Genetic alterations of triple negative breast cancer by targeted next-generation sequencing and correlation with tumor morphology

Abstract

Triple negative breast cancer represents a heterogeneous group of breast carcinomas, both at the histologic and genetic level. Although recent molecular studies have comprehensively characterized the genetic landscape of these tumors, few have integrated a detailed histologic examination into the analysis. In this study, we defined the genetic alterations in 39 triple negative breast cancers using a high-depth targeted massively parallel sequencing assay and correlated the findings with a detailed morphologic analysis. We obtained representative frozen tissue of primary triple negative breast cancers from patients treated at our institution between 2002 and 2010. We characterized tumors according to their histologic subtype and morphologic features. DNA was extracted from paired frozen primary tumor and normal tissue samples and was subjected to a targeted massively parallel sequencing platform comprising 229 cancer-associated genes common across all experiments. The average number of non-synonymous mutations was 3 (range 0-10) per case. The most frequent somatic alterations were mutations in TP53 (74%) and PIK3CA (10%) and MYC amplifications (26%). Triple negative breast cancers with apocrine differentiation less frequently harbored TP53 mutations (25%) and MYC gains (0%), and displayed a high mutation frequency in PIK3CA and other PI3K signaling pathway-related genes (75%). Using a targeted massively parallel sequencing platform, we identified the key somatic genetic alterations previously reported in triple negative breast cancers. Furthermore, our findings show that triple negative breast cancers with apocrine differentiation constitute a distinct subset, characterized by a high frequency of PI3K pathway alterations similar to luminal subtypes of breast cancer.

Figure 1. Special histologic subtypes and special morphologic features of triple negative breast cancers

A) Carcinoma with apocrine differentiation (apocrine triple negative breast cancer); B) metaplastic carcinoma; C) tumor with a large central acellular zone of necrosis or fibrosis; D) tumor with prominent tumor infiltrating lymphocytes.

Figure 2. The landscape of somatic genetic alterations in triple negative breast cancers

Top panels: the repertoire of somatic non-synonymous mutations in our triple negative breast cancer cohort as compared to the corresponding genes in triple negative breast cancers in the Cancer Genome Atlas Cohort. Bottom panels: the genes amplified in at least 10% of our triple negative breast cancer cohort or deleted in at least 5% of our triple negative breast cancer cohort, as compared to the same genes in triple negative breast cancers in the Cancer Genome Atlas cohort. Abbreviations: TNBC, triple negative breast cancer; TCGA, the Cancer Genome Atlas; NOS, not otherwise specified; LCAZ, large central acellular zone; TILs, prominent tumor infiltrating lymphocytes.

Figure 3. Frequency of copy number aberrations in our triple negative breast cancer cohort as compared to triple negative breast cancers in the Cancer Genome Atlas cohort

Abbreviations: TNBC, triple negative breast cancer; MSKCC, Memorial Sloan Kettering Cancer Center; TCGA, the Cancer Genome Atlas.

Figure 4. Frequency of copy number aberrations in our triple negative breast cancer cohort, by morphologic group

A) amplifications and deletions; B) gains and losses Abbreviations: TNBC, triple negative breast cancer; NOS, not otherwise specified; LCAZ, large central acellular zone; TILs, prominent tumor infiltrating lymphocytes.

Figure 5. Immunohistochemical staining for androgen receptor in triple negative breast cancer with apocrine differentiation (apocrine triple negative breast cancer)

A and B) An example of androgen receptor positive apocrine triple negative breast cancer, H&E and androgen receptor immunohistochemical stain; C and D) The apocrine triple negative breast cancer that is androgen receptor negative but harboring a PIK3CA mutation, H&E and androgen receptor immunohistochemical stain.