Merkel cell polyomavirus DNA detection in a patient with Merkel cell carcinoma and multiple other skin cancers

To the Editor: With great interest, we studied the recently published article from Duncavage et al1 “Merkel Cell Polyomavirus-A Specific Marker for Merkel Cell Carcinoma in Histologically Similar Tumors”. We agree that molecular pathologic investigation for Merkel cell polyomavirus (MCPyV) of tumors, which are histologically similar to Merkel cell carcinoma (MCC) is a very interesting and important issue. It is beyond debate that analysis of MCPyV prevalence in other skin conditions than MCC is necessary to get insight in the relevance of MCPyV infection. Our group is intensively working in this field, and we discuss the value of MCPyV detection as a diagnostic tool in routine histopathology in the light of the latest literature. Duncavage et al1 screened 74 cases of visceral high-grade neuroendocrine tumors (which are histologically similar to MCC) for MCPyV DNA by means of polymerase chain reaction (PCR). They detected MCPyV DNA just in 1 case (1.4%). Interestingly this case is, “upon detailed review, actually a misclassified case of metastatic MCC”, displaying the problem of distinguishing these entities. Therefore they suggest PCR-based testing for MCPyV DNA as helpful and specific marker in differentiating those histologically similar tumors. Furthermore, they report and discuss frequent absence of MCPyV DNA in several cutaneous and extracutaneous neoplasms.1 We agree with Duncavage et al1 that detection of MCPyV DNA is highly dependent upon a number of different methodical parameters such as tissue fixation, methods of DNA extraction and specific sequence detection, such as PCR method, selection of PCR primer sets, and last but not least dependent upon the amount of available tissue. Among others, Duncavage et al1 investigated 16 small cell carcinomas of the lung reporting absence of MCPyV DNA. We analyzed in a prior study, 30 small cell carcinomas of the lung and detected MCPyV DNA in 2 of 30 patients (7.5%), in 3 of 35 samples (2 of these 3 samples were from the same patient), respectively.2 Furthermore, there is enough evidence that MCPyV DNA is a common and frequent finding in tissues of immunosuppressed and immunocompetent individuals as well. MCPyV DNA was found in several benign, semimalignant and malignant skin tumors as well as in inflammatory skin diseases and clinical healthy skin from non-MCC patients.3-12Table 1 summarizes an incomplete selection of results from various research groups analyzing MCPyV DNA prevalence in different entities. For example, prevalence of MCPyV DNA in basal cell carcinomas was investigated from 4 independent research groups and taken together MCPyV DNA was detected in 66 of 221 samples (29.9%), ranging from 0% to 72%.3,5,6,8 We think, this situation nicely shows that MCPyV DNA analysis is most likely dependent on various before mentioned technical parameters, resulting in variable MCPyV DNA detection rates. Nevertheless, almost any of these studies point to the fact that MCPyV is ubiquitously present, even detectable in healthy and non-MCC tissue. Of special interest is that various groups reported significant differences in MCPyV viral load with usually higher copy numbers in MCC tissues and lower ones in non-MCC tissues, representing a possible parameter for differentiation.5,6,9 However, one must consider that most routine pathology laboratories use standard PCR-based methods for MCPyV detection as a qualitative test without analyzing quantitative viral load.TABLE 1: Prevalence of MCPyV DNA in Different Entities Analyzed by PCR-Based MethodsIn summary, latest reports proved that MCPyV DNA is abundantly detectable in tissues of various entities including clinical healthy skin (Table 1). Therefore, a qualitative proof of MCPyV DNA in a tissue sample is by far not specific for MCC. To us, the value of an ancillary MCPyV DNA-analysis in the differential diagnosis of MCC and similar tumors is questionable, and results have to be interpreted in the context of the recent literature suggesting a high prevalence of MCPyV. ACKNOWLEDGMENTS This study was in part supported by the Dr. H. Legerlotz, the R. Bartling and the M. Lackas Foundation. Christian Andres, MD* Benedetta Belloni, MD* *Department of Dermatology and Allergy, Ludwig-Maximilians-Universität München, Munich, Germany Michael J. Flaig, MD† †Department of Dermatology and Allergy Biederstein, Technische Universität München, Munich, Germany

