Abstract PO2-15-05: Mesenchymal-like immune-activated: the 4th robust triple-negative breast cancer molecular subtype
Authors: Campone Mario , Jézéquel Pascal , Azzouz Fadoua Ben , Gouraud Wilfried , Bassevile Agnes , Michel Bertrand , Lasla Hamza , FRENEL Jean Sebastien 
Published: May 2024

Abstract
Triple-negative breast cancer (TNBC) accounts for 15-20% of primary BC. This heterogeneous disease is the most aggressive BC subtype. TNBC is characterized by young age at onset, high-grade tumors, high risk of early recurrence and poor prognosis. Until recently, TNBC patients were considered as one group and all patients were treated with conventional chemotherapy. With the emergence of new therapies targeting distinct biological pathways, personalized medicine is now accessible by defining finer TNBC subtypes, e.g. molecular subtypes, and identifying their specific targetable pathways. Several studies based on transcriptomics have identified three to six TNBC molecular subtypes. Today, there is a clear consensus on three TNBC robust subtypes: luminal androgen receptor (LAR), basal-like immune-activated (BLIA) and basal-like immune-suppressed (BLIS). The debate about the robustness of other subtypes is still open, notably mesenchymal-like (M) and mesenchymal stem-like (MSL or MES).
The goal of this study was to establish a robust molecular classification of TNBC that allow to determine the best treatment options. To optimize this process, we harvested as much transcriptomic data as possible. Available clinicopathological data linked to these data were also imported. To our knowledge, this is the largest study aimed at subtyping TNBC; it exploits a total of 1942 TNBC patient data collected from 19 datasets: 1243 samples from microarray technology (14 datasets) and 699 from RNAseq technology (five datasets including a new one from our institution [GSE225002]).
We performed independent unsupervised analyses on these two large cohorts with consensus k-means clustering including selection of optimal number of clusters and optimal number of variables. Both consensus clustering analyses separated TNBC microarray and RNAseq TNBC cohorts into four optimal clusters. These clusters were then characterized using clinicopathological features, gene ontology enrichment analyses (GOEA), gene set enrichment analysis (GSEA), 70 gene expression signatures (GES) and expression of 47 immune checkpoint genes.
We concluded that TNBC are composed of four robust subtypes: LAR, mesenchymal-like immune-activated (MLIA), BLIA and BLIS. Importantly, we propose that MLIA acronym should replace M or MSL, because it highlights the two main biological characteristics of this subtype, i.e. mesenchymal-like features and a high IR comparable to that of BLIA. Furthermore, we showed that each subtype shares common biological characteristics with another one: LAR and MLIA (androgen receptor pathway activation); MLIA and BLIA (immune activation, notably high levels of immune checkpoints); BLIA and BLIS (basal-like features); BLIS and LAR (immune suppression).
As regards microarray cohort, LAR included 19.23% (n = 239) of TNBC, MLIA 17.38% (n = 216), BLIA 32.50% (n = 404) and BLIS 30.89% (n = 384). Concerning RNAseq cohort, LAR included 19.60% (n = 137) of TNBC, MLIA 17.31% (n = 121), BLIA 28.47% (n = 199) and BLIS 34.62% (n = 242). Distributions of patients among the four clusters in the two cohorts were similar (p = 0.3583). Distributions were also similar between studies for microarray cohort (p = 0.2479) and RNAseq cohort (p = 0.1775). Pooled data (n = 1942 patients) gave the following distribution: 19.36% for LAR; 17.35% for MLIA; 31.06% for BLIA; 32.23% for BLIS, confirming that TNBC are largely but not exclusively basal-like (63.29%).
In brief, according to the TNBC subtype molecular characterisation, anti-androgen therapy could be prescribed for LAR, immune checkpoint inhibitors for MLIA and BLIA, alone or in combination, and TAM-focused and antineurogenic therapies for BLIS.
As an extension of this work, TNBC transcriptomic data associated with their clinicopathological features and TNBC subtype annotations were included in bc-GenExMiner v5.0 web tool (http://bcgenex.ico.unicancer.fr).
Citation Format: Mario Campone, Pascal Jézéquel, Fadoua Ben Azzouz, Wilfried Gouraud, Agnes Bassevile, Bertrand Michel, Hamza Lasla, Jean Sebastien FRENEL. Mesenchymal-like immune-activated: the 4th robust triple-negative breast cancer molecular subtype [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-05.