Analysis of Genomic Alterations Associated with Recurrence in Early Stage HER2-Positive Breast Cancer

Abstract

We aimed to compare gene expression in primary tumors of patients with recurrence and nonrecurrence to gain insight into the biology of high-risk HER2-positive early breast cancer. Patients who underwent curative resection and received adjuvant trastuzumab for HER2-positive early breast cancer were evaluated. Gene expression analyses were performed using NanoString Technologies’ nCounter Breast Cancer 360 Panel. PAM50 intrinsic subtypes and Breast Cancer Signatures including tumor inflammation signature (TIS) were evaluated. Of 247 patients, 28 (11.3%) had recurrence at a median follow-up of 54.2 months. Patients with pathological stage III, tumor size > 5 cm, axillary lymph node metastases, and hormone receptor-negativity were more frequently observed in the recurrent group compared with the nonrecurrent group. In patients with recurrence, seven genes were upregulated significantly, including WNT11, HAPLN1, FGF10, BBOX1, CXADR, NDP, and EREG, and two genes were downregulated, including CXCL9 and GNLY. TIS score was significantly lower in patients with recurrence compared with controls without recurrence. These findings suggest that activation of oncogenic signaling pathways related to cell proliferation, adhesion, cancer stemness, and noninflamed tumor microenvironment are associated with the risk of recurrence in early stage, HER2-positive breast cancer.

Keywords: breast cancer; human epidermal growth factor receptor 2; recurrence; tumor inflammation signature.

Figure 1

nCounter Breast Cancer 360 Panel analysis (n = 34). (A) Volcano plot showing differentially expressed genes comparing nonrecurrence and recurrence groups. (B) Relative mRNA expression of nine genes with an absolute value of log2-fold change ≥ 2.0 and p-value ≤ 0.05. (C) Nine genes significantly differentially expressed in nonrecurrence and recurrence groups to categorize the biological processes. (D) Heatmap of NanoString gene expression analysis by nine genes associated with recurrence. The heatmap indicates upregulation (red), downregulation (green), and mean gene expression (black).

Figure 2

Comparative boxplots of tumor inflammation signature (TIS) score. (A) TIS score was higher in the basal-like and HER2-enriched subtypes than in the luminal subtypes. (B) TIS score was significantly lower in patients with recurrence (blue) compared to controls without recurrence (red) (p = 0.046). Mann–Whitney U test.

Figure 3

Heatmap for the 18 genes in the tumor inflammation signature (TIS). Heatmap showing the individual TIS genes normalized expression, intrinsic molecular subtypes, and recurrence. The heatmap indicates upregulation (red), downregulation (blue), and mean gene expression (yellow). The columns represent individual tissue samples: luminal A (blue), luminal B (sky blue), basal-like (red), and HER2-enriched (pink). The rows represent individual genes.

Figure 4

Comparative boxplots of seven breast cancer signature scores, which were statistically different between patients with recurrence (blue) and without recurrence (red). (A) CD8 T cells, (B) cytotoxic cells, (C) cytotoxicity, (D) indoleamine 2,3-dioxygenase 1 (IDO1), (E) interferon-gamma, (F) programmed cell death protein 1 (PD1), and (G) SRY-box transcription factor 2 (SOX2). Mann–Whitney U test.