Clinical Application of Multiple Reaction Monitoring-Mass Spectrometry to Human Epidermal Growth Factor Receptor 2 Measurements as a Potential Diagnostic Tool for Breast Cancer Therapy.

Abstract

Human epidermal growth factor receptor 2 (HER2) is often overexpressed in breast cancer and correlates with a worse prognosis. Thus, the accurate detection of HER2 is crucial for providing the appropriate measures for patients. However, the current techniques used to detect HER2 status, immunohistochemistry and fluorescence in situ hybridization (FISH), have limitations. Specifically, FISH, which is mandatory for arbitrating 2+ cases, is time-consuming and costly. To address this shortcoming, we established a multiple reaction monitoring-mass spectrometry (MRM-MS) assay that improves on existing methods for differentiating HER2 status. We quantified HER2 expression levels in 210 breast cancer formalin-fixed paraffin-embedded (FFPE) tissue samples by MRM-MS. We aimed to improve the accuracy and precision of HER2 quantification by simplifying the sample preparation through predicting the number of FFPE slides required to ensure an adequate amount of protein and using the expression levels of an epithelial cell-specific protein as a normalization factor when measuring HER2 expression levels. To assess the correlation between MRM-MS and IHC/FISH data, HER2 quantitative data from MRM-MS were divided by the expression levels of junctional adhesion molecule A, an epithelial cell-specific protein, prior to statistical analysis. The normalized HER2 amounts distinguished between HER2 2+/FISH-negative and 2+/FISH-positive groups (AUROC = 0.908), which could not be differentiated by IHC. In addition, all HER2 status were discriminated by MRM-MS. This MRM-MS assay yields more accurate HER2 expression levels relative to immunohistochemistry and should help to guide clinicians toward the proper treatment for breast cancer patients, based on their HER2 expression.