Circulating tumor DNA (ctDNA) as a real-time biomarker in marker-negative germ cell tumors (GCT): A proof-of-concept study.

619 Background: Traditional GCT biomarkers include AFP, β-HCG, and LDH. Marker-negative GCTs—comprising seminoma, embryonal carcinoma, and teratoma with or without somatic transformation (SM)—pose diagnostic and monitoring challenges. While miRNA assays have shown promise, they are not widely available. ctDNA has emerged as a robust, dynamic biomarker across cancers, but its role in marker-negative GCT remains underexplored. Methods: A prospectively maintained institutional database of patients with GCT and available ctDNA results was analyzed. ctDNA detection was correlated with radiologic and pathologic evidence of disease across four treatment phases: orchiectomy, RPLND, initial chemotherapy, and salvage therapy. Results were considered concordant when ctDNA status matched radiologic or pathologic evidence of disease (positive/positive or negative/negative); all others were classified as discordant. Descriptive statistics were applied. Results: From December 2024 to September 2025, 33 ctDNA samples were obtained from 21 patients: 18 (86%) with testicular primaries and 6 (29%) with stage I disease. Histologies included seminoma (11, 52%), embryonal carcinoma (4, 19%), SM (4, 19%), mature teratoma (1, 5%), and mixed (1, 5%). Across all treatment phases, ctDNA detection showed 100% concordance with disease status. Serial measurements were available for 8 patients, 5 of whom had results before and after interventions (surgery, radiation, or chemotherapy). (See Table) Four patients were monitored serially during surveillance—2 maintained undetectable ctDNA with no relapse, while 2 showed ctDNA re-emergence preceding relapse. In 6 instances with indeterminate imaging, ctDNA provided adjunctive diagnostic clarity: 3/3 patients with detectable ctDNA had subsequent biopsy-proven active disease, whereas 3/3 with undetectable ctDNA remained disease-free on surveillance. Conclusions: This study provides proof-of-concept for ctDNA as a real-time biomarker in marker-negative GCTs. ctDNA trends mirrored treatment response and disease dynamics across all phases and served as a sensitive adjunct when imaging was inconclusive. To our knowledge, this is the first report of ctDNA utility in SM, warranting further exploration in monitoring and early detection of transformed teratoma. Patient Histology Pre-intervention value (MTM/ml) Intervention Post-intervention value (MTM/ml) Current Status 1 Transformed teratoma 83.77 Salvage surgery 0 (No evidence of disease)NED 2 Seminoma 0.2 RPLND 0 NED 3 Transformed teratoma 18.44 Salvage Chemotherapy 0.23 3 contd. 0.23 Salvage Surgery 0 NED 4 Seminoma 7.88 Salvage Radiation 0.24 On treatment 5 Seminoma 0.25 Orchiectomy 0 NED