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The classical serum tumor markers used routinely in the management of testicular germ cell tumor (TGCT) patients-alpha fetoprotein (AFP) and human chorionic gonadotropin (HCG)-show important limitations. miR-371a-3p is the most recent promising biomarker for TGCTs, but it is not sufficiently informative for detection of teratoma, which is therapeutically relevant. We aimed to test the feasibility of hypermethylated RASSF1A ( RASSF1A M  ) detected in circulating cell-free DNA as a non-invasive diagnostic marker of testicular germ cell tumors, combined with miR-371a-3p. A total of 109 serum samples of patients and 29 sera of healthy young adult males were included, along with representative cell lines and tumor tissue samples. We describe a novel droplet digital polymerase chain reaction (ddPCR) method for quantitatively assessing RASSF1A M  in liquid biopsies. Both miR-371a-3p (sensitivity = 85.7%) and RASSF1A M  (sensitivity = 86.7%) outperformed the combination of AFP and HCG (sensitivity = 65.5%) for TGCT diagnosis. RASSF1A M  detected 88% of teratomas. In this representative cohort, 14 cases were negative for miR-371a-3p, all of which were detected by RASSF1A M  , resulting in a combined sensitivity of 100%. We have described a highly sensitive and specific panel of biomarkers for TGCT patients, to be validated in the context of patient follow-up and detection of minimal residual disease.

cancers

Combining Hypermethylated RASSF1A Detection Using ddPCR with miR-371a-3p Testing: An Improved Panel of Liquid Biopsy Biomarkers for Testicular Germ Cell Tumor Patients