To detect MCPyV-LT, IHC along with a monoclonal antibody, CM2B4, is commonly used by several pathologists and researchers to determine infection in MCCs
[14, 18]. However, Kuwamoto et al.
 pointed imperfect in sensitivity and specificity of CM2B4. Some researchers also reported CM2B4-pseudonegative or -pseudopositive MCC cases that were immunonegative for CM2B4 despite the detection of MCPyV-DNA using conventional PCR or real-time PCR
[3, 12, 14] or immunopositive for CM2B4 despite no detection of MCPyV-DNA using conventional PCR and real-time PCR
. The CM2B4 antibody is extremely useful but not ideal in both sensitivity and specificity to determine accurate MCPyV infection in MCCs, because CM2B4 IHC data were not necessarily compatible with those of real-time PCR, which is the most reliable method. Recently, Leitz et al.
 also emphasized that combined use of ST-specific antibody or a more sensitive LT-specific antibody (Ab3) and PCR using multiple primer pairs need to be applied to increase sensitivity, because CM2B4 IHC does not detect all MCPyV-positive MCC cases.
In this study, we prepared each 16 cases of MCPyV-DNA-positive and -negative MCCs which were determined by real-time PCR for MCPyV-LT. The sensitivity and specificity in all methods used in this study to detect MCPyV-ST DNA, −ST mRNA or -ST were summarized in Table
2, comparing with those of the popularized CM2B4 IHC. MCPyV-ST DNA was detected in 15 of the total 32 MCC cases (15/16 MCPyV-LT DNA-positive MCCs) using conventional PCR with the ST primers (sensitivity, 0.94; specificity, 1). Differences in the sensitivity of real-time or conventional PCR with the different primers may be caused by some mutations in the target region, making amplification of DNA fragments difficult. ST-1 antibody, which was newly developed to detect MCPyV-ST (aa: 164–177), was immunoreactive for the nuclei and/or cytoplasm of MCC tumor cells. According to the sensitivity and specificity data of ST-1 IHC (Nuclear reaction: sensitivity, 0.69, specificity, 1.0; cytoplasmic reaction: sensitivity, 0.875, specificity, 0), evaluation of nuclear ST (ST-1 antibody)-immunoreactivity was significant and useful for diagnosis of MCPyV-infected MCCs but cytoplasmic ST immunoreactivity was not significant. Shuda et al. also reported
 that expression of MCPyV-ST was detected in both nuclei and cytoplasm by IHC. MCPyV-LT region has a nuclear localization signal (NLS) that is located between the retinoblastoma protein (RB)-binding and DNA helicase domains
. Therefore, nuclear LT (CM2B4 antibody)-immunoreactivity is evaluated as significant. On the other hand, NLS of MCPyV-ST region has not been revealed until now. Therefore, it may be possible that cytoplasmic staining of MCPyV-positive MCCs may be a partly true immunoreaction, although cytoplasmic immunoreactions in both MCPyV-positive MCCs and -negative MCCs suggest that they are not significant. In each case of MCPyV-pseudonegative (MCC UK11) or -pseudopositive (MCC64) MCC determined by CM2B4 IHC, nuclear ST-1 imunoreactivity corresponded to the data of real-time PCR for LT-DNA, conventional PCR for ST-DNA, and real-time PCR or ISH for ST mRNA. This was useful for correcting the misdiagnosis for MCPyV infection evaluated by CM2B4 IHC. As another method of detecting MCPyV-ST mRNA expression, we developed a new ISH approach using the ST probe recognizing MCPyV-ST (nt: 196–756). ISH revealed nuclear positive staining demonstrated in all MCPyV-positive MCCs except in one case (sensitivity, 0.94; specificity, 1.0). This ISH is also a good method and the first application to detect the MCPyV infection in FFPE samples of MCCs. The reason why one ST mRNA could not be detected by ISH was because of the old issue of FFPE. Real-time PCR for quantifying ST mRNA expression demonstrated the best sensitivity (1.0) and is comparable with the sensitivity (1.0) of real-time PCR for LT DNA. However, quantities of MCPyV-ST mRNA expression using real-time PCR in MCPyV-infected MCCs did not necessarily correlate with the expression levels of ST mRNA-ISH or ST-1 IHC. With regard to the studies of MCCs, MCPyV-LT is the main analytic region in the studies of MCPyV-infected MCCs because MCPyV-DNA with truncated mutations in the LT region is integrated into the MCC host genome
. The interaction of MCPyV-LT and RB
 is essential for sustaining tumor growth. A recent study
 indicated that MCPyV-ST expression was required for the Merkel cell tumor growth in vitro and was observed to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation and that 4E-BP1 inhibition is required for MCPyV transformation. The function of MCPyV-ST has not been fully elucidated and in order to analyze this in the future, ST-1 IHC and ST mRNA-ISH will be useful methods.