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.
For more than 50 years it has been known that some polyomavirus types can induce cancer in experimental animals. However, associations between the various polyomaviruses known to chronically infect most humans and the development of cancer have been difficult to uncover. Last year, DNA from a new human polyomavirus, called Merkel cell polyomavirus (MCV), was found embedded in an uncommon form of skin cancer called Merkel cell carcinoma. Emerging evidence indicates that most adults display detectable immune responses to MCV, suggesting that most individuals eventually become infected with the virus. In this study, we investigate antibodies that directly bind the protein coat of MCV, thereby obstructing its ability to penetrate cultured cells. We found that the magnitude of antibody responses against MCV varies dramatically among normal adults. Interestingly, patients suffering from MCV-associated Merkel cell carcinoma display uniformly strong antibody responses against the virus. This suggests that the development of Merkel cell carcinoma is preceded by an unusually robust MCV infection. It is currently unclear whether MCV infection may also be associated with additional diseases aside from Merkel cell carcinoma. Quantitation of immune responsiveness to the virus, using techniques reported here, could help identify such links.
A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen.
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.
Merkel cell polyomavirus (MCPyV) is a recently discovered virus highly associated with a rare skin cancer, Merkel cell carcinoma (MCC). The causal role of MCPyV in cancer is suggested by integration of viral sequences into the cell genome and by a specific molecular signature. We looked for and compared molecular species of MCPyV in tumour and non tumour samples of 33 MCC patients. We showed that a tumour viral load greater than 1 copy per cell was associated with a better outcome, and that detection of the virus in blood but not in urine correlated with a shorter overall survival. A tumour–specific molecular signature was found in the blood of two patients with metastatic disease, but did not occur in their respiratory nor urine samples. We propose that molecular analysis of MCPyV in tumour and blood be used as a biomarker of infection and cancer progression in MCC patients.
Merkel cell carcinoma is a skin cancer with 30% mortality and an incidence that has tripled in the past 15 years. There is agreement that surgical excision with negative margins is an appropriate therapeutic first step and that sentinel lymph node biopsy is a powerful prognostic indicator. Following excision of detectable cancer, optimal adjuvant therapy is not well established. A role for adjuvant radiotherapy is increasingly supported by retrospective data suggesting a nearly four-fold decrease in local recurrences if radiation is added to surgery. In contrast, a role for adjuvant chemotherapy is not well supported. The rationale for chemotherapy in this disease is based on small-cell lung cancer, a more common neuroendocrine tumor for which chemotherapy is the primary treatment modality. Several issues call into question the routine use of adjuvant chemotherapy in Merkel cell carcinoma: lack of evidence for improved survival; the associated morbidity and mortality; important differences between small-cell lung cancer and Merkel cell carcinoma; and rapid development of resistance to chemotherapy. Importantly, chemotherapy suppresses immune function that plays an unusually large role in defending the host from the development and progression of Merkel cell carcinoma. Taken together, these arguments suggest that adjuvant chemotherapy for Merkel cell carcinoma patients should largely be restricted to clinical trials.
Merkel cell carcinoma is a neuroendocrine cancer that typically presents as a rapidly growing non-specific nodule on sun exposed skin in people over 65 years of age. The recent increase in incidence to over 1000 cases a year in the United States has led Merkel cell carcinoma to become the second most common cause of non-melanoma skin cancer death.1,2 Optimal management for Merkel cell carcinoma beyond surgical excision is not agreed on, and no randomized trials have been carried out. Sentinel lymph node biopsy has been shown to be powerful in predicting subsequent recurrences as well as in determining if further nodal treatment is indicated.3,4
Merkel cell polyomavirus (MCPyV) has been detected in approximately 75% of patients with the rare skin cancer Merkel cell carcinoma. We investigated the prevalence of antibodies against MCPyV in the general population and the association between these antibodies and Merkel cell carcinoma.
Multiplex antibody-binding assays were used to assess levels of antibodies against polyomaviruses in plasma. MCPyV VP1 antibody levels were determined in plasma from 41 patients with Merkel cell carcinoma and 76 matched control subjects. MCPyV DNA was detected in tumor tissue specimens by quantitative polymerase chain reaction. Seroprevalence of polyomavirus-specific antibodies was determined in 451 control subjects. MCPyV strain–specific antibody recognition was investigated by replacing coding sequences from MCPyV strain 350 with those from MCPyV strain w162.
