Merkel cell polyomavirus in Merkel cell carcinoma of Italian patients.
ABSTRACT: BACKGROUND: Merkel cell carcinoma (MCC) is a rare but very aggressive human malignancy of elderly or immunosuppressed patients. Clonal integration of a new human polyomavirus, the Merkel cell polyomavirus (MCPyV), has been reported in MCC patients. The main objective of the study was the detection of MCPyV and viral expression in clinical samples of Italian patients who were diagnosed MCC. FINDINGS: DNA and RNA were extracted from nine MCCs to detect the presence of MCPyV. Viral large T gene (LT1 and LT3), and viral capsid gene (VP1) were detected by polymerase chain reaction (PCR) based methods, and the amplified PCR products were subjected to direct sequencing. The presence of viral T antigen and/or viral capsid DNA sequences was demonstrated in eight of the nine MCC lesions, whereas RNA transcripts were detected in three MCCs. CONCLUSIONS: These findings indicate a potential role of MCPyV in the pathogenesis of at least a subset of MCCs.
Project description:INTRODUCTION:Merkel cell carcinoma (MCC) is a rare and highly aggressive malignancy of the skin which occurs mainly in old people and is very uncommon in young individuals. A new tumor virus belonging to the Polyomaviridae family; Merkel Cell Polyomavirus (MCPyV) has recently been identified in more than 80% of MCCs. CASE PRESENTATION:We conducted a retrospective review on the archives of the Department of Pathology; Imam Khomeini Hospital Cancer Institute affiliated to Tehran University of Medical Sciences to confirm the MCC samples and we found medical records and samples of a young case with MCC who developed leg skin and scalp tumor six and seven years after bone marrow transplantation, respectively. We analyzed patient formalin-fixed paraffin-embedded samples for the presence of MCPyV DNA using polymerase chain reaction (PCR) method, and the PCR amplicons were subjected to DNA sequencing. Merkel Cell Polyomavirus DNA was detected in both tumors from patient and sequence analysis of the viral LT3 region showed a close homology to strains circulating worldwide. CONCLUSIONS:The findings of this study are consistent with the hypothesis that local, systemic, or tumor-induced immunosuppression may allow the MCPyV to initiate skin aggressive cancer. It is necessary to maintain regular check over patients taking immunosuppressive medications for MCPyV infection. Since there is not any information about detection and molecular biology analysis of MCPyV among Iranian patients with MCC, this study provides more information about MCC and MCPyV in Iran.
Project description:Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor with high mortality rates. Merkel cell polyomavirus (MCPyV), identified in the majority of MCC, may drive tumorigenesis via viral T antigens. However, mechanisms underlying pathogenesis in MCPyV-negative MCC remain poorly understood. To nominate genes contributing to pathogenesis of MCPyV-negative MCC, we performed DNA microarray analysis on 30 MCCs. MCPyV status of MCCs was determined by PCR for viral DNA and RNA. 1593 probe-sets were differentially expressed between MCPyV-negative and -positive MCC, with significant differential expression defined as at least 2-fold change in either direction and p-value of ≤ 0.05. MCPyV-negative tumors showed decreased RB1 expression, whereas MCPyV-positive tumors were enriched for immune response genes. Validation studies included immunohistochemistry demonstration of decreased RB protein expression in MCPyV-negative tumors and increased peritumoral CD8+ T lymphocytes surrounding MCPyV-positive tumors. In conclusion, our data suggest that loss of RB1 expression may play an important role in tumorigenesis of MCPyV-negative MCC. Functional and clinical validation studies are needed to determine whether this tumor suppressor pathway represents an avenue for targeted therapy. We used microarrays to characterize global gene expression patterns related to Merkel cell polyomavirus status in Merkel cell carcinoma. Furthermore, we compared Merkel cell carcinoma to less aggressive primary cutaneous carcinomas. We utilized flash-frozen tumor tissue from primary Merkel cell carcinomas, metastatic Merkel cell carcinomas, primary cutaneous squamous cell carcinomas, and basal cell carcinomas. Merkel cell carcinoma cell lines, which represent a pure population of tumor cells, were also included. Merkel cell polyomavirus status was determined at the DNA and RNA level using multiple primers for viral T-antigen and capsid protein sequences. This Series represents two analyses - one with new Samples normalized together, and another with some of the new Samples re-normalized with Samples previously submitted under Series GSE13355. The latter group contain 'renormalized' in the titles.
