Background: Estimating quantitative circulating tumor fraction in liquid biopsy samples is a promising area of clinical development for monitoring therapeutic molecular response and correlates with patient outcomes. Here we introduce a sensitive measure of estimating ctDNA tumor fraction (ctDNA TF) using a novel combination of tissue-informed CGP with a
non-bespoke, blood-based liquid biopsy panel.

Methods: Advanced pan-solid tumor samples were sequenced with both the Tempus xF/xF+ (105/523 gene, liquid biopsy) and Tempus xT (648 genes, solid-tumor with matched buffy coat) NGS assays (samples collected within 90 days). A normal distribution was fit against the somatic variants detected in solid-tissue and somatic variants with variant allele fractions (VAF) falling within two standard deviations were used as selected biomarkers in the corresponding liquid biopsy to calculate a tumor-informed ctDNA TF for each sample.

Results: In the initial validation set (n=79 samples also sequenced with low-pass whole genome sequencing), the linearity of tumor-informed ctDNA TF was compared to mean VAF (R2=0.26, slope=0.55), ichorCNA (R2=0.84, slope=0.84), and tumor-naive ctDNA TF (R2=0.71, slope=0.90). In a larger validation set (n=12,080), 37.5% samples had no variants detected in both xF and/or xT. Of the remaining samples, the linearity of tumor-informed ctDNA TF was compared to mean VAF (R
2=0.21, slope=0.55) and tumor-naive ctDNA TF (R2=0.65, slope=0.81). In a follow-up analysis, we required that 5 or more variants be detected in both xF and xT (n=1,659 samples) and compared the linearity of tumor-informed ctDNA TF to mean VAF (R2=0.37, slope=0.64) and tumor-naive ctDNA TF (R2=0.68, slope=0.98). In samples that received xF+ profiling (n=567), 24.3% had no variants detected in both xF+ and xT. The linearity of tumor-informed ctDNA TF of the remaining samples was compared to mean VAF (R2=0.01, slope=0.26) and tumor-naive ctDNA TF (R2=0.63, slope=1.02). Increasing the required number of variants to 5 or more had marginal improvement in linearity of tumor-informed ctDNA TF to mean VAF (R2=0.12, slope=0.47) and tumor-naive ctDNA TF (R2=0.67, slope=1.13). The limit-of-blank of the tumor-informed ctDNA TF approach was established to be 0% in both xF and xF+, and the limit-of-detection was ≤0.1% demonstrating that tumor informed ctDNA TF is a sensitive measure in detecting tumor fraction at low concentration levels.

Conclusion: Here we introduce a novel sensitive and specific tumor-informed, non-bespoke approach for estimating ctDNA TF. Linearity improves with increased panel size and increased variant number. These results suggest that a tumor-informed ctDNA TF can be utilized to improve the sensitivity of existing methods for estimating tumor fraction to help in treatment decisions using Tempus’ tissue and liquid comprehensive NGS genomic profiling platform.

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