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Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor–immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments. Acquired resistance is a major problem in cancer immunotherapy. Here the authors report a study of two patients with Merkel cell carcinoma under immunotherapy treatment who develop resistance after deep responses for >1 year and identified a novel mechanism of acquired, gene-specific transcriptional suppression of HLAs.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer associated with advanced age and immunosuppression. Over the past decade, an association has been discovered between MCC and either integration of the Merkel cell polyomavirus, which likely drives tumorigenesis, or somatic mutations owing to ultraviolet-induced DNA damage. Both virus-positive and virus-negative MCCs are immunogenic, and inhibition of the programmed cell death protein 1 (PD-1)–programmed cell death 1 ligand 1 (PD-L1) immune checkpoint has proved to be highly effective in treating patients with metastatic MCC; however, not all patients have a durable response to immunotherapy. Despite these rapid advances in the understanding and management of patients with MCC, many basic, translational and clinical research questions remain unanswered. In March 2018, an International Workshop on Merkel Cell Carcinoma Research was held at the US National Cancer Institute, at which academic, government and industry experts met to identify the highest-priority research questions. Here, we review the biology and treatment of MCC and report the consensus-based recommendations agreed upon during the workshop.

Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor frequently associated with clonal integration of a polyomavirus, Merkel cell polyomavirus (MCPyV), and MCC tumor cells express putative polyomavirus oncoprotein small T antigen (sTAg) and truncated large T antigen. Here, we show robust transforming activity of sTAg in vivo in a panel of transgenic mouse models. Epithelia of preterm sTAg-expressing embryos exhibited hyperplasia, impaired differentiation, increased proliferation, and apoptosis, and activation of a DNA damage response. Epithelial transformation did not require sTAg interaction with the protein phosphatase 2A protein complex, a tumor suppressor in some other polyomavirus transformation models, but was strictly dependent on a recently described sTAg domain that binds Fbxw7, the substrate-binding component of the Skp1/Cullin1/F-box protein ubiquitin ligase complex. Postnatal induction of sTAg using a Cre-inducible transgene also led to epithelial transformation with development of lesions resembling squamous cell carcinoma in situ and elevated expression of Fbxw7 target proteins. Our data establish that expression of MCPyV sTAg alone is sufficient for rapid neoplastic transformation in vivo, implicating sTAg as an oncogenic driver in MCC and perhaps other human malignancies. Moreover, the loss of transforming activity following mutation of the sTAg Fbxw7 binding domain identifies this domain as crucial for in vivo transformation.

Background Merkel cell carcinoma (MCC) is associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV antibodies (MCPyV-Ab) in plasma correlate with survival, while MCPyV-Ab within the tumor has never been investigated. This study evaluated plasma MCPyV-Ab and tumor MCPyV-Ab titers to evaluate their role in outcomes and prognostication. Methods A single-institution, prospective database was retrospectively reviewed for patients diagnosed with MCC from 2014 to 2021. MCPyV-Ab plasma and tumor titers, as well as patient and treatment factors, were collected. Two-year overall survival (OS) and disease-free survival (DFS) were examined based on MCPyV-Ab presence in tumor. Results Forty patients were identified, with a median follow-up of 27.6 months. Patients were stratified into four groups based on the presence of MCPyV-Ab in plasma (P+, P−) and tumor (T+, T−). Most patients (60.0%) were P−/T−. Of the remaining patients, 22.5% were P+/T+, 12.5% were P−/T+, and 5.0% were P+/T−. Two-year DFS of the P−/T− group was 16.6 months, which was not different from the other groups ( p  = 0.79). Two-year OS of P−/T− was 18.3 months, and 2-year OS of P−/T+ was 28.1 months, which was similar between groups ( p  = 0.80). Conclusions Most patients P+ for MCPyV had antibody-positive tumors (T+), and P− patients were also T−; however, there was a subset of patients where plasma and tumor antibody findings were incongruent. Patients with MCPyV-Ab in either plasma or tumor had a trend toward improved 2-year DFS and OS, but was limited by a small cohort. This study offers an exploratory investigation into the relationship between plasma and tumor antibodies to MCPyV on which to base future work.

Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of [almost equal to]80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a \"passenger virus\" that secondarily infects MCC tumors.

