DNA methylation regulates TIGIT expression within the melanoma microenvironment, is prognostic for overall survival, and predicts progression-free survival in patients treated with anti-PD-1 immunotherapy.

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.

DNA Methylation and mRNA Expression of OX40 (TNFRSF4) and GITR (TNFRSF18, AITR) in Head and Neck Squamous Cell Carcinoma Correlates With HPV Status, Mutational Load, an Interferon-γ Signature, Signatures of Immune Infiltrates, and Survival.

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 HPV-negative 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.

The DNA methylation landscape of PD-1 (PDCD1) and adjacent lncRNA AC131097.3 in head and neck squamous cell carcinoma.

Aims:PD-1 expression is associated with DNA methylation in head and neck squamous cell carcinomas (HNSCCs). We performed methylation analysis at single CpG site resolution in order to understand epigenetic regulation. Materials and methods: CpG methylation analysis of PD-1 and long non-coding RNA (lncRNA) AC131097.3 was performed in n = 528 HNSCCs and n = 50 normal adjacent tissues provided by The Cancer Genome Atlas and in isolated leukocytes. Results:PD-1 mRNA and AC131097.3 lncRNA expression correlated inversely with promoter and positively with gene body CpG methylation. PD-1 and AC131097.3 are co-expressed. Methylation was sequence-contextually associated with human papillomavirus prognosis, mutational load, and immune infiltrates. Conclusions: The significance of PD-1 and AC131097.3 methylation is highly sequence-contextual. AC131097.3 might play a role in HNSCC.

Molecular, clinicopathological, and immune correlates of LAG3 promoter DNA methylation in melanoma.

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.

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.

Detailed analysis of adenosine A2a receptor (ADORA2A) and CD73 (5'-nucleotidase, ecto, NT5E) methylation and gene expression in head and neck squamous cell carcinoma patients.

Background: The adenosine A2a receptor (A2aR) and the adenosine synthesizing enzyme CD73 have recently evolved as a novel immunotherapeutic target. However, little is known about epigenetic modification of the encoding genes ADORA2A and NT5E. Methods: In the present study, we evaluated methylation at 23 loci of ADORA2A and 17 loci of NT5E with regard to transcriptional activity, human papilloma virus (HPV) status, immune cell infiltration, and outcome in a cohort of 279 head and neck squamous carcinoma (HNSCC) patients obtained from The Cancer Genome Atlas (TCGA). Methylation and mRNA expression were generated by the Infinium HumanMethylation450 BeadChip and Illumina HiSeq 2000 RNA Sequencing Version 2 analysis (Illumina, Inc., San Diego, CA, USA). HPV status was assessed by RNA-Seq data analysis of the viral genes E6 and E7. Results: Thirteen out of 23 ADORA2A loci and 15/17 NT5E loci were significantly correlated with mRNA levels (p < 0.05). Inverse correlations were predominately found in promoter regions, while positive correlations were more profound at intragenic loci. ADORA2A hypermethylation was significantly associated with poor overall survival (OS, p ≤ 0.030), whereas NT5E hypomethylation was associated with decreased OS in HPV-positive tumors (p ≤ 0.024) and increased OS in HPV-negative HNSCC (p ≤ 0.029). Further, we found significant correlations between methylation and immune cell infiltrates. Conclusion: Our data might point towards a significant role of the A2aR/CD73 axis during cancer progression in HNSCC.

CTLA4 methylation predicts response to anti-PD-1 and anti-CTLA-4 immunotherapy in melanoma patients.

Recent years have witnessed the groundbreaking success of immune checkpoint blockage (ICB) in metastasized malignant melanoma. However, biomarkers predicting the response to ICB are still urgently needed. In the present study, we investigated CTLA4 promoter methylation (mCTLA4) in 470 malignant melanoma patients from The Cancer Genome Atlas (non-ICB cohort) and in 50 individuals with metastasized malignant melanomas under PD-1/CTLA-4-targeted immunotherapy (ICB cohort). mCTLA4 levels were quantified using the Infinium HumanMethylation450 BeadChip (non-ICB cohort) and methylation-specific quantitative real-time PCR in DNA formalin-fixed and paraffin-embedded tissues (ICB cohort). Methylation levels were associated with molecular and clinicopathological variables and analyzed with respect to response (irRECIST) and overall survival. CTLA-4 mRNA and mCTLA4 showed a significant inverse correlation (non-ICB cohort: Spearman's ρ = -0.416, P < 0.001). In ICB-treated melanoma patients, low mCTLA4 was further strongly correlated with response to therapy (P = 0.009, ANOVA) and overall survival (hazard ratio = 2.06 [95% CI: 1.29-3.29], P = 0.003). Our data strongly support the assumption that mCTLA4 predicts response to both anti-PD-1 and anti-CTLA-4 targeted ICB in melanoma and provides paramount information for the selection of patients likely to respond to ICB.