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

BackgroundMerkel cell carcinoma (MCC) is an increasingly common neuroendocrine cancer of the skin. Merkel cell polyomavirus (MCPyV) is one of the causative agents of MCC. The prevalence of MCPyV in primary MCC and sun-exposed non-MCC tumors has been known to have different results depending on where it was investigated.ObjectiveThis study assesses the prevalence of MCPyV from primary MCC and sun-exposed non-MCC tumors in Korea.MethodsA molecular pathology study was performed on 7 tissue specimens of MCC, 1 tissue specimen of metastatic small cell carcinoma of the lung, and 32 tissue specimens of non-MCC tumors occurring from sun-exposed areas [8 basal cell carcinomas (BCCs), 8 squamous cell carcinomas (SCCs), 8 actinic keratoses (AKs), and 8 seborrheic keratoses (SKs)]. All specimens were analyzed to determine the presence of MCPyV-DNA using both polymerase chain reaction (PCR) and real-time quantitative PCR. Immunohistochemistry with monoclonal antibody of MCPyV large T antigen (CM2B4) was also conducted.ResultsUsing both PCR, MCPyV sequences were detected in six of seven MCC tissue specimens (85.7%). Five (71%) of seven MCC tumors were immunoreactive for CM2B4. All five immunoreactive cases were positive for MCPyV. However, there was no association of MCPyV with BCC, SCC, AK, and SK.ConclusionOur results implicate that MCPyV may contribute to the pathogenesis of primary MCC, not of non-MCC skin tumors in Korea, and the persons with MCPyV infection are unusual in Korea compared to other areas.

Multiple Merkel cell carcinomas: Late metastasis or multiple primary tumors? A molecular study

Merkel cell carcinoma is a rare malignancy of the skin. Integration of Merkel cell polyomavirus (MCPyV) DNA to the tumor genome is frequent in these cancers. The clinical consequences of MCPyV infection are unknown. We analyzed formalin-fixed paraffin-embedded Merkel cell carcinoma tissue samples from 114 of 207 patients diagnosed with Merkel cell carcinoma in Finland from 1979 to 2004 for the presence of MCPyV DNA with the use of polymerase chain reaction (PCR), quantitative PCR, and DNA sequencing and examined associations between tumor MCPyV DNA status and histopathologic factors and survival. The median follow-up time after Merkel cell carcinoma diagnosis for subjects who were alive was 9.9 years (range = 4.9-21.9 years). All P values are two-sided. MCPyV DNA was present in 91 carcinomas (79.8%). Compared with MCPyV DNA-negative cancers, MCPyV DNA-positive cancers were more often located in a limb (40.7% vs 8.7%, P = .015) and less frequent in patients who had regional nodal metastases at diagnosis (6.6% vs 21.7%, P = .043). Patients with MCPyV DNA-positive tumors had better overall survival than those with MCPyV DNA-negative tumors (5-year survival: 45.0% vs 13.0%, respectively; P < .001, two-sided log-rank test). MCPyV infection is associated with clinical outcomes in patients with Merkel cell carcinoma. These findings lend support to the hypothesis that viral infection is frequently associated with the pathogenesis of Merkel cell carcinoma.

