Comprehensive genomic analysis identifies novel subtypes and targets of triple-negative breast cancer

Abstract

Purpose: Genomic profiling studies suggest that triple-negative breast cancer (TNBC) is a heterogeneous disease. In this study, we sought to define TNBC subtypes and identify subtype-specific markers and targets.

Experimental design: RNA and DNA profiling analyses were conducted on 198 TNBC tumors [estrogen receptor (ER) negativity defined as Allred scale value ≤ 2] with >50% cellularity (discovery set: n = 84; validation set: n = 114) collected at Baylor College of Medicine (Houston, TX). An external dataset of seven publically accessible TNBC studies was used to confirm results. DNA copy number, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed independently using these datasets.

Results: We identified and confirmed four distinct TNBC subtypes: (i) luminal androgen receptor (AR; LAR), (ii) mesenchymal (MES), (iii) basal-like immunosuppressed (BLIS), and (iv) basal-like immune-activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (log-rank test: P = 0.042 and 0.041, respectively) and DSS (log-rank test: P = 0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA) and suggested that gene amplification drives gene expression in some cases [FGFR2 (BLIS)]. Putative subtype-specific targets were identified: (i) LAR: androgen receptor and the cell surface mucin MUC1, (ii) MES: growth factor receptors [platelet-derived growth factor (PDGF) receptor A; c-Kit], (iii) BLIS: an immunosuppressing molecule (VTCN1), and (iv) BLIA: Stat signal transduction molecules and cytokines.

Conclusion: There are four stable TNBC subtypes characterized by the expression of distinct molecular profiles that have distinct prognoses. These studies identify novel subtype-specific targets that can be targeted in the future for the effective treatment of TNBCs.

Figure 1. Classification of TNBCs by mRNA profiling reveals four stable molecular phenotypes

84 (discovery set) and 114 TN breast tumors (validation set) both demonstrate 4 stable clusters by NMF of mRNA expression across the top 1000 genes (IQR summarized) selected by DEDS aggregate rank of median absolute deviations (see complete methods) of the discovery set. A & D. Cophenetic and dispersion metrics for NMF across 2-10 clusters with 50 runs suggest 4 stable clusters. Full metrics are available for each set in Supplemental Figures 1 and 2. B & E. Silhouette analyses and consensus plots for rank basis 4 NMF clusters (1000 runs, nsNMF factorization). Average silhouette widths worsened with increasing clusters beyond the 4 shown. SigClust was significant for all pairwise comparisons with this feature set. C & F. PAM50 intrinsic subtypes and TNBCType distributions by 4 NMF clusters.

Figure 2. Gene signature defines four subtypes of TNBC with prognostic differences

Discovery, validation, and external sets tumors with intermediate grade, high ESR1, PGR, and ERBB2 expression, activated ER downstream targets, and luminal A/B subtypes are enriched in subtype 1. A. The four assigned subtypes in both the discovery (84/84) and validation sets (114/114). B. Gene signature applied successfully to 220 of 221 external set TNBCs. C. Clinical outcomes from independent sets classified by the discovery set-based signature. Subtype 4 has a better prognosis for both disease-free and disease-specific survival.

Figure 3. Molecular pathways enriched in the four identified subtypes of TNBCs

Significant pathways from the discovery set also found in validation and external sets are listed for the LAR, MES, BLIS and BLIA subtypes.

Figure 4. DNA Copy Number Analysis identifies focal changes in TNBC subtypes

DNA copy number changes observed in each subtype are listed. A. Focal gains (red) and losses (blue) detected by GISTIC 2.0 are plotted by log10(q-value) and reported by cytoband. Adjacent numbers are percentages of subtype specific cases (n = 24, 17, 33, 34, respectively) with this focal aberration. Presence of a colored square demonstrates this region was detected by subtype-specific GISTIC 2.0 analysis as well. All structural events for each subtype and set are available in the supplemental. B. Broad copy number events distinguish the LAR subtype from all others. Gains (red) and losses (blue) are plotted along the genome, with darker colors representing a region enriched to the displayed subtype by Fisher Exact Test.