Merkel cell polyomavirus small tumor antigen contributes to immune evasion by interfering with type I interferon signaling.

Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment.

The prognostic value of the Merkel cell polyomavirus serum antibody test: A dual institutional observational study.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive cancer with often poor outcomes. Limited biomarkers exist for predicting clinical outcomes. The Merkel cell polyomavirus (MCPyV) serum antibody test (AMERK) has shown potential for indicating better recurrence-free survival in a single-institution study. The study aimed to evaluate the link between initial AMERK serostatus and survival. Secondary objectives included examining the relationship between initial AMERK titer levels and tumor burden. METHODS: A retrospective cohort study across two institutions analyzed patients tested with AMERK within 90 days of MCC diagnosis. Regression models assessed the association of survival outcomes with serostatus, considering various factors. The relationship between AMERK titer and tumor burden indicators was evaluated using ANOVA. Significance testing was exploratory, without a fixed significance level. RESULTS: Of 261 MCC patients tested, 49.4% were initially seropositive (titer ≥75). Multivariable analysis showed that seropositivity improved recurrence, event-free, overall, and MCC-specific survival rates. Strong associations were found between initial AMERK titer and clinical, tumor, and nodal stages, tumor size, and disease extent. Notably, improved survival with seropositivity was observed only in patients with localized disease at initial presentation. CONCLUSION: Circulating antibodies to MCPyV oncoproteins, as indicated by the AMERK test, are linked with better survival in MCC patients with localized disease at presentation. This could enhance patient risk profiling and treatment personalization. The study's retrospective nature and exploratory analysis are key limitations. PLAIN LANGUAGE SUMMARY: Merkel cell carcinoma (MCC) is a potentially aggressive skin cancer, and tools to predict patient outcomes are limited. A blood test called anti-Merkel cell panel (AMERK), which checks for specific antibodies related to this cancer, might give us some clues. In this study, we looked at 261 MCC patients who took the AMERK test within 90 days of diagnosis. We found that patients with an initial positive AMERK result tended to have better outcomes, especially if their cancer was in the early stages. However, it is important to note that this study has limitations, including using retrospective data and exploratory analyses.

Characterization of the Impact of Merkel Cell Polyomavirus-Induced Interferon Signaling on Viral Infection.

Merkel cell polyomavirus (MCPyV) has been associated with approximately 80% of Merkel cell carcinoma (MCC), an aggressive and increasingly incident skin cancer. The link between host innate immunity, viral load control, and carcinogenesis has been established but poorly characterized. We previously established the importance of the STING and NF-κB pathways in the host innate immune response to viral infection. In this study, we further discovered that MCPyV infection of human dermal fibroblasts (HDFs) induces the expression of type I and III interferons (IFNs), which in turn stimulate robust expression of IFN-stimulated genes (ISGs). Blocking type I IFN downstream signaling using an IFN-ß antibody, JAK inhibitors, and CRISPR knockout of the receptor dramatically repressed MCPyV infection-induced ISG expression but did not significantly restore viral replication activities. These findings suggest that IFN-mediated induction of ISGs in response to MCPyV infection is not crucial to viral control. Instead, we found that type I IFN exerts a more direct effect on MCPyV infection postentry by repressing early viral transcription. We further demonstrated that growth factors normally upregulated in wounded or UV-irradiated human skin can significantly stimulate MCPyV gene expression and replication. Together, these data suggest that in healthy individuals, host antiviral responses, such as IFN production induced by viral activity, may restrict viral propagation to reduce MCPyV burden. Meanwhile, growth factors induced by skin abrasion or UV irradiation may stimulate infected dermal fibroblasts to promote MCPyV propagation. A delicate balance of these mutually antagonizing factors provides a mechanism to support persistent MCPyV infection. IMPORTANCE Merkel cell carcinoma is an aggressive skin cancer that is particularly lethal to immunocompromised individuals. Though rare, MCC incidence has increased significantly in recent years. There are no lasting and effective treatments for metastatic disease, highlighting the need for additional treatment and prevention strategies. By investigating how the host innate immune system interfaces with Merkel cell polyomavirus, the etiological agent of most of these cancers, our studies identified key factors necessary for viral control, as well as conditions that support viral propagation. These studies provide new insights for understanding how the virus balances the effects of the host immune defenses and of growth factor stimulation to achieve persistent infection. Since virus-positive MCC requires the expression of viral oncogenes to survive, our observation that type I IFN can repress viral oncogene transcription indicates that these cytokines could be explored as a viable therapeutic option for treating patients with virus-positive MCC.

