Abstract 7684: Absolute quantification of 400 plasma proteins using a novel NGS-based immunoassay: Analytical validation and biomarker discovery in breast cancer

Abstract Next generation sequencing (NGS)-based immunoassays have enabled multiplex, sensitive, wide dynamic range protein measurements, but existing platforms lack absolute quantitation and a clear path for translating proteomic hits into focused assay panels on platforms suitable for down-stream studies. We introduce a novel high multiplex immunoassay platform from ProteinXI, a division of Meso Scale Diagnostics, LLC. (MSD), with accurate and reproducible absolute quantification, and demonstrate simultaneous measurement of hundreds of targets in human biofluids. We also show strong concordance with analogous assays on MSD’s high-throughput electrochemiluminescence (ECL) platform, providing an efficient route to focused validation panels. The ProteinXI Discovery 400 panel was evaluated for analytical sensitivity, precision, reproducibility and dilution linearity using a set of 40 commercially sourced plasma samples from healthy donors. Concordance with well-established commercial immunoassays was assessed for over one hundred analytes by measuring the same sample set using MSD V-PLEX or U-PLEX assays. To assess translational relevance, we measured plasma samples from a cohort of 387 subjects with breast cancer of varying stages and molecular subtypes monitored during neoadjuvant therapy (NAT). Samples were collected at baseline (pre-treatment), on day 1 of the second therapy cycle (C2D1), and prior to surgery. Tumor and lymph node biopsies were obtained before and after NAT, and treatment response was evaluated using the MD Anderson Residual Cancer Burden (RCB) score. The platform showed high dynamic range, with 90% of assays spanning at least 4 logs between lower limit of detection (LLOD) and upper limit of quantitation (ULOQ). Analytical sensitivity exceeded 1 fg/mL for some assays, with a median LLOD of 0.48 pg/mL. Most (80%) analytes were within the quantifiable range in at least 75% of healthy plasma samples. Dilution linearity was between 70% and 130% for 85% of quantifiable analytes. Median intraplate CVs of calculated concentrations across all quantifiable samples and analytes was 6.8% (IQR=3.1%-13.3%). Median Interplate and inter-run CVs of calculated concentrations were 10.3 and 11.7% respectively. Concordance with U-PLEX and V-PLEX immunoassays was high with Pearson correlations 0.8 for 84% of assays. In breast cancer samples, classical markers CA15-3, CEA, CA125 and Cytokeratin 8 were elevated relative to healthy controls and correlated with tumor stage, as did VEGF-A, VCAM-1, MMP-7 and Osteopontin. High baseline Ki67 and Leptin levels were associated with lower RCB score. PDGF-AA and PDGF-AB both decreased with NAT, while BAFF and GDF-15 increased. These results demonstrate the ProteinXI platform’s robust analytical performance and ability to reproduce expected biological patterns, supporting its translational utility. Citation Format: David Aaron Routenberg, Evan A. Gizzie, Annamaria Szabolics, Lucie Hebert, Somnath Paul, Anthony Williams, Richard J. Cote, Jacob N. Wohlstadter. Absolute quantification of 400 plasma proteins using a novel NGS-based immunoassay: Analytical validation and biomarker discovery in breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7684.