MCPyV-negative Merkel Cell Carcinoma Research Articles

LT and SOX9 expression are associated with gene setsthat distinguish Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet radiation (UVR). MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, as the former harbour few DNA mutations and are not related to UVR, and the latter usually arise in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT; coded by MCPyV_gp3) and small T antigen (sT; coded by MCPyV_gp4), promote different carcinogenic pathways. To determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC; to describe the mutational burden and the most frequently mutated genes in both MCC subtypes; and to identify the clinical and molecular factors that may be related to patient survival. Ninety-two patients with a diagnosis of MCC were identified from the medical databases of participating centres. To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter technology. For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the GATK Best Practices workflow for somatic mutations. The expression of LT enabled the series to be divided into two groups (LT positive, n = 55; LT negative, n = 37). Genes differentially expressed in LT-negative patients were related to epithelial differentiation, especially SOX9, or proliferation and the cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive patients, and differentially expressed genes in SOX9-positive patients were related to epithelial/squamous differentiation. In LT-positive patients, the mean SNV frequency was 4.3; in LT-negative patients it was 10 (P = 0.03). On multivariate survival analysis, the expression of SNAI1 [hazard ratio (HR) 1.046, 95% confidence interval (CI) 1.007-1.086; P = 0.02] and CDK6 (HR 1.049, 95% CI 1.020-1.080; P = 0.001) were identified as risk factors. Tumours with weak LT expression tend to co-express genes related to squamous differentiation and the cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.

Combined Merkel Cell Carcinoma and Squamous Cell Carcinoma: A Systematic Review.

Combined Merkel cell carcinoma (MCC) and squamous cell carcinoma (SCC) have classically been regarded as more aggressive than conventional, pure, Merkel cell polyomavirus (MCPyV)-positive MCC. It is still unknown whether combined MCC and SCC are more aggressive than pure, MCPyV-negative MCC, and the origin of both the SCC and MCC elements of these combined tumors has not been elucidated. The main objective of this systematic review was to assess whether combined MCC and SCC tumors are associated with a worse prognosis than pure MCC; the secondary goals were the characterization of the clinical and histopathological features of these combined neoplasms. A total of 38 studies, including 152 patients, were selected for review. In total, 76% of the cases were MCPyV-negative, whereas 4% were MCPyV-positive. The most frequent histopathological pattern was that of an SCC in situ combined with a dermal MCC (36%), followed by both an in situ and invasive SCC combined with a dermal MCC (20%). Forty-seven percent of all cases fitted in the morphology of the so-called "collision tumors". Three combined MCC cases that would fit in the morphological category of collision tumors presented both squamous and neuroendocrine elements in their respective nodal metastases. The mean overall survival was 36 months, comparable to that of pure, MCPyV-negative MCC. This review found similarly aggressive behavior for combined MCC and SCC and pure, MCPyV-negative MCC. Preliminary data strongly suggest that all MCPyV-negative MCC tumors, whether combined or pure, are part of a common spectrum.

The effect of GP-2250 on cultured virus-negative Merkel cell carcinoma cells: preliminary results.

Even in the novel immunotherapy era, Merkel cell carcinoma (MCC) remains challenging in its treatment. Apart from Merkel cell polyomavirus (MCPyV) associated MCC, this cancer is linked in about 20% of cases to ultraviolet-induced mutational burden frequently causing aberrations in Notch and PI3K/AKT/mTOR signalling pathways. The recently developed agent GP-2250 is capable to inhibit growth of cells of different cancers, including pancreatic neuroendocrine tumors. The objective of the present study was to investigate the effects of GP-2250 on MCPyV-negative MCC cells. MethodsWe employed three cell lines (MCC13, MCC14.2, MCC26) which were exposed to different GP-2250doses. GP-2250's effects on cell viability, proliferation, and migration were evaluated by means of MTT, BrdU, and scratch assays, respectively. Flow cytometry was performed for the evaluation of apoptosis and necrosis. Western blotting was implemented for the determination of AKT, mTOR, STAT3, and Notch1 protein expression. Cell viability, proliferation, and migration decreased with increasing GP-2250 doses. Flow cytometry revealed a dose response to GP-2250 in all three MCC cell lines. While the viable fraction decreased, the share of necrotic and in a smaller amount the apoptotic cells increased. Regarding Notch1, AKT, mTOR, and STAT3 expression a comparatively time- and dose-dependent decrease of protein expression in the MCC13 and MCC26 cell lines was observed. By contrast, Notch1, AKT, mTOR, and STAT3 expression in MCC14.2 was scarcely altered or even increased by the three dosages of GP-2250 applied. The present study indicates GP-2250 having anti-neoplastic effects in MCPyV-negative tumor cells in regard to viability, proliferation, and migration. Moreover, the substance is capable of downregulating protein expression of aberrant tumorigenic pathways in MCPyV-negative MCC cells.

