Characterization of different clinical presentations of Merkel cell carcinomas and their potential prognostic implications.

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

OBJECTIVE: The newly discovered Merkel cell polyomavirus (MCV) may play a role in the etiology of Merkel cell carcinoma (MCC), a rare neuroendocrine malignancy of the skin, as several studies have demonstrated MCV DNA in tumor tissues. However, MCV serologic data are sparse, with few studies investigating MCV DNA in tumor tissues and MCV antibodies within the same patients, and no published data on antibodies to MCV T-antigen. We conducted a pilot study to evaluate MCV antibody profiles in MCC cases versus controls and to examine the correlation between MCV DNA in tumors and MCV seroreactivity. METHODS: Plasma samples were obtained from 33 patients diagnosed and/or treated for MCC at the Moffitt Cancer Center in 2006-08, including 25 males and 8 females, ages 53-88 years. Controls were comprised of 37 patients undergoing routine skin cancer screening exams who had no history of skin cancer and were determined to be negative for all types of non-melanoma skin cancer (NMSC) by a nurse practitioner. Fresh-frozen tumor tissue was obtained from 15 MCC patients, including 9 from whom plasma was also obtained. Virus-like particle-based enzyme linked immunosorbent assays (ELISA) were used to measure IgG and IgA antibodies to the MCV VP1 capsid protein. Levels of IgG directed against MCV large T-antigen were also measured using ELISA. MCV DNA was measured in MCC tumor tissues using real-time polymerase chain reaction for the amplification of the small T-antigen region of the MCV genome. Differences in antibody levels across groups were assessed using Wilcoxon rank sum test. RESULTS: Levels of IgG directed against the MCV capsid protein were higher among MCC cases than healthy controls (mean (SD)= 1876 (4001) in cases, 1521 (4889) in controls; p=0.005), as were levels of MCV capsid IgA (mean (SD) = 0.21 (0.25) and 0.09 (0.15) p=0.005). Although levels of IgG antibodies directed against the large T-antigen were slightly higher among MCC cases compared to healthy controls (mean (SD) =0.25 (0.27) vs. 0.19(0.07)), this difference was not statistically significant. MCV DNA was observed in 10 (67%) of 15 tumor tissues tested, with viral loads ranging from 7.1 to 15.6 viral copies per cell equivalent. Levels of IgG antibodies directed against MCV capsid and T-antigen were higher among the 6 MCV DNA-positive cases than the 3 MCV DNA-negative cases for whom plasma was available (mean (SD): capsid IgG: 1040 (1210) in MCV DNA-positive, 384 (494) in MCV DNA-negative, p=0.52; T-antigen IgG: 0.26 (0.26) in MCV DNA-positive, 0.15 (0.15) in MCV DNA-negative, p=0.52). CONCLUSION: MCV seroreactivity is associated with MCC, and MCV antibody levels are higher among MCC patients with MCV DNA-positive tumor tissues. A larger study is needed to confirm these associations with greater statistical power and provide the evidence needed to establish causality between MCV infection and MCC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3772A.