OBJECTIVE: The newly discovered Merkel cell polyomavirus (MCV) may play a role in the etiology of Merkel cell carcinoma (MCC), a rare neuroendocrine malignancy of the skin, as several studies have demonstrated MCV DNA in tumor tissues. However, MCV serologic data are sparse, with few studies investigating MCV DNA in tumor tissues and MCV antibodies within the same patients, and no published data on antibodies to MCV T-antigen. We conducted a pilot study to evaluate MCV antibody profiles in MCC cases versus controls and to examine the correlation between MCV DNA in tumors and MCV seroreactivity. METHODS: Plasma samples were obtained from 33 patients diagnosed and/or treated for MCC at the Moffitt Cancer Center in 2006-08, including 25 males and 8 females, ages 53-88 years. Controls were comprised of 37 patients undergoing routine skin cancer screening exams who had no history of skin cancer and were determined to be negative for all types of non-melanoma skin cancer (NMSC) by a nurse practitioner. Fresh-frozen tumor tissue was obtained from 15 MCC patients, including 9 from whom plasma was also obtained. Virus-like particle-based enzyme linked immunosorbent assays (ELISA) were used to measure IgG and IgA antibodies to the MCV VP1 capsid protein. Levels of IgG directed against MCV large T-antigen were also measured using ELISA. MCV DNA was measured in MCC tumor tissues using real-time polymerase chain reaction for the amplification of the small T-antigen region of the MCV genome. Differences in antibody levels across groups were assessed using Wilcoxon rank sum test. RESULTS: Levels of IgG directed against the MCV capsid protein were higher among MCC cases than healthy controls (mean (SD)= 1876 (4001) in cases, 1521 (4889) in controls; p=0.005), as were levels of MCV capsid IgA (mean (SD) = 0.21 (0.25) and 0.09 (0.15) p=0.005). Although levels of IgG antibodies directed against the large T-antigen were slightly higher among MCC cases compared to healthy controls (mean (SD) =0.25 (0.27) vs. 0.19(0.07)), this difference was not statistically significant. MCV DNA was observed in 10 (67%) of 15 tumor tissues tested, with viral loads ranging from 7.1 to 15.6 viral copies per cell equivalent. Levels of IgG antibodies directed against MCV capsid and T-antigen were higher among the 6 MCV DNA-positive cases than the 3 MCV DNA-negative cases for whom plasma was available (mean (SD): capsid IgG: 1040 (1210) in MCV DNA-positive, 384 (494) in MCV DNA-negative, p=0.52; T-antigen IgG: 0.26 (0.26) in MCV DNA-positive, 0.15 (0.15) in MCV DNA-negative, p=0.52). CONCLUSION: MCV seroreactivity is associated with MCC, and MCV antibody levels are higher among MCC patients with MCV DNA-positive tumor tissues. A larger study is needed to confirm these associations with greater statistical power and provide the evidence needed to establish causality between MCV infection and MCC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3772A.

Merkel cell polyomavirus (MCPyV) has recently been identified in Merkel cell carcinoma (MCC), an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR) and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n = 9) and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n = 45], actinic keratosis [AK, n = 52], Bowen’s disease [n = 34], seborrheic keratosis [n = 5], primary cutaneous anaplastic large-cell lymphoma [n = 5], malignant melanoma [n = 5], and melanocytic nevus [n = 6]). In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%), BCC (1 case; 2%), and AK (3 cases; 6%). We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119–42.8) and AK (0.02–0.07) groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662). Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4) demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC), but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection.

The prevalence of Merkel cell polyomavirus in Japanese patients with Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive tumour for which an increasing incidence has been reported. A new human polyomavirus, Merkel cell polyomavirus (MCV), was recently isolated from these tumours by applying digital transcriptome subtraction methodology. To detect the presence or absence of MCV in MCCs and other, randomly selected neoplasms. Nine primary or recurrent MCCs from seven patients were examined; 29 other tumours (squamous cell, basal cell and basosquamous carcinomas and malignant melanomas) were examined for comparative purposes. Viral large T protein (LT1 and LT3), and viral capsid protein (VP1) were detected by primer-directed amplification, using a polymerase chain reaction (PCR)-based method, and the amplified PCR products were analysed by agarose gel electrophoresis and subsequent sequence analysis. The presence of viral T antigen and/or viral capsid DNA sequences was demonstrated in seven of the eight MCC lesions. None of the comparative samples contained MCV DNA. Our findings strongly support the hypothesis that MCV infection may well be specific for MCC, and MCV may play a role in the pathogenesis of MCC.

