Merkel cell carcinoma (MCC) is a highly aggressive, often lethal neuroendocrine cancer. Its carcinogenesis may be either caused by the clonal integration of the Merkel cell polyomavirus into the host genome or by UV-induced mutations. Notably, virally-encoded oncoproteins and UV-induced mutations affect comparable signaling pathways such as RB restriction of cell cycle progression or p53 inactivation. Despite its low incidence, MCC recently received much attention based on its exquisite immunogenicity and the resulting major success of immune modulating therapies. Here, we summarize current knowledge on epidemiology, biology and therapy of MCC as conclusion of the project ‘Immune Modulating strategies for treatment of Merkel Cell Carcinoma’, which was funded over a 5-year period by the European Commission to investigate innovative immunotherapies for MCC.

Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally Hansen et al. Neoepitopes in ccRCC represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.

Forty-one isolates of Fusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains of F. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remaining F. sambucinum strains produced T-2 toxin, TB1 and TB2. Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB. Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.

BackgroundMerkel cell carcinoma (MCC) is an aggressive human skin cancer primarily induced by Merkel Cell Polyomavirus (MCPyV) driven by expression of the oncogenic T antigens (T-Ags): Large T and Small T antigen. Checkpoint inhibition therapy blocking the programmed cell death protein-1 (PD-1) pathway has proven effective with a clinical response rate up to 58%,1 highlighting the critical role of immune surveillance for tumor control. Yet, evidence for the impact of T-Ag-specific T cells following immunotherapy is still limited.MethodsPotential CD8+ T cell epitopes derived from the T-Ags and the Viral capsid protein 1 (VP1) were predicted with netMHCpan 4.0 as 9- and 10-mer peptides with a rank score <2 for binding to 33 different HLA class I types. Peripheral blood mononuclear cells (PBMC) were obtained from 24 MCPyV+ MCC patients prior and post anti-PD-1 therapy initiation (CITN-09/Keynote-017). T cell recognition of the MCPyV-derived peptides during therapy was evaluated using the high-throughput technology of DNA barcode labeled pMHC multimers. Phenotypic characteristics of multimer-binding T cells were evaluated for selected patients.ResultsAcross all patients, 40 T-Ag-specific CD8+ T-cell populations were detected recognizing 31 epitopes restriction to 14 different HLA class I types. T-Ag-specific CD8+ T cells were detected in responders (complete and partial response, n=17) during therapy with a trend of increased number of responses observed after therapy initiation. Whereas only a single T-Ag-specific population was detectable in non-responders (stable and progressive disease, n=7). Moreover, the T cell repertoire after therapy initiation was significantly increased in the responder group compared to non-responder with the T-Ag-specific T cells showing an activated effector memory phenotype.ConclusionsThe current study indicates that the T-Ag-specific T cells are associated with clinical benefit to checkpoint inhibitor therapy. Furthermore, the broad identification of novel T-Ag-derived T cell epitopes could potentially facilitate the use of targeted T cell therapy to enhance T cell recognition and clearance of MCC in combination with checkpoint inhibition.AcknowledgementsSupported by the Cancer Immunotherapy Trials Network (CITN)ReferenceNghiem P, Bhatia S, Lipson E. Three-year survival, correlates and salvage therapies in patients receiving first-line pembrolizumab for advanced Merkel cell carcinoma. J Immunother cancer 2021;9:e002478.

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Mutation-derived neoantigens are important targets of T-cell mediated reactivity towards tumors. Their unique tumor-restriction poses an advantage compared to shared tumor antigens in that they are in principle both foreign and tumor specific, hence presumably less impacted by T-cell tolerance and for therapeutic applications less prone to mediate immune-related destruction of noncancerous tissue. Moreover, the mutational burden and predicted number of neoantigens correlate to favorable clinical outcome and benefit from immune checkpoint therapy. Neoantigen-reactive T-cells have been detected across a number of solid cancers, ranging from immunogenic tumors such as melanoma and non-small cell lung cancer to less immunogenic tumors such as breast cancer. Renal cell carcinomas (RCCs) are among medium-range mutational burden tumors and present with the highest pan-cancer number and proportion of frameshift mutations, a mutation type considered to be highly immunogenic. However, to our knowledge, yet no reports have described neoantigen-specific T-cells in this malignancy. In this study, the mutational landscape and HLA (human leukocyte antigen) profile of tumors from six renal cell carcinoma patients were analyzed by whole-exome sequencing (WXS) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs) and tumor-infiltrating lymphocytes (TILs, germline reference). Hereafter the online MuPeXi tool was used to predict binding of mutated peptide sequences of 9-11mer length to the HLAs of each patient, using a rank score &lt; 2 for selection of peptide binding, hereby creating patient-specific libraries of putative neo-peptides. TILs extracted from the patients tumors were screened for T-cell recognition of the peptide libraries by use of a novel high-throughput platform based on DNA barcode labeled peptide-MHC multimers, and responses were verified by conventional fluorochrome labeled MHC multimers. In four of six patients WXS was performed on both TF and TCL, in two of six patients only on TF. The average mutational burden of the six patients was 271 for TF (range 146–381, n=6) and 289 for TCL (range 182-404, n=4). Prediction of HLA-restricted peptides within the mutated sequences resulted in patient specific libraries of average 269 peptides for TF and TCL combined (range 126-443, n=6). Half of the peptides were predicted from both sources (52%, range 28-74%, n=4) compared to 20% (range 8-31%, n=4) predicted solely from TF and 29% (range 18-41%, n=4) predicted solely from TCL. The proportion of predicted peptides derived from frameshift mutations out of total mutations was 16% (range 7-24%, n=6). A total of 67 neoantigen-specific T-cell responses were detected across all patients by use of a novel high-throughput DNA barcode screening platform, with the number of detected responses ranging from 4-30 and spanning 3-5 HLA restrictions per patient. Of note, we detected a number of T-cell responses towards HLA-C restricted peptides, which have previously been poorly described. For several patients, the number of HLA-C restricted T-cell responses observed was substantially higher than for both HLA-A and -B, highlighting the importance of including this HLA type for neoepitope analyses. In the four patients in whom peptides were predicted from both TF and TCL, the distribution of responses was 37% on TF (range 14-60%, n=4), 36% on TCL (range 29-43%, n=4) and 27% (range 0-43%) on TF+TCL combined. The proportion of responses towards frameshift mutations was 17% (range 0-24%, n=6) of total responses. The identification of neoantigen-specific T-cells within tumors from RCC patients is an important step towards the use of neoantigens as therapeutic targets and predictors of response to immunotherapy in this cancer subtype. Moreover, our study points toward the importance of broad peptide prediction platforms covering multiple sources for WXS and mutational analyses covering both point and frameshift mutations. Citation Format: Sofie Ramskov, Ulla Kring Hansen, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Marco Donia, Rikke Andersen, Zoltan Szallasi, Inge Marie Stentoft Svane, Aron Charles Eklund, Sine Reker Hadrup. Tumor infiltrating T-cells from renal cell carcinoma patients recognize neoantigens derived from point and frameshift mutations [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B092.

S. R. (2017). T cell recognition of large T and small T antigen in Merkel cell polyomavirus-associated cancer. Abstract from 44th Scandinavian Society for Immunology Meeting, Stockholm, Sweden.