Abstract 4068: Development and evaluation of standardized samples for multiomic ctDNA assays across multiple cancer types

Abstract Background: Multiomic circulating tumor DNA (ctDNA) assays offer several benefits over tumor-dependent ones, including being more rapid, less invasive, and cost-effective for monitoring tumor recurrence or therapy response through serial testing. Despite these advantages, identifying ultra-low frequency ctDNA alterations, whether genetic or epigenetic, from limited input material is highly challenging. Furthermore, complex assays are often more susceptible to experimental and computational variables and artifacts. This poses a pressing need for standardized samples to assess the analytical performance and enable unbiased comparisons between different technology platforms. Methods: We have developed contrived standards using cell line, tissue, plasma, and PBMC samples that were either commercially purchased or provided with informed consent. The 'ground truth' for tumor fraction (TF) was defined by the dilution ratios and validated through in silico reads-mixing simulations. Subsequently, estimates of tumor allele fraction (TAF) using different biomarkers were analyzed and compared. (i) Mutation-based TAF: Both tumor-agnostic (somatic variants allele frequency, SVAF) and tumor-informed (circulating tumor allele fraction, cTAF) ones were characterized by a validated 520-/168-gene panel (10, 000-35, 000X) or an individualized mutation tracking panel (100, 000X depth) following a WES scan on paired tumors. (ii) Methylation-based TAF: Both tumor-agnostic (tumor methylated fraction, TMeF) and tumor-informed (methylated tumor allele fraction, mTAF) ones were characterized by a validated targeted methylation assay. Results: We have tested >400 contrived and clinical samples across different cancer types. Sample dilutions at pre-determined levels of TAF (0.001%-1%) are suitable for evaluating the limits of detection (LoDs) for most mainstream tumor-informed/-agnostic ctDNA assays. Samples with TAF near the LoD and background plasma were tested with replicates for precision and accuracy studies. Overall, reliable estimates of TAF were observed across different biomarkers, although the measurement of ultra-low fractions (i.e. TAF<0.01%) still remains a challenge. Notably, the number of unique patients/healthy donors is crucial for generating methylation-based standards, as demographic factors such as age, sex, and ethnicity have been shown to influence DNA methylation patterns. Conclusions: With clinical adoption underway, it is critical to understand and evaluate the variables that impact the analytical performance of complex multiomic ctDNA assays. Here, we report a systematic evaluation of the analytical features and clinical relevance of standardized samples, thereby providing a versatile tool for assay optimization, validation, and cross-platform comparison. Citation Format: Qiancheng You, Jiayue Xu, Jing Su, Xingyu Yang, Sa Zhang, Zeliang Deng, Xiaoling Li, Guangyou Li, Xinyue Kang, Si Zhang, Meifang Wu, Bingsi Li. Development and evaluation of standardized samples for multiomic ctDNA assays across multiple cancer types [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 4068.