Presence Of Merkel Cell Polyomavirus Research Articles

Is the presence of Merkel cell polyomavirus and humanpapillomavirus DNA in keratinocyte cancers andprecancers associated with HIV status? A case-control study.

The epidemiology and potential pathogenic roles of human papillomavirus (HPV) and Merkel cell polyomavirus (MCV) in keratinocyte cancers (KCs) arising in people living with HIV (PLWH) compared with HIV-negative individuals are poorly understood. These issues were investigated by a case-control study in which the presence of MCV and HPV DNA was identified by polymerase chain reaction in microdissected formalin-fixed paraffin-embedded tissue from PLWH and HIV-negative individuals. The samples comprised 190 cutaneous and genital KCs/precancers (actinic keratoses, n = 43; cutaneous squamous cell carcinoma (cSCC) in situ, n = 24; basal cell carcinoma, n = 78; cSCC, n = 34; penile carcinoma in situ, n = 9; penile SCC, n = 2 from 104 individuals (PLWH, n = 51; HIV-negative, n = 53). Almost one-quarter of samples were positive for MCV: this was not significantly associated with either HIV status (P = 0.06) nor lesion type. Overall, 36% (16/44) of MCV-positive lesions were coinfected with HPV; this was also not associated with HIV status. These findings indicate that if these viruses do contribute to the pathogenesis of KCs, it is likely to be independent of HIV status.

Merkel cell polyomavirus and its etiological relationship with skin tumors

Viruses have been frequently identified in several human neoplasms, but the etiological role of these viruses in some tumors is still a matter of controversy. Polyomaviruses stand out among the main viruses with oncogenic capacity, specifically the Merkel cell polyomavirus (MCPyV). Recent revisions in the taxonomy of polyomaviruses have divided the Polyomaviridae family into six genera, including 117 species, with a total of 14 currently known human-infecting species. Although the oncogenicity of polyomaviruses has been widely reported in the literature since 1950, the first description of a polyomavirus as an etiological agent of a neoplasm in humans was made only in 2008 with the description of MCPyV, present in approximately 80% of cases of Merkel cell carcinoma (MCC), with the integration of its genome to that of the tumor cells and tumor-specific mutations, and it is considered the etiological agent of this neoplasm since then. MCPyV has also been detected in keratinocyte carcinomas, such as basal cell carcinoma and squamous cell carcinoma of the skin in individuals with and without immunosuppression. Data on the occurrence of oncogenic viruses potentially involved in oncogenesis, which cause persistence and tissue injury, related to the Merkel cell polyomavirus are still scarce, and the hypothesis that the Merkel cell polyomavirus may play a relevant role in the genesis of other cutaneous carcinomas in addition to MCC remains debatable. Therefore, the present study proposes to explore the current knowledge about the presence of MCPyV in keratinocyte carcinomas.

Prevalence of Merkel Cell Polyomavirus in Primary Eyelid Merkel Cell Carcinomas and Association With Clinicopathological Features

Merkel cell polyomavirus (MCPyV) infection is a known to be a critical risk factor for the development of Merkel cell carcinoma (MCC). Various reports on cutaneous MCC have shown that the differences in clinicohistopathological characteristics depend on the presence of MCPyV, but the situation in eyelid MCC is unknown. This study aimed to assess the prevalence of MCPyV in patients with eyelid MCC and examine the clinicohistopathological characteristics of MCPyV-associated eyelid MCC. Retrospective observational case series with laboratory investigations. Ten patients treated for eyelid MCC were included. Histopathological characteristics were examined by immunohistochemical staining using 12 antibodies. MCPyV infection was evaluated by PCR using primer sets targeting large T antigens of the MCPyV genome and by immunohistochemical staining using CM2B4 and Ab3 monoclonal antibodies. The MCPyV viral load was also quantified by PCR using 3 primer sets. All patients (4 males and 6 females) were Japanese with mean age of 79 (range: 63 to 87) years. One patient died due to distant metastasis 8 months after surgery for MCC. Immunohistochemical studies showed typical MCC findings in all cases, including CK20 and neuroendocrine marker positivity. PCR and immunohistochemistry with CM2B4 and Ab3 detected MCPyV antigen in all tumors. Quantitative PCR using sT, LT4, and TAg primers yielded 0.94, 1.72, and 1.05 copies per cell, respectively. Clinical and histopathological characteristics of 10 patients with eyelid MCC were elucidated. MCPyV infection was detected in all eyelids. These results provide insight for understanding the tumorigenesis of eyelid MCC.