We found that 36 (88%) of 41 patients with Merkel cell carcinoma carried antibodies against VP1 from MCPyV w162 compared with 40 (53%) of the 76 control subjects (odds ratio adjusted for age and sex = 6.6, 95% confidence interval [CI] = 2.3 to 18.8). MCPyV DNA was detectable in 24 (77%) of the 31 Merkel cell carcinoma tumors available, with 22 (92%) of these 24 patients also carrying antibodies against MCPyV. Among 451 control subjects from the general population, prevalence of antibodies against human polyomaviruses was 92% (95% CI = 89% to 94%) for BK virus, 45% (95% CI = 40% to 50%) for JC virus, 98% (95% CI = 96% to 99%) for WU polyomavirus, 90% (95% CI = 87% to 93%) for KI polyomavirus, and 59% (95% CI = 55% to 64%) for MCPyV. Few case patients had reactivity against MCPyV strain 350; however, indistinguishable reactivities were found with VP1 from strain 350 carrying a double mutation (residues 288 and 316) and VP1 from strain w162.
Infection with MCPyV is common in the general population. MCPyV, but not other human polyomaviruses, appears to be associated with Merkel cell carcinoma.
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer of unknown etiology that develops on sun-exposed areas in individuals who are over 50 or immunosuppressed. DNA from a new polyomavirus, MCPyV, was recently shown to be clonally integrated into the host genome in several MCC cases. In this issue, Becker et al. show in a larger study that MCPyV DNA can be isolated from 85% of primary European MCC specimens and their metastases, while Gerneski et al. present data suggesting that the percentage of Australian MCC cases containing MCPyV may be lower than in North American cases. These reports strengthen the possibility that MCPyV may be etiologically involved in at least some cases of MCC.
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.
Viruses must interact with specific receptor molecules on their host cells in order to first attach to the cell and second gain entry into it. Therefore, a viral entry pathway is a sequence of precisely regulated binding events between viral proteins and their cellular receptors, which can be proteins or other biomolecules. In the present study, we investigated the human Merkel cell polyomavirus (MCPyV or MCV) and show that it uses complex carbohydrates containing sialic acid as receptors for entry. MCPyV was discovered in 2008, is widespread in humans and can cause aggressive skin tumors termed Merkel cell carcinomas in immunosuppressed individuals. We determined the crystal structures of the MCPyV capsid protein bound to sialylated carbohydrates, describing the contacts needed for receptor recognition in molecular detail. When we introduced targeted mutations that abolished sialic acid binding into the virus, it was unable to infect cells although it could still attach to them. Earlier studies showed that the virus uses a different group of carbohydrates called glycosaminoglycans for initial attachment to the cell surface. Thus, its entry pathway involves sequential binding to two distinct classes of carbohydrates. Our structures can be used as a starting point to develop antivirals against MCPyV.
Immunosuppression and Merkel-cell polyomavirus (MCPyV) infection may have a role in the pathogenesis of Merkel-cell carcinoma (MCC), a rare neuroendocrine carcinoma of the skin.
We studied incidence of chronic lymphocytic leukaemia (CLL) and MCC from the files of the Finnish Cancer Registry and the largest hospital of Finland, Helsinki University Central Hospital, from 1979 to 2006. Presence of MCPyV DNA in MCCs was investigated by quantitative PCR.
We identified 4164 patients diagnosed with CLL and 172 diagnosed with MCC. Six patients diagnosed with both diseases were found; CLL was the first diagnosis in four cases and MCC in two. The standardised incidence ratio (SIR) for CLL after the diagnosis of MCC was highly elevated, 17.9 (95% confidence interval (CI), 2.2–64.6; P<0.001), and the SIR for MCC after the diagnosis of CLL was also elevated, 15.7 (3.2–46.0, P<0.01). Merkel-cell polyomavirus DNA was present in all five MCCs with tumour tissue available for analysis.
We conclude that patients diagnosed with CLL have a substantially increased risk for MCC, and vice versa. Merkel-cell polyomavirus DNA is frequently present in MCCs that occur in CLL patients. Immunosuppression related with CLL and viral infection might explain the association between CLL and MCC.
chronic lymphatic leukaemia; Merkel-cell carcinoma; Merkel-cell polyomavirus; incidence; immunosuppression
The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.