Merkel cell carcinoma (MCC) has recently been associated with a novel polyomavirus. This rare but highly aggressive skin malignancy has been increasing in incidence over the past two decades.1 It is most common in elderly Caucasians as well as immunocompromised patients. Feng et al.2 was the first to discover that the Merkel cell polyomavirus (MCPyV) was integrated within the tumor genome, suggesting that it may be a contributing factor in the pathogenesis of MCC. Since then other studies have confirmed that approximately 80% of MCC are associated with MCPyV.3–7 Further studies have demonstrated that MCPyV is clonally integrated at various sites in the genome of MCC tumors, with truncating mutations that interrupt viral replication; therefore, demonstrating that the virus is not a passenger virus that secondarily infects MCC tumors, but is an etiological agent.8,9 This recent discovery has since sparked controversy, as there have been conflicting reports of the virus contributing to other skin cancers. Recent studies have shown a high prevalence of MCPyV DNA in MCC, supporting a role for the virus in tumorigenesis; however, a high prevalence of the virus has also been identified in non-melanoma skin cancers as well as non-lesional skin.10–13 One hypothesis suggests that the polyomavirus is ubiquitous, and when the virus is detected in basal cell carcinoma (BCC) or squamous cell carcinoma (SCC), it represents a coincidental infection rather than an etiological agent. Detection of MCPyV DNA polymerase chain reaction (PCR) positivity in BCC and SCC tumors is likely to represent loss of immune control over viral replication leading to enhanced coincidental detection. Because detecting viral DNA by qualitative PCR cannot differentiate incidental infection from causal infections, more studies are needed to demonstrate active viral protein expression of MCPyV small T antigen. Using a monoclonal antibody to detect MCPyV large T antigen, Reisinger et al.14 demonstrated MCPyV protein expression in 75% of MCC tumors but detected no large T antigen positivity in BCC or SCC from the same patients. In our case, we were able to test several lesions for the presence of MCPyV using both qualitative and quantitative PCR. A 73-year old Caucasian male presented to the skin cancer clinic for a full skin examination. He had a history of multiple actinic keratoses treated with cryotherapy but no history of skin cancer. His past medical history was significant for ulcerative colitis, currently treated with prednisone, mercaptopurine, and mesalamine, coronary artery disease status post bypass grafting, gastroesophageal reflux, deep vein thrombosis following a knee replacement, and osteoarthritis. The patient complained of a recurrent lesion on his left mid-cheek that had been previously treated with cryotherapy. Physical examination revealed Fitzpatrick skin type I with marked dermatoheliosis on sun-exposed areas as well as a 3 mm hyperkeratotic pink papule within a scar on the left mid-cheek and a 12 × 8-mm irregular dark brown patch on the inferior left mid-cheek. Skin biopsies were performed on the two facial lesions, revealing nodular BCC of the left mid-cheek and melanoma in situ of the inferior left mid-cheek, respectively. The lesions were removed with elliptical excision and intermediate linear layered closure four days after the initial visit. Two months after the patient’s skin examination, he developed a new, tender red nodule on his posterior right arm, which did not respond to antibiotic therapy and local heat application. His primary care physician removed the nodule by elliptical excision. The pathology report revealed a 2.2-cm MCC with focal necrosis and positive margins with evidence of vascular invasion. Immunohistochemical studies demonstrated the tumor cells were positive for pankeratin, CK20, chromogranin, and synaptophysin, and negative for LCA, HMB-45, keratin-7, and TTF-1. Due to his new diagnosis of MCC, he returned to the skin cancer clinic. At that time, he had a new pink papule on his nose, which was biopsied and found to be a BCC. There was also a new hyperkeratotic papule on his left antitragus, which was biopsied and found to be an actinic keratosis. He underwent a wide local excision of the MCC and an axillary lymph node dissection, which revealed 1/27 positive lymph nodes. Basic blood work was performed, which showed no evidence of hematological malignancy. A human