Examination There was an exophytic, redish tumor of the lateral wall of the hypopharynx. The mobility of the vocal chords was not impaired. The glottic lumen was subtotaly obstructed by the tumor. A CT scan of the neck and a panendoscopy with an excision biopsy were performed. A Kaposi's sarcoma was diagnosed.

Ein 52-jähriger, homosexueller Patient stellte sich mit seit vier Monaten bestehender Dysphagie und Dyspnoe ohne Stridor vor. Eine weitere Symptomatik bestand nicht.

BACKGROUND:Tumorous SEPT9 (septin 9, SEPTIN9) circulating cell-free DNA (ccfDNA) methylation in blood plasma is a powerful biomarker for diagnosis, molecular staging, prognosis, and recurrence monitoring in head and neck squamous cell carcinoma (HNSCC) patients. The present study aimed to evaluate the clinical performance of SEPT9 ccfDNA methylation to detect post-surgical minimal residual disease (MRD) in patients with localized or locally advanced HNSCC treated with curative intent.METHODS:We applied quasi-digital methylation-specific real-time PCR to quantify SEPT9 ccfDNA methylation levels 2 to 30 days post-surgically in plasma from n = 219 prospectively enrolled HNSCC patients. We tested the associations of SEPT9 ccfDNA methylation with clinicopathological parameters and used Kaplan-Meier and Cox proportional hazards analyses for univariate, pairwise bivariate, and multivariate analyses of disease-free survival.RESULTS:Of 219 patients, 26.5% (58/219) were post-surgically SEPT9 ccfDNA methylation positive. SEPT9 ccfDNA methylation positivity was significantly associated with tumor site, American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC; 8th edition) tumor stage, nodal category and extracapsular extension, lymphatic and vascular invasion, and surgical margin. Bivariate Cox proportional hazards analysis proved post-surgical SEPT9 ccfDNA methylation positivity to be an independent prognostic factor tested together with AJCC/UICC tumor stage (SEPT9: hazard ratio [HR] = 2.43, 95% CI, 1.37-4.30, P = 0.002; AJCC/UICC stage: HR = 1.48, 95% CI, 1.11-1.98, P = 0.008).CONCLUSIONS:Post-surgical SEPT9 ccfDNA methylation may aid to identify high-risk HNSCC patients who could benefit from an intensified adjuvant treatment and surveillance.

Background The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value. Methods We analyzed CTLA4 promoter methylation in tumors of HNSCC patients ( N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort ( N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine. Results Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3 + , CD4 + , CD8 + , and CD45 + immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines. Conclusions Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.

Liquid biopsies allow for the non-invasive detection of tumorous circulating cell-free DNA (ccfDNA) in blood plasma of head and neck squamous cell carcinoma (HNSCC) patients. The current study tested the performance of post-surgical SEPT9 ccfDNA methylation as a molecular staging parameter in HNSCC as adjunct to established prognostic factors. A cohort of n = 219 prospectively enrolled HNSCC patients suffering from localized or locally advanced disease were tested for post-surgical SEPT9 ccfDNA methylation in their blood plasma via quantitative methylation specific real-time PCR. SEPT9 ccfDNA methylation positivity was then correlated with established prognostic factors, namely 8th edition of AJCC/UICC stage, surgical margin, lymphatic and vascular invasion. Post-surgical SEPT9 DNA methylation positivity was detected in 26.5 % (58/219) of all analyzed cases and is significantly associated with AJCC/UICC stage (P = 0.001), lymphatic invasion (P = 0.032), vascular invasion (P = 0.012), and a histopathologically verified positive surgical margin (P < 0.001) in localized and locally advanced HNSCC.Post-surgical SEPT9 ccfDNA methylation in blood is a promising molecular staging parameter in localized and locally advanced HNSCC patients. Future studies are necessary to evaluate the clinical relevance of post-surgical SEPT9 ccfDNA methylation positivity to personalize patients’ adjuvant treatment.

Einleitung Immuncheckpoint (IC) Inhibitoren erweiterten das Therapiespektrum für rekurrente und metastasierte Plattenepithelkarzinome im Kopf-Hals-Bereich (HNSCC). Eine Begrenzung stellt die niedrige Ansprechrate von 10-15% dar. Die DNA-Methylierung ist ein vielversprechender Biomarker, um Patienten zu identifizieren, die von einer Immuntherapie profitieren. Bedeutende Ergebnisse wurden für die prädiktive Rolle der DNA Methylierung des IC Gens T lymphocyte-associated protein 4 (CTLA4) beobachtet.

Introduction The tumor necrosis factor receptor superfamily member 7 (TNFRSF7, also known as CD27) is an immune checkpoint that is currently investigated as a target for immunotherapy of cancers, including head and neck squamous cell carcinomas (HNSCCs). A comprehensive understanding of the CD27 regulation, in particular on an epigenetic level, might aid in the development of companion predictive biomarkers.

Einleitung Dank des Erfolges von Immuntherapien werden eine Vielzahl von Immun-Checkpoints, wie B- and T-Lymphocyte Attenuator (BTLA), als Ziele für die Therapie von Tumoren, einschließlich des Kopf-Hals-Plattenepithelkarzinoms (HNSCC), untersucht. Es bedarf dringend an prädiktiven Biomarkern, um Patienten zu identifizieren, die auf derartige Therapien ansprechen. Das umfangreiche Verständnis der BTLA Regulation auf einer epigenetischen Ebene könnte den Weg zur Entwicklung solcher Marker bereiten. Deshalb zielte diese Studie auf eine umfassende Untersuchung der DNA Methylierung von BTLA im HNSCC im Hinblick auf die mRNA Expression, den humanen Papillomavirus-Status (HPV), die Immunzellinfiltration, isolierte Immunzellen, sowie das Überleben.

