Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel...

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis.