Abstract B063: Investigation of tumor heterogeneity using integrated single-cell RNA sequence data based on HER2 status in patients with breast cancer
Authors: Sho Shiino, Momoko Tokura, Jun Nakayama, Masayuki Yoshida, Akihiko Suto, Yusuke Yamamoto
Journal: Cancer Research (2024)

Abstract [Introduction] Human epidermal growth factor receptor 2 (HER2), which is characterized by ERBB2 amplification, is one of the important markers for treatment decision related to breast cancer. Many targeted therapies for HER2-positive (immunohistochemistry [IHC] scores of 3+ or 2+ with an in situ hybridization [ISH] gene amplification) or HER2-low (IHC score 1+ or 2+ with no ISH gene amplification) breast cancers have been extensively developed thus far. Meanwhile, tumoral heterogeneity is considered one of the mechanisms for drug resistance; however, it has not been fully elucidated on each HER2 status at single-cell level. Therefore, an integrated analysis of the breast cancer single-cell gene expression data of both public datasets and our cohort was used to investigate tumor heterogeneity based on HER2 status. [Methods] We collected 21 and 6 scRNA-seq samples of primary breast cancer from the public Gene Expression Omnibus (GEO) and our institution, respectively. A total of 27 samples included HER2-positive cases (pure HER2 cases: estrogen receptor [ER]-negative/HER2-positive and Luminal-HER2 cases: ER-positive/HER2-positive) and Luminal cases (ER-positive/HER2-negative). These datasets were imported into R software version 4.2.0. and transformed into Seurat objects with the package Seurat version 4.3.0. UMAP plots, which is a non-linear dimension reduction method, were used for clustering analyses. Seurat in R was used to generate UMAP, feature, and violin plots. Additionally, we performed pathway enrichment analysis with the significant gene list from each cluster between the ERBB2-high and ERBB2-low groups. [Results] Clustering analysis revealed heterogeneous distribution in each gene related to breast cancer (ESR1, PGR, ERBB2, and MKI67). One of the clusters revealed high MKI67 expression. ERBB2 expressions were diffusely distributed on each cluster of pure HER2 cases; however, the expressions were considerably higher in one of the clusters in cases of Luminal-HER2. The ERBB2 expression in Luminal cases, which included HER2-low status, was lower than that in HER2-positive cases, albeit slight expression was observed. Cell proliferation factors, including IGF, IGF1R, and EGF, were included in the ERBB2-high expression group compared with the ERBB2-low expression group for pathway analysis. Further, we examined typical gene expressions which are associated with markers related to breast cancer, cancer stem cell, and epithelial-to-mesenchymal transition in each case and revealed heterogeneous patterns across patients. [Conclusion] Heterogeneous ERBB2 expression distribution was observed in HER2-positive cases, and slight ERBB2 expression was identified in the Luminal cases. Moreover, Luminal-HER2 cases could be considered a more heterogeneous subtype compared with pure HER2 cases. Additionally, gene expressions of typical gene markers varied across patients. These results indicated that breast cancer displays heterogeneous patterns on each HER2 status not only intra-tumoral heterogeneity but also inter-patient heterogeneity. Citation Format: Sho Shiino, Momoko Tokura, Jun Nakayama, Masayuki Yoshida, Akihiko Suto, Yusuke Yamamoto. Investigation of tumor heterogeneity using integrated single-cell RNA sequence data based on HER2 status in patients with breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B063.