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

BackgroundThe 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.MethodsMethylation 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.FindingsSignificant 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.InterpretationOur 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.

Background Few diagnostic and prognostic biomarkers are available for head-and-neck squamous cell carcinoma (HNSCC). Long non-coding RNAs (lncRNAs) have shown promise as biomarkers in other cancer types and in some cases functionally contribute to tumor development and progression. Here, we searched for lncRNAs useful as biomarkers in HNSCC. Methods Public datasets were mined for lncRNA candidates. Two independent HNSCC tissue sets and a bladder cancer tissue set were analyzed by RT-qPCR. Effects of lncRNA overexpression or downregulation on cell proliferation, clonogenicity, migration and chemosensitivity were studied in HNSCC cell lines. Results Data mining revealed prominently CASC9, a lncRNA significantly overexpressed in HNSCC tumor tissues according to the TCGA RNAseq data. Overexpression was confirmed by RT-qPCR analyses of patient tissues from two independent cohorts. CASC9 expression discriminated tumors from normal tissues with even higher specificity than HOTAIR, a lncRNA previously suggested as an HNSCC biomarker. Specificity of HNSCC detection by CASC9 was further improved by combination with HOTAIR. Analysis of TCGA pan-cancer data revealed significant overexpression of CASC9 across different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. By RT-qPCR analysis we furthermore detected stronger CASC9 overexpression in pure SCC of the urinary bladder and mixed urothelial carcinoma with squamous differentiation than in pure urothelial carcinomas. Thus, CASC9 might represent a general diagnostic biomarker and particularly for SCCs. Unexpectedly, up- or downregulation of CASC9 expression in HNSCC cell lines with low or high CASC9 expression, respectively, did not result in significant changes of cell viability, clonogenicity, migration or chemosensitivity. Conclusions CASC9 is a promising biomarker for HNSCC detection. While regularly overexpressed, however, this lncRNA does not seem to act as a major driver of development or progression in this tumor. Electronic supplementary material The online version of this article (10.1186/s12885-019-6021-6) contains supplementary material, which is available to authorized users.

The tumor necrosis factor receptor superfamily members 4 (TNFRSF4, OX40) and 18 (TNFRSF18, GITR, AITR) are under investigation as targets for immunotherapy of various cancers, including head and neck squamous cell carcinomas. Understanding the regulation of OX40 and GITR, particularly on an epigenetic level, might help to develop companion predictive biomarkers. We conducted broad correlation analyses of DNA methylation of 46 CpG sites within the GITR/ OX40 gene locus in head and neck squamous cell carcinomas and normal adjacent tissues provided by The Cancer Genome Atlas (TCGA) Research Network. We analyzed methylation levels with regard to transcriptional gene activity (mRNA expression), human papillomavirus (HPV) infection, differential methylation between tumors and normal adjacent tissues, signatures of immune cell infiltrates, an interferon-γ signature, mutational load, and overall survival. Moreover, we investigated methylation levels in HPV-positive and HPVnegative cell lines and in isolated monocytes, granulocytes, CD8 + and CD4 + T cells, and B cells from peripheral blood from healthy donors. Our results revealed a complex and sequence-contextual methylation pattern in accordance with features of epigenetic regulated genes. We detected significant methylation differences between normal adjacent and tumor tissues, between HPV-positive and HPV-negative tumors, between tumor and immune cells, and significant correlations between methylation and mRNA expression. We further found significant correlations of CpG methylation with overall survival, signatures of immune cell infiltrates, an interferon-γ signature, and mutational load.Our study provides a framework to prospectively test specific CpG sites as biomarkers, in particular in the context of immunotherapies.