Proteogenomic Analysis of Clinical Trial Samples Identifies New Biomarkers

A comprehensive proteogenomic analysis of the CALGB 40601 clinical trial has identified two protein biomarkers, GPRC5A and TPBG, that strongly predict resistance to anti-HER2 therapy in HER2+ breast cancer patients. Using mass spectrometry to comprehensively evaluate the proteome/phosphoproteome for 80 frozen core biopsies from 54 patients, the study was able to uncover resistance mechanisms not fully apparent from standard genomic or transcriptomic analyses alone. 

Addressing Diagnostic Limitations and Uncovering Pathway-Level Resistance 

A key initial finding was the identification of a subset of tumors clinically designated as HER2-positive which lacked proteogenomic evidence of ERBB2 gene amplification. These cases, representing potential "false positives," were uniformly associated with a lack of pathological complete response (pCR) suggesting diagnostic limitations may contribute to resistance in a significant number of cases (~7%) when treated with anti-HER2 therapy.

After excluding “false positives,” pathway-level analyses of the HER2+ tumors with ERBB2 gene amplification revealed noteworthy differences between responsive and resistant tumors. In resistant tumors, there was an elevation of pathways related to the extracellular matrix, epithelial-mesenchymal transition, and WNT-beta-catenin signaling. In contrast, responsive tumors showed higher activity in immune and cell cycle-related pathways. 

Dr. Meenakshi Anurag, Assistant Professor at BCM and co-corresponding author added, "Proteogenomics offers a powerful tool to refine biomarker discovery and uncover key mechanisms of drug resistance in HER2+ breast cancer. By incorporating proteomics, this study enhances our understanding of protein expression and signaling pathways that drive treatment response and resistance, with the potential to improve precision medicine strategies." 

Discovery and Validation of Key Biomarkers

Differential expression analysis comparing resistant and responsive samples resulted in the identification of 4 proteins of interest: GPRC5A, TPBG, NEU1, and SP140L. These protein-level findings correlated with the transcriptomic data--Higher baseline levels of two cell surface proteins GPRC5A and TPBG were consistently found in treatment-resistant tumors and were validated at both protein and RNA levels. In the full CALGB 40601 cohort, elevated mRNA levels of these markers were associated with treatment failure and worse overall survival. 

“We identified GPRC5A and TPBG as key biomarkers associated with treatment resistance, presenting new opportunities for targeted therapies. Cell surface proteins hold significant therapeutic potential, particularly in the context of treatment pressure. To help realize this opportunity, the Broad Institute Proteomics Platform, continues to refine technologies to advance the identification of such proteins in relevant clinical samples, including patient-derived tissues acquired in the context of clinical trials,” writes co-authors Drs. Shankha Satpathy, Michael Gillette and Steven Carr from the Broad Institute.

Confirming Clinical Relevance and Biological Function

To establish clinical relevance, researchers conducted a meta-analysis across four independent studies consisting of ten neoadjuvant anti-HER2 treatment regimens. This large-scale analysis confirmed that elevated GPRC5A and TPBG expression was robustly associated with high rates of treatment failure. A composite scoring system combining GPRC5A, TPBG, and HER2 expression levels provided superior prediction compared to any single marker, achieving predictive accuracy up to 79%. 

"A key challenge in studying underpowered clinical cohorts is distinguishing true biomarkers from study-specific associations, and this study demonstrates how meta-analysis of similar datasets can help identify the most reproducible biomarkers for clinical application," explains Dr. Eric Jaehnig, Staff Scientist at BCM and first author. 

Biologically, the study linked GPRC5A and TPBG expression to pathways distinct from HER2 and ESR1 signaling. Their protein levels were positively correlated with TGF-beta signaling and negatively correlated with a T-cell infiltration signature, suggesting a role in modulating the tumor microenvironment and immune response. TPBG is of particular interest as an established oncofetal antigen and a target for antibody-drug conjugates and immunotherapies already in development. 

The Power of Translational Research

"This study exemplifies the power of persistence in translational research," says Dr. Matthew J Ellis, formerly at BCM, now at Guardant Health and one of the senior authors of this paper. "The CALGB 40601 trial was approved in 2001, and the proteogenomic technologies applied in this paper were not even conceived at the time of patient accrual. Yet, the ability of frozen samples to preserve deep cancer biology is vividly demonstrated, enabling the discovery of new plasma membrane targets in trastuzumab-resistant HER2+ breast cancer that would have been missed without mass spectrometry-based proteomics."