive clinical course that harbors integrated Merkel Cell Polyomavirus (MCPyV) in up to 80% of cases. While mutations in the integrated MCPyV large T antigen sequence have been well-documented, little is known about human genomic mutations in MCC, beyond Rb1, TP53, and PIK3CA. We sought to determine the human genomic landscape of MCC by exome sequencing.
Design: From a set of 8 previously characterized MCC cases, exome sequencing was performed using Agilent V4 reagents and HiSeq 2000 paired-end sequencing. DNA from 6 MCPyV-positive and 2 MCPyV-negative cases was extracted from formalin-fixed paraffin-embedded (FFPE) blocks. Data was mapped to hg19 reference and all known SNPs and non-coding variants removed from the files.
Results: The average number of reads generated per case was 130,477,404 (26GBases). On average, 94.1% of the exome for each case had ≥ 25x coverage. There were 1,514 genes identified with non-SNP mutations found in at least 1/8 cases; 9 genes mutated (all 8/8 cases); 4 genes (7/8 cases); 9 genes (6/8 cases); 21 genes (5/8 cases); 75 genes (4/8 cases); 232 genes (3/8 cases); 902 genes (2/8 cases); and 262 genes (1/8 cases). Some of the most frequently mutated genes (in at least 7/8 cases) with predicted protein damaging mutations include UGT2B15, KLF14, ACVR2A, and CCNE1. Few cases showed mutations in Rb1 (3/8), TP53 (2/8), and none in PIK3CA. There was no significant difference in the average numbers of mutations between MCPyV-positive and -negative cases. There were 15 genes with non-SNP SNVs identified in only the MCPyV negative cases (2/2) and 575 non-SNP SNVs present in only the MCPyV-positive cases (at least 2/6). The most frequently mutated gene unique to the MCPyV-positive group was PLEC, found in 6/6 MCPyV-positive cases. In 4/6 MCPyV-positive cases we had previously determined the MCPyV integration site, and saw no evidence of human genomic DNA mutation flanking these sites.
Conclusions: Whole exome sequencing from archival FFPE tissue demonstrated no-significant difference in the overall number of mutations in MCPyV-positive versus -negative MCC, however we identified several gene mutations that appeared specific to MCPyV-positive or -negative MCC. In cases with known MCPyV integration sites, we saw no evidence of coding region insertional mutagenesis. Our sequencing results identify several mutations in genes not previously associated with MCC that may play a role in disease development. Overall, these findings expand our understanding of frequently mutated human genes in MCC.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 63, Monday Morning