Purpose Genomic profiling studies suggest 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. Patients and Methods RNA and DNA profiling analyses were conducted on 198 TNBC tumors (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. An external data set of 7 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: (1) Luminal-AR (LAR); 2) Mesenchymal (MES); 3) Basal-Like Immune-Suppressed (BLIS), and 4) Basal-Like Immune-Activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (logrank test p=0.042 and 0.041, respectively) and DSS (logrank test p=0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA), and suggested gene amplification drives gene expression in some cases (FGFR2 (BLIS)). Putative subtype-specific targets were identified: 1) LAR: androgen receptor and the cell surface mucin MUC1; 2) MES: growth factor receptors (PDGF receptor A; c-Kit); 3) BLIS: an immune suppressing molecule (VTCN1); and 4) 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 effective treatment of TNBCs.

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Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of SCID/Beige and NOD/SCID/IL2γ-receptor null (NSG) mice, under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (~21% and ~19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing 25 unique patients. Most tumors yielding xenografts were “triple-negative” (ER-PR-HER2+) (n=19). However, we established lines from three ER-PR-HER2+ tumors, one ER+PR-HER2−, one ER+PR+HER2− and one “triple-positive” (ER+PR+HER2+) tumor. Serially passaged xenografts show biological consistency with the tumor of origin, are phenotypically stable across multiple transplant generations at the histologic, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses as those observed clinically. Xenografts representing 12 patients, including two ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis.