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Circulating tumor DNA (ctDNA) is a promising cancer biomarker, but accurately detecting tumor mutations in cell-free DNA (cfDNA) is challenging due to their low frequency and sequencing errors. Our study benchmarked Mutect2, VarScan2, shearwater, and DREAMS-vc using deep targeted sequencing of cfDNA with Unique Molecular Identifiers (UMIs) from 111 colorectal cancer patients. Performance was assessed at both the mutation level (distinguish tumor variants from errors) and the sample level (detect if an individual has cancer). Additionally, we investigated the effects of various UMI grouping and consensus strategies. The shearwater-AND variant calling method demonstrated the highest precision in detecting tumor-derived mutations from plasma, and reached the highest ROC-AUC of 0.984 for sample classification in tumor-informed cfDNA analyses. DREAMS-vc exhibited the highest ROC-AUC of 0.808 for sample classification in tumor-agnostic studies. We also found that sequencing depth differences in PBMCs could lead to false positives, particularly with VarScan2 and Mutect2, which was addressed by downsampling to equivalent mean depths. Additionally, network-based UMI grouping methods outperformed those using identical UMIs when all reads were retained. Our findings emphasize that the optimal variant caller depends on the study context—whether focused on mutation or sample classification, and whether conducted under tumor-informed or tumor-agnostic conditions. [ABSTRACT FROM AUTHOR]

Tumor‐informed mutation‐based approaches are frequently used for detection of circulating tumor DNA (ctDNA). Not all mutations make equally effective ctDNA markers. The objective was to explore if prioritizing mutations using mutational features—such as cancer cell fraction (CCF), multiplicity, and error rate—would improve the success rate of tumor‐informed ctDNA analysis. Additionally, we aimed to develop a practical and easily implementable analysis pipeline for identifying and prioritizing candidate mutations from whole‐exome sequencing (WES) data. We analyzed WES and ctDNA data from three tumor‐informed ctDNA studies, one on bladder cancer (Cohort A) and two on colorectal cancer (Cohorts I and N). The studies included 390 patients. For each patient, a unique set of mutations (median mutations/patient: 6, interquartile 13, range: 1–46, total n = 4023) were used as markers of ctDNA. The tool PureCN was used to assess the CCF and multiplicity of each mutation. High‐CCF mutations were detected more frequently than low‐CCF mutations (Cohort A: odds ratio [OR] 20.6, 95% confidence interval [CI] 5.72–173, p = 1.73e−12; Cohort I: OR 2.24, 95% CI 1.44–3.52, p = 1.66e−04; and Cohort N: OR 1.78, 95% CI 1.14–2.79, p = 7.86e−03). The detection‐likelihood was additionally improved by selecting mutations with multiplicity of two or above (Cohort A: OR 1.55, 95% CI 1. 14–2.11, p = 3.85e−03; Cohort I: OR 1.78, 95% CI 1.23–2.56, p = 1.34e−03; and Cohort N: OR 1.94, 95% CI 1.63–2.31, p = 2.83e−14). Furthermore, selecting the mutations for which the ctDNA detection method had the lowest error rates, additionally improved the detection‐likelihood, particularly evident when plasma cell‐free DNA tumor fractions were below 0.1% (p = 2.1e−07). Selecting mutational markers with high CCF, high multiplicity, and low error rate significantly improve ctDNA detection likelihood. We provide free access to the analysis pipeline enabling others to perform qualified prioritization of mutations for tumor‐informed ctDNA analysis. [ABSTRACT FROM AUTHOR]

The presence of circulating tumor DNA (ctDNA) in patients with colorectal adenomas remains uncertain. Studies using tumor-agnostic approaches report ctDNA in 10-15% of patients, though with uncertainty as to whether the signal originates from the adenoma. To obtain an accurate estimate of the proportion of patients with ctDNA, a sensitive tumor-informed strategy is preferred, as it ensures the detected signal originates from the adenoma. Here, tumor-informed whole-genome sequencing-based ctDNA analysis (MRD-EDGE SNV ) was applied to two independent cohorts. Cohort 1, comprising 93 patients with stage III colorectal cancer (CRC) and 40 healthy individuals, was used to establish the signal threshold at 95% specificity. This threshold was then applied to Cohort 2, consisting of 22 patients with symptomatic and 20 with asymptomatic adenomas. In stage III, MRD-EDGE SNV had an area under the curve of 0.98. ctDNA was detected in 50% and 25% of patients with symptomatic and asymptomatic adenomas, respectively. The median adenoma plasma tumor fraction was 5.9 × 10 -5 . These finding not only demonstrate the feasibility of ctDNA detection in patients with colorectal adenomas, but also provides an estimate of the necessary sensitivity required to detect these lesions, paving the way for future ctDNA-based screening strategies., (© 2025 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)