Abstract 3237: Advancing non-invasive multiomics for early breast cancer detection: Transitioning from cfMeDIP-seq to single-molecule sequencing of cell-free DNA in the RENOVATE study
Authors: Martina Dameri, Francesco Ravera, Pamela Becherini, Mario Stabile, Alberto Tagliafico, Marilena Iorio, Gianmaria Frigè, Luca Mazzarella, Giovanni Scala, Michele Ceccarelli, Massimo Calabrese, Michele Piana, Alberto Ballestrero, Lorenzo Ferrando, Gabriele Zoppoli
Journal: Cancer Research (2025)

Abstract Introduction: Tissue biopsy for suspicious breast lesions (BI-RADS 3-5) is invasive, costly, anxiety-inducing, and often yields false positives, with benign lesions (BLs) like fibroadenomas identified in approximately one-third of cases. Liquid biopsy, which analyzes the genomic and epigenomic features of cell-free DNA (cfDNA), offers a promising alternative. Techniques such as cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) and cfDNA profiling using single-molecule Nanopore sequencing (NP-seq) could leverage the potential of cfDNA multiomics in early cancer detection. Methods: In the RENOVATE trial (NCT04781062) conducted at IRCCS Ospedale Policlinico San Martino (Genoa, IT), we collected PAXgene plasma samples from 366 women. Over a recruitment period of 18 months, we included in the final analysis, per protocol criteria, 64 patients with pT1N1/pT2N0 breast cancer (BC) and 115 women with BLs. After extracting cfDNA from these samples, we prepared cfMeDIP-seq and low-pass whole genome sequencing (WGS) libraries following the protocol described by Shen et al. (Nat Prot, 2019) with minor modifications. Libraries were sequenced on a NovaSeq6000 (Illumina). Differentially methylated regions were analyzed using the MEDIPS package. Additionally, a subset of RENOVATE cfDNA samples was used to implement NP-seq. Libraries were prepared according to Ligation Sequencing V14 Human cfDNA multiplex protocol and sequenced on the Oxford Nanopore Technologies P2 Solo device. Results: CfMeDIP-seq libraries achieved an average of 45 MegaReads per sample (3-5x coverage), while low-pass WGS libraries were sequenced to obtain 1-2x coverage. CfMeDIP-seq analysis identified 2, 133 hypomethylated regions and 1, 299 hypermethylated regions, allowing us to determine methylomic variables that distinguish BC from BLs. For representative RENOVATE samples, we obtained NP-seq data showing that an average of 30 million basecalled reads and 11 GB of output data can be achieved for each sample. Preliminary data indicate that NP-seq can replicate genomic and epigenomic features while also enabling complex analyses due to the richness of information generated. Conclusions: The assessment of cfDNA multiomics to differentiate BLs from BC holds great potential in early BC management. Advanced sequencing technologies like cfMeDIP-seq and NP-seq extract key cfDNA features, including copy number variations (CNVs), fragment length, and 5mC for both methods, but unprecedented capability to simultaneously capture native fragment features, as well as previously poorly characterized epigenetic modifications for NP-seq, such as 5hmC and 8-oxodG. This approach could have clinically relevant implications, reducing unnecessary biopsies and benefiting both patients and healthcare systems globally. Citation Format: Martina Dameri, Francesco Ravera, Pamela Becherini, Mario Stabile, Alberto Tagliafico, Marilena Iorio, Gianmaria Frigè, Luca Mazzarella, Giovanni Scala, Michele Ceccarelli, Massimo Calabrese, Michele Piana, Alberto Ballestrero, Lorenzo Ferran. Advancing non-invasive multiomics for early breast cancer detection: Transitioning from cfMeDIP-seq to single-molecule sequencing of cell-free DNA in the RENOVATE study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3237.