Abstract 4562: Bespoke sequencing of enriched and normalized amplicons increases on-target rate and reduces background noise for circulating tumor DNA detection and quantification

Abstract Background: Analysis of circulating tumor DNA (ctDNA) using tumor-guided bespoke sequencing can achieve high accuracy for minimal residual disease detection. However, bespoke laboratory workflows are often complex, difficult to scale, and costly. Multiplexed PCR methods have lower costs and preserve input DNA but instill higher background noise, variable on-target rates and unpredictable relative representation of amplicons. Locus- and mutation-specific hybrid capture can overcome these challenges but with higher costs of patient-specific custom baits and greater loss of input DNA. Here, we describe a workflow that combines targeted amplification with fixed content exome-wide hybrid capture to overcome these challenges. Methods: We performed tumor-guided plasma DNA analysis using a novel method based on enrichment and normalization of amplicons and targeted sequencing. Using tumor and germline exome sequencing, we identified somatic founder mutations, and designed primers for multiplexed amplification. Targeted sequencing libraries were prepared using a combination of single-stranded adapter ligation, targeted PCR and hybrid capture. Sequencing reads were grouped in read families based on fragment size and unique molecular identifiers, and analyzed to improve specificity for detection of known mutations. We evaluated assay performance using reference samples (Seracare Life Sciences) as well as plasma samples from patients with cancer. Results: We analyzed 8 replicates of reference samples at 10 ng input each for variant allele fractions (VAFs) of 0.5%, 0.25% and 0.125%, using an 18 mutation panel, and detected mutated DNA in 8/8 samples for all VAFs. In addition, we prepared a dilution at 0.01% VAF and analyzed 16 replicates of this sample at 40 ng input each, and detected mutated DNA in 15/16 samples. Using the same analytical thresholds, 1/8 wild-type reference samples were called positive. A comparison of observed and expected tumor fractions across all samples showed a correlation coefficient of 0.97 (p<0.001, n=48). In addition, we developed 44 patient-specific panels targeting a median of 30 mutations per patient (range 5 to 125 mutations) for patients with pancreatic cancer, breast cancer and glioblastoma. Using a median input of 6.7 ng (range 1.1 to 24 ng), we analyzed 112 plasma DNA samples across 44 panels and observed a median on-target rate of 89% using the first round of panel design. ctDNA was detected in 62 plasma samples (55%) at a median tumor fraction of 0.013% (range 0.009% to 5.8%). Conclusion: Our results demonstrate analytical performance and assay stability across 44 patients using bespoke sequencing of enriched amplicons for circulating tumor DNA (enACT-DNA) analysis. On-going studies are evaluating the clinical relevance of perioperative ctDNA detection using enACT-DNA in patients with cancer. Citation Format: Tania Contente-Cuomo, Devin Dinwiddie, Mojca Stampar, Adriana Marshall, Michael E. Berens, Stephanie J. Pond, Jeffrey M. Trent, Muhammed Murtaza. Bespoke sequencing of enriched and normalized amplicons increases on-target rate and reduces background noise for circulating tumor DNA detection and quantification [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 4562.