Density of tertiary lymphoid structures predicts clinical outcome in breast cancer brain metastasis

Abstract

Background: Patients with breast cancer brain metastases (BCBM) experience a rapid decline in their quality of life. Recently, tertiary lymphoid structures (TLSs), analogs of secondary lymphoid organs, have attracted extensive attention. However, the potential clinical implications of TLSs in BCBMs are poorly understood. In this study, we evaluated the density and composition of TLSs in BCBMs and described their prognostic value.

Methods: Clinicopathological data were collected from 98 patients (2015-2021). TLSs were evaluated, and a TLS scoring system was constructed. Differences in progression-free survival (PFS) and overall survival (OS) between groups were calculated using the Kaplan-Meier method. Immunohistochemistry and multiplex immunofluorescence (mIF) were used to assess TLSs heterogeneity.

Results: TLSs were identified in 47 patients with BCBM. High TLSs density indicated favorable survival (OS, p=0.003; PFS, p<0.001). TLS was positively associated with OS (p=0.0172) and PFS (p=0.0161) in the human epidermal growth factor receptor type 2-positive subtype, and with prolonged OS (p=0.0482) in the triple-negative breast cancer subtype. The mIF results showed significant differences in the percentages of T follicular helper (Tfh) cells, M2 macrophages, cytotoxic T lymphocytes, and CD8⁺TIM-3⁺ T lymphocytes between the groups of TLS scores 0-3 (cytotoxic T lymphocytes, p=0.044; Tfh, p=0.021; M2 macrophages, p=0.033; CD8⁺TIM-3⁺ T lymphocytes, p=0.018). Furthermore, novel nomograms incorporating the TLS scores and other clinicopathological predictors demonstrated prominent predictability of the 1-year, 3-year, and 5-year outcomes of BCBMs (area under the curve >0.800).

Conclusion: Our results highlight the impact of TLSs abundance on the OS and PFS of patients with BCBM. Additionally, we described the immune composition of TLSs and proposed novel nomograms to predict the prognosis of patients with BCBM.

Keywords: Central Nervous System Cancer; Relapse; Survivorship; Tumor Microenvironment.

Figure 1

Characteristics and density of TLS in BCBMs. (A) Representative images of H&E staining for TLS scoring system (scores 0–3). TLSs are highlighted in green. (B, C) The number and percentage of scores 0–3 of TLS in all cases was determined. (D) Heat map of TLS relative gene expression in GSE2034 and GSE14020. BCBM, breast cancer brain metastases; TLS, tertiary lymphoid structure.

Figure 2

Characteristics of TLSs maturation heterogeneity in BCBMs. IHC assay to evaluate aggregates of lymphocytes that have histological features analogous to lymphoid tissues with CD3⁺ T cells, CD20⁺ follicular B cells, CD21⁺ follicular DCs, or CD23⁺ germinal center cells in serial sections. (A) Representative images of early TLSs. (B) Representative images of primary follicle-like TLSs. BCBM, breast cancer brain metastases; CD, cluster of differentiation; DC, dendritic cells; IHC, immunohistochemistry; TLS, tertiary lymphoid structure.

Figure 3

Prognostic value of TLSs presence and density in BCBM patients and correlation between TLSs and different breast subtype. (A, B) Kaplan-Meier and log-rank tests identifying the predictive value of the presence of TLSs for OS and PFS. (C, D) Kaplan-Meier curves showing OS and PFS of BCBM patients stratified by TLS scores. (E, F) Kaplan-Meier curves for OS and PFS in the HER2+ subtype according to the presence of TLSs. (H, I). Kaplan-Meier curves for OS and PFS in the TNBC subtype according to the presence of TLSs. BCBM, breast cancer brain metastases; HER2, human epidermal growth factor receptor type 2; OS, overall survival; PFS, progression-free survival; TLS, tertiary lymphoid structure; TNBC, triple-negative breast cancer.

Figure 4

An intratumor TLS nomogram predicts clinical outcome of BCBMs. (A–C) A TLS nomogram was established to predict the probability of 1-year, 3-year, and 5-year overall survival using Kaplan-Meier and ROC analyses. (D–E) A TLS nomogram was constructed to predict the probability of 1-year, 3-year, and 5-year progression-free survival using Kaplan-Meier and ROC analyses. AUC, area under the curve; BCBM, breast cancer brain metastases; KPS, Karnofsky performance status; ROC, receiver operating characteristic; TLS, tertiary lymphoid structure; **, p<0.01; ***, p<0.001.

Figure 5

The heterogeneous distribution of immune cells within TLSs. (A) Representative multiplex immunofluorescence images showing the positivity of CD3, CD8, CD20, CD68, CD163, Foxp3, PD-1, PD-L1, TIM-3, and CK in intratumoral TLSs. (B) Comparison of the percentage of specific cell types among DAPI-positive cells using one-way ANOVA. ANOVA, analysis of variance; CD, cluster of differentiation; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; TIM-3, T cell immunoglobulin domain and mucin domain-3; TLS, tertiary lymphoid structure.