A new in situ hybridization and immunohistochemistry with a novel antibody to detect small T-antigen expressions of Merkel cell polyomavirus (MCPyV)

Background Approximately 80% of Merkel cell carcinomas (MCCs) harbor Merkel cell polyomavirus (MCPyV) which monoclonally integrates into the genome and has prognostic significance. The presence or absence of MCPyV is usually diagnosed using CM2B4 immunohistochemistry (IHC) for MCPyV-large T antigen (LT) protein. However, this method poses a risk of misdiagnosis. Methods In this study, we determined MCPyV infection in MCCs using real-time PCR for MCPyV-LT DNA and prepared 16 cases of MCPyV-DNA-positive and -negative groups. Diagnostic sensitivity and specificity of conventional PCR for MCPyV-small T antigen (MCPyV-ST), IHC using a newly developed polyclonal antibody (ST-1) for MCPyV-ST protein (MCPyV-ST) (aa: 164–177), and in situ hybridization (ISH) as well as real-time PCR for MCPyV-ST mRNA were compared against CM2B4-IHC for sensitivity (0.94, 15/16) and specificity (0.94, 15/16). Results The followings are the respective sensitivity and specificity results from examinations for MCPyV-ST gene: conventional PCR for the MCPyV-ST (0.94, 1.0), ST-1-IHC (0.69, 1.0), real-time PCR for ST mRNA (1.0, no data), ST mRNA ISH (0.94, 1.0). Each of the MCPyV-pseudonegative (1/16) and -pseudopositive (1/16) diagnoses evaluated using CM2B4-IHC were accurately corrected by examinations for MCPyV-ST or its expression as well as real-time PCR for MCPyV-LT. Sensitivity of CM2B4-IHC (0.94) was superior to that of ST-1-IHC (0.69) but equal to that of ST mRNA-ISH (0.94). Specificities of ST-1-IHC (1.0) and ST mRNA-ISH (1.0) were superior to that of CM2B4-IHC (0.94). Conclusions Therefore, combined application of ST mRNA-ISH and ST-IHC as well as CM2B4-IHC is recommended and will contribute to the diagnostic accuracy for MCPyV infection in MCCs. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9966295741144834


Background
Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer and Merkel cell polyomavirus (MCPyV) is monoclonally integrated into the genome of approximately 80% of MCCs [1]. The MCPyV genome contains smallT antigen (ST) and largeT antigen (LT) that encode nonstructural proteins and are responsible for viral replication and viral proteins (VPs) that constitute viral particles [2]. Although the exact pathogenesis has not yet been elucidated in MCCs, it is considered that pathogenesis of MCPyV-positive and -negative MCCs is different [2][3][4][5]. Moreover, MCPyV-positive MCCs are reported to have a better prognosis than MCPyV-negative MCCs [6][7][8][9][10], although these findings are some controversial [11]. Therefore, accurate diagnosis of the presence of MCPyV in MCCs is clinically important. Immunohistochemistry (IHC) using a monoclonal antibody CM2B4 that detects MCPyV-LT protein (MCPyV-LT) is currently the most common and prevailed method for diagnosis of MCPyV infection in MCCs, although realtime PCR is the most reliable method for confirming MCPyV-DNA and MCPyV infection in MCCs. The only commercially available antibody used for MCPyV infection diagnosis is CM2B4 antibody. IHC with CM2B4 antibody displays high sensitivity and good specificity for MCPyV detection and is usually sufficient for practical diagnosis, but it is not ideal for determining the presence or absence of MCPyV, based on reported MCC cases with pseudonegative and pseudopositive staining [3,[12][13][14]. The ST gene harbors fewer mutations than the LT gene in MCPyV from MCCs [15], and the MCPyV-ST protein (MCPyV-ST) was detected in human MCC tumors more commonly than was MCPyV LT [16].
In this study, we aimed to raise the diagnostic accuracy in determining MCPyV infection in MCCs and developed a new polyclonal antibody (ST-1) for detecting MCPyV-ST (aa: 164-177) and established a new in situ hybridization (ISH) as well as real-time PCR for MCPyV-ST mRNA expression. The sensitivity and specificity of the newly developed methods to detect MCPyV-ST expressions were compared with those of CM2B4-IHC.

Detection of MCPyV-DNA and quantification of MCPyV-ST mRNA expression
Real-time PCR was performed as previously described to detect and quantify MCPyV-LT DNA [13,17]. In addition, conventional PCR was performed using ST primer sets to detect MCPyV-ST DNA [14]. To quantify expression of MCPyV-ST mRNA, we used the Universal Probe Library Human TBP Gene Assay (Roche, Switzerland) as an internal control. After converting RNAs to cDNAs, cDNA fragments from MCPyV-ST mRNA and control TBP gene were amplified by real-time PCR using the following primer sets and probe: qST forward primer; 5′-AGTGTTTTTGCTAT CAGTGCTTTATTCT-3′, qST reverse primer; 5′-CC ACCAGTCAAAACTTTCCCA-3′ and fluorogenic ST probe; 5′-FAM-TGGTTTGGATTTCCTC-MGB-3′.

