Quantitative analysis of viral load per haploid genome revealed the different biological features of Merkel cell polyomavirus infection in skin tumor

Abstract

Merkel cell polyomavirus (MCPyV) has recently been identified in Merkel cell carcinoma (MCC), an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR) and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n = 9) and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n = 45], actinic keratosis [AK, n = 52], Bowen's disease [n = 34], seborrheic keratosis [n = 5], primary cutaneous anaplastic large-cell lymphoma [n = 5], malignant melanoma [n = 5], and melanocytic nevus [n = 6]). In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%), BCC (1 case; 2%), and AK (3 cases; 6%). We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119-42.8) and AK (0.02-0.07) groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662). Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4) demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC), but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection.

Figure 1. Polymerase chain reaction (PCR) amplification of the Merkel cell polyomavirus in the skin tumors.

Six MCPyV gene fragments were detected in Merkel cell carcinoma, basal cell carcinoma (BCC), and actinic keratosis (AK). Cases involving synchronous or metachronous metastases are marked with an asterisk. Specific PCR fragments, including large T (LT)2, VP1, and VP2, were not amplified constantly in AK cases 2 and 3 (see text). To clarify, we replaced this part with a picture of successful amplification in another trial. Abbreviations: BCC, basal cell carcinoma; AK, actinic keratosis; 293T, polyomavirus SV40 T antigen-positive 293 cells. The lower panel indicates the single PCR proliferation band of the CDC25 gene.

Figure 2. Morphology and immunohistochemical staining.

Representative cases of Merkel cell carcinoma (MCC; A, B), a basal cell carcinoma (BCC)-positive case (C, D), and a BCC-negative case (E). Immunohistochemical staining with the anti-MCPyV large T-antigen antibody (CM2B4) (B, D, E). Heterogeneous and diffuse staining was observed in MCC (B), and strong diffuse positivity (D) and total negativity (E) was detected in BCC. Inset: Nuclear staining of MCPyV in MCC (B) and BCC (D,E).

Figure 3. MCPyV copy number in various skin tumors.

(A) Digital PCR software-generated composite heat maps showing chambers with positive signals for both control RNaseP genes (blue) and MCPyV (red). Digital PCR heat maps are indicated in the upper panel for Merkel cell carcinoma case 6, in the middle panel for basal cell carcinoma (positive case), and in the lower panel for actinic keratosis (case 1). (B) Scatter plot of the MCPyV copy number of Merkel cell carcinoma, basal cell carcinoma, and actinic keratosis. Immunohistochemically positive cases are shown as black dots (▪), and negative cases are indicated by triangles (▴).