Merkel Cell Polyomavirus Infection Induces an Antiviral Innate Immune Response in Human Dermal Fibroblasts.

Merkel cell polyomavirus (MCPyV) infects most of the human population asymptomatically, but in rare cases it leads to a highly aggressive skin cancer called Merkel cell carcinoma (MCC). MCC incidence is much higher in aging and immunocompromised populations. The epidemiology of MCC suggests that dysbiosis between the host immune response and the MCPyV infectious cycle could contribute to the development of MCPyV-associated MCC. Insufficient restriction of MCPyV by normal cellular processes, for example, could promote the incidental oncogenic MCPyV integration events and/or entry into the original cell of MCC. Progress toward understanding MCPyV biology has been hindered by its narrow cellular tropism. Our discovery that primary human dermal fibroblasts (HDFs) support MCPyV infection has made it possible to closely model cellular responses to different stages of the infectious cycle. The present study reveals that the onset of MCPyV replication and early gene expression induces an inflammatory cytokine and interferon-stimulated gene (ISG) response. The cGAS-STING pathway, in coordination with NF-κB, mediates induction of this innate immune gene expression program. Further, silencing of cGAS or NF-κB pathway factors led to elevated MCPyV replication. We also discovered that the PYHIN protein IFI16 localizes to MCPyV replication centers but does not contribute to the induction of ISGs. Instead, IFI16 upregulates inflammatory cytokines in response to MCPyV infection by an alternative mechanism. The work described herein establishes a foundation for exploring how changes to the skin microenvironment induced by aging or immunodeficiency might alter the fate of MCPyV and its host cell to encourage carcinogenesis. IMPORTANCE MCC has a high rate of mortality and an increasing incidence. Immune-checkpoint therapies have improved the prognosis of patients with metastatic MCC. Still, a significant proportion of the patients fail to respond to immune-checkpoint therapies or have a medical need for iatrogenic immune-suppression. A greater understanding of MCPyV biology could inform targeted therapies for MCPyV-associated MCC. Moreover, cellular events preceding MCC oncogenesis remain largely unknown. The present study aims to explore how MCPyV interfaces with innate immunity during its infectious cycle. We describe how MCPyV replication and/or transcription elicit an innate immune response via cGAS-STING, NF-κB, and IFI16. We also explore the effects of this response on MCPyV replication. Our findings illustrate how healthy cellular conditions may allow low-level infection that evades immune destruction until highly active replication is restricted by host responses. Conversely, pathological conditions could result in unbridled MCPyV replication that licenses MCC tumorigenesis.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and ß-TrCP.

Merkel cell polyomavirus (MCPyV) accounts for 80% of all Merkel cell carcinoma (MCC) cases through expression of two viral oncoproteins: the truncated large T antigen (LT-t) and small T antigen (ST). MCPyV ST is thought to be the main driver of cellular transformation and has also been shown to increase LT protein levels through the activity of its Large-T Stabilization Domain (LSD). The ST LSD was reported to bind and sequester several ubiquitin ligases, including Fbw7 and ß-TrCP, and thereby stabilize LT-t and several other Fbw7 targets including c-Myc and cyclin E. Therefore, the ST LSD is thought to contribute to transformation by promoting the accumulation of these oncoproteins. Targets of Fbw7 and ß-TrCP contain well-defined, conserved, phospho-degrons. However, as neither MCPyV LT, LT-t nor ST contain the canonical Fbw7 phospho-degron, we sought to further investigate the proposed model of ST stabilization of LT-t and transformation. In this study, we provide several lines of evidence that fail to support a specific interaction between MCPyV T antigens and Fbw7 or ß-TrCP by co-immunoprecipitation or functional consequence. Although MCPyV ST does indeed increase LT protein levels through its Large-T Stabilization domain (LSD), this is accomplished independently of Fbw7. Therefore, our study indicates a need for further investigation into the role and mechanism(s) of MCPyV T antigens in viral replication, latency, transformation, and tumorigenesis.