Merkel cell polyomavirus (MCV or MCPyV) is a recently discovered member of the viral family Polyomaviridae. The virus plays a causal role in Merkel cell carcinoma, a highly lethal form of skin cancer. MCV encodes a major capsid protein, VP1, which forms the non-enveloped surface of the virion. Other polyomavirus species encode two minor capsid proteins, VP2 and VP3, which associate with the inner surface of the capsid and facilitate infectious entry. In this report we show that MCV does not have a VP3 minor capsid protein. Sequence analyses suggest that more than a quarter of known polyomavirus species share MCV's lack of a VP3 protein. In contrast to VP3, VP2-knockout MCV mutants displayed dramatically reduced infectivity. Consistent with native virion findings, MCV pseudovirions lacking VP2 or carrying mutations in the VP2 myristoylation motif displayed reduced infectivity on several cell lines. Puzzlingly, MCV pseudoviruses lacking VP2 successfully transduced other cell lines with high efficiency. Taken together, the data show that the lone MCV minor capsid protein, VP2, plays an important role during infectious entry into some cell types, but is dispensable for entry into other cell types.
Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC.
IMPORTANCE Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin by its known association with the cellular phosphatase catalytic subunit PP4C. These findings highlight stathmin as a possible biomarker of MCC and as a target for novel antitumoral therapies.
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.
Diseases that occur exclusively in immunocompromized patients are often of an infectious nature. Trichodysplasia spinulosa (TS) is such a disease characterized by development of papules, spines and alopecia in the face. Fortunately this disease is rare, because facial features can change dramatically, as in the case of an adolescent TS patient who was on immunosuppressive drugs because of heart-transplantation. A viral cause of TS was suspected already for some time because virus particles had been seen in TS lesions. In pursuit of this unknown virus, we isolated DNA from collected TS spines and could detect a unique small circular DNA suggestive of a polyomavirus genome. Additional experiments confirmed the presence in these samples of a new polyomavirus that we tentatively called TS-associated polyomavirus (TSPyV or TSV). TSV shares several properties with other polyomaviruses, such as genome organization and proteome composition, association with disease in immunosuppressed patients and occurence in individuals without overt disease. The latter indicates that TSV circulates in the human population. Future studies have to show how this newly identified polyomavirus spreads, how it causes disease and if it is related to other (skin) conditions as well.
Merkel cell carcinoma is a rare malignant cutaneous neoplasm that is locally invasive and frequently metastasizes to lymph nodes, liver, lungs, bone and brain. The incidence of Merkel cell carcinoma has increased in the past three decades.
A 65-year-old Caucasian man presented with a sudden onset of severe headache and a three-month history of balance disturbance. Magnetic resonance imaging revealed a large meningeal metastasis. The radiologic workup showed retroperitoneal and inguinal lymph node metastases. Biopsy of the inguinal lymph nodes showed metastases of Merkel cell carcinoma. Biopsy from three different suspected skin lesions revealed no Merkel cell carcinoma, and the primary site of Merkel cell carcinoma remained unknown. Leptomeningeal metastases, new axillary lymph node metastases, and intraspinal (epidural and intradural) metastases were detected within six, seven and eight months, respectively, from the start of symptoms despite treating the intracranial metastasis with gamma knife and the abdominal metastases with surgical dissection and external radiotherapy. This indicates the aggressive nature of the disease.
To the best of our knowledge, this is the first report in the literature of an intracranial meningeal metastasis of Merkel cell carcinoma treated with gamma knife and of intraspinal intradural metastases of Merkel cell carcinoma. Despite good initial response to radiotherapy, recurrence and occurrence of new metastases are common in Merkel cell carcinoma.
Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein.
IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential.
Merkel cell polyomavirus (MCPyV) is a common infectious agent that is likely involved in the etiology of most Merkel cell carcinomas (MCCs). Serum antibodies recognizing the MCPyV capsid protein, VP1, are detectable at high titer in nearly all MCC patients, and remain stable over time. Although antibodies to the viral capsid indicate prior MCPyV infection, they provide limited clinical insight into MCC because they are also detected in more than half of the general population. We investigated whether antibodies recognizing MCPyV large and small tumor-associated antigens (T-Ags) would be more specifically associated with MCC. Among 530 population control subjects, these antibodies were present in only 0.9% and were of low titer. In contrast, among 205 MCC cases, 40.5% had serum IgG antibodies that recognize a portion of T-Ag shared between small and large T-Ags. Among cases, titers of T-Ag antibodies fell rapidly (approximately 8 fold/year) in patients whose cancer did not recur, while they rose rapidly in those with progressive disease. Importantly, in several patients who developed metastases, the rise in T-Ag titer preceded clinical detection of disease spread. These results suggest that antibodies recognizing T-Ag are relatively specifically associated with MCC, do not effectively protect against disease progression, and may serve as a clinically useful indicator of disease status.
