Abstract 5895: Development and analytical validation of a highly sensitive tumor fraction estimation method using blood samples without matched normal controls

Abstract Background: Tumor fraction (TF) in cell-free DNA (cfDNA) is a quantitative indicator of circulating tumor DNA (ctDNA) and has become an important biomarker for disease monitoring and tracking therapeutic responses. We developed a longitudinal TF estimation method that does not require matched normal control samples and analytically validated its ability to accurately estimate TF when longitudinal blood samples are available. Methods: Both reference materials and clinical blood samples were used in the analytical validation. We first determined the limit of detection (LoD) of TF for our PredicineCARE cfDNA assay at targeted sequencing depth of 20, 000x using serially diluted reference materials with 12 gradient values from 0 to 4% TF. We further used a sequentially diluted clinical sample (with known TF) with healthy donor cfDNA to a gradient of expected TF values. We evaluated the variant calling and TF calculation algorithm by treating each dilution separately or treating the immediately nearby samples as longitudinal samples. We further evaluated our longitudinal-aware analysis method on 20 clinical subjects tested with PredicineCARE cfDNA assay with ≥2 time points to evaluate the improvement in TF estimation compared to individual estimation. Results: With the titrated reference materials and manufacturer-labelled somatic variants, we achieved an LoD of 0.01% TF for 30ng cfDNA input and a sequencing depth at 20, 000x using PredicineCARE cfDNA assay. When approaching the LoD, the number of reads for the somatic variants becomes more stochastic, which resulted in big variances in the calculated TF values. When independently estimating the TF without referring to matched normal or longitudinal samples, the LoD was 0.5% TF. When the longitudinal sample information was included, the LoD went down to 0.01% TF, contingent on the number of trackable variants. Our simulated results showed that when there are 4 or more tracking variants between longitudinal samples, the LoD can go down to 0.01% TF most of the time. We further applied this longitudinal sample aware dynamic TF estimation method to 20 clinical subjects tested with PredicineCARE cfDNA assay and with ≥2 time points, most showed improved TF estimation with ≥50% of the subjects having questionable somatic/germline calls if called separately. Conclusions: We developed a dynamic TF estimation methodology that leverages longitudinal samples to improve somatic/germline variant calling and exclude CHIP mutations. Our results demonstrated that with longitudinal samples, the accuracy in TF estimation can approach the ideal situation where all somatic/germline status of the variants are known. Notably, longer interval between time points and a higher number of tracking somatic mutations (≥2) in longitudinal samples generally enhance the effectiveness of this dynamic estimation methodology. Citation Format: Junmei Wang, Wanshu He, Lisha Zhu, Yong Huang, Xiaohong Wang, Shidong Jia, Pan Du. Development and analytical validation of a highly sensitive tumor fraction estimation method using blood samples without matched normal controls [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 5895.