Project description:BACKGROUND: Prokineticin-1 (PROK1) and prokineticin-2 (PROK2) are chemokine-like proteins that may influence cancer growth by regulating host defence and angiogenesis. Their significance in viral infection-associated cancer is incompletely understood. We studied prokineticins in Merkel cell carcinoma (MCC), a skin cancer linked with Merkel cell polyomavirus (MCPyV) infection. METHODS: Carcinoma cell expression of PROK1 and PROK2 and their receptors (PROKR1 and PROKR2) was investigated with immunohistochemistry, and tumour PROK1 and PROK2 mRNA content with quantitative PCR from 98 MCCs. Subsets of tumour infiltrating leukocytes were identified using immunohistochemistry. RESULTS: Merkel cell polyomavirus-positive MCCs had higher than the median PROK2 mRNA content, whereas MCPyV-negative MCCs contained frequently PROK1 mRNA. Cancers with high tumour PROK2 mRNA content had high counts of tumour infiltrating macrophages (CD68+ and CD163+ cells). Patients with higher than the median PROK2 mRNA content had 44.9% 5-year survival compared with 23.5% among those with a smaller content (hazard ratio (HR): 0.53; 95% confidence interval (CI): 0.34-0.84; P=0.005), whereas the presence of PROK1 mRNA in tumour was associated with unfavourable survival (P=0.052). CONCLUSIONS: The results suggest that prokineticins are associated with MCPyV infection and participate in regulation of the immune response in MCC, and may influence outcome of MCC patients.
Project description:Merkel cell polyomavirus (MCPyV) is present in approximately 80% of human Merkel cell carcinomas (MCCs). A previous in silico prediction suggested MCPyV encodes a microRNA (miRNA) that may regulate cellular and viral genes.To determine the presence and prevalence of a putative MCPyV-encoded miRNA in human MCC tumors.Over 30 million small RNAs from 7 cryopreserved MCC tumors and 1 perilesional sample were sequenced. 45 additional MCC tumors were examined for expression of an MCPyV-encoded mature miRNA by reverse transcription real-time PCR.An MCPyV-encoded mature miRNA, "MCV-miR-M1-5p", was detected by direct sequencing in 2 of 3 MCPyV-positive MCC tumors. Although a precursor miRNA, MCV-miR-M1, had been predicted in silico and studied in vitro by Seo et al., no MCPyV-encoded miRNAs have been directly detected in human tissues. Importantly, the mature sequence of MCV-miR-M1 found in vivo was identical in all 79 reads obtained but differed from the in silico predicted mature miRNA by a 2-nucleotide shift, resulting in a distinct seed region and a different set of predicted target genes. This mature miRNA was detected by real-time PCR in 50% of MCPyV-positive MCCs (n = 38) and in 0% of MCPyV-negative MCCs (n = 13).MCV-miR-M1-5p is expressed at low levels in 50% of MCPyV-positive MCCs. This virus-encoded miRNA is predicted to target genes that may play a role in promoting immune evasion and regulating viral DNA replication.
Project description:Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor with high mortality rates. Merkel cell polyomavirus (MCPyV), identified in the majority of MCCs, may drive tumorigenesis via viral T antigens. However, the mechanisms underlying pathogenesis in MCPyV-negative MCCs remain poorly understood. To nominate genes contributing to the pathogenesis of MCPyV-negative MCCs, we performed DNA microarray analysis on 30 MCCs. The MCPyV status of MCCs was determined by PCR for viral DNA and RNA. A total of 1,593 probe sets were differentially expressed between MCPyV-negative and MCPyV-positive MCCs, with significant differential expression defined as at least a 2-fold change in either direction and a P-value ?0.05. MCPyV-negative tumors showed decreased RB1 expression, whereas MCPyV-positive tumors were enriched for immune response genes. Validation studies included immunohistochemistry demonstration of decreased RB protein expression in MCPyV-negative tumors and increased peritumoral CD8+ T lymphocytes surrounding MCPyV-positive tumors. In conclusion, our data suggest that loss of RB1 expression may have an important role in the tumorigenesis of MCPyV-negative MCCs. Functional and clinical validation studies are needed to determine whether this tumor-suppressor pathway represents an avenue for targeted therapy.