immunodeficiency virus (HIV) test was negative. Chest x-ray showed stable pulmonary fibrosis with no evidence of metastases. He was started on radiation therapy and is scheduled to receive adjuvant chemotherapy with four cycles of cisplatin and etoposide. All tissue specimens obtained from the patient were tested for the presence of MCPyV DNA. DNA quality assessment was first performed using a beta-globin reference gene PCR of the DNA extracted from the samples. MCPyV detection was performed with PCR utilizing a primer set designed in our lab within the “small T” region of the virus. In four samples, putative MCPyV-PCR products were detected. The MCC, two samples of the Merkel cell-positive lymph node, and the actinic keratosis tested positive for MCPyV DNA. The melanoma in situ and the two BCC were negative for the polyomavirus (Fig. 1). MCPyV copy number determination with real-time PCR technology, designed within the “small T” viral region, was then performed on the samples found positive by the qualitative PCR. MCPyV copy number/nanogram tissue DNA was highest in the primary MCC (769) compared with the two metastatic lymph node samples (20, 81). MCPyV copy number was very low in the actinic keratosis (0.15) compared with the samples from the primary MCC and lymph node metastases. Detection of MCPyV DNA by PCR in different lesions from a patient with MCC. PCR products were analyzed on 2.0% agarose gel electrophoresis and visualized on a UV transilluminator. Lanes, M: φX174RF DNA marker (Promega); 1: BCC; 2: actinic keratosis; 3: primary MCC; 4–6: melanoma in situ and adjacent skin edges; 7 and 8: sentinel lymph nodes with MCC metastases; C+: MCPyV positive control (plasmid with MCPyV DNA insert from the small T viral); C−: MCPyVC negative control DNA extracted from PBMC (Promega); R: reagent control. 150-bp MCPyV PCR products can be seen in lanes 2, 3, 7 and 8, as well as in the positive control Our case further confirms the association of MCPyV and MCC. The virus was detected in the MCC and MCC-positive lymph node but was not detected in the melanoma in situ or the two BCC. Our case, however, also demonstrates the complexity and controversial nature of the issue as the virus was also detected in the actinic keratosis. The copy number determination demonstrated a very low number in the actinic keratosis as compared with the MCC and positive lymph node. This strengthens the hypothesis that the polyomavirus did not have an etiological role in the actinic keratosis but that the lesion was likely coincidentally infected by the ubiquitous virus. There are abundant data providing evidence of an association between MCC and immunosuppression. Patients with HIV have a relative risk of MCC of 13.4 compared with the general population.15 An increased rate of other malignancies in patients with MCC further supports an impaired immune status in the pathogenesis of MCC. An increased risk of MCC as a second primary has been identified in patients with multiple myeloma, chronic lymphocytic leukemia, non-Hodgkin’s lymphoma SCC, and melanoma.16,17 Recognizing this association of MCC with other malignancies may lead to earlier detection of MCC and therefore earlier treatment and improved survival. Our case is also consistent with the fact that immunosuppression is a risk factor for MCC. Although the workup for HIV and hematological malignancies was negative, our patient was iatrogenically immunosuppressed as he had been on 10 mg of prednisone daily for the past four years for ulcerative colitis. While most studies have shown an association with patients receiving post-transplantation immunosuppression or chemotherapy, it may be possible that our patient’s four-year history of prednisone therapy was sufficient enough to contribute to the pathogenesis of his MCC.18–20 The newly discovered virus appears to be widespread, so it is unclear why it only causes MCC in very few people. Other factors such as ultraviolet radiation, immunogenetics, and immunosuppression likely also contribute to carcinogenesis. More studies are needed to determine viral oncoprotein expression to further delineate the ubiquitous virus’ role in MCC tumorigenesis from a possible passenger virus in non-lesional skin and non-melanoma skin cancer. Although the role of this polyomavirus is controversial, the discovery of the virus in association with MCC will hopefully lead to more effective therapy for the highly aggressive tumor.

