Distinct Merkel Cell Polyomavirus Molecular Features in Tumour and Non Tumour Specimens from Patients with Merkel Cell Carcinoma

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.     

Gene expression profile identifies distinct molecular subtypes and potential therapeutic genes in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare primary cutaneous neoplasm of neuroendocrine carcinoma of the skin. About 80% of the MCC occurs due to Merkel cell polyomavirus (MCPyV) and 20% of the tumors usually occur due to severe UV exposure which is a more aggressive type of MCC. It tends to have an increased incidence rate among elderly and immunosuppressed individuals. On therapeutic level, sub-classification of MCC through molecular subtyping has emerged as a promising technique for MCC prognosis. In current study, two consistent distinct molecular subtypes of MCCs were identified using gene expression profiling data. Subtypes I MCCs were associated with spliceosome, DNA replication and cellular pathways. On the other hand, genes overexpressed in subtype II were found active in TNF signalling pathway and MAPK signalling pathway. We proposed different therapeutic targets based on subtype specificity, such as PTCH1, CDKN2A, AURKA in case of subtype I and MCL1, FGFR2 for subtype II. Such findings may provide fruitful knowledge to understand the intrinsic subtypes of MCCs and the pathways involved in distinct subtype oncogenesis, and will further advance the knowledge in developing a specific therapeutic strategy for these MCC subtypes.     

Quantitative analysis of viral load per haploid genome revealed the different biological features of Merkel cell polyomavirus infection in skin tumor

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.     

An update on Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare cancer of the skin characterized by a neuroendocrine phenotype and an aggressive clinical behavior. It frequently originates in sun-exposed body areas, and its incidence has steadily increased in the last three decades. Merkel cell polyomavirus (MCPyV) and ultraviolet (UV) radiation exposure are the main causative agents of MCC, and distinct molecular features have been documented in virus-positive and virus-negative malignancies. Surgery remains the cornerstone of treatment for localized tumors, but even when integrated with adjuvant radiotherapy is able to definitively cure only a fraction of MCC patients. While characterized by a high objective response rate, chemotherapy is associated with a short-lasting benefit of approximately 3 months. On the other hand, immune checkpoint inhibitors including avelumab and pembrolizumab have demonstrated durable antitumor activity in patients with stage IV MCC, and investigations on their use in the neoadjuvant or adjuvant setting are currently underway. Addressing the needs of those patients who do not persistently benefit from immunotherapy is currently one of the most compelling unmet needs in the field, and multiple clinical trials of new tyrosine kinase inhibitors (TKIs), peptide receptor radionuclide therapy (PRRT), therapeutic vaccines, immunocytokines as well as innovative forms of adoptive cellular immunotherapies are under clinical scrutiny at present.     

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.     

T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.     

Seroprevalence of Merkel Cell Polyomavirus in the General Rural Population of Anyang,China

Background

Despite the probably causal link between Merkel cell polyomavirus (MCPyV) infection and Merkel cell carcinoma (MCC), a rare but aggressive skin malignancy, little is known about the seroepidemiology of MCPyV among healthy adults in China.

Methods

Serum antibodies against MCPyV were evaluated by multiplex serology in a population-based study of 5548 adults (including 1587 heterosexual couples) aged 25–65 years who were enrolled from rural Anyang, China in 2007–2009. Univariate and multivariate logistic regression analyses were performed to assess the risk factors for the seropositivity of MCPyV.

Results

The seroprevalence for MCPyV was 61.0%. MCPyV seropositivity was significantly higher in males than in females (64.5% vs. 57.7%, P<0.001), and for both genders, showed a trend of increase with age (Male: P _trend<0.001; Female: P _trend<0.001). Furthermore, among antibody positives, antibody levels of MCPyV increased with advancing age (P _trend = 0.017). MCPyV seropositivity of one spouse was significantly associated with that of the other partner (Adjusted OR = 1.32, 95% CI: 1.07–1.62). However, there was no association between sexual behaviors and the seropositivity of MCPyV.

Conclusions

High seroprevalence of MCPyV was observed in healthy Chinese individuals. Serological evidence suggests that nonsexual horizontal spread of MCPyV can occur among family members, and further research in this regard is needed.     