Project description:Merkel cell carcinoma (MCC) is a rare but often deadly skin cancer that is typically caused by the Merkel cell polyomavirus (MCPyV). Polyomavirus T-antigen oncoproteins are persistently expressed in virus-positive MCCs (˜80% of cases), while remarkably high numbers of tumor-associated neoantigens are detected in virus-negative MCCs, suggesting that both MCC subsets may be immunogenic. Here we review mechanisms by which these immunogenic tumors evade multiple levels of host immunity. Additionally, we summarize the exciting potential of diverse immune-based approaches to treat MCC. In particular, agents blocking the PD-1 axis have yielded strikingly high response rates in MCC as compared with other solid tumors, highlighting the potential for immune-mediated treatment of this disease.
Project description:Previous studies have reached conflicting conclusions regarding the proportion of Merkel cell carcinomas (MCCs) that contain the Merkel cell polyomavirus (MCPyV) and the clinical significance of tumor viral status. To address these controversies, we detected MCPyV large T antigen using immunohistochemistry with two distinct antibodies and MCPyV DNA using quantitative PCR. Tumors were called MCPyV-positive if two or more of these three assays indicated presence of this virus. A total of 53 of 282 (19%) MCC tumors in this cohort were virus-negative using this multimodal system. Immunohistochemistry with the CM2B4 antibody had the best overall performance (sensitivity = 0.882, specificity = 0.943) compared with the multimodal classification. Multivariate analysis including age, sex, and immunosuppression showed that, relative to MCC patients with virus-positive tumors, virus-negative MCC patients had significantly increased risk of disease progression (hazard ratio = 1.77, 95% confidence interval = 1.20-2.62) and death from MCC (hazard ratio = 1.85, 95% confidence interval = 1.19-2.89). We confirm that approximately 20% of MCCs are not driven by MCPyV and that such virus-negative MCCs, which can be quite reliably identified by immunohistochemistry using the CM2B4 antibody alone, represent a more aggressive subtype that warrants closer clinical follow-up.
Project description:Metastatic Merkel cell carcinoma (MCC) is a lethal, Merkel cell polyomavirus (MCPyV) cancer with no currently available effective therapy. Harnessing the immune system through an immune checkpoint blockade is an attractive option because the immune system appears to be dysfunctional in the Merkel cell tumor microenvironment. Although MCPyV is expressed in 80% of MCCs and serves as a powerful antigen for stimulating host immune response, intratumoral CD8+ T-cell infiltration is seen only in 18% of MCCs. In contrast, about 50% of MCPyV-positive MCCs express the programmed death-ligand 1 (PD-L1) on multiple cell types in the tumor microenvironment. We present a case of a young patient with MCC involving the heart and pancreas that showed an impressive response after treatment with four cycles of the anti-PD-1 monoclonal antibody, nivolumab.
Project description:BACKGROUND: A new virus called the Merkel Cell Polyomavirus (MCPyV) has recently been found in Merkel Cell Carcinoma (MCC). MCC is a rare aggressive small cell neuroendocrine carcinoma primarily derived from the skin, morphologically indistinguishable from small cell lung carcinoma (SCLC). So far the actual presence of the virus in MCC tumour cells on a morphological level has not been demonstrated, and the presence of MCPyV in other small cell neuroendocrine carcinomas has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: We investigated MCC tissue samples from five patients and SCLCs from ten patients for the presence of MCPyV-DNA by PCR and sequencing. Electron microscopy was used to search ultrastructurally for morphological presence of the virus in MCPyV-DNA positive samples. MCPyV was detected in two out of five primary MCCs. In one MCC patient MCPyV-DNA was detected in the primary tumour as well as in the metastasis, strongly suggesting integration of MCPyV in the cellular DNA of the tumour in this patient. In the primary MCC of another patient viral particles in tumour cell nuclei and cytoplasm were identified by electron microscopy, indicating active viral replication in the tumour cells. In none of the SCLCs MCPyV-DNA was detected. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that MCPyV is an oncogenic polyomavirus in humans, and is potentially causally related to the development of MCC but not to the morphological similar SCLC.