Background: Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of Merkel cell carcinomas (MCC). A number of previous studies have shown that MCC patients are at a significantly increased risk to develop chronic lymphocytic leukemia (CLL) and vice versa. Until recently, clonal integration and truncating mutations of the Large T antigen (LTAg) of MCPyV were restricted to MCC. We have recently reported the presence of the MCPyV in highly purified tumor cells of CLL (n = 19/70, 27.1%) (Blood. 2010 Sep 3). Of these, six revealed a novel 246 bp deletion in the helicase gene of the LTAg. The presence of MCPyV was confirmed by immunohistochemistry. Material and Method: Here we aimed to determine the presence of MCPyV by fluorescence in situ hybridization (FISH) analysis in CLL cells in order to evaluate whether MCPyV was integrated or episomal. For this purpose we performed FISH analysis as previously described (Int J Cancer. 2005, 115:419-428) using the MCPyV genome as FISH probe. We tested 2 of the previously reported MCPyV positive CLL cases (EDTA decalcified bone marrow trephines) and MCPyV-positive MCC (n = 5). In addition, we tested MCPyV-negative tumors, e.g. breast and colon cancers. All tissues were formalin-fixed and paraffin-embedded. Results: Specific MCPyV DNA by FISH analysis was detected in the nuclei of MCPyV-positive CLL and MCC cells. In contrast to particularly punctate FISH signals in MCC indicating viral integration, the nuclear FISH signals of the CLL cases revealed especially granular signals, indicative for integrated MCPyV DNA together with transcribed viral RNA (Int J Cancer. 2008, 122:2656-2664). No signals were obtained by MCPyV FISH in breast or colon cancer specimens. Conclusion: The specific detection of MCPyV in CLL cells further supports our previous report of a possible involvement of MCPyV in a significant subset of CLL. The specific but rather granular nuclear FISH signals in MCPyV positive CLL cells needs further study to evaluate if it is also generated by co-existing episomal virus copies, by viral RNA or by the decalcification process. In addition, more CLL cases will be analyzed for the presence of MCPyV. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2717. doi:10.1158/1538-7445.AM2011-2717