Detection of Merkel cell polyomavirus in cervical squamous cell carcinomas and adenocarcinomas from Japanese patients

ABSTRACT: BACKGROUND: Merkel cell polyomavirus (MCPyV) was identified originally in Merkel cell carcinoma (MCC), a rare form of human skin neuroendocrine carcinoma. Evidence of MCPyV existence in other forms of malignancy such as cutaneous squamous cell carcinomas (SCCs) is growing. Cervical cancers became the focus of our interest in searching for potentially MCPyV-related tumors because: (i) the major histological type of cervical cancer is the SCC; (ii) the uterine cervix is a common site of neuroendocrine carcinomas histologically similar to MCCs; and (iii) MCPyV might be transmitted during sexual interaction as demonstrated for human papillomavirus (HPV). In this study, we aimed to clarify the possible presence of MCPyV in cervical SCCs from Japanese patients. Cervical adenocarcinomas (ACs) were also studied. RESULTS: Formalin-fixed paraffin-embedded tissue samples from 48 cervical SCCs and 16 cervical ACs were examined for the presence of the MCPyV genome by polymerase chain reaction (PCR) and sequencing analyses. PCR analysis revealed that 9/48 cervical SCCs (19 %) and 4/16 cervical ACs (25 %) were positive for MCPyV DNA. MCPyV-specific PCR products were sequenced to compare them with reference sequences. The nucleotide sequences in the MCPyV large-T (LT)-sequenced region were the same among MCPyV-positive cervical SCCs and AC. Conversely, in the MCPyV viral protein 1 (VP1)-sequenced, two cervical SCCs and three cervical ACs showed several nucleotide substitutions, of which three caused amino acid substitutions. These sequencing results suggested that three MCPyV variants of the VP1 were identified in our cases. Immunohistochemistry showed that the LT antigen was expressed in tumor cells in MCPyV-positive samples. Genotyping of human HPV in the MCPyV-positive samples revealed that infected HPVs were HPV types 16, 31 and 58 for SCCs and HPV types 16 and 18 for ACs. CONCLUSIONS: This study provides the first observation that MCPyV coexists in a subset of HPV-associated cervical cancers from Japanese patients. The prevalence of MCPyV in these lesions was close to that observed in the cutaneous SCCs. Further worldwide epidemiological surveys are warranted to determine the possible association of MCPyV with pathogenesis of cervical cancers.     

Structures of Merkel Cell Polyomavirus VP1 Complexes Define a Sialic Acid Binding Site Required for Infection

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.     

Second cancers following the diagnosis of Merkel cell carcinoma: A nationwide cohort study

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin. MCCs and some other skin cancers, such as basal cell carcinomas, frequently harbour Merkel cell polyomavirus DNA. The purpose of the study was to investigate the frequency of second cancers following the diagnosis of MCC. We studied the incidence of second primary cancers after the diagnosis of MCC from the files of the Finnish Cancer Registry in 1979–2006. Among the 172 MCC patients identified a total of 34 second primary cancers were detected in 30 individuals after the diagnosis of MCC. Female MCC patients were diagnosed with 25 subsequent cancers (SIR, 2.35; 95% CI, 1.52–3.47; p < 0.001) and male patients with 9 cancers (SIR, 2.32, 95% CI, 1.06–4.40; p < 0.05). The MCC patients had an increased risk for a subsequent cancer (any site) compared to age-, gender- and calendar period-matched general population (standardized incidence ratio [SIR] 2.34; 95% confidence interval [CI], 1.62–3.27). The risks for basal cell carcinoma of the skin (O = 11), SIR, 3.48; 95% CI, 1.74–6.22 and chronic lymphocytic leukemia (O = 2), SIR, 17.9; 95% CI, 2.16–64.6 were significantly elevated. The SIRs for an overall second primary cancer risk did not change markedly with time since the diagnosis of MCC. We conclude that patients diagnosed with MCC have an increased risk for a second cancer. This risk may in part result from shared etiological factors between MCC and other tumour types, such as immunosuppression or possibly Merkel cell polyomavirus infection.     

Response of Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Xenografts to a Survivin Inhibitor

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ~80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs.     

The T Antigen Locus of Merkel Cell Polyomavirus Downregulates Human Toll-Like Receptor 9 Expression