Expression of Genes Associated With Epithelial-Mesenchymal Transition in Merkel Cell Polyomavirus–Negative Merkel Cell Carcinoma

Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-β signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. To identify overexpressed oncogenes as potential drug targets for MCC. NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Clinical Next-Generation Sequencing Panels Reveal Molecular Differences Between Merkel Cell Polyomavirus-Negative Merkel Cell Carcinomas and Neuroendocrine Carcinomas.

We aim to determine molecular differences between Merkel cell polyomavirus (MCPyV)-negative Merkel cell carcinomas (MCCs) and neuroendocrine carcinomas (NECs). Our study included 56 MCCs (28 MCPyV negative, 28 MCPyV positive) and 106 NECs (66 small cell NECs, 21 large cell NECs, and 19 poorly differentiated NECs) submitted for clinical molecular testing. APC, MAP3K1, NF1, PIK3CA, RB1, ROS1, and TSC1 mutations, in addition to high tumor mutational burden and UV signature, were frequently noted in MCPyV-negative MCC in comparison to small cell NEC and all NECs analyzed, while KRAS mutations were more frequently noted in large cell NEC and all NECs analyzed. Although not sensitive, the presence of either NF1 or PIK3CA is specific for MCPyV-negative MCC. The frequencies of KEAP1, STK11, and KRAS alterations were significantly higher in large cell NEC. Fusions were detected in 6.25% (6/96) of NECs yet in none of 45 analyzed MCCs. High tumor mutational burden and UV signature, as well as the presence of NF1 and PIK3CA mutations, are supportive of MCPyV-negative MCC, whereas KEAP1, STK11, and KRAS mutations are supportive of NEC in the appropriate clinical context. Although rare, the presence of a gene fusion is supportive of NEC.

The Antineoplastic Effect of Dimethyl Fumarate on Virus-Negative Merkel Cell Carcinoma Cell Lines: Preliminary Results.

Merkel cell carcinoma (MCC) is a rare, difficult-to-treat skin cancer once immunotherapy has failed. MCC is associated either with the clonal integration of the Merkel cell polyomavirus (MCPyV) or mutagenic UV-radiation. Fumaric acid esters, including dimethyl fumarate (DMF), have been shown to inhibit cell growth in cutaneous melanoma and lymphoma. We aimed to explore the effects of DMF on MCPyV-negative MCC cell lines. Three MCC cell lines (MCC13, MCC14.2, and MCC26) were treated with different doses of DMF. The cytotoxic effects and cell proliferation were assessed by the MTT cytotoxicity assay and BrdU proliferation assay at different time points. A significant reduction in cell viability and proliferation were demonstrated for all the cell lines used, with DMF proving to be effective.