Merkel cell carcinoma (MCC) is a rare skin cancer characterized by neuroendocrine differentiation. Its carcinogenesis is based either on the integration of the Merkel cell polyomavirus or on ultraviolet (UV) mutagenesis, both of which lead to high immunogenicity either through the expression of viral proteins or neoantigens. Despite this immunogenicity resulting from viral or UV-associated carcinogenesis, it exhibits highly aggressive behavior. However, owing to the rarity of MCC and the lack of epidemiologic registries with detailed clinical data, there is some uncertainty regarding the spontaneous course of the disease. Historically, advanced MCC patients were treated with conventional cytotoxic chemotherapy yielding a median response duration of only 3 months. Starting in 2017, four programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors—avelumab, pembrolizumab, nivolumab (utilized in both neoadjuvant and adjuvant settings), and retifanlimab—have demonstrated efficacy in treating patients with disseminated MCC on the basis of prospective clinical trials. However, generating clinical evidence for rare cancers, such as MCC, is challenging owing to difficulties in conducting large-scale trials, resulting in small sample sizes and therefore lacking statistical power. Thus, to comprehensively understand the available clinical evidence on various immunotherapy approaches for MCC, we also delve into the epidemiology and immune biology of this cancer. Nevertheless, while randomized studies directly comparing immune checkpoint inhibitors and chemotherapy in MCC are lacking, immunotherapy shows response rates comparable to those previously reported with chemotherapy but with more enduring responses. Notably, adjuvant nivolumab has proven superiority to the standard-of-care therapy (observation) in the adjuvant setting.

BackgroundMerkel cell carcinoma (MCC) is an aggressive skin cancer in which PD-1/PD-L1 blockade has shown remarkable response rates. However, a significant proportion of patients shows primary or secondary resistance against PD-1/PD-L1 inhibition, with HLA class-I downregulation and insufficient influx of CD8+ T cells into the tumor as possible immune escape mechanisms. Histone deacetylase inhibitors (HDACi) have been demonstrated to reverse low HLA class-I expression caused by epigenetic downregulation of the antigen machinery (APM) in vitro and in pre-clinical models in vivo.Case presentationsWe report four cases of patients with metastatic MCC who did not respond to immunotherapy by PD-1/PD-L1 blockade. Two of the patients received, subsequently, the HDACi panobinostat in combination with PD-1/PD-L1 blockade. Tumor biopsies of the patients were analyzed for cellular and molecular markers of antigen processing and presentation as well as the degree of T-cell infiltration.Results and conclusionLow expression of APM-related genes associated with low HLA class-I surface expression was observed in all MCC patients, progressing on PD-1/PD-L1 blockade. In one evaluable patient, of the two treated with the combination therapy of the HDACi, panobinostat and PD-1/PD-L1 blockade, reintroduction of HLA class-I-related genes, enhanced HLA class-I surface expression, and elevated CD8+ T-cell infiltration into the MCC tumor tissue were observed; however, these changes did not translate into a clinical benefit. Our findings suggest that HDACi may be useful to overcome HLA class-I downregulation as a resistance mechanism against anti-PD-1/PD-L1 antibodies in MCC patients. Prospective clinical trials are needed to evaluate this notion.

Merkel cell carcinoma (MCC) is a lethal skin cancer that metastasizes rapidly. Few effective treatments are available for patients with metastatic MCC. Poor intratumoral T cell infiltration and activation are major barriers that prevent MCC eradication by the immune system. However, the mechanisms that drive the immunologically restrictive tumor microenvironment remain poorly understood. In this study, we discovered that the innate immune regulator stimulator of IFN genes (STING) is completely silenced in MCCs. To reactivate STING in MCC, we developed an application of a human STING mutant, STINGS162A/G230I/Q266I, which we found to be readily stimulated by a mouse STING agonist, DMXAA. This STING molecule was efficiently delivered toMCC cells via an AAV vector. Introducing STINGS162A/G230I/Q266I expression and stimulating its activity by DMXAA in MCC cells reactivates their antitumor inflammatory cytokine/chemokine production. In response to MCC cells with restored STING, cocultured T cells expressing MCPyV-specific T cell receptors (TCRs) showincreased cytokine production,migration toward tumor cells, and tumor cell killing. Our study therefore suggests that STING deficiency contributes to the immune suppressive nature of MCCs. More importantly, DMXAA stimulation of STINGS162A/G230I/Q266I causes robust cell death in MCCs as well as several other STING-silenced cancers. Because tumor antigens and DNA released by dying cancer cells have the potential to amplify innate immune response and activate antitumor adaptive responses, our finding indicates that targeted delivery and activation of STINGS162A/G230I/Q266I in tumor cells holds great therapeutic promise for the treatment of MCC and many other STING-deficient cancers.

BackgroundMerkel cell carcinoma (MCC) is an aggressive skin cancer associated with poor survival. Programmed cell death-1 (PD-1) pathway inhibitors have shown high rates of durable tumor regression compared with chemotherapy for MCC. The current study was undertaken to assess baseline and on-treatment factors associated with MCC regression and 3-year survival, and to explore the effects of salvage therapies in patients experiencing initial non-response or tumor progression after response or stable disease following first-line pembrolizumab therapy on Cancer Immunotherapy Trials Network-09/KEYNOTE-017.MethodsIn this multicenter phase II trial, 50 patients with advanced unresectable MCC received pembrolizumab 2 mg/kg every 3 weeks for ≤2 years. Patients were followed for a median of 31.8 months.ResultsOverall response rate to pembrolizumab was 58% (complete response 30%+partial response 28%; 95% CI 43.2 to 71.8). Among 29 responders, the median response duration was not reached (NR) at 3 years (range 1.0+ to 51.8+ months). Median progression-free survival (PFS) was 16.8 months (95% CI 4.6 to 43.4) and the 3-year PFS was 39.1%. Median OS was NR; the 3-year OS was 59.4% for all patients and 89.5% for responders. Baseline Eastern Cooperative Oncology Group performance status of 0, greater per cent tumor reduction, completion of 2 years of treatment and low neutrophil-to-lymphocyte ratio were associated with response and longer survival. Among patients with initial disease progression or those who developed progression after response or stable disease, some had extended survival with subsequent treatments including chemotherapies and immunotherapies.ConclusionsThis study represents the longest available follow-up from any first-line anti-programmed death-(ligand) 1 (anti-PD-(L)1) therapy in MCC, confirming durable PFS and OS in a proportion of patients. After initial tumor progression or relapse following response, some patients receiving salvage therapies survived. Improving the management of anti-PD-(L)1-refractory MCC remains a challenge and a high priority.Trial registration numberNCT02267603.

BackgroundMerkel cell carcinoma (MCC) is a rare, aggressive skin cancer associated with a high risk of metastasis. In 2017, avelumab (anti–programmed death-ligand 1 (PD-L1)) became the first approved treatment for patients with metastatic MCC (mMCC), based on the occurrence of durable responses in a subset of patients. Here, we report long-term efficacy and safety data and exploratory biomarker analyses in patients with mMCC treated with avelumab.MethodsIn a cohort of this single-arm, phase 2 trial (JAVELIN Merkel 200), patients with mMCC and disease progression after prior chemotherapy received avelumab 10 mg/kg intravenously every 2 weeks. The primary endpoint was confirmed objective response rate (ORR) by independent review per Response Evaluation Criteria in Solid Tumors V.1.1. Other assessments included duration of response, progression-free survival, overall survival (OS), safety and biomarker analyses.ResultsAs of 14 September 2018, 88 patients had been followed up for a median of 40.8 months (range 36.4–49.7 months). The ORR was 33.0% (95% CI 23.3% to 43.8%), including a complete response in 11.4% (10 patients), and the median duration of response was 40.5 months (95% CI 18.0 months to not estimable). As of 2 May 2019 (≥44 months of follow-up), the median OS was 12.6 months (95% CI 7.5 to 17.1 months) and the 42-month OS rate was 31% (95% CI 22% to 41%). Of long-term survivors (OS >36 months) evaluable for PD-L1 expression status (n=22), 81.8% had PD-L1+ tumors. In exploratory biomarker analyses, high tumor mutational burden (≥2 non-synonymous somatic variants per megabase) and high major histocompatibility complex class I expression (30% of tumors with highest expression) were associated with trends for improved ORR and OS. In long-term safety assessments (≥36 months of follow-up), no new or unexpected adverse events were reported, and no treatment-related deaths occurred.ConclusionsAvelumab showed continued durable responses and meaningful long-term survival outcomes in patients with mMCC, reinforcing avelumab as a standard-of-care treatment option for this disease.Trial registration number NCT02155647