Merkel cell carcinoma; Merkel cell polyomavirus; MCPyV; antibody; biomarker
Polyomaviruses are a family of small non-enveloped DNA viruses that encode oncogenes and have been associated, to greater or lesser extent, with human disease and cancer. Currently, twelve polyomaviruses are known to circulate within the human population. To further examine the diversity of human polyomaviruses, we have utilized a combinatorial approach comprised of initial degenerate primer-based PCR identification and phylogenetic analysis of nonhuman primate (NHP) polyomavirus species, followed by polyomavirus-specific serological analysis of human sera. Using this approach we identified twenty novel NHP polyomaviruses: nine in great apes (six in chimpanzees, two in gorillas and one in orangutan), five in Old World monkeys and six in New World monkeys. Phylogenetic analysis indicated that only four of the nine chimpanzee polyomaviruses (six novel and three previously identified) had known close human counterparts. To determine whether the remaining chimpanzee polyomaviruses had potential human counterparts, the major viral capsid proteins (VP1) of four chimpanzee polyomaviruses were expressed in E. coli for use as antigens in enzyme-linked immunoassay (ELISA). Human serum/plasma samples from both Côte d'Ivoire and Germany showed frequent seropositivity for the four viruses. Antibody pre-adsorption-based ELISA excluded the possibility that reactivities resulted from binding to known human polyomaviruses. Together, these results support the existence of additional polyomaviruses circulating within the human population that are genetically and serologically related to existing chimpanzee polyomaviruses.
Polyomaviruses are able to cause severe disease in immunocompromised individuals. The discovery of Merkel cell polyomavirus and its association with Merkel cell carcinoma has increased interest in these viruses, resulting in the identification of several novel human polyomaviruses in recent years. The existence of one of these recently identified viruses, human polyomavirus 9 (HPyV9), had been predicted nearly 30 years prior due to the ability of human sera to neutralize infection of an African green monkey polyomavirus (Lymphotropic polyomavirus; LPyV). HPyV9 and LPyV are now known to be antigenically and phylogenetically closely related. We hypothesized that nonhuman primate (NHP) polyomaviruses, in particular those of the closely related chimpanzee, may serve as genetic and immunological predictors for the existence of yet unknown human polyomaviruses. In the present study, we discovered 20 novel NHP polyomaviruses, six of which were isolated from chimpanzees. Of the 9 chimpanzee polyomaviruses now known, 5 do not presently have a closely related human counterpart. Serologic reactivity against these novel chimpanzee viruses was observed in humans from European and African populations. From these data we predict that additional human polyomaviruses exist which are genetically and serologically related to the novel chimpanzee polyomaviruses.
Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with poorly characterized genetics. We performed high-resolution comparative genomic hybridization on 25 MCC specimens using a high-density oligonucleotide microarray. Tumors frequently carried extra copies of chromosomes 1, 3q, 5p, and 6 and lost chromosomes 3p, 4, 5q, 7, 10 and 13. MCC tumors with less genomic aberration were associated with improved survival (p=0.04). Tumors from 13 of 22 MCC patients had detectable Merkel cell polyomavirus DNA, and these tumors had fewer genomic deletions. Three regions of genomic alteration were of particular interest: a deletion of 5q12-21 occurred in 26% of tumors, a deletion of 13q14-21 was recurrent in 26% of tumors and contains the well-characterized tumor suppressor RB1, and a novel focal amplification at 1p34 was present in 39% of tumors and centers on L-Myc (MYCL1). L-Myc is related to the c-Myc proto-oncogene, has transforming activity, and is amplified in the closely related small cell lung cancer. Normal skin showed no L-Myc expression, while 4/4 MCC specimens tested expressed L-Myc RNA in relative proportion to the DNA copy number gain. These findings suggest several genes that may contribute to MCC pathogenesis, most notably L-Myc.