Evidencing the presence of merkel cell polyomavirus in papillary thyroid cancer

Merkel cell polyomavirus (MCPyV) infects most people asymptomatically, but recent reports indicate that the virus may be related to carcinogenesis. This study aimed to evaluate the impact of MCPyV on the development of papillary thyroid cancer (PTC). Totally, 1057 samples, including 412 fresh biopsy samples (FBS) and 645 paraffin-embedded PTC biopsy samples (PEBS), and 1057 adjacent non-cancerous samples were assessed for the presence of MCPyV DNA and RNA. MCPyV DNA was positive in 215 (20.3%) of samples, including 126 (30.6%) in FBS and 89 (13.8%) in PEBS. In MCPyV-positive samples, the mean MCPyV copy number was higher in the patients with FBS (2.3 × 10–1 ± 0.5 × 10–1 copies/cell) compared to PEBS (0.7 × 10–4 ± 0.1 × 10–4 copies/cell) and adjacent non-PTC normal samples (0.3 × 10–5 ± 0.02 × 10–5 copies/cell), indicating a statistically significant difference (P < 0.001). The LT-Ag RNA expression was higher in FBS compared to PEBS, while VP1 gene transcript was not detected in any samples. Although our findings showed the presence of MCPyV in a subset of PTC Iranian patients, further research is required to confirm these findings.

From Merkel Cell Polyomavirus Infection to Merkel Cell Carcinoma Oncogenesis.

Merkel cell polyomavirus (MCPyV) infection causes near-ubiquitous, asymptomatic infection in the skin, but occasionally leads to an aggressive skin cancer called Merkel cell carcinoma (MCC). Epidemiological evidence suggests that poorly controlled MCPyV infection may be a precursor to MCPyV-associated MCC. Clearer understanding of host responses that normally control MCPyV infection could inform prophylactic measures in at-risk groups. Similarly, the presence of MCPyV in most MCCs could imbue them with vulnerabilities that-if better characterized-could yield targeted intervention solutions for metastatic MCC cases. In this review, we discuss recent developments in elucidating the interplay between host cells and MCPyV within the context of viral infection and MCC oncogenesis. We also propose a model in which insufficient restriction of MCPyV infection in aging and chronically UV-damaged skin causes unbridled viral replication that licenses MCC tumorigenesis.

Neuroendocrine small cell carcinoma of the cervix: A case report.

Merkel cell polyomavirus (MCPyV) has been found in patients with Merkel cell carcinoma and respiratory tract infections. Merkel cell carcinoma is a primary aggressive neuroendocrine carcinoma of the skin. It has been demonstrated that MCPyV can be transmitted during sexual activity and may be present in the oral and anogenital mucosa. The aim of the present study was to evaluate whether MCPyV coexisted with HPV in three cases of neuroendocrine small cell carcinoma of the cervix using PCR and immunohistochemical analysis Three cases of NSC of the cervix were identified in the pathology archives of Parma University (Italy). Of these, two cases were associated with an adenocarcinomatous component. A set of general primers from the L1 region (forward, L1C1 and reverse, L1C2 or L1C2M) was PCR amplified to detect the broad-spectrum DNA of genital HPV. The presence of MCPyV was investigated via immunohistochemistry using a mouse monoclonal antibody against the MCPyV LT antigen and through PCR analysis to separate viral DNA. HPV DNA was present in all three neuroendocrine carcinomas and in the adenocarcinoma component of the two mixed cases. None of the cases were immunoreactive to CM2B4 and did not contain viral DNA in either their neuroendocrine or adenocarcinomatous component. Whilst it is difficult to draw definitive conclusions from such a small sample size, these data suggested that MCPyV does not coexist with HPV in the cervix. However, in the present study, the absence of detectable MCPyV may have been due to the presence of a genotype that was not detected by the primers used in the PCR analysis or by the antibody used for the immunohistochemical study. MCPyV microRNA may also have been present, inhibiting LT expression. Additional studies with larger cohorts and more advanced molecular biology techniques are required to confirm the hypothesis of the current study.