Merkel cell polyomavirus small tumour antigen activates the p38 MAPK pathway to enhance cellular motility.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with high rates of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases. MCPyV-induced tumourigenesis is largely dependent on the expression of the small tumour antigen (ST). Recent findings implicate MCPyV ST expression in the highly metastatic nature of MCC by promoting cell motility and migration, through differential expression of cellular proteins that lead to microtubule destabilisation, filopodium formation and breakdown of cell-cell junctions. However, the molecular mechanisms which dysregulate these cellular processes are yet to be fully elucidated. Here, we demonstrate that MCPyV ST expression activates p38 MAPK signalling to drive cell migration and motility. Notably, MCPyV ST-mediated p38 MAPK signalling occurs through MKK4, as opposed to the canonical MKK3/6 signalling pathway. In addition, our results indicate that an interaction between MCPyV ST and the cellular phospatase subunit PP4C is essential for its effect on p38 MAPK signalling. These results provide novel opportunities for the treatment of metastatic MCC given the intense interest in p38 MAPK inhibitors as therapeutic agents.

Identification of the Merkel Cell Polyomavirus Large Tumor Antigen Ubiquitin Conjugation Residue.

Merkel cell polyomavirus (MCPyV) large tumor (LT) antigen is a DNA binding protein essential for viral gene transcription and genome replication. MCPyV LT interacts with multiple E3 ligases in a phosphorylation-dependent manner, limiting its own viral replication by enhancing LT protein degradation, which is a unique mechanism for MCPyV latency. Thus, identifying LT ubiquitination sites is an important step toward understanding the biological role of these virus-host interactions that can potentially result in viral oncogenesis. The ubiquitin (Ub) attachment sites in LT were predicted by using Rapid UBIquitination (RUBI), a sequence-based ubiquitination web server. Using an immunoprecipitation approach, the lysine (Lys, K) 585 residue in LT is identified as the ubiquitin conjugation site. Lysine 585 is deleted from tumor-derived truncated LTs (tLTs), resulting in stable expression of tLTs present in cancers. Substitution of lysine 585 to arginine (Arg, R) increased LT protein stability, but impaired MCPyV origin replication, due to a loss of ATP hydrolysis activity. These findings uncover a never-before-identified ubiquitination site of LT and its importance not only in the regulation of protein turnover, but also in MCPyV genome replication.

Polyomavirus-driven Merkel cell carcinoma: Prospects for therapeutic vaccine development.

Great strides have been made in cancer immunotherapy including the breakthrough successes of anti-PD-(L)1 checkpoint inhibitors. In Merkel cell carcinoma (MCC), a rare and aggressive skin cancer, PD-(L)1 blockade is highly effective. Yet, ~50% of patients either do not respond to therapy or develop PD-(L)1 refractory disease and, thus, do not experience long-term benefit. For these patients, additional or combination therapies are needed to augment immune responses that target and eliminate cancer cells. Therapeutic vaccines targeting tumor-associated antigens, mutated self-antigens, or immunogenic viral oncoproteins are currently being developed to augment T-cell responses. Approximately 80% of MCC cases in the United States are driven by the ongoing expression of viral T-antigen (T-Ag) oncoproteins from genomically integrated Merkel cell polyomavirus (MCPyV). Since T-Ag elicits specific B- and T-cell immune responses in most persons with virus-positive MCC (VP-MCC), and ongoing T-Ag expression is required to drive VP-MCC cell proliferation, therapeutic vaccination with T-Ag is a rational potential component of immunotherapy. Failure of the endogenous T-cell response to clear VP-MCC (allowing clinically evident tumors to arise) implies that therapeutic vaccination will need to be potent ansd synergize with other mechanisms to enhance T-cell activity against tumor cells. Here, we review the relevant underlying biology of VP-MCC, potentially applicable therapeutic vaccine platforms, and antigen delivery formats. We also describe early successes in the field of therapeutic cancer vaccines and address several clinical scenarios in which VP-MCC patients could potentially benefit from a therapeutic vaccine.

