Rapid detection of genetic mutations in individual breast cancer patients by next-generation DNA sequencing

Abstract

Breast cancer is the most common malignancy in women and the leading cause of cancer deaths in women worldwide. Breast cancers are heterogenous and exist in many different subtypes (luminal A, luminal B, triple negative, and human epidermal growth factor receptor 2 (HER2) overexpressing), and each subtype displays distinct characteristics, responses to treatment, and patient outcomes. In addition to varying immunohistochemical properties, each subtype contains a distinct gene mutation profile which has yet to be fully defined. Patient treatment is currently guided by hormone receptor status and HER2 expression, but accumulating evidence suggests that genetic mutations also influence drug responses and patient survival. Thus, identifying the unique gene mutation pattern in each breast cancer subtype will further improve personalized treatment and outcomes for breast cancer patients. In this study, we used the Ion Personal Genome Machine (PGM) and Ion Torrent AmpliSeq Cancer Panel to sequence 737 mutational hotspot regions from 45 cancer-related genes to identify genetic mutations in 80 breast cancer samples of various subtypes from Chinese patients. Analysis revealed frequent missense and combination mutations in PIK3CA and TP53, infrequent mutations in PTEN, and uncommon combination mutations in luminal-type cancers in other genes including BRAF, GNAS, IDH1, and KRAS. This study demonstrates the feasibility of using Ion Torrent sequencing technology to reliably detect gene mutations in a clinical setting in order to guide personalized drug treatments or combination therapies to ultimately target individual, breast cancer-specific mutations.

Figure 1

Sequence read distribution across 189 amplicons generated from 80 breast cancer samples, normalized to 300,000 reads per sample. (A) Average number of reads observed for each amplicon. (B) Number of targets with a given read depth, sorted in bins of 100 reads.

Figure 2

Summary of mutated genes detected in 80 breast cancer samples. Thirty-two samples harbor mutations in PIK3CA, TP53, KRAS, BRAF, PTEN, GNAS, and IDH1. Samples are classified by four methods: 1) Immunohistochemistry of ER, PR, and HER2; 2) pathologic type (IDC2, IDC3, other); 3) AJCC/TNM-staging (2a, 2b, 3a, 3b, 3c, 4); and 4) recurrence or no recurrence.

Figure 3

Signaling pathways in breast cancer with gene mutations. Genetic alterations in breast cancer primarily occur in genes of the MAPK, p53, and PI3K/AKT signaling pathways. Alterations in oncogenes are indicated in pink, and those in cancer suppressor genes are shown in green. Percentages (%) are the frequency of mutations per gene in our study out of 80 samples. Tyrosine kinase receptors (TKRs) include HER2, EGFR, and IGF-1R in breast cancer.