Merkel cell polyomavirus is causally associated with a rare human neuroendocrine origin skin cancer, MCC [18]. Generally, in most tumors caused by polyomaviruses, viral abortive infection does occur; it is defined by virus penetration and early gene expression (viral oncogenes) but no late gene expression (viral structural genes) and virion production. At first, all cell populations can become infected, but after subsequent cell divisions, only rare cells carrying an integrated viral oncogene proliferate [21, 22]. Therefore, the presence and high copy number of polyomavirus oncogene provided important evidence in the virus's carcinogenic role in human cancers [23]. The normalized copy number of the MCPyV LTAg gene in MCC is usually high and more than one copy per cell [6]. The main objective of the current study was to investigate the presence and quantitate MCPyV viral load in patients with NMSC and compare it with adjacent non-tumoral margins. In addition, we used the human RNase P internal control gene as a normalizer to compare the level of infection in skin tumor cells with adjacent normal tissues.
In the present investigation, the frequency and median copy number of MCPyV LTAg in cancerous samples (both BCC and SCC groups) were higher than normal tumor margins. Viral LTAg was detected in 67.3% of BCC tumors and 39.1% of normal margins. Moreover, the MCPyV genome was observed in 41.6% of SCC compared with 33.3% of normal margins. The frequency of the MCPyV LTAg gene in non-MCC skin cancers (BCC and SCC) was evaluated in several studies across the globe. A large number of BCC(n = 114) and SCC (n = 53) samples by Kassem et al. study in Germany, revealed the presence of MCPyV genomic sequences (LTAg or VP1) in 72.2% and 37.5% of BCCs and 52% and 25% of SCCs in immunosuppressed and immunocompetent patients respectively [24]. According to this study, MCPyV was 2-fold more frequent in immunosuppressed compared with immunocompetent BCC and SCC patients. To determine the direct carcinogenic role of the virus, we exclude immunosuppressed patients in our investigation. The lower frequency of MCPyV in Kassem et al.'s BCC and SCC immunocompetent patients compared to ours may be explained by different sensitivity of PCR techniques (End-point PCR in Kassem et al study vs Real-Time PCR in our study). In addition, the results of the present study are inconsistent with recent reports from Iran [25], which demonstrated no MCPyV detection in SCCs and only 10% positivity in BCC samples. Moreover, in the other two studies from Japan low frequency of MCPyV genome was detected in BCCs and SCCs. In a study done by Satoshi Ota et al, only 2.2% of BCC cases were positive (22), also in the Murakami study, 13% of SCC samples had MCPyV genomic sequence (26). It should be noted that in these studies similar to Kassem et al.'s investigation, the presence of MCPyV was evaluated by conventional PCR, and normal tumor margins were not evaluated for virus sequences.
In terms of MCPyV quantitative analysis, several studies were done on MCC and non-MCC samples throughout the world. Regarding MCC samples, the investigations conducted by Shuda [16], Loyo [17], Bhatia [26], and Satoshi Ota (22) showed an MCPyV normalized viral load between one to over one hundred copies per cell. Unfortunately, in the present study, there was no access to the MCC samples for viral load determination and quantitative analysis. In the current investigation, the MCPyV LTAg normalized viral load in cancerous and normal tumor margins had a wide range; the lowest viral load was10− 4 and the highest was 50.53 copies per cell. It is worth noting that we had 4 BCC samples with more than 1 copy per cell normalized MCPyV LTAg viral load and 2 BCC samples with moderate viral load (compared to that observed in the MCCs). This finding was in agreement with Satoshi Ota's report (22) that revealed a moderate MCPyV viral load (0.662 viral copies per haploid human genome) in 1 BCC case and recommended that MCPyV could be a factor in development of the minority of BCC skin tumors in addition to MCC. The results of the current investigation are consistent with two reports indicating low MCPyV viral load in the majority of SCC and BCC samples [27, 28]. Regarding MCPyV LTAg low viral load in adjacent non-tumoral margins, Generally, low level of MCPyV is present on all skin surfaces of most healthy individuals, and this virus is considered as a part of physiological skin microbiota[29].
In non-MCC samples, the detection of low level of MCPyV LTAg might be explained by several factors. First, MCPyV could be played as a passenger virus without clear pathological consequences or viral-containing lymphocytes infiltrate within epidermis in SCC and BCC. Second, low viral load suggests the hypothesis that the virus may be involved in the early stages of tumor progression through indirect carcinogenic mechanisms such as hit and run had no role in the complete progression of malignancy [30, 31].
The findings of the present study should be interpreted with caution because of some limitations such as the lack of fresh and non-paraffin samples, the small sample size in SCC and BCC groups and the possible presence of some normal cells in SCC and BCC sections that could reduce MCPyV LTAg genome copy number per cell. Further complementary studies can shed more light on the possible role of the virus in SCC and BCC skin tumors, including the analysis of the integration of the viral genome (especially the LTAg region), identification of nonsense mutations in the LTAg region, evaluation of MCPyV oncogenes expression and investigation of the interaction between MCPyV oncoproteins and human tumor suppressor proteins.
In conclusion, the present study revealed the detection of MCPyV LTAg sequences at low viral copy numbers (less than 1 viral copy per cell) in the majority of NMSCs and the adjacent non-tumoral margins of the same patients weakening the hypothesis of the pathogenic role of MCPyV in SCC and BCC tumorigenesis. Higher levels of MCPyV LTAg normalized viral load in some BCC and SCC samples may be correlated with the role of the virus in some cases of BCC and SCC skin cancer. These findings should encourage further investigations to determine the possible role of MCPyV in NMSC.