Triple-Negative and Basal-like Carcinoma

Intrinsic Subtype and Integrative Cluster Classification

The transcriptomic profiles of ER+ and ER- breast cancers are substantially different, with multiple tumor classes for each set of hormone receptor (HR) status. The molecular classification of breast cancer is summarized here and described in detail in Chapter 20 . Hierarchical cluster analyses of gene expression in IBCs with an intrinsic gene list revealed the existence of four main intrinsic subtypes: (1) luminal A, characterized by expression of ER and genes related to the ER pathway, and correlating to ER+/HER2– tumors with low proliferation; (2) luminal B subtype, also characterized by expression of ER and genes related to the ER pathway, but correlating to ER+/HER2+ tumors or ER+/HER2– tumors with high proliferation; (3) the HER2-enriched subtype, characterized by overexpression of HER2 protein and HER2 gene amplification; and (4) basal-like cancers, which express genes normally found in the basal/myoepithelial compartment of normal breast tissue and are most often TNBC. Additional less common intrinsic subtypes are “normal breast-like” (which may represent an artifact of low tumor cellularity) and claudin-low, which are largely triple-negative low-grade spindle cell carcinomas.

TNBC constitutes a heterogeneous group of intrinsic subtypes. TNBC encompasses the majority of basal-like breast cancers, some normal breast-like tumors, and claudin-low cancers. More recently, TNBCs have been further subdivided into additional molecular classes, including a four-group classification of luminal androgen receptor (AR), mesenchymal, basal-like immune-suppressed, and basal-like immune-activated tumors, and a six-group classification of basal-like I, basal-like II, mesenchymal, mesenchymal stem-like, immunomodulatory, and luminal androgen receptor tumors. Subtype classifications of TNBC are clinically relevant and associate with distinct responses to neoadjuvant chemotherapy. TNBC forms a discrete transcriptomic entity, and the basal-like subtype comprises a group of tumors fundamentally different from ER+ and HER2+ cancers. Integrative analysis combining genomic and transcriptomic data separates breast carcinomas into 10 integrative subgroups (IntClusters) with distinct clinical behavior, including an immune-infiltrated cluster and an ER- cluster with poor prognosis.

Molecular Alterations of Basal-like Carcinomas

The existence of breast cancers with ultrastructural features of basal/myoepithelial cells of the normal breast was reported many decades ago, and gene expression profiling studies verified the existence of this subgroup of breast cancers and brought them to clinical attention. Basal-like breast cancers were so named because their transcriptomic profile, cytokeratin (CK) filament expression, lack of ER expression, and lack of HER2 overexpression were identical to that of the basal/myoepithelial cells of the normal breast (e.g., expression of CK5/6 and CK17 and epidermal growth factor receptor [EGFR]).

The molecular alterations of basal-like carcinoma demonstrate heterogeneity of gene copy numbers and a large mutational spectrum with high mutation burden. Basal-like TNBCs have complex karyotypes, usually in the form of a sawtooth microarray–based comparative genomic hybridization pattern (i.e., multiple low-level gains and deletions throughout the genome, often involving whole chromosomes or chromosomal arms). High-level gene amplifications are found in more than 70% of all basal-like breast cancers; however, each locus is amplified in a small minority of cancers. Basal-like cancers also harbor a “mutator phenotype,” characterized by multiple intrachromosomal and interchromosomal rearrangements, and they also display tumoral genetic heterogeneity.

The most frequent somatic mutations or alterations in basal-like breast cancers include TP53 (87%), PTEN (35%), RB1 (20%), and PIK3CA (7%), with smaller rates of mutations in PIK3R1 and NEK2 . The high degree of genetic instability in basal-like cancers impacts genes in different gene categories (e.g., extracellular matrix, receptor, or oncogene categories), such as p-cadherin, fatty acid–binding protein 7, c-kit, matrix metalloproteinase 7, caveolin 1 and 2, metallothionein IX, transforming growth factor–beta (TGF-β) receptor II, and hepatocyte growth factor. Moreover, these tumors display increased expression of cell growth– and cell proliferation–related genes (e.g., topoisomerase IIα , CDC2 , and PCNA ), correlating to observations that these cancers display remarkably high proliferation rates. Because the majority of basal-like cancers display high expression of proliferation-related genes, prognostic gene signatures based on proliferation are not as relevant in basal-like or TNBC as in ER+ breast cancers.

Adenoid cystic and secretory carcinomas are two special histological subtypes of breast carcinoma that are basal-like by intrinsic subtyping but are typically low-grade and have additional unique molecular alterations. In contrast to high-grade basal-like breast cancers, adenoid cystic and secretory carcinomas do not harbor recurrent TP53 mutations and are not associated with BRCA1 pathway inactivation. The majority of breast adenoid cystic carcinomas harbor the t(6;9)(q22-23;p23-24) translocation, which leads to the formation of the MYB-NFIB fusion gene and MYB overexpression. MYB overexpression does not occur in high-grade basal-like TNBC and is specific for adenoid cystic carcinomas. Secretory carcinomas are characterized by the recurrent chromosomal translocation t(12;15)(p13;q25), which leads to the ETV6-NTRK3 chimeric fusion gene. This fusion gene has oncogenic properties, is a defining feature of secretory carcinoma, and is a predictive biomarker for NTRK-targeted therapy.

BRCA1 Mutations in Basal-like Carcinomas

Tumors with a basal-like phenotype/genotype are enriched in patients with germline BRCA1 mutations. BRCA1 is involved in a large number of cellular processes, including DNA repair, cell cycle regulation, transcriptional regulation (notably, regulation of ER expression), and chromatin remodeling. Loss of BRCA1 leads to a deficiency in the repair of double-strand DNA breaks by homologous recombination, an error-free DNA repair mechanism. In the absence of BRCA1, double-strand breaks are repaired by means of nonconservative and error-prone DNA repair mechanisms, including single-strand annealing and nonhomologous end joining. These nonconservative, error-prone DNA repair mechanisms lead to a genomic instability and a high tumor mutation burden.

Tumors from patients with germline BRCA1 mutations are predominantly basal-like by gene expression profiling, suggesting that BRCA1 germline mutations predispose to the development of this molecular subtype of breast cancer. Inactivation of BRCA1 and TP53 in different components of the mouse mammary gland leads to the development of tumors that recapitulate human basal-like breast cancers by histology, immunophenotype, and gene expression profiles. The basal-like breast cancers in patients with germline BRCA1 mutations have several genomic aberrations in common, such as gains at 3q; losses at 3p, 4p, 4q, and 5q; and a high frequency of TP53 mutations.

In addition, there is a link between somatic BRCA1 dysfunction and a basal-like phenotype in sporadic breast cancers. BRCA1 mRNA and protein levels are lower in sporadic basal-like breast cancers than in histologically grade-matched ER+ or HER2 + breast carcinomas. Somatic BRCA1 mutations are relatively rare; but alternative gene aberrations lead to BRCA1 dysfunction. Subsets of sporadic basal-like cancers have copy number aberrations in BRCA1 DNA damage response genes, aberrant BRCA1 copy number and decreased BRCA1 mRNA, ID4 copy number gains and overexpression of the BRCA1 gene silencer ID4, BRCA1 promoter methylation, or upregulation of microRNAs (miRNAs) that downregulate BRCA1 , including miR-146a, miR-146b-5p, and miR-182.