Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes.

Background SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance.MethodsMethylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [meanSEPT9/SHOX2] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated.ResultsSporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61–63% sensitivity/90–92% specificity (SEPT9/training), 53–57% sensitivity/87–90% specificity (SHOX2/training), and 64–65% sensitivity/90–91% specificity (meanSEPT9/SHOX2/training). Results were confirmed in a testing cohort with 54–56% sensitivity/88–90% specificity (SEPT9/testing), 43–48% sensitivity/93–95% specificity (SHOX2/testing), and 49–58% sensitivity/88–94% specificity (meanSEPT9/SHOX2/testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals (SEPT9: AUCtraining = 0.79–0.80; AUCtesting = 0.74–0.75; SHOX2: AUCtraining = 0.78–0.81, AUCtesting = 0.77–0.79; meanSEPT9/SHOX2: AUCtraining = 0.81–0.84, AUCtesting = 0.80). The accurate prediction of overall survival was possible with all three algorithms (training cohort: HRSEPT9 = 1.23-1.90, HRSHOX2 = 1.14-1.85, HRmeanSEPT9/SHOX2 =1.19-1.89 ; testing cohort: HRSEPT9 =1.22-1.67, HRSHOX2 = 1.15-1.71, HRmeanSEPT9/SHOX2 = 1.12-1.77).ConclusionThe concordant clinical performance based on different quantification algorithms allows for the application of various diagnostic platforms for the analysis of ccfDNA methylation biomarkers.Electronic supplementary materialThe online version of this article (10.1186/s13148-017-0425-4) contains supplementary material, which is available to authorized users.

BackgroundThe 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.ResultsMethylation 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.ConclusionsOur 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.

BackgroundBiomarkers that facilitate the prediction of disease recurrence in head and neck squamous cell carcinoma (HNSCC) may enable physicians to personalize treatment. In the current study, DNA promoter methylation of programmed cell death 1 (PDCD1, PD-1) was evaluated as a prognostic biomarker in HNSCC patients.ResultsHigh PDCD1 methylation (mPDCD1) was associated with a significantly shorter overall survival after surgical resection in both the discovery (HR = 2.24 [95%CI: 1.08–4.64], p = 0.029) and the validation cohort (HR = 1.54 [95%CI: 1.08–2.21], p = 0.017). In multivariate Cox proportional hazards analysis, PDCD1 methylation remained a significant prognostic factor for HNSCC (HR = 2.14 [95%CI: 1.19–3.84], p = 0.011). Further, mPDCD1 was strongly associated with the human papilloma virus (HPV) status.Materials and MethodsmPDCD1 was assessed retrospectively in a discovery cohort of 120 HNSCC patients treated at the University Hospital of Bonn and a validation cohort of 527 HNSCC cases analyzed by The Cancer Genome Atlas Research Network.ConclusionsPDCD1methylation might aid the identification of HNSCC patients potentially benefitting from a radical or alternative treatment, particularly in the context of immunotherapies targeting PD-1/PD-L1.

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