Background A novel polyomavirus, the Merkel cell polyomavirus (MCPyV), has recently been identified in Merkel cell carcinoma (MCC). Objectives To investigate the specificity of this association through the detection, quantification and analysis of MCPyV DNA in lesional and nonlesional skin biopsies from patients with MCC or with other cutaneous diseases, as well as in normal skin from clinically healthy individuals. Methods DNA was extracted from lesional and nonlesional skin samples of patients with MCC or with other cutaneous diseases and from normal-appearing skin of clinically healthy subjects. MCPyV DNA was detected by polymerase chain reaction (PCR) and quantified by real-time PCR. Additionally, the T antigen coding region was sequenced in eight samples from seven patients. Results MCPyV DNA was detected in 14 of 18 (78%) patients with MCC, five of 18 (28%) patients with other skin diseases (P = 0.007) and one of six (17%) clinically healthy subjects. In patients with MCC, viral DNA was detected in nine of 11 (82%) tumours and in 10 of 14 (71%) nontumoral skin samples (P = 0.66). MCPyV DNA levels were higher in MCC tumours than in nontumoral skin from patients with MCC, and than in lesional or nonlesional skin from patients with other cutaneous disorders. Signature mutations in the T antigen gene were not identified in the two MCC tumour specimens analysed. Conclusions High prevalence and higher levels of MCPyV DNA in MCC supports a role for MCPyV in tumorigenesis. However, the high prevalence of MCPyV in the nontumoral skin and in subjects without MCC suggests that MCPyV is a ubiquitous virus.

Merkel cell carcinoma (MCC) is a rare but aggressive primary cutaneous carcinoma with high mortality rates. The present study intends to delineate the epidemiological profile of patients with MCC seen at the Clinics Hospital of the Medical School at the University of São Paulo, Brazil, and its association with Merkel cell polyomavirus (MCPyV). This is a retrospective study. A search was performed in the hospital's medical index for all cases of MCC from January 1994 to December 2012. Among patients with MCC, the available tumoral skin specimens were analyzed with two different techniques of polymerase chain reaction (PCR) (conventional and real-time) for detection of MCPyV DNA. Additionally, paraffin-embedded samples of patients with non-MCC skin cancers were also analyzed. Analyses suitable for categorical data (i.e., x² of Fisher) were used to compare the proportion of patients in each group. Nineteen patients with MCC and 20 patients with non-MCC skin cancers entered the study. All MCC samples available (13) tested positive for the presence of MCPyV DNA; however, in the non-MCC skin cancer samples, the MCPyV DNA was detected in 4 of 20 samples (20%). MCPyV DNA detection rate was higher in patients with MCC than in the other group, and its analysis was statistically significant (P<0.01). This study demonstrates the association of MCPyV in Brazilian patients with MCC. However, further studies are necessary to determine the exact involvement of MCPyV in MCC pathogenesis and to define the significance of viral DNA detection in non-MCC skin cancers.

Lack of evidence for basal or squamous cell carcinoma infection with Merkel cell polyomavirus in immunocompetent patients with Merkel cell carcinoma