Intragenic DNA methylation of PITX1 and the adjacent long non-coding RNA C5orf66-AS1 are prognostic biomarkers in patients with head and neck squamous cell carcinomas.

BACKGROUND: Patients with squamous cell cancer of the head and neck region (HNSCC) are at risk for disease recurrence and metastases, even after initial successful therapy. A tissue-based biomarker could be beneficial to guide treatment as well as post-treatment surveillance. Gene methylation status has been recently identified as powerful prognostic biomarker in HNSCC. We therefore evaluated the methylation status of the homeobox gene PITX1 and the adjacent long intergenic non-coding RNA (lincRNA) C5orf66-AS1 in publicly available datasets. METHODS: Gene methylation and expression data from 528 patients with HNSCC included in The Cancer Genome Atlas (TCGA, there obtained by using the Infinium HumanMethylation450 BeadChip Kit) were evaluated and methylation and expression levels of PITX1 and lincRNA C5orf66-AS1 was correlated with overall survival and other parameters. Thus, ten beads targeting PITX1 exon 3 and three beads targeting lincRNA C5orf66-AS1 were identified as significant candidates. The mean methylation of these beads was used for further correlation and the median was employed for dichotomization. RESULTS: Both PITX1 exon 3 and lincRNA C5orf66-AS1 were significantly higher methylated in tumor tissue than in normal adjacent tissue (NAT) (PITX1 exon 3: tumor tissue 58.1%, NAT: 31.7%, p<0.001; lincRNA C5orf66-AS1: tumor tissue: 27.4%, NAT: 18.9%, p<0.001). In a univariate analysis, hypermethylation of both loci was significantly associated with the risk of death (univariate: exon 3: Hazard ratio (HR): 4.97 [1.78-16.71], p = 0.010, lincRNA C5orf66-AS1: Hazard ratio (HR): 12.23 [3.01-49.74], p<0.001). PITX1 exon 3 and lincRNA C5orf66-AS1 methylation was also significantly correlated with tumor localization, T category, human papilloma virus (HPV)-negative and p16-negative tumors and tumor grade. Kaplan-Meier analysis showed, that lincRNA C5orf66-AS1 hypomethylation was significantly associated with overall survival (p = 0.001) in the entire cohort as well in a subgroup of HPV-negative tumors (p = 0.003) and in patients with laryngeal tumors (p = 0.022). CONCLUSION: Methylation status of PITX1 and even more so of lincRNA C5orf66-AS1 is a promising prognostic biomarker in HNSCC, in particular for HPV-negative patients. Further prospective evaluation is warranted.

PD-L1 (CD274) and PD-L2 (PDCD1LG2) promoter methylation is associated with HPV infection and transcriptional repression in head and neck squamous cell carcinomas.

BACKGROUND: DNA methylation of the immune checkpoint gene PD-L1 has recently been shown to be associated with PD-L1 mRNA expression in various malignancies. This study aimed to investigate the association of PD-L1 and PD-L2 methylation with mRNA expression, immune cell infitration, protein expression and human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) patients. RESULTS: DNA methylation of PD-L1 and PD-L2 correlates inversely with mRNA expression (PD-L1: p ≤ 0.002; PD-L2: p ≤ 0.014). Methylation of specific CpG-sites of both PD-L1 and PD-L2 were further significantly associated with HPV infection in the TCGA cohort. Immune cell infiltrates correlated significantly with PD-L1 and PD-L2 methylation. In the validation cohort, PD-L1 protein expression was associated with PD-L1 hypomethylation (p = 0.012). CONCLUSIONS: DNA methylation of PD-L1 and PD-L2 is associated with transcriptional silencing and HPV infection in HNSCCs. Additional studies are warranted to test PD-L1 and PD-L2 methylation as predictive biomarkers for response to immunotherapies (e.g. pembrolizumab and nivolumab) that target the PD-L1/PD-L2/PD-1 immune checkpoint axis. MATERIALS AND METHODS: PD-L1 and PD-L2 promoter methylation and its mRNA expression were analyzed based on Infinium HumanMethylation450 BeadChip and RNA-Seq (both Illumina, Inc.) data in a representative HNSCC patient cohort (n = 528) enrolled by The Cancer Genome Atlas (TCGA) Research Network. A validation cohort consisting of 168 HNSCC patients treated at the University Hospital Bonn was analyzed regarding PD-L1 and PD-L2 promoter methylation by means of methylation-specific quantitative real-time PCR. PD-L1 protein expression in the validation cohort was quantified via immunohistochemistry (PD-L1 antibody clone 22C3, Dako/Agilent Technologies, Inc.).