PROJECT SUMMARY The overarching goal of this research is to utilize a plasma-based genomic biomarker of cell-free DNA (cfDNA) to guide therapy in metastatic breast and other cancers. In our approach, we use a cfDNA ‘ultra low pass whole genome sequencing’ (ULP-WGS) assay with computational pipeline (ichorCNA) to determine the amount of tumor DNA in circulation (‘tumor fraction’; TFx). Changes in TFx may serve as an early identifier for patients responding – or failing to respond – to therapy and cfDNA ULP-WGS provides a cost-effective, minimally-invasive approach to determine TFx. Our ULP-WGS cfDNA assay allows rapid, precise quantitation of TFx from a single blood sample without prior knowledge of tumor mutations. While most liquid biopsy approaches to date have focused on tracking known alterations or commonly mutated genes in cancer, our approach is mutation agnostic and broadly applicable across advanced cancers. This proposal developed through deep collaboration between three primary investigators working on concert on the development and application of a cell-free DNA assay to guide prognosis and therapy in advanced cancers. To date, we have performed the research version of this assay on over 3000 patients samples and, in three publications to date, we demonstrate clinical utility in metastatic breast and prostate cancer as well as multiple myeloma. We bring expertise in clinical breast cancer, sequencing assay development, and CLIA sequencing at scale, with a strong track record of collaboration with multiple shared publications. All three PIs are dedicated to the success of this proposal, with distinct yet complementary roles and responsibilities. In this proposal, we will analytically validate our cfDNA ULP-WGS assay (UH2 portion) then establish clinical validity and prospectively evaluate performance in therapeutic clinical trials (UH3 portion). In the UH2 portion, we will determine the sensitivity and specificity of ULP-WGS using serial dilutions of patient samples, then assess reproducibility, repeatability, and reportable range. We will then determine performance in the context of ‘real world’ pre-analytic variability including blood collection tube type, amount of plasma, and detection thresholds with respect to DNA input quantity. In the UH3 portion, we will establish clinical validity by evaluating TFx in 700 clinical plasma specimens from patients with metastatic breast cancer then advance our understanding of cfDNA by evaluating the association of TFx with patient response to therapy and survival outcomes. Finally, we will evaluate ULP-WGS in two prospective therapeutic clinical trials of metastatic triple- negative breast cancer, evaluating association with response to therapy.