The landscape of CD28, CD80, CD86, CTLA4, and ICOS DNA methylation in head and neck squamous cell carcinomas.

CTLA-4 blocking therapeutic antibodies are currently under investigation in head and neck squamous cell carcinoma (HNSCC). A better understanding of the epigenetic regulation of the CD28 superfamily members CD28, CTLA-4, and ICOS and their B7 ligands, CD80 and CD86, could support the development of biomarkers for response prediction to anti-CTLA-4 immunotherapy. We investigated methylation of the encoding genes CD28, CTLA4, ICOS, CD80, and CD86 at single CpG resolution (51 CpG sites) in a cohort of HNSCC (N = 528) and normal adjacent tissue samples (N = 50) provided by The Cancer Genome Research Atlas, in isolated blood leukocytes from healthy individuals (N = 28), and HNSCC cell lines (N = 39). We analysed methylation levels with regard to mRNA expression, overall survival, mutational load, interferon-γ signature, and signatures of immune cell infiltrates. Depending on the location of the CpG sites (promoter, promoter flank, gene body, and intergenic sites), we found significant differences in methylation levels among isolated leukocytes, between leukocytes and HNSCC cell lines, and among HNSCCs. Methylation of all analysed genes correlated inversely or positively with mRNA expression, depending on the CpG site. CD28, CTLA4, and ICOS revealed almost identical correlation patterns. Furthermore, we found significant correlations with survival and features of response to immunotherapy, i.e. interferon-γ signature, signatures of tumour infiltrating immune cells, and mutational load. Our results suggest CD28, CTLA4, ICOS, CD80, and CD86 expression levels are epigenetically co-regulated by DNA methylation. This study provides rationale to test their DNA methylation as potential biomarker for prediction of response to CTLA-4 immune checkpoint inhibitors.

LAG3 (LAG-3, CD223) DNA methylation correlates with LAG3 expression by tumor and immune cells, immune cell infiltration, and overall survival in clear cell renal cell carcinoma.

BACKGROUND: Lymphocyte activating 3 (LAG3, LAG-3, CD223) is a promising target for immune checkpoint inhibition in clear cell renal cell carcinoma (KIRC). The aim of this study was to investigate the epigenetic regulation of LAG3 in KIRC by methylation. METHODS: We correlated quantitative LAG3 methylation levels with transcriptional activity, immune cell infiltration, and overall survival in a cohort of n=533 patients with KIRC and n=160 normal adjacent tissue (NAT) samples obtained from The Cancer Genome Atlas (TCGA). Furthermore, we analyzed LAG3 methylation in peripheral blood mononuclear cells (PBMCs) and KIRC cell lines. We validated correlations between LAG3 expression, immune cell infiltrates, survival, and methylation in an independent KIRC cohort (University Hospital Bonn (UHB) cohort, n=118) by means of immunohistochemistry and quantitative methylation-specific PCR. RESULTS: We found differential methylation profiles among PBMCs, NAT, KIRC cell lines, and KIRC tumor tissue. Methylation strongly correlated with LAG3 mRNA expression in KIRCs (TCGA cohort) and KIRC cell lines. In the UHB cohort, methylation correlated with LAG3-positive immune cells and tumor-intrinsic LAG3 protein expression. Furthermore, LAG3 methylation strongly correlated with signatures of distinct immune cell infiltrates, an interferon-y signature (TCGA cohort), and immunohistochemically quantified CD45+, CD8+, and CD4+ immune cell infiltrates (UHB cohort). LAG3 mRNA expression (TCGA cohort), methylation (both cohorts), and tumor cell-intrinsic protein expression (UHB cohort) was significantly associated with overall survival. CONCLUSION: Our data suggest an epigenetic regulation of LAG3 expression in tumor and immune cells via DNA methylation. LAG3 expression and methylation is associated with a subset of KIRCs showing a distinct clinical course and immunogenicity. Our study provides rationale for further testing LAG3 DNA methylation as a predictive biomarker for response to LAG3 immune checkpoint inhibitors.

Comprehensive analysis of tumor necrosis factor receptor TNFRSF9 (4-1BB) DNA methylation with regard to molecular and clinicopathological features, immune infiltrates, and response prediction to immunotherapy in melanoma.

