Fluorescencein situhybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas

Abstract

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumorspecific 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 5 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.

Age and Gender Associations of Virus Positivity in Merkel Cell Carcinoma Characterized Using a Novel RNA In Situ Hybridization Assay

“…However, CM2B4 has been shown to have limited sensitivity relative to qPCR (14,31). Other methods that have also been reported in the literature include fluorescence in situ hybridization (FISH) and IsHyb ISH (32)(33)(34). However, the interpretation/reporting of FISH results can be challenging.…”

Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors

“…MCPyV FISH was performed as previously described . In brief, deparaffinized 3 μm thick sections were pretreated with 0.2 M hydrochloric acid, incubated with 1 M NaSCN and digested with 1 mg/mL pepsin (2500–3500 U/mg, Sigma Chemical, St. Louis, MO, USA).…”

“…MCPyV FISH was performed as previously described. 22,23 In brief, deparaffinized 3 μm thick sections were pretreated with 0.2 M hydrochloric acid, incubated with 1 M NaSCN and digested with 1 mg/mL pepsin (2500-3500 U/mg, Sigma Chemical, St. Louis, MO, USA). The biotin labeled "specific" MCPyV DNA probe was added to the samples at a concentration of 5 ng/μL, followed by denaturation of DNA (five minutes, 80 C) and hybridization overnight (37 C, humid chamber; ThermoBrite System, Abbott Molecular, Abbot Park, IL, USA).…”

Polyomavirus BK and prostate cancer: a complex interaction of potential clinical relevance

“…These mutations do not affect the oncogenic potential of MCPyV LTag, because the LXCXE binding motif required for binding the retinoblastoma tumor suppressor protein is maintained . Although MCPyV‐related carcinogenesis has been experimentally proved in vitro , new techniques, such as the fluorescence in situ hybridization, are employed to improve the quality of the molecular‐based testing for MCPyV detection with the aim to elucidate MCPyV‐related carcinogenesis in humans .…”