Background: Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous neuroendocrine tumor, often metastatic at presentation, with a median survival time of 6.8 months for stage IV disease. Current chemotherapeutic regimens are largely ineffective. It is still unknown whether established oncogenes or tumor suppressors are major players in the pathogenesis of MCC. Recently, a new polyomavirus has been identified in 50 to 80% of MCC, but a mechanistic role for this virus in the pathogenesis of MCC has not yet been demonstrated. We hypothesized that deregulation of signaling pathways that are commonly activated in cancer may contribute to MCC tumorigenesis and may provide insights into targeted therapy approaches for this malignancy. Design: We retrospectively profiled 60 primary MCC samples diagnosed at the MGH from 1995 to 2010 using a recently developed SNaPshot genotyping assay to screen for the presence of common mutations in 13 cancer genes, many of which are targeted by FDA approved drugs or by targeted agents undergoing clinical trials. In addition, all MCC samples were tested for the presence of Merkel cell polyomavirus (MCPyV) using PCR. Results: The SnaPshot assay identified mutations in 15% (9/60) of MCC primary tumors. The TP53 tumor suppressor gene was mutated in 3 of 60 cases and activating mutations in the p110 alpha subunit of the phosphatidylinositol 3-kinase (PIK3CA) gene were found in 6 of 60 cases. Sanger sequencing of the primary MCC tumors identified one additional PIK3CA mutation (R19K) that has not been previously described in cancer. In primary MCC cell lines, we observed that the presence of a PI3KCA activating mutation was associated with sensitivity to treatment with NVP-BEZ-235, a dual PI3K-mTOR inhibitor currently under active clinical development. Clinical correlations with PIK3CA mutational status and the presence of MCPyV are ongoing. Conclusions: We discovered that a subset of patients with MCC (10%) carry activating mutations in PIK3CA. Furthermore, we found that a MCC cell line harboring a PIK3CA mutation is selectively sensitive to a PI3K inhibitor under clinical development. Our results suggest the relevance of screening patients with MCC for activation of the PI3K pathway, as these cancers may be particularly sensitive to PI3K pathway inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2210. doi:10.1158/1538-7445.AM2011-2210

The prevalence of Merkel cell polyomavirus(MCPyV) in Merkel cell carcinoma(MCC) and non-MCC skin lesions and its possible role in the etiology of other skin diseases remain controversial. To systematically assess the association between MCPyV infection and MCC, non-MCC skin lesions, and normal skin. For this systematic review and meta-analysis, a comprehensive search for eligible studies was conducted using Medline Ovid, Pubmed, Web of Science, and the Cochrane CENTRAL databases until August 2021; references were searched to identify additional studies. Observational studies that investigated the association between MCPyV infection and MCC, non-MCC skin lesions, and normal skin using polymerase chain reaction(PCR) as a detection method and provided sufficient data to calculate the prevalence of MCPyV positivity. A total of 50 articles were included in the study after exclusion criteria were applied. Two reviewers independently reviewed and assessed the eligibility of the studies, and all disagreements were resolved by consensus. To determine the association between MCPyV and MCC, overall odds ratio (OR) were calculated with 95% CI using a random-effects model. Single-arm meta-analyses were performed to examine the prevalence rate of MCPyV+ in MCC, non-MCC skin lesions, and normal skin. The primary analysis was the prevalence rate of MCPyV+ in MCC. Secondary outcomes included the prevalence rate of MCPyV+ in non-MCC skin lesions and normal skin. A total of 50 studies involving 5428 patients were reviewed based on our inclusion and exclusion criteria. Compared with the control group, MCPyV infection was significantly associated with MCC (OR = 3.51, 95% CI = 2.96 - 4.05). The global prevalence of MCPyV+ in MCC, melanoma, squamous cell carcinoma, basal cell carcinoma, Bowen’s disease, actinic keratosis, keratoacanthoma, seborrheic keratosis, and normal skin was 80%, 4%, 15%, 15%, 21%, 6%, 20%, 10%, and 11%, respectively. The current results suggest that MCPyV infection is significantly associated with an increased risk of MCC. However, the low prevalence rate of MCPyV+ in non-MCC skin lesions does not exclude a pathogenic association of this virus with the development of non-MCC skin lesions.

Merkel cell polyomavirus in naevoid basal cell carcinoma syndrome-associated basal cell carcinomas and sporadic trichoblastomas