726P Circulating cell-free DNA-based biomarkers as a new tool for prognosis and surveillance of adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare aggressive endocrine cancer with heterogeneous behaviour. Disease surveillance relies on frequent imaging, which comes with significant radiation exposure. We investigated the role of circulating cell-free DNA (ccfDNA)-related biomarkers (BM) for prognostication and monitoring of ACC. We studied 33 patients with primary ACC and 23 healthy subjects (HS) as controls. ccfDNA was extracted by commercial kits from pre-surgical and serial EDTA plasma samples and quantified by fluorimeter (BM1). All ccfDNA samples passed the quality check showing a desired fragment length of 150-200 bp. Primary tumour DNA was isolated from paraffin-embedded tissue for 25/33 patients. Next-generation sequencing was performed using a customised panel of 30 ACC-specific genes (Cell3TM Target Nonacus). Leucocyte DNA was sequenced to discriminate germline from somatic variants (BM2). BM1 and BM2 were merged into a combined ccfDNA-BM score to be correlated with clinical outcome. ccfDNA concentrations were higher in ACC than HS (median 0.40 vs 0.05 ng/μl, P<0.001) and correlated with tumour stage and ki67 index (R=0.48 and 0.55, respectively). 94% of ACC were positive for BM1 (cut off 0.146 ng/μl, median HS+2SD) and 45.5% for BM2 (at least one somatic mutation in ccfDNA, variant allele frequency 1.0-30.5%). Mutational status at ccfDNA matched with tumour DNA in 60% of cases (additional variants in 16% and missed variants in 12%). ccfDNA-BM score (0-1=negative, 2-3=positive) was strongly associated with progression-free and overall survival (P=0.0004, HR=8.68, 95%CI=2.88-26.5, and P=0.003, HR=3.35, 95%CI=1.34-8.37, respectively). Among 13 patients followed up for at least 3 months (range 1-6), 10 tumour-free at last imaging and 1/3 with early relapse had negative ccfDNA-BM score (100% vs 33%). In two recurrent cases, somatic mutations in ACC-specific genes remained detectable during monitoring. ccfDNA-related BMs are frequently detected in patients with ACC, representing a promising non-invasive tool to predict early disease recurrence and complement imaging for monitoring. Our findings will be validated in a larger cohort of ACCs with long-term follow up.