Predictive significance of HER2 intratumoral heterogeneity, determined by simultaneous gene and protein analysis, for resistance to trastuzumab-based treatments for HER2-positive breast cancer

Gene-protein assay (GPA), a combination of immunohistochemistry and dual in situ hybridization, allows simultaneous visualization of HER2 protein and gene on a single slide. We aimed to clarify the clinical significance of HER2 intratumoral heterogeneity (ITH) using GPA. We investigated the relationships between various HER2 ITH indicators and clinical course in 102 patients with HER2-positive breast cancer, treated with neoadjuvant trastuzumab and chemotherapy. Five representative microscopic images were captured from each GPA slide of pre-therapeutic biopsy materials. All evaluable cancer cells in the images were individually assessed for HER2 gene copy number and protein expression. Mean and coefficient of variation (CV) of both gene copy number and protein category were calculated, and each was divided into negative, equivocal, and positive. Based on their combined status, cancer cells were classified into nine types. Pathological complete response (pCR) to neoadjuvant treatments showed positive relationships to mean gene copy number ( P < 0.001), mean protein category ( P < 0.001), and proportion of gene- and protein-positive tumor cells ( P < 0.001) and showed negative relationships to the CV of protein category ( P < 0.001) and the proportion of gene-amplified but protein-negative tumor cells ( P = 0.002). Two diagnostic models, created by combining clinicopathological factors and ITH indicators, showed excellent potential diagnostic ability for pCR (mean gene copy number and protein category CV; AUC = 0.837, proportion of gene- and protein-positive tumor cells; AUC = 0.831). HER2 ITH quantified by GPA is a potential predictive indicator for efficacy of HER2-targeted treatment.