Abstract P4-09-02: A molecular classification system for basal-like breast cancer based on the tumor microenvironment is prognostic for survival
Authors: Khorkova Svetlana , Shamsutdinova Diana , Kushnarev Vladimir , Popyvanov Lev , Dymov Daniil , Zotova Anastasia , Valiev Ivan , Antysheva Zoya , Love Anna , Brown Jessica H. , Bagaev Alexander , Kotlov Nikita , Fowler Nathan 
Published: March 2023

Abstract
Recent advancements in molecular profiling have revealed distinct breast cancer subtypes, but many clinical NGS assays rely on gene panels, such as PAM50, limiting their clinical utility. Basal-like breast cancer (BLBC), one of the most aggressive subtypes, has highly variable molecular and clinical characteristics. Now, the tumor microenvironment (TME) is recognized as a vital participant in tumor progression and therapeutic response. The development of more refined classifications based on the TME, capable of accounting for tissue heterogeneity, may improve NGS clinical utility for BLBC. Here, we apply our transcriptomic-based approach, recently described by Bagaev et. al., to classify the BLBC TME into discrete immune portraits, to potentially improve clinical outcomes and facilitate therapeutic decisions. We collected a cohort of 1,708 BLBC samples based on the expression levels of 50 genes (PAM50) from 10 publicly available datasets, with clinical outcomes available (n = 819). Using methodology described by Bagaev et. al., 31 functional gene expression signatures (Fges) were selected, and unsupervised dense Louvain clustering was performed to identify TME subtypes. A novel RNA-seq deconvolution algorithm was used to determine the cell types within the TME. Validation of the histological features, including stroma, tumor infiltrating lymphocytes (TILs), and tertiary lymphoid structures (TLS), relative to gene expression patterns in the TME subtypes was performed by automated and manual annotation of BLBC H&E slides (n = 146) from an independent TCGA cohort. Overall survival (OS) analysis was performed using Kaplan-Meier and Cox regression methods.
We revealed 5 BLBC subtypes with distinct expression patterns: immune-enriched, non-fibrotic (IE, 19%), B-cell–enriched, TLS-like (TLS, 25.5%), granulocyte-enriched (G, 12.8%), fibrotic (F, 28%), and immune desert (D, 17.7%) (Table). IE tumors featured an active immune TME, with high immune checkpoint expression and T cell activity. The TLS subtype also had an immune-rich TME, presenting high levels of B cells, T helper cells, and TLS (p &lt; 0.001). The TLS subtype exhibited the highest number of stromal TILs on TCGA H&E slides. The G subtype was characterized by high expression of granulocytes and granulocyte traffic molecules. In accordance with our findings, deconvolution predicted the highest percent of neutrophils in the G subtype (p &lt; 0.001). The F subtype demonstrated the highest levels of angiogenesis, stromal Fges, and VEGFR1-3, FGFR1, and EGFR expression. By histological evaluation, 84% of F subtype samples demonstrated a medium or high level of fibrosis. The D subtype showed a high proliferation rate and low stromal and immune Fges. Indicative of proliferation rate, CCNB1 and cyclin B1 were highest in G, D, and IE subtypes. OS analysis revealed a significant association between TME subtypes (TLS = baseline; log HR G = 0.87, p &lt; 0.05; IE = 0.39, p = 0.18; F = 0.99, p &lt; 0.05; D = 1.21, p &lt; 0.05), and survival outcomes. The immune-enriched subtypes, IE and TLS, demonstrated good prognosis and higher expression of immune checkpoint genes, while immune desert D and granulocyte-enriched G subtypes exhibited the worst OS.
Using our transcriptomic-based approach, BLBC was classified into 5 distinct subtypes, each with unique therapeutic vulnerabilities. Further investigation of these TME subtypes may lead to potential clinical utility as a prognostic tool to improve clinical decision making.
Table. Characteristics of Basal-like breast cancer (BLBC) tumor microenvironment (TME) subtypes. TLS - Tertiary lymphoid structures; CAF - cancer-associated fibroblasts
Citation Format: Svetlana Khorkova, Diana Shamsutdinova, Vladimir Kushnarev, Lev Popyvanov, Daniil Dymov, Anastasia Zotova, Ivan Valiev, Zoya Antysheva, Anna Love, Jessica H. Brown, Alexander Bagaev, Nikita Kotlov, Nathan Fowler. A molecular classification system for basal-like breast cancer based on the tumor microenvironment is prognostic for survival [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-02.