Epidemiology of Merkel Cell Polyomavirus Infection and Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is a ubiquitous virus replicating in human dermal fibroblasts. MCPyV DNA can be detected on healthy skin in 67-90% of various body sites, and intact virions are regularly shed from the skin. Infection occurs early in life, and seropositivity increases from 37 to 42% in 1- to 6-year-olds to 92% in adults. Merkel cell carcinoma (MCC) is a rare but very aggressive neuroendocrine tumor of the skin. It develops mainly on sun-exposed areas as a fast-growing, reddish nodule. Two MCC entities exist: about 80% of MCC are MCPyV-associated. Tumorigenesis is driven by viral integration into the host genome and MCPyV oncogene expression. In MCPyV-negative MCC, UV radiation causes extensive DNA damage leading to the deregulation of the cell cycle. In recent decades, MCC incidence rates have increased worldwide, e.g., in the United States, from 0.15 in 1986 to 0.7/100,000 in 2016. Risk factors for the development of MCC include male sex, older age (&gt;75 years), fair skin, intense UV exposure, and immunosuppression. Projections suggest that due to aging populations, an increase in immunosuppressed patients, and enhanced UV exposure, MCC incidence rates will continue to rise. Early diagnosis and prompt treatment are crucial to reducing high MCC morbidity and mortality.

The differential expression of long interspersed nuclear elements‐1 as a marker for hypomethylation in Merkel cell carcinoma

The protein long interspersed nuclear elements-1 (LINE-1) serves as a useful surrogate marker of global methylation but little is known for Merkel cell carcinoma. LINE-1 expression was found only in Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinomas but not in MCPyV-negative Merkel cell carcinomas, suggesting that epigenetic dysregulation may be associated with MCPyV expression.

Selinexor is a novel inhibitor of DNA damage response in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly lethal cutaneous carcinoma, which in ~80% of cases in the USA is aetiologically linked to Merkel cell polyomavirus (MCPyV). Immune checkpoint inhibitors (ICIs) can successfully treat ~50% of patients with metastatic MCC, but some MCCs are refractory to ICIs, possibly due to altered DNA damage response (DDR). Selinexor, an anticancer therapy that is currently approved in combination with chemotherapy for multiple myeloma, downregulates the small T and large T tumour antigens in MCC through selective inhibition of nuclear exportin 1 (XPO1). We examined the effect of varying doses of selinexor on DDR protein expression in MCPyV-positive and MCPyV-negative MCC cells. Selinexor was found to inhibit DDR protein expression in both MCPyV-positive and MCPyV-negative cells. Addition of selinexor alone or combined with ICI may be a promising treatment for MCC, but further in vivo research and clinical trials are required to validate these findings.

The Role of Histone Post-Translational Modifications in Merkel Cell Carcinoma.

Merkel Cell Carcinoma (MCC) is a rare but highly aggressive form of non–melanoma skin cancer whose 5-year survival rate is 63%. Merkel cell polyomavirus (MCPyV), a small DNA tumor virus, is the etiological agent of MCC. Although representing a small proportion of MCC cases, MCPyV-negative MCCs have also been identified. The role of epigenetic mechanisms, including histone post-translational modifications (PTMs) in MCC, have been only partially determined. This review aims to describe the most recent progress on PTMs and their regulative factors in the context of MCC onset/development, providing an overview of current findings on both MCC subtypes. An outline of current knowledge on the potential employment of PTMs and related factors as diagnostic and prognostic markers, as well as novel treatment strategies targeting the reversibility of PTMs for MCC therapy is provided. Recent research shows that PTMs are emerging as important epigenetic players involved in MCC onset/development, and therefore may show a potential clinical significance. Deeper and integrated knowledge of currently known PTM dysregulations is of paramount importance in order to understand the molecular basis of MCC and improve the diagnosis, prognosis, and therapeutic options for this deadly tumor.

Tertiary Lymphoid Structures and Chemokine Landscape in Virus-Positive and Virus-Negative Merkel Cell Carcinoma.

