Quantitative Assays for TROP2 Measurement in Breast Cancer and Comparison to H-Score

Area of Science:

• Oncology
• Biomarker Development
• Translational Research

Background:

• Trophoblast cell surface antigen 2 (TROP2) is a key target for antibody-drug conjugates in various solid tumors, including breast cancer.
• Recent FDA approvals highlight the clinical success of TROP2-targeting therapies, yet response rates vary, suggesting a need for precise TROP2 expression measurement.
• Current H-score assessments lack the sensitivity and objectivity required for optimal patient stratification in TROP2-targeted therapy.

Purpose of the Study:

• To develop and analytically validate quantitative immunofluorescence (QIF) and quantitative hematoxylin-DAB (QH-DAB) assays for measuring TROP2 protein expression.
• To establish TROP2 peptide concentration equivalents for QIF and QH-DAB assay readouts using mass spectrometry.
• To evaluate the performance of the developed assays in breast cancer patient cohorts and compare them with the standard H-score method.

Main Methods:

• Development of QIF and QH-DAB assays for TROP2 protein quantification.
• Mass spectrometry-based calibration to convert assay signals to TROP2 peptide concentrations (amol/mm²).
• Utilized QuPath-based image analysis (Qymia) for automated quantification and assessment in breast cancer patient samples (N=264 + N=100).
• Comparative analysis of QIF and QH-DAB assay results against H-scores from 5 certified pathologists on 68 breast cancer biopsies.

Main Results:

• The QIF and QH-DAB assays demonstrated robust performance with defined limits of detection and quantification.
• Mass spectrometry enabled accurate conversion of assay signals to TROP2 protein concentrations.
• Broad dynamic range of TROP2 expression was observed across breast cancer and triple-negative breast cancer patient cohorts.
• Quantitative assays showed overall agreement with pathologist-evaluated H-scores but offered superior sensitivity and reduced subjectivity.

Conclusions:

• Developed QIF and QH-DAB assays provide accurate and reproducible TROP2 protein quantification.
• These assays have the potential to serve as companion diagnostics for TROP2-targeted therapies.
• The quantitative approach may improve patient selection for therapies targeting TROP2, enhancing treatment efficacy.