Abstract 3433: Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer
Authors: Roy Roopali , Schunkert Elisa , Olivova Petra , Gilar Martin , Geromanos Scott , Li Guo-Zhong , Gebler John , Dagher Adelle , El-Hayek Andrew , Aldakhlallah Rama , Zurakowski David , Pories Susan , Moses Marsha A. 
Published: April 2023

Abstract
Introduction: Breast cancer (BC) is a complex heterogenous disease and is a leading cause of death in women. Following primary BC therapy ~40% of patients will develop metastatic BC (MBC) and 60-70% of the recurrent disease will occur at distant sites. Median survival of MBC patients with distant recurrence is ~2-3 yr. While the early detection and monitoring of BC progression are essential to improve prognosis and reduce BC-related mortality, there is a lack of surveillance strategies for monitoring patients for distant recurrence of MBC. The aim of our study was to identify urinary biomarkers for detection and monitoring of MBC.
Method: We have conducted a comparative label-free LC-MS/MS analysis of the urinary proteome of patients with MBC and healthy age-matched controls (HC). A hybrid quadrupole time of flight (Q-Tof™) mass spectrometer was used for urine analysis via liquid chromatography (LC) with tandem mass spectrometry (MS/MS). Three different database search algorithms were used for peptide identification including MASCOT, Protein Lynx Global Server and Ion Accounting software. Only peptides detected in at least two out of three replicated LC-MS experiments were counted towards protein identification. Identified candidate biomarkers were validated via ELISA assays.
Results: Using this approach, we identified ~400 urinary proteins of which ~104 and 89 were unique to the MBC and HC groups, respectively. Upregulated proteins in the MBC cohort were associated with angiogenesis, apoptosis, proteolysis, ECM regulation, and cell adhesion pathways. Using bioinformatic and in silico approaches, we identified a specific metastasis signature comprised of CALB1, S100A8, ZAG, VTN and TN. Diagnostic accuracies of these candidate markers were validated using independent training and validation sets according to the REMARK criteria. Urinary VTN (uVTN) and uTN levels correlated significantly with disease status in MBC patients when compared to patients with locally invasive BC and HC. Interestingly, a small subset of patients with bone metastasis also had significantly higher uVTN levels. A multiplexed marker panel of uVTN and uTN could discriminate between HC and MBC groups (AUC=0.831, P&lt;0.001). Blinded testing indicated that uVTN levels (cutoff&gt;1018 ng/ml) could discriminate (AUC=0.782, P&lt;0.006) between MBC and HC. Longitudinal analysis of samples from MBC patients indicated a strong correlation between uVTN levels and disease status.
Conclusions: Our findings suggest that uVTN and uTN have the potential to serve as biomarkers for the detection of MBC. While validation in larger cohorts is necessary, these results may be useful in the development of noninvasive diagnostic tests for monitoring BC progression and recurrence. [Supported by: the Advanced Medical Research Foundation, the Jo Ann Webb Fund for Angiogenesis Research and the S. Elizabeth O’Brien Trust]
Citation Format: Roopali Roy, Elisa Schunkert, Petra Olivova, Martin Gilar, Scott Geromanos, Guo-Zhong Li, John Gebler, Adelle Dagher, Andrew El-Hayek, Rama Aldakhlallah, David Zurakowski, Susan Pories, Marsha A. Moses. Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3433.