Project Summary/Abstract (Biomarker Reference Laboratory (BRL), RFA-CA-22-040) We propose to develop a blood-based test whose indicated use is to complement mammography in the early detection of breast cancers. Although mammography saves lives through early detection, it is imperfect. Approximately one in seven breast cancers goes undetected despite screening mammography, and interval cancers that manifest within a year of a normal mammogram remain a vexing problem, especially (although not exclusively) for the >27 million women in the United States with heterogeneously or extremely dense breasts at high risk for interval cancers. We propose to develop a blood test that could be used as an adjunct to mammography to improve early detection by improving sensitivity and/or specificity of mammography. Using a novel biomarker discovery approach leveraging human-in-mouse breast cancer patient-derived xenograft models and state-of-the-art mass spectrometry methods, we prioritized a subset of 162 candidate breast cancer protein biomarkers (verified in human plasma from breast cancer cases vs. controls) for follow up EDRN phase 2 validation studies in this proposed BCC. Our project will clinically validate and deploy (in our CLIA laboratories) a novel multiplex immuno-multiple reaction monitoring mass spectrometry (immuno-MRM-MS) assay to perform EDRN phase 2 biomarker validation studies using strongly unbiased sets of plasma samples obtained from existing biorepositories. The Biomarker Reference Laboratory will contribute to the proposed EDRN phase 2 biomarker validation studies by: (i) validating a CLIA-compliant standard operating procedure for an immuno-MRM assay to quantify up to 162 candidate plasma protein biomarkers of early-stage breast cancer that will serve as the basis for our biomarker validation studies, (ii) performing a phase 2 case-control biomarker validation study using “strongly unbiased” plasma collections collected using EDRN PRoBE study design, and (iii) providing reference laboratory support for the EDRN network, including high-throughput targeted mass spectrometric analyses, flow cytometry, next-gen sequencing, and 96-well plate ELISA assays, as needed.