Introduction: Merkel cell carcinoma (MCC) is rare and often aggressive skin cancer that manifests mainly in elderly white people. The majority of MCCs contain Merkel cell polyomavirus (MCPyV) DNA. Patients with MCPyV-positive MCC have favorable survival as compared to those with MCPyV-negative tumor. The reasons for this remain speculative, but host immune defence might influence outcome of patients with MCPyV infection. Material and Methods: Tumor infiltrating T-lymphocytes (CD3+ cells) and macrophages (CD68+ cells) were stained using immunohistochemistry from 116 MCCs. The numbers of stained cells were counted at 400× magnification of the microscope. Presence of MCPyV infection was assessed by detecting MCPyV DNA using quantitative PCR and the viral large T antigen using immunohistochemistry. The associations between the tumor immune cell counts, presence of MCPyV DNA, clinicopatholocial factors and patient survival were examined using the Mann-Whitney test and the Kaplan-Meyer survival analysis. Results: MCPyV-positive tumors contained more CD3+ and CD68+ cells as compared to MCPyV-negative cancers (5.3 vs. 3.3 cells/high-power field [HPF], p=0.014; and 5.3 vs. 4.0 cells/HPF, p=0.028, respectively). Similar results were obtained when presence of MCPyV infection was efined by expression of the viral large T-antigen (5.9 vs. 2.7 cells/HPF, p<0.001; and 5.6 vs. 3.9 cells/HPF, p=0.003, respectively). Patients with regional nodal metastases had fewer CD3+ cells in the primary tumor as compared to those without metastases (2.3 vs. 5.3 cells/HPF, p=0.006). Patients who had MCC with a higher than the median number of CD3+ cells had better MCC-specific survival (55.2% vs. 82.1%, log-rank test p=0.003) and overall survival (22.4% vs. 50.0%, p=0.002) as compared to those with a smaller than the median tumor CD3+ cell count. Tumor CD3+ count was an independent prognostic factor for overall survival in a Cox multivariable analysis that included the presence of regional nodal etastases, gender and MCPyV large T-antigen expression as covariates (HR=0.55, 95% CI, 0.35 to 0.88, p=0.012). Conclusions: MCCs with evidence of MCPyV infection harbor high numbers of tumor infiltrating T-lymphocytes. A high number of CD3+ cells in the tumor is associated with generally favorable survival. Host immune response to MCPyV might explain in part the favorable survival associated with MCCs with MCPyV infection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C237.

