Droplet Digital PCR Assay for Detection and Monitoring of Universally Methylated Circulating Tumor DNA in Sarcoma Patients

Abstract Purpose: No universal circulating biomarker exists for soft tissue (STS) and bone sarcoma (BS). We report the translational relevance of a droplet digital PCR (ddPCR) assay allowing universal, specific and dynamic detection of sarcoma-related hypermethylated circulating tumor DNA (ctDNA). Experimental Design: In-silico analysis (TCGA/GEO datasets, n=8330) identified hypermethylated DNA positions in STS/BS, unmethylated in non-sarcoma tissues or white blood cells releasing circulating plasma cell-free DNA (cfDNA). A ddPCR assay following bisulfite conversion of cfDNA was developed. The methylation signature performances were evaluated in independent in-silico cohorts (TCGA/GEO, n=1342). The ddPCR assay was applied to cfDNA from healthy donors, patients with metastatic STS (METASARC cohort, n=49, 13 histotypes), and patients with STS/BS treated with neoadjuvant chemotherapy (NEOSARC cohort, n=42, 10 histotypes). Results: A ddPCR assay targeting seven methylated genomic positions distinguished sarcoma samples versus non-neoplastic mesenchymal and endothelial/liver tissues (AUC=0.95; in silico validation set). Sensitivity allowed methylated DNA detection at 1:1000 dilution in genomic DNA, with a methylated allele frequency of 0.06%. CtDNA was positively detected in 45% of METASARC (22/49) and 74% of NEOSARC (31/42) patients, across all histotypes. CtDNA detection correlated with poor overall survival in METASARC patients with STS (p=0.039). Increasing ctDNA during neoadjuvant chemotherapy was associated with poor outcomes in NEOSARC (composite criteria with poor histological response, radiological progression or relapse within 6 months; p=0.0095). Conclusions: This sensitive ddPCR assay for universally methylated ctDNA enables precise detection, prognostication, and real-time monitoring of tumor burden in patients with high-grade and advanced sarcoma, regardless of histotype or origin.