Droplet Digital PCR Assay for Detection and Monitoring of Universally Methylated ctDNA in Patients with Sarcoma

PURPOSE: No universal circulating biomarker exists for soft-tissue sarcoma (STS) and bone sarcoma. We report the translational relevance of a Droplet Digital PCR (ddPCR) assay allowing universal, specific, and dynamic detection of sarcoma-related hypermethylated ctDNA. EXPERIMENTAL DESIGN: In silico analysis (The Cancer Genome Atlas/Gene Expression Omnibus datasets, n = 8,330) identified hypermethylated DNA positions in STS/bone sarcoma, unmethylated in nonsarcoma 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 (The Cancer Genome Atlas/Gene Expression Omnibus, n = 1,342). The ddPCR assay was applied to cfDNA from healthy donors, patients with metastatic STS (METASARC cohort, n = 49, 13 histotypes), and patients with STS/bone sarcoma treated with neoadjuvant chemotherapy (NEOSARC cohort, n = 42, 10 histotypes). RESULTS: A ddPCR assay targeting seven methylated genomic positions distinguished sarcoma samples from nonneoplastic mesenchymal and endothelial/liver tissues (AUC = 0.95; in silico validation set). Sensitivity allowed methylated DNA detection at a 1:1,000 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 histologic 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.