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.

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The authors would like to thank Ms. Tomoyo Kakita, Japanese Foundation for Cancer Research, and Mr. Motoyoshi Iwakoshi, Japanese Foundation for Cancer Research, for their excellent technical support and to thank Dr. Masaaki Matsuura, Teikyo University, and Dr. Toshinari Yamashita, Kanagawa Cancer Center, for their advice.


This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant No. 17K10567).

Author information




RH: conceptualization, data curation, funding acquisition, investigation, methodology, writing of the original draft, and writing of review and editing. HN: conceptualization, investigation, methodology, visualization, and writing of review and editing. MN: statistical analysis and writing of review and editing. RM: conceptualization and writing-review and editing. TU: conceptualization and writing of review and editing. YI: supervision and writing of review and editing. SO: supervision and writing of review and editing. PB: writing of review and editing. HK: supervision and writing of review and editing. FA: conceptualization, methodology, project administration and writing of review and editing.

Corresponding author

Correspondence to Rie Horii.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the ethical committee of the Japanese Foundation for Cancer Research on August fourth, 2014 (No. 2014-1033). General consent for the use of specimens was obtained preoperatively from all patients participating in this study.

Consent for publication

This work has not been published before, and it is not under consideration for publication anywhere else. Its publication has been approved by all authors.

Conflict of interest

RH reports receiving honoraria from Chugai Pharmaceutical Co., Ltd. and US-Lead Inc. HN is an employee of Roche Tissue Diagnostics. MN reports receiving honoraria from Chugai Pharmaceutical Co., Ltd. TU reports receiving honoraria from Chugai Pharmaceutical Co., Ltd., Eisai Co., Ltd. and Novartis Pharma K.K. YI reports receiving research grants from Chugai Pharmaceutical Co., Ltd., AstraZeneca K.K., PAREXEL International Corp., Novartis Pharma K.K., Eli Lilly Japan K.K., and MSD K.K. SO reports receiving honoraria from Chugai Pharmaceutical Co., Ltd., AstraZeneca K.K. and Eisai Co., Ltd. FA reports receiving a consulting fee from Sysmex Corporation and research grants from Roche Tissue Diagnostics and Hitachi, Ltd. No potential conflicts of interest were disclosed by the other authors.

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Supplementary Figure 1.

Quantification of HER2 diversity comparing between pCR and non-pCR patient groups (TIF 116 kb). Heat maps of all patients showed the magnitude of the cell proportions as contrasting densities of red color. The maps were arranged from most homogeneous to most heterogeneous, with patients who achieved pCR separated from those who did not.

Supplementary Figure 2.

Relationships between the proportion of each cell type and hormone receptor (HR) status Patients with HR-positive cancer had significantly more type C (P = 0.037), E (P = 0.025), F (P = 0.009), H (P = 0.023) and I cells (P = 0.012) compared to those with HR-negative cancer, while patients with HR-positive cancer included significantly fewer type D (P = 0.047) and G cells (P = 0.021). (TIF 45 kb)

Supplementary Figure 3.

Outcome analyses using the novel diagnostic models Patients with pCR showed a significantly better prognosis compared to those with non-pCR (disease free survival (DFS): P = 0.010, overall survival (OS): P = 0.049). However there was no significant relationship between the novel diagnostic models and prognosis (Model 2: DFS, P = 0.513, OS, P = 0.753; Model 3: DFS, P = 0.206, OS, P = 0.681; Model 4: DFS, P = 0.142, OS, P = 0.579). Only Model 4 tended to be associated with DFS. (TIF 108 kb)


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Horii, R., Nitta, H., Nojima, M. et al. Predictive significance of HER2 intratumoral heterogeneity, determined by simultaneous gene and protein analysis, for resistance to trastuzumab-based treatments for HER2-positive breast cancer. Virchows Arch (2021).

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  • Intratumoral heterogeneity
  • Human epidermal growth factor receptor 2 (HER2)
  • Breast cancer
  • Gene-protein assay
  • Pathology
  • Predictive factor