Introduction Due to the success of immunotherapy, several immune checkpoints, such as the immunosuppressive B- and T-Lymphocyte Attenuator (BTLA), are investigated as potential therapeutic targets for therapy of various cancers, including head and neck squamous cell carcinoma (HNSCC). Predictive biomarkers are desperately needed in order to identify patients responding to such treatments. The comprehensive understanding of BTLA regulation on an epigenetic level might provide an avenue in the development of such markers.

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.

Uveal melanoma (UM) is an aggressive disease with poor response to oncological treatment, including immunotherapy. Loss of the epigenetic modifier BRCA1-associated protein 1 (BAP1) function drives UM oncogenesis and is associated with an immune-suppressive tumor microenvironment, poor prognosis, and a distinct DNA methylation and gene expression profile. Our study aimed to analyze comprehensively the DNA methylation status of the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4, TIM-3 ( HAVCR2 ), TIGIT , and LAG3 and its association with mRNA expression, BAP1 -aberrancy, and patients' survival. We analyzed the DNA methylation landscape of immune checkpoint genes at single CpG resolution in N=80 UM samples provided by The Cancer Genome Atlas. We analyzed CpG methylation levels of the immune checkpoints with regard to their transcriptional signatures and patient outcomes.Methylation of specific CpG sites within the immune checkpoint genes PD-1 , PD-L1 , PD-L2 , CTLA4 , TIM-3 , TIGIT , and LAG3 correlated strongly with mRNA expression levels, indicating a strong regulation of gene expression through DNA methylation. Moreover, immune checkpoint gene methylation was strongly associated with BAP1 -mutation status and associated with overall survival in UM. Our data indicate an epigenetic regulation of immune checkpoints through DNA methylation in UM. Further, our data highlight the prognostic significance of DNA methylation of immune checkpoint genes in UM thereby providing a rationale for methylation testing as predictive biomarkers for immunotherapy response.

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.

Background Immune checkpoint (IC) inhibitors extended the range of therapies for recurred or metastasized head and neck squamous cell carcinoma (HNSCC). A limitation is the low response rate of only 10-15%. DNA methylation is a promising biomarker to select patients benefitting from immunotherapy. Substantiated results were observed for DNA methylation of the IC gene Tlymphocyte-associated protein 4 (CTLA4) and its predictive value.

Flüssigbiopsien („Liquid Biopsies”) ermöglichen die nicht-invasive Bestimmung tumoröser zellfreier zirkulierender DNA (ccfDNA) im Blutplasma von Kopf-Hals-Karzinom (HNSCC) Patienten. Die aktuelle Studie untersucht die Leistungsfähigkeit der postoperativ gemessenen SEPT9 ccfDNA-Methylierung als molekularen Staging-Parameter für HNSCC-Patienten ergänzend zu etablierten prognostischen Parametern. Eine prospektiv zusammengestellte HNSCC-Patientenkohorte, bestehend aus n=219 Patienten mit lokalisierter oder lokal fortgeschrittener Tumorerkrankung, wurde postoperativ mittels methylierungsspezifischer real-time PCR auf die SEPT9 ccfDNA-Methylierung im Blutplasma untersucht. Anschließend wurde der statistische Zusammenhang zwischen der SEPT9 ccfDNA-Methylierung und den etablierten prognostischen Faktoren UICC-TNM Stadium (8. Auflage), dem Resektionsrand sowie der Lymphgefäß- und Veneninvasion analysiert. Postoperative Positivität der SEPT9 ccfDNA-Methylierung wurde in 26,5% (58/219) aller untersuchten Proben nachgewiesen. Die SEPT9 ccfDNA-Methylierungs-Positivität korrelierte signifikant mit dem UICC-TNM Stadium (P=0,001), der Lymphgefäß- (P=0,032) sowie Veneninvasion (P=0,012) und dem Resektionsrand (P<0,001). Die postoperative Bestimmung der SEPT9 ccfDNA-Methylierung im Blutplasma ist ein vielversprechender molekularer Staging-Parameter für Patienten mit lokalisierten oder lokal fortgeschrittenen Plattenepithelkarzinomen des Kopf-Hals-Bereiches. Zukünftige Studien sind erforderlich um die klinische Relevanz der postoperativ bestimmten SEPT9 ccfDNA-Methylierung für die Personalisierung der adjuvanten Therapie der Patienten zu untersuchen.

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.

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.

Immunotherapy is a promising treatment for head and neck squamous cell carcinomas (HNSCC) patients. TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a promising target for a novel immune checkpoint inhibition. Therefore, this study aimed at investigating DNA methylation of TIGIT with regard to mRNA expression, molecular features, immune cell infiltrates and outcome of HNSCC patients.

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.

Immuntherapien haben in den letzten Jahren verstärkt Einzug in die Behandlung der Plattenepithelkarzinome des Kopf-Hals-Bereichs (HNSCC) gehalten. Ein vielversprechendes Target einer neuartigen Immuntherapie ist TIGIT (T cell immunoreceptor with Ig and ITIM domains). Im Rahmen dieser Arbeit wurde die DNA-Methylierung von TIGIT hinsichtlich der Assoziation mit der mRNA-Expression, molekularer Eigenschaften, Immunzellinfiltraten und dem Überleben von Patienten mit Kopf-Hals-Plattenepithelkarzinomen untersucht.

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. 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. 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.