The spectrum of Merkel cell polyomavirus expression in Merkel cell carcinoma, in a variety of cutaneous neoplasms, and in neuroendocrine carcinomas from different anatomical sites

Background: Merkel cell polyoma virus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). The clonal integration and tumor specific mutations in the large T Antigen (LTAg) gene strongly implicate an oncogenic impact of the virus. To date the relationship between the viral presence and cancer induction, development or clinical prognosis is discussed controversial. Yet almost all studies are based on quantitative virus detection e.g. PCR or qPCR. Material and Method: In order to gain additional information about the quality of the viral presence on the single cell level we performed FISH analysis of formalin fixed and paraffin embedded (FFPE) MCCs (n= 62) on tissue micro arrays (TMA), determined the FISH pattern and correlated the results of the FISH analysis with the qPCR data. We grouped the MCCs according to different FISH evaluations and correlated them with the respective qPCR data on the basis of a determined cut-off. MCPyV FISH was established using the MKL-1 cell line which harbors integrated copies of MCPyV DNA. For MCPyV-qPCR the LT3 primer pair was used on whole tissue sections. Results: MCPyV-FISH on FFPE MKL-1 cells revealed punctate signals compatible with viral integration. The MCPyV FISH positive MCC cores (76%) mainly revealed two different signal patterns: a punctate pattern (85%) which correlated with a moderate relative viral presence and in some areas the punctate pattern was combined with a diffuse pattern (15%) indicating the episomal presence of the virus which is linked to viral replication. If a pattern mixture was seen this was associated with very high qPCR values. Comparing MCPyV FISH and qPCR data the results correlated highly with 83 % in the MCPyV positive evaluated group, whereas the negative group showed a concordance of 93 %. The mean of the qPCR values of all MCPyV positive cores differed significantly from the negative cores (p= 0.0076). The FISH signals were in some tumor areas from the same patient heterogenic in intensity, pattern and localization. Conclusion: While the qPCR using the LT3 primer pair was highly sensitive to detect MCPyV sequences in the respective MCC cases, the MCPyV FISH analysis highly concordantly revealed the quality of the viral presence, i.e. viral integration or episomal presence and morphological sublocalization in the tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 557. doi:1538-7445.AM2012-557

Multiple Merkel cell carcinomas: Late metastasis or multiple primary tumors? A molecular study

<div>Abstract<p><b>Purpose:</b> Merkel cell carcinoma (MCC) is rare skin cancer that is often associated with Merkel cell polyomavirus (MCPyV) infection. Polyomaviruses repress tumor suppressor proteins, thus influencing cell-cycle progression, but the effect of MCPyV on the key cell-cycle regulating proteins is poorly understood.</p><p><b>Experimental Design:</b> We evaluated expression of the MCPyV large T-antigen (LTA), Ki-67, and the key putative tumor suppressor proteins, the retinoblastoma protein (RB and phospho-RB) and p53, and their regulatory proteins (cyclin D1, cyclin E, p16, p21, p27, and MDM2) by using immunohistochemistry from tumors of 91 MCC patients identified from a population-based nationwide cohort. Tumor MCPyV DNA was measured by using quantitative PCR, and <i>TP53</i> mutations were identified with sequencing.</p><p><b>Results:</b> MCPyV LTA expression was strongly associated with presence of MCPyV DNA in tumor, and it was almost invariably associated with tumor RB expression (<i>P</i> < 0.0001 for both comparisons). Both MCC LTA and RB expression were strongly associated with favorable MCC-specific and overall survival in univariable analyses (<i>P</i> ≤ 0.01 for all four analyses). Presence of MCPyV LTA was also associated with the female gender, the intermediate type of tumor histology, location of the tumor in a limb, cell proliferation rate, and absence of p53 expression. <i>TP53</i> mutations were detected only in MCPyV DNA–negative tumors.</p><p><b>Conclusions:</b> MCPyV DNA–positive MCC has several clinical and molecular features that differ from MCPyV DNA-negative cancers. MCPyV-associated MCCs express RB, but may not harbor <i>TP53</i> mutations. These findings provide further support that MCPyV causes the majority of MCCs. <i>Clin Cancer Res; 17(14); 4806–13. ©2011 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> Merkel cell carcinoma (MCC) is rare skin cancer that is often associated with Merkel cell polyomavirus (MCPyV) infection. Polyomaviruses repress tumor suppressor proteins, thus influencing cell-cycle progression, but the effect of MCPyV on the key cell-cycle regulating proteins is poorly understood.</p><p><b>Experimental Design:</b> We evaluated expression of the MCPyV large T-antigen (LTA), Ki-67, and the key putative tumor suppressor proteins, the retinoblastoma protein (RB and phospho-RB) and p53, and their regulatory proteins (cyclin D1, cyclin E, p16, p21, p27, and MDM2) by using immunohistochemistry from tumors of 91 MCC patients identified from a population-based nationwide cohort. Tumor MCPyV DNA was measured by using quantitative PCR, and <i>TP53</i> mutations were identified with sequencing.</p><p><b>Results:</b> MCPyV LTA expression was strongly associated with presence of MCPyV DNA in tumor, and it was almost invariably associated with tumor RB expression (<i>P</i> < 0.0001 for both comparisons). Both MCC LTA and RB expression were strongly associated with favorable MCC-specific and overall survival in univariable analyses (<i>P</i> ≤ 0.01 for all four analyses). Presence of MCPyV LTA was also associated with the female gender, the intermediate type of tumor histology, location of the tumor in a limb, cell proliferation rate, and absence of p53 expression. <i>TP53</i> mutations were detected only in MCPyV DNA–negative tumors.</p><p><b>Conclusions:</b> MCPyV DNA–positive MCC has several clinical and molecular features that differ from MCPyV DNA-negative cancers. MCPyV-associated MCCs express RB, but may not harbor <i>TP53</i> mutations. These findings provide further support that MCPyV causes the majority of MCCs. <i>Clin Cancer Res; 17(14); 4806–13. ©2011 AACR</i>.</p></div>

