Development and validation of a digital PCR assay for detection and monitoring of universally methylated circulating tumor DNA in patients with high-grade sarcoma

BackgroundNo universal circulating biomarker is available for high-grade soft tissue (STS) and bone sarcoma (BS). We aimed to identify universally methylated DNA positions in sarcoma and to develop a highly sensitive digital droplet PCR (ddPCR) assay allowing specific, quantitative, and dynamic detection of sarcoma-related hypermethylated circulating tumor DNA (ctDNA) in patients (pts) with high-grade sarcoma.MethodsDNA positions hypermethylated in STS/BS and unmethylated in non-sarcoma tissues (cancerous and normal tissues) or white blood cells releasing circulating cell-free DNA in plasma were identified in silico (TCGA/Gene Expression Omnibus (GEO) datasets, training, n=7228 samples). A ddPCR assay following bisulfite conversion of DNA extracted from plasma was developed on STILLA® system. Performances were evaluated in an independent in silico cohort (GEO, validation, n = 1342), and using plasma from 20 healthy donors, 49 pts with metastatic STS treated with pazopanib (METASARC, various histotypes), and 42 pts with localized sarcoma treated with neoadjuvant chemotherapy (NEOSARC, high-grade STS/BS (osteosarcoma, Ewing)).ResultsA ddPCR assay was developed and validated for detection of DNA methylation at 7 genomic positions. Mean methylation value of this signature identified sarcoma samples versus non-cancerous mesenchymal tissues with area under the curve = 0.95 in the in-silico validation set. The ddPCR assay showed technical sensitivity reaching 0.1% and high specificity. ctDNA was detected in 21/49 pts (43%) in the METASARC cohort and in 19/29 pts (66%) in NEOSARC cohort, across all histotypes. ctDNA detection was associated with poor OS in METASARC (p = 0.026). ctDNA rise during neoadjuvant chemotherapy was associated with poor outcomes in the NEOSARC cohort (poor histological response, radiological progression or relapse within 6 months; p = 0.0096).ConclusionsddPCR-based detection of a universally methylated ctDNA allows detection, prognostication, and dynamic quantification of tumor burden in patients with high grade sarcoma, regardless of histotype and tumor origin.Legal entity responsible for the studyG. Beinse.FundingAssociation pour la Recherche contre le Cancer, Institut du Cancer Paris CARPEM.DisclosureV. Taly: Financial Interests, Personal, Research Grant: Biomnis; Financial Interests, Personal, Ownership Interest: Emulseo; Financial Interests, Personal, Advisory Role: Emulseo; Financial Interests, Personal, Speaker, Consultant, Advisor: Raindance technologies, Boehringer Ingelheim. A. Gaudet-Chardonnet: Financial Interests, Personal, Advisory Board: GSK. P. Boudou Rouquette: Financial Interests, Personal, Invited Speaker, speaker Journée du Centre Hépato-Biliaire (2022): Ipsen; Other, travel fees for ESMO sarcoma congress (2023) travel fees for GSF GETO congress (2022): PharmaMar; Other, Travel fees for ASCO congress (2021): Pfizer; Other, travel fees for GSF GETO congress (2023): PharmaMar. F. Goldwasser: Financial Interests, Personal, Advisory Board: Nutricia, Frenesius Kabi; Financial Interests, Personal, Research Grant: Baxter. P. Laurent-Puig: Financial Interests, Personal, Invited Speaker: Amgen, Sanofi; Financial Interests, Personal, Advisory Board: Biocartis, Pierre Fabre; Financial Interests, Personal, Ownership Interest: Methys DX; Financial Interests, Institutional, Research Grant, PI of translational research: Federation francophone de cancerologie digestive; Non-Financial Interests, Leadership Role: President of canceropole ile de france. J. Alexandre: Financial Interests, Personal, Advisory Board: Eisai, MSD, GSK, Janssen, Pfizer, Seagen; Financial Interests, Personal, Invited Speaker: Eisai, MSD, AstraZeneca, GSK, Novartis; Financial Interests, Institutional, Research Grant: Janssen, GSK, MSD; Financial Interests, Institutional, Local PI: MSD, Eisai, Agenus, GSK, Immunogen, Incyte; Financial Interests, Institutional, Coordinating PI: Kartos. All other authors have declared no conflicts of interest.