Circulating Cell-Free SEPT9 DNA Methylation in Blood Is a Biomarker for Minimal Residual Disease Detection in Head and Neck Squamous Cell Carcinoma Patients.

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.

CTLA4 , PD-1 , PD-L1 , PD-L2 , TIM-3 , TIGIT , and LAG3 DNA Methylation Is Associated With BAP1 -Aberrancy, Transcriptional Activity, and Overall Survival in Uveal 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.