Establishment of a chronic infection is a key event in virus-mediated carcinogenesis. Several cancer-associated, double-stranded DNA (dsDNA) viruses act via their oncoproteins to downregulate Toll-like receptor 9 (TLR9), a key receptor in the host innate immune response that senses viral or bacterial dsDNA. A novel oncogenic virus, Merkel cell polyomavirus (MCPyV), has been recently identified that causes up to 80% of Merkel cell carcinomas (MCCs). However, it is not yet known whether this oncogenic virus also disrupts immune-related pathways. We find that MCPyV large T antigen (LT) expression downregulates TLR9 expression in epithelial and MCC-derived cells. Accordingly, silencing of LT expression results in upregulation of mRNA TLR9 levels. In addition, small T antigen (sT) also appears to inhibit TLR9 expression, since inhibition of its expression also resulted in an increase of TLR9 mRNA levels. LT inhibits TLR9 expression by decreasing the mRNA levels of the C/EBPβ transactivator, a positive regulator of the TLR9 promoter. Chromatin immunoprecipitation reveals that C/EBPβ binding at a C/EBPβ response element (RE) in the TLR9 promoter is strongly inhibited by expression of MCPyV early genes and that mutation of the C/EBP RE prevents MCPyV downregulation of TLR9. A survey of BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI polyomavirus (KIPyV), MCPyV, simian virus 40 (SV40), and WU polyomavirus (WUPyV) early genes revealed that only BKPyV and MCPyV are potent inhibitors of TLR9 gene expression. MCPyV LT targeting of C/EBP transactivators is likely to play an important role in viral persistence and potentially inhibit host cell immune responses during MCPyV tumorigenesis.     

Entry Tropism of BK and Merkel Cell Polyomaviruses in Cell Culture

Merkel Cell Polyomavirus (MCV or MCPyV) was recently discovered in an aggressive form of skin cancer known as Merkel cell carcinoma (MCC). Integration of MCV DNA into the host genome likely contributes to the development of MCC in humans. MCV infection is common and many healthy people shed MCV virions from the surface of their skin. MCV DNA has also been detected in samples from a variety of other tissues. Although MCC tumors serve as a record that MCV can infect the Merkel cell lineage, the true tissue tropism and natural reservoirs of MCV infection in the host are not known. In an effort to gain insight into the tissue tropism of MCV, and to possibly identify cellular factors responsible for mediating infectious entry of the virus, the infection potential of human cells derived from a variety of tissues was evaluated. MCV gene transfer vectors (pseudoviruses) carrying reporter plasmid DNA encoding GFP or luciferase genes were used to transduce keratinocytes and melanocytes, as well as lines derived from MCC tumors and the NCI-60 panel of human tumor cell lines. MCV transduction was compared to transduction with pseudoviruses based on the better-studied human BK polyomavirus (BKV). The efficiency of MCV and BKV transduction of various cell types occasionally overlapped, but often differed greatly, and no clear tissue type preference emerged. Application of native MCV virions to a subset of highly transducible cell types suggested that the lines do not support robust replication of MCV, consistent with recent proposals that the MCV late phase may be governed by cellular differentiation in vivo. The availability of carefully curated gene expression data for the NCI-60 panel should make the MCV and BKV transduction data for these lines a useful reference for future studies aimed at elucidation of the infectious entry pathways of these viruses.     

Discovery of a New Human Polyomavirus Associated with Trichodysplasia Spinulosa in an Immunocompromized Patient

The Polyomaviridae constitute a family of small DNA viruses infecting a variety of hosts. In humans, polyomaviruses can cause infections of the central nervous system, urinary tract, skin, and possibly the respiratory tract. Here we report the identification of a new human polyomavirus in plucked facial spines of a heart transplant patient with trichodysplasia spinulosa, a rare skin disease exclusively seen in immunocompromized patients. The trichodysplasia spinulosa-associated polyomavirus (TSV) genome was amplified through rolling-circle amplification and consists of a 5232-nucleotide circular DNA organized similarly to known polyomaviruses. Two putative “early” (small and large T antigen) and three putative “late” (VP1, VP2, VP3) genes were identified. The TSV large T antigen contains several domains (e.g. J-domain) and motifs (e.g. HPDKGG, pRb family-binding, zinc finger) described for other polyomaviruses and potentially involved in cellular transformation. Phylogenetic analysis revealed a close relationship of TSV with the Bornean orangutan polyomavirus and, more distantly, the Merkel cell polyomavirus that is found integrated in Merkel cell carcinomas of the skin. The presence of TSV in the affected patient''s skin was confirmed by newly designed quantitative TSV-specific PCR, indicative of a viral load of 10⁵ copies per cell. After topical cidofovir treatment, the lesions largely resolved coinciding with a reduction in TSV load. PCR screening demonstrated a 4% prevalence of TSV in an unrelated group of immunosuppressed transplant recipients without apparent disease. In conclusion, a new human polyomavirus was discovered and identified as the possible cause of trichodysplasia spinulosa in immunocompromized patients. The presence of TSV also in clinically unaffected individuals suggests frequent virus transmission causing subclinical, probably latent infections. Further studies have to reveal the impact of TSV infection in relation to other populations and diseases.