Genomic Characterization of Merkel Cell Polyomavirus Integration Sites in Merkel Cell Carcinoma
Eric J Duncavage, Ryan Demeter, Todd Wylie, Vince Magrini, John Pfeifer. Washington University, St. Louis, MO
Background: Merkel Cell Carcinoma (MCC) is a rare cutaneous tumor that has a strong association with Merkel Cell Polyoma Virus (MCPyV). Given its relative rarity, little high-quality frozen tissue is available for traditional genomic characterization. We used targeted next generation sequencing (NGS) to identify viral sequence alterations and human genomic integration sites from formalin-fixed cases of MCC.
Design: We identified 25 cases of previously characterized MCC cases, including 7 cases that tested negative for MCPyV by PCR. 500ng of genomic DNA was extracted from each case and indexed NGS libraries created. DNA was then captured using custom synthesized, biotinylated oligonucleotides that spanned the 5.3Kb MCPyV genome. The enriched DNA was sequenced on both an Illumina GAII and Illumina MiSeq as two indexed pools of 13 samples using 100bp and 150bp paired-end reads, respectively. Data were analyzed using freely-available software.
Results: The 18 MCPyV positive cases yielded an average coverage of 3084x with the GAII and 676x with the MiSeq. Sufficient coverage was obtained in 11 cases for further analysis. In 7 of these cases (63%), MCPyV genomic deletions >100bp were detected. In 8 cases (72%), human integration sites were identified, including 4 within introns (UBAC2, SEMA3D, XRCC2,FAM53A) and 4 within non-coding regions. In 5 of 7 (71%) MCPyV-negative cases we detected small fragments of MCPyV, all of which included the same 100bp segment of the large T antigen. Alignments of this area across all organisms demonstrated that region was unique to MCPyV.
Conclusions: The finding that none of the viral integration sites occurred in coding or known regulatory regions argues against a role for insertional mutagenesis of MCPyV in the pathogenesis of MCC. The identification of a small 100bp region within the MCPyV large T region in the majority of PCR-negative cases suggests that this minimal viral region is involved in MCC pathogenesis; it also indicates that a higher percentage of cases of MCC harbor MCPyV than currently recognized and provides a target for more sensitive PCR-based tests to detect the virus. In a broader context, our results indicate that NGS can detect unique, non-recurring deletion and insertion events, demonstrating the utility of the methodology for clinical applications focused on these types of genetic aberrations.
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 113, Tuesday Morning