Cellular sheddases are induced by Merkel cell polyomavirus small tumour antigen to mediate cell dissociation and invasiveness.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is recognised as the causative factor in the majority of MCC cases. The MCPyV small tumour antigen (ST) is considered to be the main viral transforming factor, however potential mechanisms linking ST expression to the highly metastatic nature of MCC are yet to be fully elucidated. Metastasis is a complex process, with several discrete steps required for the formation of secondary tumour sites. One essential trait that underpins the ability of cancer cells to metastasise is how they interact with adjoining tumour cells and the surrounding extracellular matrix. Here we demonstrate that MCPyV ST expression disrupts the integrity of cell-cell junctions, thereby enhancing cell dissociation and implicate the cellular sheddases, A disintegrin and metalloproteinase (ADAM) 10 and 17 proteins in this process. Inhibition of ADAM 10 and 17 activity reduced MCPyV ST-induced cell dissociation and motility, attributing their function as critical to the MCPyV-induced metastatic processes. Consistent with these data, we confirm that ADAM 10 and 17 are upregulated in MCPyV-positive primary MCC tumours. These novel findings implicate cellular sheddases as key host cell factors contributing to virus-mediated cellular transformation and metastasis. Notably, ADAM protein expression may be a novel biomarker of MCC prognosis and given the current interest in cellular sheddase inhibitors for cancer therapeutics, it highlights ADAM 10 and 17 activity as a novel opportunity for targeted interventions for disseminated MCC.

Antibody response to polyomavirus primary infection: high seroprevalence of Merkel cell polyomavirus and lymphoid tissue involvement.

Human polyomaviruses (HPyVs) asymptomatically infect the human population establishing latency in the host, and their seroprevalence can reach 90% in healthy adults. Few studies have focused on the pediatric population, and there are no reports regarding the seroprevalence of all the newly isolated HPyVs among Italian children. Therefore, we investigated the frequency of serum antibodies against 12 PyVs in 182 immunocompetent children from Northeast Italy, by means of a multiplex antibody detection system. Additionally, secondary lymphoid tissues were collected to analyze the presence of HPyV DNA sequences using a specific real-time PCRs or PCRs. Almost 100% of subjects were seropositive for at least one PyV. Seropositivity ranged from 3% for antibodies against simian virus 40 (SV40) in children from 0 to 3 years, to 91% for antibodies against WU polyomavirus (WUPyV) and HPyV10 in children from 8 to 17 years. The mean number of PyV for which children were seropositive increased with the increasing of age: 4 standard deviations (SD) 1.8 in the 0-3-year group, 5 (SD 1.9) in the 4-7-year group, and 6 (SD 2.2) in the 8-17-year group. JC polyomavirus (JCPyV) DNA was detected in 1% of the adenoids, WUPyV in 12% of the tonsils, and 28% of the adenoids, and Merkel cell polyomavirus (MCPyV) was present in 6 and 2% of the tonsils and adenoids, respectively. Our study gives new insights on the serological evidence of exposure to PyVs during childhood, and on their possible respiratory route of transmission.

A monoclonal antibody against SV40 large T antigen (PAb416) does not label Merkel cell carcinoma.

AIMS: Merkel cell carcinoma represents poorly differentiated neuroendocrine carcinoma of cutaneous origin. In most studies, the vast majority of Merkel cell carcinomas are Merkel cell polyomavirus (MCPyV)-associated. SV40 polyomavirus immunohistochemistry is typically used in the diagnosis of other polyomavirus-associated diseases, including tubulointerstitial nephritis and progressive multifocal leukoencephalopathy, given cross-reactivity with BK and JC polyomaviruses. MCPyV-specific immunohistochemistry is commercially available, but, if antibodies against SV40 also cross-reacted with MCPyV, that would be advantageous from a resource-utilisation perspective. METHODS AND RESULTS: Tissue microarrays were constructed from 39 Merkel cell carcinomas, 24 small-cell lung carcinomas, and 18 extrapulmonary visceral small-cell carcinomas. SV40 large T antigen immunohistochemistry (clone PAb416) was performed; MCPyV large T antigen immunohistochemistry (clone CM2B4) had been previously performed. UniProt was used to compare the amino acid sequences of the SV40, BK, JC and MCPyV large T antigens, focusing on areas recognised by the PAb416 and CM2B4 clones. SV40 immunohistochemistry was negative in all tumours; MCPyV immunohistochemistry was positive in 38% of Merkel cell carcinomas and in 0% of non-cutaneous poorly differentiated neuroendocrine carcinomas. UniProt analysis revealed a high degree of similarity between SV40, BK, and JC viruses in the region recognised by PAb416. There was less homology between SV40 and MCPyV in this region, which was also interrupted by two long stretches of amino acids unique to MCPyV. The CM2B4 clone recognises a unique epitope in one of these stretches. CONCLUSIONS: The PAb416 antibody against the SV40 large T antigen does not cross-react with MCPyV large T antigen, and thus does not label Merkel cell carcinoma.