Prognostic and predictive value of EZH2 expression and the tumor immune microenvironment in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and lethal type ofskin cancer characterized by frequent recurrences and metastases. In view ofthe lack ofa proven treatment option for MCC, we immunochemically evaluated the presence ofMerkel cell polyomavirus (MCPyV), PD-1, PD-L1, CD8, and EZH2 on slides prepared from tumor tissues of13 patients with MCC, and examined their association with disease progression and overall survival. PD-1 was expressed on tumor infiltrating lymphocytes (TILs) in 92.3% ofthe patients. None ofthe tumor cells expressed PD-L1. CD8 levels were higher in MCPyV-positive tumors. Interestingly, higher CD8 levels correlated with better overall survival (p=0.025), while higher EZH2 expression correlated with metastasis/recurrence (z=-1.396, p=0.089). However, low EZH2 expression was associated with poor overall survival (χ2=3.745, Cramer V=0.537, p=0.086). These findings suggest that EZH2 plays a significant role in MCC and may be a promising therapeutic target.

The presence of Merkel cell carcinoma polyomavirus is associated with a distinct phenotype in neoplastic Merkel cell carcinoma cells and their tissue microenvironment.

AimsMerkel cell carcinoma (MCC) is an aggressive primary neuroendocrine tumor of the skin, associated with Merkel cell polyomavirus (MCPyV) in 49–89% of cases, depending on the country of origin and the techniques of detection. The presence of MCPyV defines heterogeneity in MCC; MCPyV-negative cases bear a much higher mutational load, with a distinct ultraviolet signature pattern featuring C > T transitions, as a consequence of exposure to ultraviolet light radiation. MCC stroma has not been thoroughly studied, although MCC patients benefit from therapy targeting PD1/PDL1.Methods and resultsIn this study, using Tissue Microarrays and immunohistochemistry, we have analyzed a series of 219 MCC cases in relation to the presence of MCPyV, and confirmed that the presence of MCPyV is associated with changes not only in the neoplastic cells, but also in the composition of the tumor stroma. Thus, MCPyV, found in 101/176 (57,4%) analyzable cases, exhibits changes in its tumor morphology, the density of the inflammatory infiltrate, the phenotype of the neoplastic cells, and the cell composition of the tumor stroma. MCPyV presence is negatively correlated with a higher level of p53 expression, and associated with a very high frequency (86%) of HLA-I expression loss, a higher apoptotic index, and a stroma richer in T-cells, cytotoxic T-cells, macrophages, PDL1-positive macrophages, and B-cells.ConclusionsOur findings provide evidence of the basic heterogeneity of MCC, supporting the hypothesis that the presence of MCPyV may induce a rich inflammatory response, which is at least partially avoided through loss of HLA-I antigen expression. On the other hand, MCPyV-negative cases show a much higher frequency of stronger p53 expression and, probably, p53 alterations.