BackgroundMerkel 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.MethodsMultiplex 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.ResultsWe 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.ConclusionInfection with MCPyV is common in the general population. MCPyV, but not other human polyomaviruses, appears to be associated with Merkel cell carcinoma.

BackgroundMerkel cell carcinoma (MCC) is a rare and aggressive skin cancer with neuroendocrine features, and it is associated with elevated mortality. The pathogenesis is associated with presence of clonally integrated Merkel cell polyomavirus (MCPyV) or ultraviolet light (UV) exposure.1 The MCPyV causes up to 80% of MCC tumors in North America and Europe.2–4 Recently immunotherapy is having good results,5 the phase 2 trial JAVELIN Merkel 200 indicated that treatment with Avelumab (PDL1 inhibitor) in patients with metastatic MCC pre-treated have a meaningful long-term survival outcomes respect chemotherapy. Moreover, ORRs were highest in patients with high TMB that were also MCPyV−, PD-L1+ or had a greater CD8+ T cell density at the invasive margin.6 In this study, we investigated the biological signatures in patients with MCPyV or not.MethodsFrom April 2011 to June 2018, we collected retrospectively 50 FFPE (Formalin-Fixed Paraffin-Embed) from 37 patients with metastatic MCC and 13 tissues from a secondary metastatic site. All patients have appropriately signed informed consent. We performed an immunohistochemistry assays (IHC) for MCPyV and PDL1. In addition, through the NanoString GeoMx DSP (Digital Spatial Profiling), we analysed 11 patients (6 MCPyV+; 5 MCPyV-) with cutaneous metastasis using a 44-plex antibody cocktail. For each slide we selected three different areas: Intratumoral, extratumoral and tumour border, in each area we selected CD4+ and CD8+ cells in 4 different ROIs (Region of Interest). Statistical analysis was performed via Bonferroni correction, P< 0.05 was considered statistically significant for median stratification.ResultsThe DSP analysis showed that the tumour border cells have an overexpression of IDO respect intratumoral area (adj. p<0.01). Instead, extratumoral area of MCPyV- patients have a higher expression of B7-H3 respect MCPyV+ as well as FOXP3 is higher in the tumour border of MCPyV+ patients and EpCAM in the intratumoral area (p<0.05). PDL1 is overexpressed in MCPyV+ CD4+ cells respect CD8+ (p<0.05). The IHC assay shown that viral status does not change in multiple metastases and PDL1 is elevated in the tumour border (p<0.05).ConclusionsIn this retrospective study, our preliminary data shown that tumour edge have an important role in the modulations of immune infiltrate and patients with Merkel cell polyomavirus could have a different pathway of immunosuppression compared to patients with non-virus related etiology. Further investigations are needed to get additional information.AcknowledgementsThe study was supported by the Institutional Project ”Ricerca Corrente” of Istituto Nazionale Tumori IRCCS Fondazione ”G. Pascale” of Napoli, Italy.ReferencesKaae J, Hansen AV, Biggar RJ, et al. Merkel cell carcinoma: incidence, mortality, and risk of other cancers. J Natl Cancer Inst 2010 June 2;102(11):793–801.Feng H, Shuda M, Chang Y, et al. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 2008 February 22;319(5866):1096–100.Garneski KM, Warcola AH, Feng Q, et al. Merkel cell polyomavirus is more frequently present in North American than Australian Merkel cell carcinoma tumors. J Invest Dermatol 2009 January;129(1):246–8.Goh G, Walradt T, Markarov V, et al. Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy. Oncotarget 2016 January 19;7(3):3403–15.Bichakjian CK, Olencki T, Aasi SZ, et al. Merkel cell carcinoma, version 1.2018, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2018 June;16(6):742–774.D’Angelo SP, Bhatia S, Brohl AS, et al. Avelumab in patients with previously treated metastatic Merkel cell carcinoma: long-term data and biomarker analyses from the single-arm phase 2 JAVELIN Merkel 200 trial. J Immunother Cancer 2020 May;8(1):e000674.Ethics ApprovalThe study was approved by internal ethics board of the Istituto Nazionale Tumori IRCCS Fondazione ”G. Pascale” of Napoli Italy, approval number of registry 33/17 OSS.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV LT-positive nuclear foci. Such a phenotype was recapitulated using our previously established system for visualizing MCPyV replication complexes in cells. By combining immunofluorescent staining, fluorescence in situ hybridization, and BrdU incorporation analysis, we demonstrate that DDR proteins are important for maintaining robust MCPyV DNA replication. This study not only provides the first look into the microscopic details of DDR factor/LT replication complexes at the MCPyV origin but also provides a platform for further studying the mechanistic role of host DDR factors in the MCPyV life cycle and virus-associated oncogenesis.

Association of Merkel cell polyomavirus infection with clinicopathological differences in Merkel cell carcinoma

Recently, it has been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus, thought to be a carcinogenic agent. However, it is not fully elucidated whether Merkel cell carcinomas differ with regard to the presence or absence of Merkel cell polyomavirus. To address this, we investigated morphologic differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas by morphometry. Using polymerase chain reaction and real-time quantitative polymerase chain reaction, Merkel cell polyomavirus was detected in 20 (77%) of 26 Merkel cell carcinoma cases, including 4 Merkel cell carcinomas combined with squamous cell carcinomas. Interestingly, Merkel cell polyomavirus was detected only in ordinary (pure) Merkel cell carcinomas; none of the 4 combined Merkel cell carcinomas + squamous cell carcinomas was positive for Merkel cell polyomavirus (P = .001). Morphometric analyses revealed that Merkel cell polyomavirus-negative Merkel cell carcinomas had more irregular nuclei (P < .001) and more abundant cytoplasm (P = .001) than Merkel cell polyomavirus-positive Merkel cell carcinomas, which had uniform round nuclei and scant cytoplasm. Reliability of the morphometry was confirmed using intraobserver and interobserver reliability tests. These results demonstrated statistically significant differences in tumor cell morphology between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas and reconfirmed the absence of Merkel cell polyomavirus in combined tumors. Furthermore, the results strongly suggest fundamental biological differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas, supporting that Merkel cell polyomavirus plays an important role in the pathogenesis of Merkel cell polyomavirus-positive Merkel cell carcinoma.