Detection of 'Merkel Cell Polyoma Virus' in Cases of Paraffin-Embedded Merkel Cell Carcinoma
EJ Duncavage, JD Pfeifer. Washington University, Saint Louis, MO
Background: It was recently shown that the majority of cases of Merkel Cell Carcinoma (MCC), a rare and often lethal cutaneous malignancy, harbor a novel, clonally-integrated polyoma virus aptly named Merkel Cell Polyoma Virus (MCPyV). Using both established primers and a novel primer set we studied the prevalence of MCPyV in cases of MCC from routine paraffin-embedded tissue blocks.
Design: We identified 41 cases of MCC in our archives from 1989 to 2008, including 28 cases from unique patients. Of the 41 cases, 20 were primary cutaneous excisions, 17 from metastases, and 4 from recurrences. To ensure similar cellularity and viability across all cases, 2mm representative areas from each tissue block were punched and DNA was extracted following a 48 hour digestion. PCR using two previous published primer sets, LT1 (440bp amplicon) and LT3 (308bp amplicon) as well as a novel primer set MCVPS1 (109bp amplicon) was performed on all samples. Amplification controls including beta globin and a size control ladder were run in tandem. PCR products of the predicted size were detected by 3% agarose gel electrophoresis. Selected PCR products were sequenced to confirm amplicon identity. In addition, the MCVPS1 products were digested with BamH1, yielding an 83bp product that was easily detected by electrophoresis.
Results: A 110bp beta globin control was amplified in all 41 cases. Detection rates of MCPyV for each of the three primer sets was 32 of 41 cases, 78%, (21 of 28 unique cases) for MCVPS1, 17 of 41, 41%, for LT3, and 9 of 41, 22%, for LT1. The variation between primer set detection rates was due to poor DNA quality as supported by low amplification of the higher molecular weight markers in the size control ladder products. All cases that were positive by LT1 and LT3 were positive by MCVPS1.
Conclusions: Previous reports indicate detectable MCPyV in 77% to 80% of MCC cases using the LT1 and LT3 primer sets. We achieved similar findings with a primer set optimized for formalin-fixed tissue that is less prone to the effects of degradation and has the advantage of product verification by restriction fragment length analysis.
Wednesday, March 11, 2009 9:30 AM
Poster Session V # 55, Wednesday Morning