Deletions in Merkel Cell Polyomavirus Are Frequent, and Spare the Retinoblastoma-Binding Region
EJ Duncavage, SR Tripp, JD Pfeifer. University of Utah, Salt Lake City, UT; Washington University, Saint Louis, MO
Background: Merkel cell carcinoma (MCC) is a rare cutaneous tumor that was recently shown to harbor a novel polyomavirus. Similar to other polyomaviruses, the Merkel cell polyomavirus (MCPyV) encodes a viral large T antigen. In other carcinogenic polyomaviruses, the large T antigen effects control of cell proliferation via inhibition of Rb. It is unclear, however, if the MCPyV large T-antigen plays a role in carcinogenesis of MCC. We provide evidence that the Retinoblastoma (Rb) binding domain of MCPyV large T antigen is important in MCC and that it acts to block the normal regulatory activity of Rb.
Design: The study cohort consisted of 41 cases of MCC, 32 of which have been previously shown by PCR to harbor MCPyV. Paraffin blocks were first punched and DNA extracted. MCPyV positive MCC cases were then mapped by PCR for viral deletions using a novel set of 23 overlapping primers with an average size of 270bp that span the viral genome. The resulting PCR products were detected by gel electrophoresis. In a subset of cases, the Rb binding region (LxCxE domain) of the viral large T protein was sequenced. The function of the large T protein was evaluated via immunohistochemistry (IHC), using a phospo-specific antibody that recognizes only the Ser807/811 phosphorylated form of Rb (pRb). As a comparison, we measured the cellular proliferation rate by Ki-67 IHC. Slides were scanned at 20X resolution and image analyzed to determine the percentage of positive cells (nuclear staining) in each case.
Results: We found that viral deletions were common in MCPyV, occurring in 25 of 29 cases with amplifiable viral genomes. The most common deletions involved the large T origin-binding domain, necessary for viral replication. The deletions uniformly spare the region encoding the LxCxE Rb-binding domain of the large T protein (not mutated in 16 of 16 cases tested by direct sequencing). The pRB/Ki-67 ratio was decreased in MCPyV positive compared to MCPyV negative cases (mean 1.2 vs 13.6, respectively).
Conclusions: MCPyV genomic deletions are frequent in MCC, but spare the Rb binding domain. They generally result in a truncated large T protein, but spare the LxCxE Rb-binding domain. These results are consistent with a model in which MCPyV exerts its oncogenic effects by large T LxCxE binding of Rb, leading to release of E2F and subsequent circumvention of the normal cyclinD mediated G1 checkpoint. Consequently, pRb levels remain low despite a high proliferative index.
Monday, March 22, 2010 1:00 PM
Platform Session: Section F, Monday Afternoon