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Droplet Digital PCR Assay for Detection and Monitoring of Universally Methylated ctDNA in Patients with Sarcoma
Elisabeth Ashton1, Valérie Taly1,2, Camille Tlemsani3
1Centre de recherche des Cordeliers, Inserm UMRS1138, CNRS, Sorbonne Université, Université Paris Cité, « Équipe Labellisée Ligue Nationale Contre le Cancer », CNRS SNC 5096, Paris, France.
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.
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