Evaluation of a Methylated Circulating Tumour DNA Panel for Detection and Disease Stratification in Prostate Cancer

Prostate cancer (PCa) exhibits marked biological heterogeneity, complicating accurate identification of aggressive disease at initial diagnosis. This study investigated whether circulating tumour DNA (ctDNA) methylation markers enhance detection and risk stratification across the PCa disease continuum. Plasma samples from the initial hospital visit were obtained from 280 participants in the PerPros prostate biobank (Vejle, Denmark), all referred with suspected PCa. Following diagnostic work-up, participants were classified across the PCa disease spectrum or as biopsy-confirmed PCa-free controls. Five methylated CpG regions (DOCK2, HAPLN3, ACTRT2, EVX1, HOXD13) were analysed using multiplex ddPCR. Performance was assessed using ROC analysis and logistic regression and compared with prostate-specific antigen (PSA). Methylation markers were detectable across the PCa disease spectrum, most frequently in metastatic castration-sensitive PCa (mCSPC) (85/97; 88%). The ctDNA methylation panel discriminated mCSPC from controls with an AUC (area under the curve) of 0.88 (95% CI: 0.83-0.93). In this cohort, combining PSA (cut-off 20 µg/L) with the ctDNA methylation panel significantly improved discrimination between patients with mCSPC and locally advanced PCa compared with PSA alone (AUC 0.88; 95% CI 0.83-0.94; p < 0.001) vs. 0.78 (95% CI 0.71-0.84). In contrast, discrimination between localised/locally advanced PCa and patients with biopsy-confirmed controls was limited (AUC 0.56; 95% CI: 0.51-0.61). The ctDNA methylation panel demonstrates robust identification of metastatic PCa and can effectively differentiate mCSPC from locally advanced PCa. However, its diagnostic abilities in localised disease appear limited.