LAUSR

Abstract Disclosure: D. Tourigny: None. L. Tucci: None. Y. Elhassan: None. K. Skordilis: None. M. Asia: None. A. Prete: None. A. Crastin: None. E. Onesimu: None. A.E. Taylor: None. V. Chortis: None. C.L. Ronchi: HRA Pharmaceuticals. Adrenocortical carcinoma (ACC) is a rare cancer with heterogeneous but generally aggressive outcome. Close disease monitoring is essential but relies on radiological imaging that comes with significant radiation exposure. Circulating cell-free DNA (ccfDNA) can contain tumour-derived variants, representing a non-invasive tool for cancer monitoring. Similarly, tumour-derived steroid metabolites can be detected in urine from patients with ACC and may provide an additional surveillance tool. We aimed to evaluate the role of combined ccfDNA sequencing and urine steroid metabolomics (USM) to monitor disease recurrence in ACC. We investigated 6 patients (1M/5F, median age 37.5yrs) with histologically confirmed ACC. Plasma and 24h urine samples were collected before primary tumour resection (baseline), early post-operatively (28-42 days) and at 3-monthly follow-ups. ccfDNA and germline DNA (gDNA) were isolated with commercially available kits. Tumour DNA (tDNA) was isolated from paraffin-embedded tissue. ccfDNA/gDNA/tDNA were sequenced using a customized ACC-specific panel and by shallow (0.1x) whole genome sequencing (sWGS). Gene variants and copy number variations (CNV) were called following standard bioinformatic protocols. gDNA was used to discriminate somatic variants. 32 distinct adrenocortical steroid metabolites were quantified using gas chromatography/mass spectrometry and a previously developed generalised matrix learning vector quantisation algorithm was used to detect the presence of ACC. Disease recurrence was evaluated at periodical 3-monthly radiology scans by expert radiologists. At tDNA level, 3/6 cases (50%) presented point mutations while all 6 cases (100%) had an altered CNV pattern at sWGS. tDNA-derived somatic alterations were detected in baseline ccfDNA from 4/6 patients (71%). Pre-operative USM demonstrated steroid profiles for ACC in 5/5 patients. Three patients developed radiological recurrence at 3 or 6 months, which coincided with detection of somatic alterations in follow-up ccfDNA samples in 2/3 cases (67%). In one case, sWGS gave a clear signal for recurrence that would otherwise be missed by targeted sequencing alone. USM detected ACC-diagnostic steroids at recurrence in all cases with available urine samples, one 3 months before radiological evidence. The other three patients remain tumour free at 2-year follow up. One case presented somatic alterations at baseline tDNA/ccfDNA that disappeared in follow-up ccfDNA. USM reliably showed no evidence of ACC-diagnostic steroids in both cases with available samples during follow up. In conclusion, integrating molecular signatures from ccfDNA and USM analysis could complement standard imaging surveillance in monitoring of patients with ACC. sWGS had an additional value beyond targeted sequencing alone. Validation in a larger cohort is required to confirm these promising findings. Presentation: Monday, July 14, 2025