Merkel cell carcinoma; comparative genomic hybridization; L-Myc; Merkel cell polyomavirus; genetics
Merkel cell polyomavirus (MCPyV) is a DNA virus expressing transcripts similar to the large T (LT) and small T (ST) transcripts of SV40, which has been implicated in the pathogenesis of Merkel cell carcinoma (MCC), a rare and highly aggressive neuroendocrine skin cancer. MCPyV LT antigen expression was found to be a requirement for MCC tumor maintenance and ST protein also likely contributes to the carcinogenesis of MCC. Previously, we have identified the probable immunodominant epitope of MCPyV LT and developed a DNA vaccine encoding this epitope linked to calreticulin. The LT-targeting DNA vaccine generated prolonged survival, decreased tumor size and increased LT-specific CD8+ T cells in tumor-bearing mice.
In this study, we developed a MCPyV ST-expressing tumor cell line from B16 mouse melanoma cells. We then utilized this ST-expressing tumor cell line to test the efficacy of a DNA vaccine encoding ST. In ST-expressing tumor-bearing mice, this vaccine, pcDNA3-MCC/ST, generated a significant number of ST antigenic peptide-specific CD8+ T cells and experienced markedly enhanced survival compared to mice vaccinated with empty vector.
The formation of an effective vaccine against MCPyV has the potential to advance the field of MCC therapy and may contribute to the control of this severe malignancy through immunotherapy. Both of the innovative technologies presented here provide opportunities to develop and test MCPyV-targeted therapies for the control of Merkel cell carcinoma.
DNA vaccine; Gene therapy; Merkel cell polyomavirus; Small T antigen; Merkel cell carcinoma
Merkel cell carcinoma (MCC) is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV). The MCPyV-encoded large T (LT) antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT) encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT), as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells.
The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the immunodominant LT epitope as aa19-27 (IAPNCYGNI) and found that it is H-2kb-restricted.
The results of this study can facilitate the development of other modes of MCC treatment such as peptide-based vaccines and adoptive transfer of LT-specific CD8+ T cells. Likewise, the MCC DNA vaccine has great potential for clinical translation as the immunologic specificity is high and the treatment strategy can be exported to address other virus-induced tumors.
DNA vaccine; Gene therapy; Merkel cell polyomavirus; Cryptic CTL Epitope; Large T antigen
Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region.
A marked escalation in the rate of discovery of new types of human polyomavirus has occurred over the last five years largely owing to recent technological advances in their detection. Among the newly discovered viruses, Merkel Cell Polyomavirus (MCPyV or MCV) has gained the most attention due to its link with a rare human cancer. Infection with MCPyV is common in the human population, and the virus is detected in several anatomical locations, but most frequently in skin. Study of MCPyV molecular virology has been complicated by the lack of straightforward cell culture models, but recent in vitro studies are making strides towards understanding the virus life cycle, its cellular tropism, and mode of transmission. While MCPyV shares several traditional traits with other human polyomaviruses, the burst of research since its discovery reveals insight into a virus with many unique genetic and mechanistic features. The evidence for a causal link between MCPyV and the rare neuroendocrine cancer, Merkel Cell Carcinoma (MCC), is compelling. A majority of MCCs contain clonally integrated viral DNA, express viral T antigen transcripts and protein, and exhibit an addiction to the viral large T and small t antigen oncoproteins. The MCPyV large T antigen contains MCC tumor-specific mutations that ablate its replication capacity but preserve its oncogenic functions, and the small t antigen promotes an environment favorable for cap-dependent translation. The mechanisms of MCPyV-induced transformation have not been fully elucidated, but the likely etiological role of this new polyomavirus in human cancer provides a strong opportunity to expand knowledge of virus-host interactions and viral oncology.
Merkel cell polyomavirus (MCV or MCPyV) is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4) interacts with MCV large T antigen (LT) and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+) leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus.
MCV is a novel human polyomavirus that has recently been discovered in Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer. Several independent studies have confirmed that MCV is present in ∼80% of MCC tumors. However, very little is known about how the interaction between MCV and its human hosts contributes to the virus-induced cancers. Many aspects of the infectious life cycle of MCV are largely unexplored. In this study, we demonstrate that the MCV-encoded large T antigen can bind to host protein Brd4, which in turn serves as a scaffold that functionally recruits cellular DNA replication factors for replication of MCV viral DNA in host cells. This study is the first report to demonstrate mechanistic details of MCV's recruitment of the host cell DNA replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus. Importantly, our work demonstrates that blocking the Brd4 and MCV LT interaction can prevent MCV from replicating in host cells. This study identifies the Brd4-MCV LT interaction as an important target for potential development of effective therapeutic strategies to treat MCV infection.
Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown.
Here we analyzed the presence and characteristics of newly described human polyomaviruses in urban sewage and river water in order to assess the excretion level and the potential role of water as a route of transmission of these viruses. Nested-PCR assays were designed for the sensitive detection of the viruses studied and the amplicons obtained were confirmed by sequencing analysis. The viruses were concentrated following a methodology previously developed for the detection of JC and BK human polyomaviruses in environmental samples. JC polyomavirus and human adenoviruses were used as markers of human contamination in the samples. Merkel cell polyomavirus was detected in 7/8 urban sewage samples collected and in 2/7 river water samples. Also one urine sample from a pregnant woman, out of 4 samples analyzed, was positive for this virus. KI and WU polyomaviruses were identified in 1/8 and 2/8 sewage samples respectively. The viral strains detected were highly homologous with other strains reported from several other geographical areas. Lymphotropic polyomavirus was not detected in any of the 13 sewage neither in 9 biosolid/sludge samples analyzed.
This is the first description of a virus isolated from sewage and river water with a strong association with cancer. Our data indicate that the Merkel cell polyomavirus is prevalent in the population and that it may be disseminated through the fecal/urine contamination of water. The procedure developed may constitute a useful tool for studying the excreted strains, prevalence and transmission of these recently described polyomaviruses.
Background: Merkel cell carcinoma is a high-grade neuroendocrine carcinoma of skin that is characterized by immature cells which, because of its striking morphologic similarity, may be confused with other small round blue cell tumors such as pulmonary small cell carcinoma or lymphoblastic leukemia/lymphoma. Immunohistochemistry is therefore paramount to ensuring accurate diagnostic distinction between these tumors. The aim of our study was to evaluate and compare the expression of PAX5 and Terminal deoxynucleotidyl transferase (TdT), in Merkel cell carcinoma and pulmonary small cell carcinoma. Design: PAX5 and TdT immunohistochemical stains were performed on 27 Merkel cell carcinomas and 10 pulmonary small cell carcinomas. Results: PAX5 was expressed in 24/27 (89%) Merkel cell carcinomas and 0/10 (0%) pulmonary small cell carcinomas. TdT was expressed in 21/27 (78%) Merkel cell carcinomas and 9/10 (90%) pulmonary small cell carcinomas. Conclusions: Our study confirms that PAX5 and TdT expression can be expressed in a high percentage of Merkel cell carcinomas and so when positive are not diagnostic of lymphoblastic leukemia/lymphoma. When dealing with metastatic lesions, PAX5 negativity would favor a diagnosis of pulmonary small cell carcinoma over Merkel cell carcinoma. In addition, TTF-1 negative pulmonary small cell carcinoma is to be differentiated from Merkel cell carcinoma.
Merkel cell carcinoma; PAX5; TdT; small cell carcinoma; lung
Human polyomaviruses are known to cause persistent or latent infections, which are reactivated under immunosuppression. Polyomaviruses have been found to immortalize cell lines and to possess oncogenic properties. Moreover, the recently discovered Merkel cell polyomavirus shows a strong association with human Merkel cell carcinomas. Another novel human polyomavirus, WU polyomavirus (WUPyV), has been identified in respiratory specimens from patients with acute respiratory tract infections (ARTI). WUPyV has been proposed to be a pathogen in ARTI in early life and immunocompromised individuals, but so far its role as a causative agent of respiratory disease remains controversial.
The objective of our study was to determine the prevalence of WUPyV infections in adult hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) and to establish its potential clinical relevance by comparison to patients with stable COPD hospitalized for other reasons than acute exacerbation of COPD (AE-COPD).
A total of 378 respiratory specimens, each 189 induced sputum and nasal lavage samples from 189 patients, who had been recruited in a prospective 2:1 ratio case-control set-up between 1999 and 2003, were evaluated for the presence of WUPyV DNA by real-time PCR.
In the present study we could not detect WUPyV DNA in 378 respiratory specimens from 189 adult hospitalized patients with AE-COPD and stable COPD in four consecutive years.
Persistence of viral replication or reactivation of latent WUPyV infection did not occur. WUPyV may not play a major role in adult immunocompetent patients with AE-COPD and stable COPD.
Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus.
Merkel cell carcinoma; lymphocyte; polyomavirus; immunohistochemistry; confocal microscopy