Merkel cell polyomavirus detected in head and neck carcinomas from Chile

BackgroundThe role of human polyomaviruses (HPyVs) in epithelial tumors such as head and neck carcinomas (HNSCCs) including oral and oropharyngeal carcinomas has not been established. In this study, we evaluated for the first time the presence of Merkel cell polyomavirus (MCPyV), BK human polyomavirus (BKPyV), and JC human polyomavirus (JCPyV) in HNSCCs from Chilean subjects.MethodsOne hundred and twenty HNSCCs were analyzed for the presence of MCPyV, BKPyV and JCPyV using real-time polymerase chain reaction procedures. In addition, 54 oral brushes from age- and sex-paired subjects were analyzed.ResultsOf the total of 120 HNSCCs, 15 were positive for MCPyV (12.5%). Only one case was positive for BKPyV (0.8%) and none for JCPyV (0%). In subjects without cancer, only one case (1.8%) resulted positive for MCPyV and none for JCPyV and BKPyV. MCPyV was associated with HNSCCs (p = 0.0239; OR = 7.571; 95% CI: 1.192–81.46). No association was found between age (p = 0.1996), gender (p = 0.7111) or differentiation status (p > 0.9999) and MCPyV presence in HNSCCs.ConclusionsMCPyVs were detected in HNSCCs from Chilean patients and were not detected in oral brushes from patients without cancer. More studies are warranted for defining an etiological role and clinical/molecular consequences of these viruses in HNSCCs.

Merkel Cell Polyomavirus Gene Expression and Mutational Analysis of Large Tumor Antigen in Non-Merkel Cell Carcinoma Tumors of Iranian Patients

Introduction: The presence of Merkel cell polyomavirus (MCPyV) was identified in Merkel cell carcinoma (MCC). However, there was sparse information on the link of other common nonmelanoma skin cancers – basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) – to MCPyV infection. The current study describes the phylogenetic information of MCPyV isolated from Iranian non-MCC (nonmelanoma skin cancers) focusing on tumorigenesis of mutations in large tumor (LT) antigen (LT-Ag) fragment. Methods: Sixty patients with BCC and 20 patients with SCC were included in this study (48 males and 32 females; average age 65 years). The MCPyV-DNA copy number in positive samples was measured by quantitative real-time PCR. Then, mutational analysis of the MCPyV LT gene was carried out by direct sequencing. Results: While MCPyV DNA was detected in 6 (10%) of 60 BCC samples, no viral genome was found in SCCs. There was no distinct association of MCPyV positivity with gender, age, or type of tumor (BCC or SCC) (p value >0.05). Quantitative real-time PCR revealed that the median number of viral DNA copies per cell was 0.7 in 6 MCPyV-positive BCC samples. Furthermore, full-length LT-Ag sequencing of positive samples indicated no stop codon or frameshift mutations compared to reference sequences. Conclusion: Considering the important role of the LT-Ag in the pathogenicity of MCPyV, non-synonymous mutations compared with the reference proteins triggered relevant amino acid substitutions. Overall, the results showed no tumor-associated mutations in the LT-Ag sequence of MCPyVs from positive samples.

A Systematic review and Meta-Analysis of the survival and clinicopathological features of p63 expression in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare skin tumour of neuroendocrine origin with aggressive behaviour. The aims of this study were to investigate the association of p63+MCC with clinicopathological features and to estimate survival through a systematic review and meta-analysis. A comprehensive search of PubMed, Embase, Scopus and Virtual Health Library following the PRISMA guidelines was conducted on September 2017. DerSimonian and Lard random-effects models were used to calculate survival-weighted means and their corresponding 95% confidence intervals (CI) among studies. Five studies met our inclusion criteria after screening 77 citations and 36 full-text articles. The included studies enrolled 413 patients with MCC. We observed that p63+MCC was significantly associated with mortality with OR 2.92 (95% CI [1.66-5.13]). The summary hazard ratio of multivariate analysis was 1.99 (95% CI [1.32-3.01]). The only clinicopathological feature associated with p63+MCC with statistical significance was the Merkel cell polyomavirus (MCPyV) status. The presence of MCPyV was associated as a protective factor for the expression of p63 (OR 0.25, 95% CI [0.08-0.73]). These results support that p63+MCC evaluated by immunohistochemistry has a poor outcome. Therefore, we suggest p63 to be performed when staging MCC.

