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

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          Abstract

          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

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          An intact retinoblastoma protein-binding site in Merkel cell polyomavirus large T antigen is required for promoting growth of Merkel cell carcinoma cells.

          Jürgen Becker, Sabrina Angermeyer, Johannes Grimm … (2012)
          Merkel cell carcinoma (MCC) is a highly aggressive skin cancer that frequently harbours Merkel cell polyomavirus (MCV) DNA integrated in the genome of the tumor cells. In our study, we elaborate our recent finding that MCV-positive MCC cell lines require the expression of the viral T antigens (TA). Indeed, in a xeno-transplantation model, we prove that TA expression is essential also in an in vivo situation, as knock down of TA leads to tumor regression. Moreover, rescuing TA short hairpin RNA (shRNA)-treated MCV-positive MCC cells by ectopic expression of shRNA-insensitive TAs clearly demonstrates that the observed effect is caused by TA knockdown. Notably, introduction of a mutation in the LTA protein interfering with LTA binding to the retinoblastoma protein (RB) ablated this rescue. The importance of this interaction was further confirmed as LTA-specific knockdown leads to explicit cell growth inhibition. In summary, the presented data demonstrate that established MCV-positive MCC tumors critically depend on TA expression, in particular the LTA and RB interaction, for sustained tumor growth. Consequently, interference with LTA/RB interaction appears as promising strategy to treat MCC. Copyright © 2011 UICC.
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            Merkel cell polyomavirus status is not associated with clinical course of Merkel cell carcinoma.

            David Schrama, Marc Zapatka, Claudia Pföhler … (2011)
            The majority of Merkel cell carcinomas (MCCs) are associated with the recently identified Merkel cell polyomavirus (MCV). However, as it is still unclear to which extent the presence of MCV impacts tumor characteristics or clinical outcome, we correlated the MCV status of tumor lesions obtained from 174 MCC patients including 38 MCC patients from Australia and 138 MCC patients from Germany with clinical characteristics, histomorphology, immunohistochemistry, and course of the disease. MCV DNA was present in 86% of MCCs and, in contrast to previous reports, no significant difference in MCV prevalence was present between Australian and German MCC cases. When patients were stratified according to their MCV status, only tumor localization (P=0.001), gender (P=0.024), and co-morbidity, i.e., frequency of patients with previous skin tumors (P=0.024), were significantly different factors. In contrast, year of birth and diagnosis, age at diagnosis, or histological type and features representing the oncogenic phenotype such as mitotic rate or expression of p16, p53, RB1, and Ki67 were not significantly different between MCV-positive and MCV-negative MCCs. MCV status also did not influence recurrence-free, overall, and MCC-specific survival significantly. In summary, although MCV-positive and MCV-negative MCCs may have different etiologies, these tumors have comparable clinical behaviors and prognosis.
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              Association of Merkel cell polyomavirus infection with clinicopathological differences in Merkel cell carcinoma.

              Hiromi Higaki-Mori, Satoshi Kuwamoto, Takeshi Iwasaki … (2012)
              Merkel cell polyomavirus is a novel polyomavirus that is monoclonally integrated into genomes of up to 80% of human Merkel cell carcinomas. Merkel cell polyomavirus-positive Merkel cell carcinomas showed less metastatic tendency and better prognosis according to some reports, whereas others disagree. In this study, we analyzed clinicopathological characteristics of 20 Merkel cell polyomavirus-positive and 6 Merkel cell polyomavirus-negative Merkel cell carcinoma cases, in which we already reported the association of Merkel cell polyomavirus infection with statistically significant morphological differences. Immunohistochemical expressions of cell cycle-related proteins, mutations of the TP53 tumor-suppressor gene (exons 4-9) and p14ARF promoter methylation status as well as detailed clinical data were analyzed and compared between Merkel cell polyomavirus-positive and Merkel cell polyomavirus-negative cases. Merkel cell polyomavirus-positive Merkel cell carcinomas showed better prognosis with one spontaneous regression case and significantly higher expression of retinoblastoma protein (P = .0003) and less p53 expression (P = .0005) compared to Merkel cell polyomavirus-negative Merkel cell carcinomas. No significant differences were found in expressions of p63, MDM2, p14ARF or MIB-1 index, and p14ARF promoter methylation status. Interestingly, frequency of TP53 non-ultraviolet signature mutation was significantly higher in Merkel cell polyomavirus-negative Merkel cell carcinomas than in Merkel cell polyomavirus-positive Merkel cell carcinomas (P = .036), whereas no significant difference was detected in TP53 ultraviolet signature mutations between two groups. These results suggest that Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas likely develop through different tumorigenic pathways and that the presence or absence of Merkel cell polyomavirus in the tumor is still an important factor that affects survival in patients with Merkel cell carcinoma. Copyright © 2012 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Michiko Matsushita
                Daisuke Nonaka
                Takeshi Iwasaki
                Satoshi Kuwamoto
                Ichiro Murakami
                Masako Kato
                Keiko Nagata
                Yukisato Kitamura
                Kazuhiko Hayashi
                Journal
                Journal ID (nlm-ta): Diagn Pathol
                Journal ID (iso-abbrev): Diagn Pathol
                Title: Diagnostic Pathology
                Publisher: BioMed Central
                ISSN (Electronic): 1746-1596
                Publication date Collection: 2014
                Publication date (Electronic): 20 March 2014
                Volume: 9
                Page: 65
                Affiliations
                [1 ]Division of Molecular Pathology, Department of Pathology, Tottori University Faculty of Medicine, Yonago, Japan
                [2 ]Department of Pathobiological Science and Technology, School of Health Science, Tottori University Faculty of Medicine, Yonago, Japan
                [3 ]Department of Histopathology, The Christie NHS Foundation Trust, Manchester, UK
                Article
                Publisher ID: 1746-1596-9-65
                DOI: 10.1186/1746-1596-9-65
                PMC ID: 3994555
                PubMed ID: 24649978
                SO-VID: 253cf2b7-a5d3-4196-9584-2189915c59e0
                Copyright © Copyright © 2014 Matsushita et al.; licensee BioMed Central Ltd.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 29 November 2013
                Date accepted : 10 March 2014
                Categories
                Subject: Methodology

                ScienceOpen disciplines: Pathology
                Keywords: merkel cell carcinoma,mcpyv-small t antigen,st-immunohistochemistry,st mrna-ish
                Data availability:
                ScienceOpen disciplines: Pathology
                Keywords: merkel cell carcinoma, mcpyv-small t antigen, st-immunohistochemistry, st mrna-ish

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