Tertiary lymphoid structures (TLSs) are used as biomarkers in many cancers for predicting the prognosis and assessing the response to immunotherapy. In Merkel cell carcinoma (MCC), TLSs have only been examined in MCPyV-positive cases. Here, we examined the prognostic value of the presence or absence of TLSs in 61 patients with MCC, including MCPyV-positive and MCPyV-negative cases. TLS-positive samples had a significantly better prognosis than TLS-negative samples. MCPyV-positive samples had a good prognosis with or without TLSs, and MCPyV-negative/TLS-positive samples had a similarly good prognosis as MCPyV-positive samples. Only MCPyV-negative/TLS-negative samples had a significantly poor prognosis. All cases with spontaneous regression were MCPyV-positive/TLS-positive. We also performed a comprehensive analysis of the chemokines associated with TLS formation using next-generation sequencing (NGS). The RNA sequencing results revealed 5 chemokine genes, CCL5, CCR2, CCR7, CXCL9, and CXCL13, with significantly high expression in TLS-positive samples compared with TLS-negative samples in both MCPyV-positive and MCPyV-negative samples. Only 2 chemokine genes, CXCL10 and CX3CR1, had significantly different expression levels in the presence or absence of MCPyV infection in TLS-negative samples. Patients with high CXCL13 or CCL5 expression have a significantly better prognosis than those with low expression. In conclusion, the presence of TLSs can be a potential prognostic marker even in cohorts that include MCPyV-negative cases. Chemokine profiles may help us understand the tumor microenvironment in patients with MCPyV-positive or MCPyV-negative MCC and may be a useful prognostic marker in their own right.

Merkel cell polyomavirus-negative Merkel cell carcinoma is associated with JAK-STAT and MEK-ERK pathway activation.

Merkel cell polyomavirus (MCPyV) is monoclonally integrated into the genomes of approximately 80% of Merkel cell carcinomas (MCCs). While the presence of MCPyV affects the clinicopathological features of MCC, the molecular mechanisms of MCC pathogenesis after MCPyV infection are unclear. This study investigates the association between MCPyV infection and activation of the MEK‐ERK and JAK‐STAT signaling pathways in MCC to identify new molecular targets for MCC treatment. The clinicopathological characteristics of 30 MCPyV‐positive and 20 MCPyV‐negative MCC cases were analyzed. The phosphorylation status of MEK, ERK, JAK, and STAT was determined by immunohistochemical analysis. The activation status of the MEK‐ERK and JAK‐STAT pathways and the effects of a JAK inhibitor (ruxolitinib) was analyzed in MCC cell lines. Immunohistochemically, the expression of pJAK2 (P = .038) and pERK1/2 (P = .019) was significantly higher in MCPyV‐negative than in MCPyV‐positive MCCs. Male gender (hazard ratio [HR] 2.882, P = .039), older age (HR 1.137, P < .001), negative MCPyV status (HR 0.324, P = .013), and advanced cancer stage (HR 2.672, P = .041) were identified as unfavorable prognostic factors; however, the phosphorylation states of JAK2, STAT3, MEK1/2, and ERK1/2 were unrelated to the prognosis. The inhibition of cell proliferation by ruxolitinib was greater in MCPyV‐negative MCC cell lines than in an MCPyV‐positive MCC cell line. The expression of pERK1/2 and pMEK was higher in MCPyV‐negative than in MCPyV‐positive cell lines. These results suggest that activation of the JAK2 and MEK‐ERK pathways was more prevalent in MCPyV‐negative than in MCPyV‐positive MCC and the JAK inhibitor ruxolitinib inhibited MEK‐ERK pathway activation. Consequently, the JAK‐STAT and MEK‐ERK signaling pathways may be potential targets for MCPyV‐negative MCC treatment.

934 Biological mechanisms in the different etiologies of Merkel cell carcinoma patients: polyomavirus or UV exposure

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&lt; 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&lt;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&lt;0.05). PDL1 is overexpressed in MCPyV+ CD4+ cells respect CD8+ (p&lt;0.05). The IHC assay shown that viral status does not change in multiple metastases and PDL1 is elevated in the tumour border (p&lt;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.

Selective Inhibition of Aurora Kinase A by AK-01/LY3295668 Attenuates MCC Tumor Growth by Inducing MCC Cell Cycle Arrest and Apoptosis.