Merkel Cell Polyomavirus DNA Detection in Respiratory Samples: Study of a Cohort of Patients Affected by Cystic Fibrosis.

Background: The role of Merkel cell polyomavirus (MCPyV) as a respiratory pathogen is controversial, and it is still unclear in patients with cystic fibrosis (CF). The aim of this study was to define the MCPyV prevalence and epidemiology in CF patients in order to gain new insights into the association between MCPyV infection and respiratory diseases. Methods: A one-year study was conducted testing oropharyngeal aspirate samples from 249 and 124 CF and non-CF patients, respectively. Detection of MCPyV was carried out by nested polymerase chain reaction (PCR). Moreover, a sequence alignment to examine viral capsid protein 1 (VP1) and a phylogenetic analysis were performed. Results: MCPyV DNA was detected in 65 out of 249 samples analyzed CF (26%), a percentage that was higher than that recorded in non-CF patients (0.8%). There were no statistically significant differences in MCPyV prevalence according to gender, while there was a correlation between MCPyV detection and age. Interestingly, an association between the presence of MCPyV and the concurrent isolation of Staphylococcus aureus was found. Sequence analysis of MCPyV VP1 and phylogenetic analysis revealed a 99% homology with the published sequences of these viruses in GenBank. Conclusions: Detection of MCPyV in CF patient specimens pointed out a possible interaction between the virus and CF. Further studies are necessary to fully understand the involvement of MCPyV in the pathogenesis of respiratory disorders.

An open label, multicenter, phase II study of KRT-232, an oral small molecule inhibitor of MDM2, for the treatment of patients with Merkel cell carcinoma (MCC) who have failed treatment with anti-PD-1/L1 immunotherapy.

TPS9602 Background: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a poor prognosis in patients with advanced disease, with mortality as high as 46%. An oncogenic Merkel cell polyomavirus (MCPyV) is present in about 80% of MCC tumors, which inactivates tumor suppressors p53 and RB. Virus-positive MCC has proven to be an immunologically responsive disease and the recently approved PD-L1 inhibitor avelumab (March 2017) has improved the prognosis of patients with advanced MCC. However, despite the addition of immune checkpoint inhibitors to the treatment paradigm, a lack of response or disease progression occurs in up to two-thirds of avelumab patients previously treated with chemotherapy, and 32-44% of first-line patients treated with the PD-1 inhibitors nivolumab or pembrolizumab. KRT-232 is a potent and selective small molecule targeted drug that binds to murine double minute chromosome 2 (MDM2) and inhibits the MDM2/tumor protein 53 (p53) protein-protein interaction. Inactivating mutations in p53 have rarely been found in MCC, and the majority of MCCs with the presence of MCPyV express p53WT. The purpose of this study is to evaluate the tolerability and efficacy of KRT-232 in p53WT MCC patients who have been treated with anti-PD-1/L1 immunotherapy. Methods: The primary objective is ORR by RECIST v1.1 after 2 cycles (42 days) as determined by independent review. A Simon two-stage design is employed. In Stage 1, up to 13 eligible patients will be enrolled and treated with 240 mg KRT-232 once daily (QD) on Days 1-7 of a 21-day cycle. If the predesignated efficacy threshold is reached in Stage 1, enrollment for Stage 2 will include an additional 14 patients (for a total of 27 patients). The study is enrolling in the U.S. Clinical trial information: NCT03787602.

Investigation of three oncogenic epitheliotropic viruses shows human papillomavirus in association with non-melanoma skin cancer.

