PROJECT SUMMARY Circulating tumor DNA (ctDNA) analysis has enabled noninvasive tumor genotyping for patients with advanced metastatic cancers. There is now growing interest in replicating this success in patients with early stage cancers. ctDNA analysis could enable blood-based minimal residual disease detection in patients receiving treatment and early detection of cancers in pre-symptomatic individuals. However, ctDNA levels are 10s-100s fold lower in localized cancers than in metastatic cancers, limiting the sensitivity of current assays. This is further confounded by pre-analytical variability such as differences in blood collection and processing and DNA extraction. For example, inappropriate or delayed blood processing can cause peripheral cell lysis and dilute ctDNA fraction in blood samples. In early stage cancer patients where ctDNA levels are already quite low, this can cause false-negative results. The effects of pre-analytical factors on ctDNA detection in patients with localized cancers are not well understood. To address this gap, we propose a study of 180 patients with early and locally advanced breast cancer to investigate three aspects of pre-analytical variation: 1) DNA extraction methods (Aim 1), 2) blood collection tubes and processing protocols (Aim 2) and 3) long-term storage of plasma and extracted DNA (Aim 3). We will investigate these factors using two assays we have recently developed: 1) a quality assessment assay that measures total cell-free DNA concentration and fragment size and 2) TARgeted DIgital Sequencing (TARDIS), an assay that simultaneously measures up to 30 patient-specific founder mutations in plasma DNA to quantify ctDNA levels. By leveraging multiple mutations for each patient, improving error suppression and minimizing loss of input DNA material, TARDIS enables sensitive detection and precise quantification of ctDNA in patients with localized cancers and improves limit of detection by 10-100 fold over current assays. Minimal residual disease detection and early detection using ctDNA analysis hold tremendous promise to individualize cancer treatment and to improve outcomes. Our study will clarify pre-analytical factors that could be critical to the success of future clinical studies across localized cancers of multiple subtypes.