Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, primarily due to failed early detection. HCC screening is recommended among individuals with cirrhosis using biannual abdominal ultrasound, for earlier tumor detection, administration of curative treatment, and improved survival. Surveillance by imaging with or without biomarkers such as alpha-fetoprotein (AFP) remains suboptimal for early stage HCC detection. Here we report on the development and assessment of methylation biomarkers from liquid biopsies for HCC surveillance in cirrhotic patients. METHODS: DNA methylation markers including the HCCBloodTest (Epigenomics AG) and a DNA-methylation panel established by next generation sequencing (NGS) were assessed using a training/testing design. The NGS panel algorithm was established in a training study (41 HCC patients; 46 cirrhotic non-HCC controls). For testing, plasma samples were obtained from cirrhotic patients (Child class A or B) with (60) or without (103) early stage HCC (BCLC stage 0, A, B). The assays were then tested using blinded sample sets and analyzed by preset algorithms. RESULTS: The HCCBloodTest and the NGS panel exhibited 76.7% and 57% sensitivities at 64.1% and 97% specificity, respectively. In a post-hoc analysis, a combination of the NGS panel with AFP (20 ng/mL) achieved 68% sensitivity at 97% specificity (AUC = 0.9). CONCLUSIONS: Methylation biomarkers in cell free plasma DNA provide a new alternative for HCC surveillance. Multiomic panels comprising DNA methylation markers with other biological markers, such as AFP, provide an option to further increase the overall clinical performance of surveillance via minimally invasive blood samples. TRIAL REGISTRATION: Test set study-ClinicalTrials.gov (NCT03804593) January 11, 2019, retrospectively registered.

An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens: sample preparation for epigenetic analysis.

OBJECTIVE: DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5 mL of plasma or urine, using reagents from the commercially available Epi BisKit. RESULTS: Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4 × 24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7 h 30 min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized ß-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.

Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy.

Prostate cancer is the most common cancer among men. The prospective discrimination of aggressive and clinically insignificant tumors still poses a significant and, as yet, unsolved problem. PITX2 DNA methylation is a strong prognostic biomarker in prostate cancer. Recently, a diagnostic microarray for prostate cancer prognosis based on PITX2 methylation has been developed and validated. Because this microarray requires nonstandard laboratory equipment, its use in a diagnostic setting is limited. This study aimed to develop and validate an alternative quantitative real-time PCR assay for measuring PITX2 methylation that can easily be established in clinical laboratories, thereby facilitating the implementation of this biomarker in clinical practice. A methylation cut-off for patient stratification was established in a training cohort (n = 157) and validated in an independent test set (n = 523) of men treated with radical prostatectomy. In univariate Cox proportional hazards analysis, PITX2 hypermethylation was a significant predictor for biochemical recurrence (P < 0.001, hazard ratio = 2.614). Moreover, PITX2 hypermethylation added significant prognostic information (P = 0.003, hazard ratio = 1.814) to the Gleason score, pathological T stage, prostate-specific antigen, and surgical margins in a multivariate analysis. The clinical performance was particularly high in patients at intermediate risk (Gleason score of 7) and in samples containing high tumor cell content. This assay might aid in risk stratification and support the decision-making process when determining whether a patient might benefit from adjuvant treatment after radical prostatectomy.

DNA methylation of the homeobox genes PITX2 and SHOX2 predicts outcome in non-small-cell lung cancer patients.

Biomarkers that facilitate prediction of disease progression in lung cancer patients might be clinically valuable in optimizing individualized therapy. In this study, the ability of the DNA methylation biomarkers PITX2 and SHOX2 to predict disease outcome in lung cancer patients has been evaluated. Quantitative, methylation-specific (HeavyMethyl), real-time polymerase chain reaction assays were used to measure DNA methylation of PITX2 and SHOX2 in bisulfite-converted DNA from formalin-fixed, paraffin-embedded tissues from 474 non-small-cell lung cancer patients. In univariate Cox Proportional Hazard analysis, high methylation of SHOX2 and PITX2 was a significant predictor of progression-free survival [SHOX2: n=465, hazard ratio (HR)=1.395 (1.130 to 1.721), P=0.002; PITX2: n=445, HR=1.312 (1.059 to 1.625), P=0.013]. Patients with low methylation of either PITX2 and/or SHOX2 (n=319) showed a significantly higher risk of disease progression as compared with patients with higher methylation of both genes [n=126; HR=1.555 (1.210 to 1.999), P=0.001]. This was particularly true for the subgroup of patients receiving no adjuvant radiotherapy or chemotherapy [n=258, HR=1.838 (1.252 to 2.698), P=0.002]. In multivariate analysis, both biomarkers added significant independent prognostic information to pT, pN, pM, and grade. Another interesting finding of this study was that SHOX2 and PITX2 DNA methylation was shown to be inversely correlated with TTF1 (also known as NKX2-1) expression (PITX2: P=0.018, SHOX2: P<0.001). TFF1 expression was previously found to be associated with improved survival in the same patient cohort. DNA methylation of PITX2 and SHOX2 is an independent prognostic biomarker for disease progression in non-small-cell lung cancer patients.