Abstract P2-06-02: Breast cancer HER2 epigenetic intratumoral heterogeneity results from lack of HER2 protein translation
Authors: Nitta H , Horii R , Murillo A , Portier B , Akiyama F 
Published: February 2018

Abstract
Research objective
Previously, we reported the negative correlation between pathological complete response (pCR) and HER2 positive breast cancer exhibiting amplified HER2 gene tumor cells without HER2 protein overexpression (HER2 epigenetic intratumoral heterogeneity) among trastuzumab-based neoadjuvant chemotherapy treated patients. Our objective in this study was to elucidate if tumor cells with HER2 epigenetic intratumoral heterogeneity express HER2 RNA using a HER2 RNA in situ hybridization (ISH) method.
Materials and methods
Formalin-fixed, paraffin-embedded (FFPE) sections of breast cancer biopsy samples were investigated for HER2 RNA expression at the individual cell level using a HER2 RNA ISH assay. RNA preservation in tissue sections was examined using a peptidylprolyl isomerase B (PPIB) RNA ISH assay.
Three groups of cases were examined:
1) HER2 negative breast cancer cases (HER2 RNA ISH negative control group).
2) HER2 positive breast cancer cases with homogeneous HER2 positive tumor cells (HER2 RNA ISH positive control; pCR group)
3) HER2 positive breast cancer cases with HER2 epigenetic intratumoral heterogeneity (a mixture of HER2 gene and protein positive tumor cells and HER2 gene positive tumor cells without HER2 protein expression; incomplete response group)
Consecutive sections of HER2 RNA ISH slides were stained for HER2 gene and protein concurrently on the same tissue section using HER2 gene-protein assay (GPA) which is a combination of FDA-approved HER2 immunohistochemical (HER2 protein) and HER2 dual ISH (HER2 gene and chromosome 17 centromere) assays. Analyses of HER2 RNA expression in individual cells was microscopically evaluated and matched to HER2 GPA slides.
Results
RNA preservation was confirmed in tissue sections of all three groups by a PPIB RNA ISH assay. Tumor cells of HER2 negative breast cancer cases (negative control group) lacked HER2 RNA ISH signal while HER2 gene and protein positive tumor cells of homogeneous breast cancer cases (positive control group) demonstrated high HER2 RNA expression levels. HER2 gene and protein positive tumor cells of HER2 positive intratumoral heterogeneity cases showed high HER2 RNA expression. However, amplified HER2 gene breast cancer cells without HER2 protein overexpression of HER2 positive intratumoral heterogeneity cases also showed high levels of HER2 RNA expression. Thus, revealing in cases with intratumoral heterogeneity, transcription of HER2 RNA occurs, but translation of HER2 protein is altered by some mechanism(s) in tumor cells.
Conclusions
Transcription of HER2 RNA was observed in breast tumor cells with amplified HER2 gene but absence of HER2 protein overexpression (HER2 epigenetic intratumoral heterogeneity) of patients who showed incomplete response to neoadjuvant trastuzumab therapy. Our study suggests that inconsistent HER2 protein translation in breast cancer with HER2 epigenetic heterogeneity might be the primary resistance mechanism to trastuzumab-based neoadjuvant chemotherapy.
Citation Format: Nitta H, Horii R, Murillo A, Portier B, Akiyama F. Breast cancer HER2 epigenetic intratumoral heterogeneity results from lack of HER2 protein translation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-06-02.