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Immunological detection of viral large T antigen identifies a subset of Merkel cell carcinoma tumors with higher viral abundance and better clinical outcome

International Journal of Cancer2009Vol. 127(6), pp. 1493–1496

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Kishor Bhatia, James J. Goedert, Rama Modali, Liliana Preiss, Leona W. Ayers

Abstract

MCPyV, a new human oncogenic polyoma virus, appears etiologic for the classic, rare, aggressive Merkel Cell Carcinomas (MCC) of the skin characterized by MCPyV DNA integration, high virus copy numbers,1 expression of the potentially transforming MCPyV large T antigen (LTA) evidenced by reactivity to monoclonal antibody CM2B4,2-4 signature mutations in LTA,5 expression of retinoblastoma protein3, 6 and a vigorous MCPyV specific neutralizing antibody response.7, 8 Prevalence of this MCC phenotype has been reported as upward of 77–84% of MCC tested.1, 3, 9, 10 Thus, 16–23% of MCC may have a different etiology. MCPyV detection in MCC tissue includes MCCs with high and low virus copy numbers, detection ranges from 24 to 100% (Table 1).1-4, 6, 9-19 Differences in relative proportions of MCC with low and high viral copies may also be significantly influenced by geographic region (Table 2).13 All MCC tumors in our study population6 with viral abundance of 0.06–1.2 DNA copies/cell conform to the reported MCC MCPyV positive phenotype with demonstrable MCPyV LTA (IHC CM2B4, kindly provided by Dr. Moore) and retinoblastoma protein (Fig. 1a). Our tumor study population additionally contained variant MCC with low viral DNA (0.0005–0.0035 copies/cell) or no detectable virus; these later 2 subgroups were negative for LTA (CM2B4) and retinoblastoma protein. These data are similar to the more recent data from Busam et al.4 who found 5/15 MCPyV positive MCC non reactive to CM2B4 and are consistent with the recent comprehensive characterization of MCC cell lines.20 (a) Immunohistochemical staining of MCC was performed for the detection of expression of retinoblastoma protein (pRb) using the commercially available antibody from Novocastra (clone 13A10) and for the detection of the LTA encoded by MCPyV using the CM2B4 mAb obtained as a gift from Dr. Patrick Moore and Yuan Chang (University of Pittsburgh). These antibodies were applied to formalin-fixed paraffin-embedded MCC tumor core samples in an MCC tissue microarray. MCPyV copies per cell (CPC) were determined by real time PCR using Taqman technology. Details of the PCR assay have been described previously.6 MCPyV CPC associates with (first row) expression of MCPyV LTA using CM2B4 mAb and (second row) expression of pRb. (b) Correlation between overall survival (Kaplan Meier) and the presence of MCPyV LTA by CM2B4 mAb staining demonstrates that patients with LTA positive tumors survived longer following a diagnosis of MCC than patients with LTA negative tumors. By Cox relative hazard (RH) analysis, likelihood of survival was lower per level (HR = 0.35, CI = 0.16–0.77, p = 0.009). The number of cases in each category is: LTA positive, viral positive, 9; LTA negative, viral positive, 8; and LTA negative, viral negative, 6. Patients with viral positive tumors tended to be younger (p = 0.07) but did not differ by sex. Additional characteristics of these patients have been described previously.6 Classic MCC viral oncogenesis characterized by abundance of MCPyV is clinically relevant as suggested by Sihto et al.,16 who demonstrated that viral abundance in MCC correlates with survival. Our result6 supports this conclusion. When our patient MCC population is segregated by high versus low or no viral copies/cell, survival is best for those with high viral copies/cell and worse for MCC likely initiated through other modes of oncogenesis (Fig. 1b). We differ from the report by Becker et al.3, 10 where high viral copy numbers were present in most of their samples. Lack of CM2B4 reactivity in a tumor with high viral copy number has been described.2 Some CM2B4 negative MCC might be ascribed to variations in large T antigenic epitopes. However, high correlation among viral load, retinoblastoma protein and CM2B4 reactivity in our data suggests that viral low-abundance or negative MCC tumor cells may have no or insufficient LTA for detection with CM2B4. Several recent reports have demonstrated presence of MCPyV in normal skin and other tumors.6, 11, 14, 21 Our results do not allow us to discriminate between the presence of MCPyV