Background: Triple negative breast cancers (TNBC) are more aggressive, have a worse prognosis, and few targeted therapies compared to other BC subtypes. TNBC is molecularly heterogeneous, with at least 4 distinct subtypes: basal-like immune-activated (BLIA), basal-like immunosuppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES). The molecular subtyping gene signature, BluePrint (BP), classifies breast tumors into Luminal, HER2, or Basal subtype based on the assessment of downstream signaling pathways and independently of IHC expression. Compared to IHC-defined TNBC, a higher frequency of BLIS or BLIA subtypes and fewer LAR or MES tumors were reported in BP-defined Basal tumors. To advance our understanding of TNBC heterogeneity, we evaluated the relationship between gene expression signatures, TNBC subtype and BluePrint, in IHC-defined TNBC.

Methods: The FLEX registry (NCT03053193) is an ongoing, prospective study evaluating primary tumors from stage I-III BC patients who receive the risk of distant recurrence gene signature, MammaPrint (MP), and BP testing and consent to clinically annotated full transcriptome data collection. This analysis includes 204 IHC-defined TNBC patients. TNBC subtypes BLIA, BLIS, LAR, and MES were derived using an adjusted version of the Burstein centroid signature. BP classified patient samples into Luminal, HER2, and Basal subtypes. A proportion of tumors may exhibit a secondary but less pronounced activated pathway or BP subtype. Therefore, each BP subtype was divided into single activated or mixed subtype based on BP indices.

Results: Of 204 TNBC tumors, 84% were classified as Basal by BP, most of which were BLIS (65%), followed by BLIA (22%), with a low frequency of MES (8%) and LAR (5%) subtypes (Table). Approximately 14% of TNBCs were reclassified as Luminal by BP, most of which were LAR (76%), whereas 24% were MES. Clustering analysis revealed similar gene expression profiles between Basal-BLIS and Basal-BLIA tumors. Interestingly, the transcriptional profile of Basal-MES and Basal-LAR tumors were similar to Luminal-MES and Luminal-LAR tumors. BP Basal indices distinguished between different TNBC subtypes. The Basal pathway was predominantly activated in 90% of BP Basals (single activated tumors), most of which were either BLIS or BLIA (96%), whereas 10% of BP Basals were mixed subtype and more likely to classify as LAR (53%) or MES (35%). Approximately 25% of the BP Basal gene signature overlapped with the TNBC subtype gene signature. Expression of 18 and 12 genes out of 28 genes that make up the BP basal signature were significantly different in Basal-BLIA/BLIS compared to LAR or MES, respectively (P < 0.05). PRR15 and CAPN13 were significantly differentially expressed between LAR and MES within Basals.

Conclusion: BP reclassified a subgroup of TNBC tumors to Luminal, explaining the discrepancy in the distribution of TNBC subtypes between IHC-defined TNBC and BP Basal tumors. Furthermore, BP indices distinguished between single activated and mixed subtypes, which correlated with different TNBC subtypes. These data suggest that molecular classification by BP adds further precision in classifying TNBC patients and sheds new light on the heterogeneity of these tumors. These findings have clinical implications in stratifying patients and identifying successful targeted treatment options. Future studies are warranted to investigate treatment response and prognosis in these molecular subgroups.

BasalLuminalHER2Total
BLIA 37 37 
BLIS 111 111 
LAR 10 22 35 
MES 14 21 
Total 172 (84.3%) 29 (14.2%) 3 (1.5%) 204 (100%) 
BasalLuminalHER2Total
BLIA 37 37 
BLIS 111 111 
LAR 10 22 35 
MES 14 21 
Total 172 (84.3%) 29 (14.2%) 3 (1.5%) 204 (100%) 

Citation Format: Virginia G. Kaklamani, Cathy Graham, Karen L. Tedesco, Abirami Sivapiragasam, Jennifer A. Crozier, Apurva N. Shah, Yuan Yuan, Josien Haan, Andrea Menicucci, Michelle L. Bolner, Shiyu Wang, Lorenza Mittempergher, Erin Yoder, William Audeh, FLEX Investigators' Group. Using blueprint to elucidate the molecular heterogeneity of triple negative breast cancers [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-05.