Non-melanoma skin cancers (NMSC) share similar risk factors with other virus-related cancers, despite the lack of proved causal association between viral infection and NMSC development. We investigated the presence of Merkel cell polyomavirus (MCPyV), Epstein-Barr virus (EBV), and human papillomavirus (HPV) DNA in 83 NMSC fresh-frozen and 16 non-cancerous skin biopsies and evaluated viral infection according to demographical data, histopathological diagnosis, and ultraviolet exposure. Our results showed that 75% of NMSC biopsies were positive for at least one out of three viruses, whereas only 38% of non-cancerous skin biopsies were positive (p = 0.02). Notably, HPV detection was frequent in NMSC (43%) and nearly absent (one sample, 6.7%) in non-cancerous biopsies (p = 0.007). MCPyV was associated with sites of higher exposure to ultraviolet radiation (p = 0.010), while EBV was associated with a compromised immune system (p = 0.032). Our study showed that HPV was strongly associated with NMSC while EBV and MCPyV with other risk factors. Though further studies are required to elucidate the role of viral infection in NMSC development and management, this study supports the possible role of oncogenic viruses in skin cancers, especially HPV.

Low prevalence of Merkel cell polyomavirus in human epithelial thymic tumors.

BackgroundThe etiology of thymic epithelial tumors is unknown. Murine polyomavirus strain PTA has been shown to induce thymomas in mice. Recently, using diverse molecular techniques, we reported the presence of human polyomavirus 7 (HPyV7) in thymic epithelial tumors. In the present study, we investigated the prevalence of Merkel cell polyomavirus (MCPyV) in thymic epithelial tumors.MethodsThirty‐six thymomas were screened for MCPyV by PCR and subsequently tested by DNA and RNA in situ hybridization and immunohistochemistry. Twenty‐six thymomas were diagnosed with myasthenia gravis (MG).ResultsMCPyV DNA was detected by PCR in 7 (19.4%) of the 36 thymic epithelial tumors and in six of these, the presence of MCPyV was confirmed by fluorescence situ hybridization. Of these, 3 (28.6%) revealed weak MCPyV LT‐antigen protein expression. In addition, one of the MCPyV positive thymomas tested positive for MCPyV LT RNA with RNAscope. Of interest, two out of the three thymomas that previously tested positive for MCPyV by immunohistochemistry also tested positive for HPyV7. One of the 11 MG‐negative and 2 of the 25 MG‐positive were positive for MCPyV.ConclusionsMCPyV DNA and MCPyV protein expression can be detected in human epithelial thymoma; however, to a far lesser extent than HPyV7. Our data strongly indicate that because of its infrequent detection and weak expression, MCPyV is unlikely to play an important role in the etiopathogenesis of human thymomas.

Detection of Merkel Cell Polyomavirus in Seborrheic Keratosis.

Seborrheic keratosis (SK) is the most common benign cutaneous neoplasm. A subset shows increased p16 expression. Since SK shares several features with verruca vulgaris, e.g., increased p16 expression, human papillomaviruses (HPV) have been suggested as possible causal agents. However, a relevant association could not be established between HPV and SK. In the present study we aimed to investigate the presence of Merkel cell polyomavirus (MCPyV) in relation to p16 expression in SK. P16 expression was investigated using immunohistochemistry (IHC). Presence of MCPyV was assessed in 23 formalin-fixed paraffin-embedded tissue samples of SK by molecular techniques (i.e., PCR and FISH) and IHC. 16/23 SK showed strong to moderate p16 expression. 6/23 of SK were MCPyV positive by PCR which was confirmed by FISH. Of interest, two samples with strong FISH signals also showed MCPyV expression as tested by IHC. Samples with weaker signal intensity were negative in IHC. P16 expression was not associated with the presence of MCPyV. Concluding, the detection of MCPyV DNA by PCR and FISH in SK reflects the widespread prevalence of MCPyV in the skin. However, low detection rates exclude MCPyV as a major pathogenic factor in SK, most likely representing a coincidental infection. P16 IHC does not appear as useful adjunctive surrogate marker for the presence of MCPyV in SK.