in MCC tumor cells versus the presence of MCPyV in surrounding non tumor cells; this issue is germane to the relevance of MCPyV mediated tumorigenesis particularly in MCC with low viral abundance. One approach to address this would be to examine whether MCPyV genome is integrated in the host genome, a process that is likely to indicate a direct role for MCPyV mediated oncogenesis. The lack of fresh/frozen tumor samples, however, makes such a study technically unfeasible. Integration of MCPyV genome is also often associated with the presence of truncating mutations in the LTA and while it is unknown whether mutations occur before integration, the presence of such mutations may provide an alternative surrogate for supporting a pathogenic role of MCPyV. However, recent data21 suggest that identical truncating mutations are detected in both tumor and adjacent normal skin tissue, thus the pathogenic relevance of detecting truncating mutations warrant additional investigation. Although technically feasible, the sensitivities for detecting mutations particularly from low abundance viral DNA pose several challenges and will still fail to directly address the issue whether the viral DNA is associated with the tumor cells or the non tumor cells in the sample. Nonetheless, the rarity of cells in MCC with low amplification of virus would be inconsistent with the presence of the viral DNA before clonal expansion in these tumors. To appreciate the differences in oncogenesis and MCC survival, the MCC study population must be heterogeneous and contain tumors with high, low and no viral detection. Although artifact related to specimen quality and differences in detection methods could add to this heterogeneity, tumor factors such as mutations, ultraviolet (UV) DNA damage and patient clinical parameters such as immunosuppression must contribute. Compared to specimens from North America, MCC tissues from Australia might be expected to be virus negative because high UV radiation exposure of melanin-deficient skin probably contributes to MCC development.13 Our data (Fig. 1a) imply that MCPyV positive and negative tumors differ in oncoprotein expression and prognosis. This may be important in establishing clinical correlates that will be useful worldwide. Clarifying the role of MCPyV in MCC will require accurate quantification of viral abundance. Houben et al.3 contend that most MCCs carry at least 1 viral copy per cell and do not find MCC with low viral abundance. In contrast, we and others6, 13, 16 have found very low viral loads (0.0001–0.0035 copies/cell) in 19 of 174 MCC tumors tested (Tables 1 and 2). Both we and Garneski et al.6, 13 observe what appear to be discrete categories of high- and low-abundance MCC tumors. Estimation of viral abundance is susceptible to differences in tissue quality, proportion of tumor and non tumor cells in the sample, primers selected for PCR amplification, target cellular genomic sequences used as a reference and other technical differences, but it appears that at least a proportion of MCCs carry viral DNA that equates to less than 1 in 300 cells infected with MCPyV. Notably, similar low abundance of viral association has been described in MCC cell lines, suggesting that mere viral association may not provide growth advantage, at least in vitro, over non viral infected MCC.20 Finally, there is the question of how expression of oncoproteins in MCC differs among the classic MCPyV DNA and variant phenotypes. We find heterogeneity in MCC as to abundance or absence of detectable MCPyV and the presence of oncoproteins as well as geographic, clinical or patient factors. All may be critically important for prognosis and therapy. To this end, the development of a multi-investigator, multi-institutional consensus study using an international, geographically diverse MCC tumor population with a pre-determined common set of reagents and methods to estimate MCPyV abundance and oncoproteins in primary, recurrent and metastatic lesions, geographic distributions of MCC types and correlations with clinical features of MCC would be an important step. The authors are grateful to Dr. Patrick Moore and Dr. Yuan Chang (University of Pittsburgh) for sharing the antibody CM2B4 before its publication. They thank Dr. Allan Hildesheim (IIB, DCEG, NCI) for useful discussions and for critical reading of the manuscript. Yours sincerely, Kishor Bhatia, James J. Goedert, Rama Modali, Liliana Preiss, Leona W. Ayers.

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