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 region. Consistent with its role as costimulatory receptor in immune cells, TNFRSF9 mRNA expression and hypomethylation positively correlated with immune cell infiltrates and an interferon-γ signature. Furthermore, elevated TNFRSF9 mRNA expression and TNFRSF9 hypomethylation correlated with superior overall survival. In patients receiving anti-PD-1 immunotherapy (mRNA ICB cohort), we found that TNFRSF9 hypermethylation and reduced mRNA expression correlated with poor PFS and response. INTERPRETATION: Our study suggests that TNFRSF9 mRNA expression is regulated via DNA methylation. The observed correlations between TNFRSF9 DNA methylation or mRNA expression with known features of response to immune checkpoint blockage suggest TNFRSF9 methylation could serve as a biomarker in the context of immunotherapies. Concordantly, we identified a correlation between TNFRSF9 DNA methylation and mRNA expression with disease progression in patients under immunotherapy. Our study provides rationale for further investigating TNFRSF9 DNA methylation as a predictive biomarker for response to immunotherapy. FUNDING: AF was partly funded by the Mildred Scheel Foundation. SF received funding from the University Hospital Bonn BONFOR program (O-105.0069). DN was funded in part by DFG Cluster of Excellence ImmunoSensation (EXC 1023). The funders had no role in study design, data collection and analysis, interpretation, decision to publish, or preparation of the manuscript; or any aspect pertinent to the study.

Molecular and immune correlates of TIM-3 (HAVCR2) and galectin 9 (LGALS9) mRNA expression and DNA methylation in melanoma.

BACKGROUND: The T cell immunoglobulin and mucin-domain containing-3 receptor TIM-3 (also known as hepatitis A virus cellular receptor 2, encoded by HAVCR2) and its ligand galectin 9 (LGALS9) are promising targets for immune checkpoint inhibition immunotherapies. However, little is known about epigenetic regulation of the encoding genes. This study aimed to investigate the association of TIM-3 and LGALS9 DNA methylation with gene expression, patients' survival, as well as molecular and immune correlates in malignant melanoma. RESULTS: Methylation of all six TIM-3 CpGs correlated significantly with TIM-3 mRNA levels (P ≤ 0.05). A strong inverse correlation (Spearman's ρ = - 0.49) was found in promoter regions, while a strong positive correlation (ρ = 0.63) was present in the gene body of TIM-3. High TIM-3 mRNA expression (hazard ratio (HR) = 0.88, 95% confidence interval (CI) [0.81-0.97], P = 0.007) was significantly associated with better overall survival. Seven of the eight LGALS9 CpG sites correlated significantly with LGALS9 mRNA levels (P ≤ 0.003). Methylation at five CpG sites showed a strong inverse correlation (Spearman's ρ = - 0.67) and at two sites a weak positive correlation (Spearman's ρ = 0.15). High LGALS9 mRNA expression was significantly associated with increased overall survival (HR = 0.83, 95%CI [0.75-0.93], P = 0.001). In addition, we found significant correlations between TIM-3 and LGALS9 methylation and mRNA expression with immune cell infiltrates and significant differences among distinct immune cell subsets. CONCLUSIONS: Our study points toward an epigenetic regulation of TIM-3 and LGALS9 via DNA methylation and might provide an avenue for the development of a predictive biomarker for response to immune checkpoint blockade.

DNA methylation of indoleamine 2,3-dioxygenase 1 (IDO1) in head and neck squamous cell carcinomas correlates with IDO1 expression, HPV status, patients' survival, immune cell infiltrates, mutational load, and interferon γ signature.

BACKGROUND: The immune checkpoint, indoleamine 2,3-dioxygenase 1, is under investigation as target of novel immunotherapies for cancers, including head and neck squamous cell carcinomas (HNSCC). The aim of our study was to analyze DNA methylation of the encoding gene (IDO1) in HNSCC. METHODS: Methylation of three CpG sites within the promoter, promoter flank, and gene body was investigated and correlated with mRNA expression, immune cell infiltration, mutational burden, human papillomavirus (HPV)-status, and overall survival in a cohort of N = 528 HNSCC patients obtained from The Cancer Genome Atlas. In addition, IDO1 immunohistochemistry and DNA methylation analysis was performed in an independent cohort of N = 138 HNSCC samples. FINDINGS: Significant inverse correlations of IDO1 methylation and IDO1 mRNA expression were found in the promoter and promoter flank region (Spearman's ρ = -0.163 and ρ = -0.377, respectively) while a positive correlation was present in the gene body (ρ = 0.502; all P < 0.001). IDO1 DNA methylation significantly correlated with IDO1 protein expressing immune cells as well as tumor cells. IDO1 promoter flank hypermethylation was significantly associated with poor overall survival (P < 0.001). In addition, we discovered significant correlations between IDO1 methylation and expression with RNA signatures of immune cell infiltrates and with HPV-status, mutational load (methylation only), and interferon γ signature. INTERPRETATION: Our results suggest IDO1 expression levels are epigenetically regulated by DNA methylation. This study provides rationale to test IDO1 methylation as potential biomarker for prediction of response to IDO1 immune checkpoint inhibitors in HNSCC.