Advances in Immunotherapy for Metastatic Merkel Cell Carcinoma: A Clinician's Guide.

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer. The clinical impact of MCC has been increasing due to steadily rising incidence rates. Since 2001, more than 24,000 cases of MCC have been reported to the US National Program of Cancer Registries database, and in 2018, more than 2,500 incident cases are expected. MCC is highly aggressive, and one-third of patients will either present with or develop metastatic disease. Outcomes in patients with metastatic MCC have historically been poor; median time to progression with cytotoxic chemotherapy is only 3 months. MCC has long been appreciated to be immunogenic, with reports of spontaneous regression and responsiveness to immunotherapy. However, the mechanisms of this immunogenicity have only been understood over the past decade, with approximately 80% of cases in the United States associated with the Merkel cell polyomavirus (MCPyV) and expression of viral antigens (virus-positive [VP] MCC), and the remaining 20% of cases caused by UV radiation-induced damage leading to a high mutational burden and expression of neoantigens (virus-negative [VN] MCC). These insights have led to multiple successful trials of immunotherapies for MCC. PD-1 axis checkpoint inhibitors are now regarded as the preferred frontline systemic therapy in eligible patients (including both VP- and VN-MCC), with impressive frequency, durability, and depth of objective responses, which compare favorably to those of most solid tumors. This article reviews the safety and efficacy data from the key clinical trials of immune checkpoint inhibitors for metastatic MCC, and discusses several issues relevant to the clinical use of these agents. Finally, emerging immunotherapies for MCC, including cellular therapies and adjuvant systemic therapies, are reviewed.

Merkel cell polyomavirus IgG antibody levels are associated with progression to AIDS among HIV-infected individuals.

The association of Merkel cell polyomavirus (MCPyV) with Merkel cell carcinoma (MCC) in immunocompromised individuals has been revealed in a number of surveys. The study of MCPyV specific antibody titers and viral loads in such patients has a great attraction for research groups interested in viral reactivation. In this cross-sectional study to evaluate MCPyV antibody titer, DNA prevalence and viral load in peripheral blood mononuclear cells (PBMCs), we examined 205 HIV-1 infected patients and 100 un-infected controls. The HIV-1 infected patients divided into two groups (HIV/AIDS and non-AIDS) according to their CD4 status. Total IgG antibody titer against MCPyV was analyzed by virus like particle (VLP)-based enzyme linked immunosorbent assay (ELISA). Presence of MCPyV-DNA in subject's PBMCs was examined by quantitative real-time PCR assay. Levels of anti-MCPyV IgG in HIV/AIDS patients were significantly higher than those in non-AIDS HIV-infected and control subjects (p value = <0.001). The prevalence rate of MCPyV-DNA in PBMCs of HIV/AIDS, non-AIDS HIV-infected and un-infected controls were 17%, 16%, and 14% respectively. The MCPyV viral load among the groups ranged between 0.15 to 2.9 copies/103cells (median, 1.9 copies/103cells), with no significant difference between the studied populations (p value = 0.3).

Serine 220 phosphorylation of the Merkel cell polyomavirus large T antigen crucially supports growth of Merkel cell carcinoma cells.

Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT(S220A) demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches.