Differentiating Merkel cell carcinoma of lymph nodes without a detectable primary skin tumor from other metastatic neuroendocrine carcinomas: The ELECTHIP criteria

Merkel cell carcinoma (MCC) can present as a cutaneous tumor or a lymph node metastasis without a primary tumor. MCC presenting without a primary tumor (MCCWOPT) can be misinterpreted on histologic examination as lymph node metastasis (LNM) from another neuroendocrine carcinoma (LNMNEC). However, this distinction is crucial for therapeutic management. To determine the discriminative criteria for the differential diagnosis of MCCWOPT, LNM from cutaneous MCC, and LNMNECs. Clinical, morphologic, and immunohistochemical data (expression of cytokeratins AE1, AE3, 7, 19, and 20; chromogranin A, synaptophysin, thyroid transcription factor-1 [TTF-1]), as well as the presence of Merkel cell polyomavirus (by immunohistochemistry and PCR) were compared in patients with MCCWOPT (n=17), LNM from a cutaneous MCC (n=11), and LNMNEC (n=20; 8 lung, 7 thyroid, 3 digestive tract, 2 other). MCC (including MCCWOPT and LNM from a cutaneous MCC) differed from LNMNEC by 7 discriminative criteria: 1) elderly age, 2) location of the tumor, 3) extent of the disease, 4) cytokeratin expression, 5) TTF-1 expression, 6) histologic type, and 7) Merkel cell polyomavirus detection, summarized under the acronym ELECTHIP. All MCC patients had ≥5 of the ELECTHIP criteria, whereas all patients with LNMNEC (except 1) had <3 criteria. The discriminant ability of the ELECTHIP criteria should be validated in a second independent set. MCCWOPT can be distinguished from other LNMNEC by the ELECTHIP criteria.

Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer with neuroendocrine features. MCC pathogenesis is associated with either the presence of Merkel cell polyomavirus or chronic exposure to ultraviolet light (UV), which can cause a characteristic pattern of multiple DNA mutations. Notably, in the Northern hemisphere, the majority of MCC cases are of viral aetiology; by contrast, in areas with high UV exposure, UV-mediated carcinogenesis is predominant. The two aetiologies share similar clinical, histopathological and prognostic characteristics. MCC presents with a solitary cutaneous or subcutaneous nodule, most frequently in sun-exposed areas. In fact, UV exposure is probably involved in both viral-mediated and non-viral-mediated carcinogenesis, by contributing to immunosuppression or DNA damage, respectively. Confirmation of diagnosis relies on analyses of histological features and immunological marker expression profiles of the lesion. At primary diagnosis, loco-regional metastases are already present in ∼30% of patients. Excision of the tumour is the first-line therapy; if not feasible, radiotherapy can often effectively control the disease. Chemotherapy was the only alternative in advanced-stage or refractory MCC until several clinical trials demonstrated the efficacy of immune-checkpoint inhibitors.

Detection and quantification of Merkel cell polyomavirus. Analysis of Merkel cell carcinoma cases from 1977 to 2015.

This study investigates the presence of Merkel cell polyomavirus (MCPyV) in skin lesions of patients with Merkel cell carcinoma (MCC). MCPyV was quantified using quantitative Real-Time-PCR (qRT-PCR) in 34 paraffinized MCC samples (resected/biopsied) originally taken between 1977 and 2015, and six non-MCC samples. In 31 (91.2%) MCC-individuals, MCPyV was detected. No virus was observed in any non-MCC tumor. Average age at diagnosis was 78.2 ± 9.35 (55-97) years for women (n = 19) and 69.5 ± 14.7 (45-91) for men (n = 15) (P = 0.04). MCC tumor location, known in 25 cases, was: 11 (44%) in the head region, 6 (24%) in upper limbs, 4 (16%) in lower limbs, and 4 (16%) in the trunk. All but one patient had received some sort of treatment: 15 (45.45%) underwent both radio and chemotherapy, 13 (39.39%) only surgery, 2 (6.06%) surgery, plus radio and chemotherapy, 2 (6.06%) surgery and chemotherapy, and 1 (3.03%) only radiotherapy. Follow up data were available for 21/34 patients: recurrence was recorded for 4 (19.04%), and metastasis for 13 (61.9%). Recorded data showed that 10 men and 5 women (total 44.1%) died during follow up, 7 (46.7%) of them within 2 years of diagnosis. Viral load was 5.8 ± 1.4 log copies/105 cells (3.1-8.6), independent of any variable. MCPyV was very frequent in MCC. It was principally associated with head and limb tumors, it more commonly affected men, who in this study were, on average, younger than women, and had high rates of recurrence and mortality. The amplification techniques described here are easily applied and suitable for detecting the presence of MCPyV virus in MCC.