Simple SummaryMerkel cell carcinoma is a deadly skin cancer with few treatment options. When the tumor has spread, less than 18% of patients survive past five years, and the mortality rate is 3-times higher than melanoma. Cancer immunotherapy is a promising field, harnessing the patient’s immune system to fight cancer and offering hope to many patients. However, ineligibility or resistance to immunotherapy is a critical challenge; half of all MCC patients are ineligible, and many treated patients either stop responding after an initial positive response or don’t respond at all. In this study, we tested a promising drug based on genomic information from MCC patient tumors. We found that it was highly effective in killing MCC cells and MCC tumors grown in mice; we also observed that MCC genetic characteristics partly predicted how well the drug worked. These results provide strong evidence for its potential clinical application in MCC patients.Merkel cell carcinoma (MCC) is an often-lethal skin cancer with increasing incidence and limited treatment options. Although immune checkpoint inhibitors (ICI) have become the standard of care in advanced MCC, 50% of all MCC patients are ineligible for ICIs, and amongst those treated, many patients develop resistance. There is no therapeutic alternative for these patients, highlighting the urgent clinical need for alternative therapeutic strategies. Using patient-derived genetic insights and data generated in our lab, we identified aurora kinase as a promising therapeutic target for MCC. In this study, we examined the efficacy of the recently developed and highly selective AURKA inhibitor, AK-01 (LY3295668), in six patient-derived MCC cell lines and two MCC cell-line-derived xenograft mouse models. We found that AK-01 potently suppresses MCC survival through apoptosis and cell cycle arrest, particularly in MCPyV-negative MCC cells without RB expression. Despite the challenge posed by its short in vivo durability upon discontinuation, the swift and substantial tumor suppression with low toxicity makes AK-01 a strong potential candidate for MCC management, particularly in combination with existing regimens.

Immunobiology of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer, which is associated in 80% of cases with the Merkel cell polyomavirus (MCPyV). Advanced stages respond to immune checkpoint inhibitors in 50% of cases. Major issues remain unanswered regarding its oncogenesis and optimal treatment. MCPyV-negative and MCPyV-positive MCCs have been hypothesized to derive from distinct cells, although the cell of origin remains a matter of debate. The crucial role the MCPyV small T oncoprotein was recently confirmed by its ability to inactivate p53, together with its contribution to the metastatic progression. In advanced cases, tumoral microenvironment may adequately predict responses to immunotherapies, and several mechanisms of primary and secondary resistance have been investigated. Identifying the mechanisms of oncogenesis allow experimentation of new therapeutic targets, which remain mandatory even at the era of immunotherapies. Although new insights in the mechanisms of primary and secondary resistance pave the way for development of further immunotherapy strategies, neoadjuvant strategies may challenge our whole approach of the disease.

Combining DNA Damage Induction with BCL-2 Inhibition to Enhance Merkel Cell Carcinoma Cytotoxicity.

Merkel cell carcinoma (MCC) is a highly lethal skin cancer. MCC tumors rapidly develop resistance to the chemotherapies tested to date. While PD-1/PD-L1 immune checkpoint blockade has demonstrated success in MCC treatment, a significant portion of MCC patients are nonresponsive. Therefore, the pressing need for effective MCC chemotherapies remains. We screened a library of natural products and discovered that one compound, glaucarubin, potently reduced the viability of Merkel cell polyomavirus (MCPyV)-positive MCCs, while remaining nontoxic to primary human fibroblasts and MCPyV-negative MCC cell lines tested. Protein array and Western blot analyses revealed that glaucarubin induces DNA damage and PARP-1 cleavage that correlates with the loss of viability in MCC cells. However, high basal expression of the antiapoptotic factor BCL-2 allowed a subpopulation of cells to survive glaucarubin treatment. Previous studies have shown that, while targeting BCL-2 family proteins significantly decreases MCC cell viability, BCL-2 antisense therapy alone was insufficient to inhibit tumor growth in patients with advanced MCC. We discovered that treatment with an FDA-approved BCL-2 inhibitor in the context of glaucarubin-induced DNA damage led to near complete killing in multiple MCPyV-positive MCC cell lines that express high levels of BCL-2. The combination of DNA damage-induced apoptosis and BCL-2 inhibition thus represents a novel therapeutic strategy for MCPyV-positive MCCs.