Prognostic value of antibodies to Merkel cell polyomavirus T antigens and VP1 protein in patients with Merkel cell carcinoma.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the main aetiological agent of Merkel cell carcinoma (MCC). Serum antibodies against the major MCPyV capsid protein (VP1) are detected in the general population, whereas antibodies against MCPyV oncoproteins (T antigens) have been reported specifically in patients with MCC. OBJECTIVES: The primary aim was to assess whether detection of serum antibodies against MCPyV proteins at baseline was associated with disease outcome in patients with MCC. The secondary aim was to establish whether evolution of these antibodies during follow-up was associated with the course of the disease. METHODS: Serum T-antigen and VP1 antibodies were assessed by enzyme-linked immunosorbent assay using recombinant proteins in a cohort of 143 patients with MCC, including 84 patients with serum samples available at baseline. RESULTS: Low titres of VP1 antibodies at baseline (< 10 000) were significantly and independently associated with increased risk of recurrence [hazard ratio (HR) 2·71, 95% confidence interval (CI) 1·13-6·53, P = 0·026] and death (HR 3·74, 95% CI 1·53-9·18, P = 0·004), whereas T-antigen antibodies were not found to be associated with outcome. VP1 antibodies did not differ between patients in remission and those with recurrence or progression during follow-up. However, T-antigen antibodies were more frequently detected in patients with recurrence or progression at 12 months (P = 0·020) and 24 months (P = 0·016) after diagnosis. CONCLUSIONS: VP1 antibodies constitute a prognostic marker at baseline, whereas T-antigen antibodies constitute a marker of disease recurrence or progression if detected > 12 months after diagnosis.

Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

Complete Spontaneous Regression of Merkel Cell Carcinoma After Biopsy: A Case Report and Review of the Literature.

Merkel cell carcinoma (MCC) is a rare primary cutaneous neuroendocrine tumor that typically occurs on the head and neck of the elderly and follows an aggressive clinical course. Merkel cell polyomavirus (MCPyV) has been identified in up to 80% of cases and has been shown to participate in MCC tumorigenesis. Complete spontaneous regression of MCC has been rarely reported in the literature. We describe a case of a 79-year-old man that presented with a rapidly growing, 3-cm mass on the left jaw. An incisional biopsy revealed MCC. Additional health issues were discovered in the preoperative workup of this patient which delayed treatment. One month after the biopsy, the lesion showed clinical regression in the absence of treatment. Wide excision of the biopsy site with sentinel lymph node dissection revealed no evidence of MCC 2 months later. The tumor cells in the patient's biopsy specimen were negative for MCPyV by polymerase chain reaction and immunohistochemistry (CM2B4 antibody, Santa Cruz, CA). The exact mechanism for complete spontaneous regression in MCC is unknown. To our knowledge, only 2 previous studies evaluated the presence of MCPyV by polymerase chain reaction in MCC with spontaneous regression. Whether the presence or absence of MCPyV correlates with spontaneous regression warrants further investigation.

Merkel cell polyomavirus T antigens promote cell proliferation and inflammatory cytokine gene expression.

Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T-antigen locus that result in deletions of C-terminal replicative functions of the LT antigen. Despite these truncations, the LT LXCXE retinoblastoma (Rb) pocket protein family binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication and genes involved in cellular movement. The LXCXE mutant LT largely reversed gene expression alterations detected with the WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.

Seroreactivity against Merkel cell polyomavirus and other polyomaviruses in chronic lymphocytic leukaemia, the MCC-Spain study.

Merkel cell polyomavirus (MCPyV) has been suspected to cause chronic lymphocytic leukaemia (CLL) but previous data are inconsistent. We measured seroreactivities of nine polyomaviruses (MCPyV, BKPyV, JCPyV, LPyV, KIPyV, WUPyV, HPyV-6, HPyV-7 and TSPyV) in 359 CLL cases and 370 controls using bead-based multiplex serology technology. We additionally tested two herpesviruses (HSV-1 and CMV). Associations between disease and viral seroreactivities were assessed using logistic regression. All human viruses showed high seroprevalences (69-99%) against structural proteins in controls but significantly lower viral seroprevalences in cases (58-94%; OR range = 0.21-0.70, P value < 0.05), except for MCPyV (OR = 0.79, 95% CI = 0.54-1.16). Lower seroreactivity levels were observed among CLL subjects, with significant differences already observed at early stages of disease, unrelated to treatment status. Seroreactivities against polyomavirus related oncoproteins were almost null. Our data suggest no association for MCPyV polyomavirus with CLL development and an unlikely association for other polyomaviruses tested.