Abstract 662: The differential association of PD-1, PD-L1, and CD8+ cells with response to pembrolizumab and presence of Merkel cell polyomavirus (MCPyV) in patients with Merkel cell carcinoma (MCC)

Abstract We recently reported a 56% objective response rate in patients with advanced MCC receiving pembrolizumab (anti-PD-1) as first-line therapy. However, a tumor biomarker predicting clinical response was not defined. The purpose of this study was to determine potential associations of anti-PD-1 response (RECIST 1.1, analysis 8/1/16) and the presence of MCPyV, with the density and distribution of CD8+, PD-1+ and PD-L1+ cell populations in the tumor microenvironment (TME). Pretreatment FFPE tumor specimens were stained for CD8 (n=23) and PD-L1 (n=16, 22C3 assay) with immunohistochemistry (IHC). Immunofluorescence (IF) was used to detect PD-1 (n=16). Intratumoral (IT), peritumoral (PT, 100 um zone) and total (PT+IT) densities of PD-1+ and CD8+ immune cells (IC) were determined with digital image analysis. PD-L1+ cases had &amp;gt;1% tumor cells (TC) expressing PD-L1. Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ cells in the IT, PT, and IT+PT regions when compared to non-responders (Mann-Whitney test, p-value=0.03, 0.06, 0.03, respectively). There was no significant association of response with CD8+ IC densities (IT, PT or total) or TC PD-L1 expression. Similarly, when we quantified the number of PD-1+ IC located within 15 um from a PD-L1+ cell (TC or IC), an association was observed between PD-1 and PD-L1 proximity and clinical response (Mann-Whitney test, p-value =0.04), but not CD8 and PD-L1 proximity. Because viral neoantigens can elicit a strong immune response, we also studied these TME factors for their potential associations with MCPyV. We found the presence of virus was significantly associated with increased densities of PT CD8+ cells (Mann-Whitney test, p-value=0.008) and TC PD-L1 expression (Fisher's test, p-value=0.04), but not PD-1+ IC. A more extensive multiplex IF panel (CD8, PD-1, PD-L1, CD68, FoxP3, NSE) analysis was performed on select pre-treatment specimens using the Vectra multispectral imaging system (Perkin Elmer) to explore this divergence. We found that only ~60% of observed total PD-1 expression was displayed by CD8+ cells. Although preliminary, our results suggest a relationship between PD-1+ cells and response to anti-PD-1 therapy and highlight that lymphocyte subsets other than CD8+ T-cells may contribute to the observed response. Further studies in larger cohorts are needed to validate these results. Citation Format: Nicolas A. Giraldo, Genevieve J. Kaunitz, Tricia R. Cottrell, Sneha Berry, Joel C. Sunshine, Peter Nguyen, Haiying Xu, Aleksandra Orgutsova, Candice D. Church, Natalie J. Miller, Jennifer H. Yearley, Evan J. Lipson, Ludmila Danilova, Paul T. Nghiem, Suzanne L. Topalian, Janis M. Taube. The differential association of PD-1, PD-L1, and CD8+ cells with response to pembrolizumab and presence of Merkel cell polyomavirus (MCPyV) in patients with Merkel cell carcinoma (MCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2017-662