ST antibody (ST-1) manufacturing and IHC for MCPyV-ST detection
We established a Japanese MCPyV consensus sequence (DDBJ, Accession number: AB811689). Based on this MCPyV consensus sequence, we synthesized 164-177 amino acids and manufactured a rabbit polyclonal affinity purified antibody against MCPyV-ST (ST-1).
Staining protocol for ST-1 is the same as the one used for LT antibody (CM2B4) except for the primary and secondary antibodies. We used our primary antibody (ST-1, dilution 1/5000) and peroxidase-conjugated goat anti-rabbit IgG as a secondary antibody.

ST probe and protocol for ISH
Probe against MCPyV-ST (nt 196-756) was produced using the CUGA ® 7 in vitro Transcription Kit (NIPPON GENE, Japan). Instead of 100 mM CTP, 100 mM UTP and 100 mM ATP provided in the kit, we used the DIG RNA Labeling Mix (Roche, Switzerland). We followed Kit manual and the ST probe was electrophoresed and verified as one band.
The IsHyb In Situ Hybridization (ISH) kit (BioChain, USA) and TSA PLUS DNP (HRP) SYSTEM (Perkin Elmer, USA) were used for ISH and we followed the user manuals. The protocol is described briefly as follows: After deparaffinization and rehydration, endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol for 5 minutes (min). The slides were fixed with 4% paraformaldehyde in diethylpyrocarbonate (DEPC) -PBS at room temperature (RT) for 20 min. Slides were then washed the twice with DEPC-PBS at RT for 5 min, treated with 10 μg/ml proteinase K at 37°C for 10 min, washed once in DEPC-PBS at RT for 5 min, and fixed them again with 4% paraformaldehyde in DEPC-PBS for 15 min. Following fixation, the slides were rinsed once with DEPC-water, pre-hybridized with ready-to-use pre-hybridization solution for 4 hours at 50°C, and hybridized using digoxingenin labeled probe (3 ng/μl) in a hybridization solution for overnight (8 to16 hours) at 45°C. After washing in Saline Sodium Citrate (SSC) buffer, the slides were blocked in Tris-NaCl-blocking (TNB) buffer for 30 min at RT, incubated slides with Anti-Digoxigeninperoxidase (POD), Fab fragments (Roche, Switzerland) for 2 hours at RT, washed in Tris-NaCl-Tween (TNT) buffer 3 times for 5 min at RT, incubated in dinitrophenyl (DNP) Amplification Reagent working solution for 10 min at RT, and washed in TNT again. The slides were incubated in anti-DNP-Horseradish Peroxidase (HRP) for 30 min at RT, washed in TNT, stained with Diaminobenzidine (DAB) for colorization, and counterstained with hematoxylin.

Detection of MCPyV-LT or -LT
1) MCPyV-LT detection and viral load quantification using real-time PCR Real-time PCR data were summarized in Table 1. MCC cases were divided into MCPyV DNA-positive and -negative groups based on the real-time PCR data of MCPyV-LT. In 16 MCPyV DNA-positive MCCs, MCPyV-LT viral copy numbers ranged from 0.06 to178.81 copies/cell.

2) Immunohistochemical detection of MCPyV-LT
The IHC results using CM2B4 were shown in Table 1 and representative cases were illustrated in Figure 1. MCPyV DNA-positive MCCs were immunoreactive for CM2B4 antibody except for one case (MCC UK11, left side of Figure 2, Figure 2C for    Table 1, MCPyV-ST DNA was amplified using conventional PCR in MCPyV

2) Immunohistochemical detection of MCPyV-ST
The IHC results using ST-1 were shown in Table 1 and representative cases were illustrated in Figure 1E.  Table 1).
ISH data for ST mRNA expression were shown in Table 1. A representative ST mRNA-positive case was indicated in Figure 1G. A nuclear ST mRNA-positive signal was observed in 15 of 16 MCPyV DNA-positive cases (sensitivity: 0.94) and in no MCPyV DNA-negative cases (0/16; specificity: 1.0, Figure 1H).
In MCPyV DNA-positive MCC cases, the frequency of ST mRNA expression by ISH (15/16, 94%) was higher than that of ST-1 nuclear expression (11/16, 69%). ST mRNA expression data by ISH did not necessarily correspond to the quantity of ST mRNA using real-time PCR or ST-1 nuclear immunoreaction data by IHC.
Detailed comparisons of sensitivity and specificity in all kinds of examinations for detecting MCPyV infection in MCCs were summarized in Table 2.

Discussion
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. [13] 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 [13]. 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. [19] 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  LT region has a nuclear localization signal (NLS) that is located between the retinoblastoma protein (RB)-binding and DNA helicase domains [20]. 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  [21]. The interaction of MCPyV-LT and RB [22] is essential for sustaining tumor growth. A recent study [16] 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.

Conclusion
In conclusion, we emphasize again that in order to precisely determine MCPyV infection of MCCs, combined application of ST-1 IHC and ST mRNA-ISH as well as CM2B4 IHC is recommended and will contribute to a better and accurate diagnosis for MCPyV infection in MCCs.