Decreased H3K27me3 Expression Is Associated With Merkel Cell Polyomavirus-negative Merkel Cell Carcinoma, Especially Combined With Cutaneous Squamous Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer, frequently infected with Merkel cell polyomavirus (MCPyV). H3K27me3 acts as a repressive histone modification that epigenetically controls gene transcription. The aim of this study was to examine H3K27me3 expression in MCC. H3K27me3 expression levels were immunohistochemically analyzed in 20 MCPyV-positive MCCs, 15 MCPyV-negative MCCs with squamous cell carcinoma (SCC) (combined MCCs), and six MCPyV-negative pure MCCs. Reduced H3K27me3 expression was variously observed in MCCs. H3K27me3 H-score was significantly lower in MCPyV-negative MCCs than in MCPyV-positive MCCs (p=0.002). H3K27me3 expression was significantly lower in MCPyV-negative combined MCC component than in MCPyV-positive MCCs (p<0.001), MCPyV-negative pure MCCs (p=0.036), or pure MCC histology (p<0.001). Kaplan-Meier analysis showed no association of H3K27me3 with outcome. Differential reduction in H3K27me3 expression was observed based on MCPyV status and morphological type. These results implicate H3K27me3-mediated epigenetic changes in tumorigenesis of MCC, especially in MCPyV-negative MCC combined with SCC.

UV Signature Mutations Reclassify Salivary High-grade Neuroendocrine Carcinomas as Occult Metastatic Cutaneous Merkel Cell Carcinomas.

Salivary high-grade neuroendocrine carcinomas (NECs) are rare, occur predominantly in the parotid gland, and are difficult to differentiate from metastatic cutaneous Merkel cell carcinomas (MCCs), which have overlapping morphologic, immunophenotypic, and molecular profiles. Oncogenic Merkel cell polyomavirus (MCPyV), found in 70% to 80% of MCCs, has also been reported in a few salivary NECs, but this is controversial. A promising biomarker to distinguish the 2 tumor types are UV signature mutations. UV signature mutations indicate a sun damage-induced mechanism of pathogenesis and recently have been found to be highly prevalent in MCPyV-negative MCCs but would be inconsistent with salivary origin. Here, we examine UV signature mutations as a molecular marker to distinguish primary salivary high-grade NEC from MCC. Whole exome DNA sequencing was performed on matched tumor-normal tissue from 4 MCPyV-negative high-grade salivary NECs with no other primary source identified, as well as 3 melanomas and 3 lung NECs as positive and negative controls, respectively. UV signature mutations were found in all salivary NECs, when defined as ≥60% of total mutations being C-to-T transitions at dipyrimidine sites, and when compared with known human cancer-related mutational signatures. The presence of UV signature mutations in salivary high-grade NECs strongly favors these to be occult metastatic MCCs. True salivary primary NECs are likely exceedingly rare. When a high-grade NEC is encountered in the salivary gland, the presence of UV signature mutations or MCPyV may be useful to exclude occult unknown primary MCC.

Expression of Notch 3 and Jagged 1 Is Associated With Merkel Cell Polyomavirus Status and Prognosis in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a rare, aggressive, neuroendocrine skin cancer and most MCCs are related to infection with Merkel cell polyomavirus (MCPyV). Notch signaling modulates cell fate in various tissues including the skin during development and homeostasis, and its aberrant activity relates to onset and progression of various malignancies. Therefore, association of NOTCH1/ NOTCH2/NOTCH3/jagged 1 (JAG1) expression with MCPyV status and prognosis in MCC was investigated. A total of 19 MCPyV-positive and 19 MCPyV-negative MCC samples from patients were stained immunohistochemically with antibodies against NOTCH1, NOTCH2, NOTCH3, and JAG1 and analyzed. Expression of NOTCH1 and NOTCH2 was not associated with MCPyV status or prognosis. However, higher JAG1 expression was found in MCPyV-negative than in MCPyV-positive MCC (p<0.001), and NOTCH3 expression was higher in MCPyV-positive MCC (p=0.062). Kaplan-Meier and multivariate analyses showed that patients with MCC with higher NOTCH3 expression had better overall survival than otherwise (p=0.001 and p=0.033, respectively). Expression of NOTCH3, as a tumor suppressor, is an independent predictor of MCC outcome.