Introduction: In clinical practice, immunohistochemistry (IHC) is combined with clinicopathologic information to stratify patients into subtypes and guide treatment decisions. In our mass spectrometry study of 116 breast cancers (BrCAs), we identified 34 significant proteins which provide additional prognostic information. This novel proteomic subtyping, combining IHC with proteomics, separates HER2 negative (Her2-) BrCA patients into Luminal-like and TN-like subtypes. A novel subtype, luminal by IHC and TN-like by proteomics (L/T), is associated with unfavorable outcome compared with the luminal subtype (L/L) by both IHC and proteomics. These IHC-based Her2- subtypes are described as L/L, L/T, T/L and T/T subtypes. We investigated the cell-of-origin of our enhanced subtypes using gene set enrichment analysis with gene sets consisting of breast cancer cell-of-origin signatures. We further identified genes representing our 34 proteins in the significant gene sets and investigated their association with survival outcomes across The Cancer Genome Atlas (TCGA) pan-cancers. Method: The single-cell RNA-sequencing data analyses of health breast samples identified 23 breast cell-of-origin signatures (Bhat-Nakshatri et al., 2021) with 46 up and down-regulated gene sets based on the provided fold changes. Using our cohort, we performed differential analysis for each enhanced subtype (L/L, L/T, T/T) versus the other subtypes separately. Pre-ranked gene set enrichment analyses were used to identify significantly enriched gene sets. The significance of enriched gene sets was reported at FDR<0.05. We further identified genes of 34 biomarkers that overlapped in the identified significant gene sets and investigated the impact of the genes on survival across TCGA pan-cancers. Univariate overall survival (OS) and progression-free interval (PFI) analyses were performed on each cancer cohort and the significance of the association with survival outcome was reported by log-rank p-value <0.05. Results: Our signature was compared to the clusters from healthy tissues. We found up-regulated genes in L/L subtype were enriched in gene sets elevated in mature luminal annotated clusters (N8 and N12). Down-regulated genes in L/L subtype were enriched in the elevated genes in the N3 (luminal progenitor) and N9 (luminal progenitor/basal) clusters. Up-regulated genes in L/T subtype were enriched in the elevated genes in the N19 (luminal progenitor) and N11 (basal) clusters. Down-regulated genes in L/T were enriched in the elevated genes in the N8 (mature luminal) cluster and down-regulated genes in the N9 (luminal progenitor/basal) cluster. Up-regulated genes in T/T subtype were enriched in genes highly expressed in the luminal progenitor (N3) and luminal progenitor/basal (N9) clusters. Down-regulated genes in T/T subtype were enriched in the elevated genes in the mature luminal annotated clusters (N8, N12). One gene, KPNA2, was up-regulated in both the N9 (luminal progenitor/basal) cluster and the Basal-like (TN-like) subtype. The higher expression of this gene had significant association with worse OS outcomes in most of the cancers. Conclusions: T/T and L/T subtypes likely arise from progenitor and basal cells. The L/L subtype may arise from mature luminal cells. The novel L/T subtype appears to have characteristics that result in poorer survival. Disclaimers The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of USUHS, HJF, the DOD or the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.

Citation Format: Guisong Wang, Punit Shah, Rick Searfoss, J. Leigh Fantacone-Campbell, Jeffrey A. Hooke, Brenda Deyarmin, Rebecca N. Zingmark, Stella Somiari, Jianfang Liu, Leonid Kvecher, Lori A. Sturtz, Praveen-Kumar Raj-Kumar, Elder Granger, Linda Vahdat, Niven R. Narain, Mary L. Cutler, Rangaprasad Sarangarajan, Hai Hu, Michael A. Kiebish, Albert J. Kovatich, Craig D. Shriver. Novel breast cancer proteomic subtyping with connection to cell of origin [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-04.