Background: Immunotherapy, including checkpoint inhibition, has remarkably improved prognosis in advanced melanoma. Despite this success, acquired resistance is still a major challenge. The T cell costimulatory receptor TNFRSF9 (also known as 4-1BB and CD137) is a promising new target for immunotherapy and two agonistic antibodies are currently tested in clinical trials. However, little is known about epigenetic regulation of the encoding gene. In this study we investigate a possible correlation of TNFRSF9 DNA methylation with gene expression, clinicopathological parameters, molecular and immune correlates, and response to anti-PD-1 immunotherapy to assess the validity of TNFRSF9 methylation to serve as a biomarker. Methods: We performed a correlation analyses of methylation at twelve CpG sites within TNFRSF9 with regard to transcriptional activity, immune cell infiltration, mutation status, and survival in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas. Furthermore, we used quantitative methylation-specific PCR to confirm correlations in a cohort of N = 115 melanoma patients' samples (UHB validation cohort). Finally, we tested the ability of TNFRSF9 methylation and expression to predict progression-free survival (PFS) and response to anti-PD-1 immunotherapy in a cohort comprised of N = 121 patients (mRNA transcription), (mRNA ICB cohort) and a case-control study including N = 48 patients (DNA methylation, UHB ICB cohort). Findings: We found a significant inverse correlation between TNFRSF9 DNA methylation and mRNA expression levels at six of twelve analyzed CpG sites (P 0.005), predominately located in the promoter flank

Background The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. Methods We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). Findings Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8 + and CD4 + T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. Interpretation Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. Funding A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Anti-CTLA-4-antibodies can induce long-lasting tumor remissions. However, only a few patients respond, necessitating the development of predictive companion biomarkers. Increasing evidence suggests a major role of epigenetics, including DNA methylation, in immunology and resistance to immune checkpoint blockade. Here, we tested CTLA4 promoter methylation and CTLA-4 protein expression as predictive biomarkers for response to anti-CTLA-4 immunotherapy. We identified retrospectively N = 30 stage IV melanoma patients treated with single-agent anti-CTLA-4 immunotherapy (ipilimumab). We used quantitative methylation-specific PCR and immunohistochemistry to quantify CTLA4 methylation and protein expression in pre-treatment samples. CTLA4 methylation was significantly higher in progressive as compared to responding tumors and significantly associated with progression-free survival. A subset of infiltrating lymphocytes and tumor cells highly expressed CTLA-4. However, CTLA-4 protein expression did not predict response to treatment. We conclude that CTLA4 methylation is a predictive biomarker for response to anti-CTLA-4 immunotherapy.

Background Resistance to immune checkpoint blockade and targeted therapy in melanoma patients is currently one of the major clinical challenges. With the approval of talimogene laherparepvec (T-VEC), oncolytic viruses are now in clinical practice for locally advanced or non-resectable melanoma. Here, we describe the usage of T-VEC in stage IVM1b-M1c melanoma patients, who achieved complete remission or stable disease upon systemic treatment but suffered from a locoregional recurrence. To our knowledge, there are no case reports so far describing T-VEC as a means to overcome acquired resistance to immune checkpoint blockade or targeted therapy. Methods All melanoma patients in our department treated with T-VEC in the period of 2016-2018 were evaluated retrospectively. Data on clinicopathological characteristics, treatment response, and toxicity were analyzed. Results Fourteen melanoma patients were treated with T-VEC in our center. Six patients (43%) received T-VEC first-line. In eight patients (57%), T-VEC followed a prior systemic therapy. Three patients with M1b stage and one patient with M1c stage melanoma were treated with T-VEC. These patients suffered from loco-regional progress, whilst distant metastases had regressed during prior systemic treatment. 64% of patients showed a benefit from therapy with T-VEC. The durable response rate was 36%. Conclusion T-VEC represents an effective and tolerable treatment option. This is true not only for loco-regionally advanced melanoma patients, but also for patients with stable or regressive systemic metastases who develop loco-regionally acquired resistance upon treatment with immune checkpoint blockade or targeted therapy. A sensible selection of suitable patients seems to be crucial.

Background TIGIT is an immune checkpoint under investigation as therapeutic target. Understanding the regulation of TIGIT on an epigenetic level might support the development of companion biomarkers. Methods We correlated TIGIT DNA methylation of single CpG sites with gene expression, signatures of immune infiltrates and interferon-γ, and survival in melanoma. We further analyzed methylation levels in immune cell subsets, melanocyte and melanoma cell lines. TIGIT expression patterns within components of the melanoma microenvironment were analyzed by single cell sequencing. We used quantitative methylation-specific PCR, flow cytometry, and immunohistochemistry for correlations between expression and methylation and to assess the effect of pharmacological demethylation of melanoma cells treated with 5‐aza‐2‐deoxycytidine (decitabine). Finally, we investigated the association of patients’ survival with TIGIT mRNA and methylation. Results Depending on the sequence context of the analyzed CpG site, we found a cell type-specific TIGIT gene locus methylation pattern and significant correlations of TIGIT methylation with mRNA expression, an interferon γ signature, and distinct immune cell infiltrates, including TIGIT+ lymphocytes. We detected a melanoma cell-intrinsic TIGIT protein expression. Pharmacological demethylation of the A375 melanoma cell line led to a constitutive TIGIT expression. Low promoter flank methylation and high mRNA expression was associated with patients’ prognosis and predicted progression-free survival in patients treated with anti-PD-1 immunotherapy. A high TIGIT+ lymphocyte score was associated with better progression-free survival under anti-PD-1 immunotherapy. Conclusions Our data demonstrate an epigenetic regulation of TIGIT expression via DNA methylation within the melanoma microenvironment. TIGIT DNA methylation and expression may serve as predictive biomarkers in the context of immunotherapies in melanoma.