Characterization of tumor-infiltrating lymphocytes and their spatial distribution in triple-negative breast cancer

Abstract

Background: The tumor immune microenvironment, particularly tumor-infiltrating lymphocytes (TILs), plays a critical role in disease progression and treatment response in triple-negative breast cancers (TNBCs). This study was aimed to characterize the composition of TILs and investigate their clinicopathological and prognostic significance with a special focus on the spatial distribution of TILs in TNBCs.

Methods: We analyzed TNBC samples through PanCancer Immune Profiling using NanoString nCounter assays to identify immune-related genes that are expressed differentially in relation to TIL levels and evaluated protein expression of selected markers through immunohistochemical staining on tissue microarrays. For a comprehensive assessment of the expression of cytotoxic T lymphocyte (CTL) and natural killer (NK) cell markers, a CTL-NK score was devised based on CD8⁺, CD56⁺, CD57⁺, GNLY⁺, and GZMB⁺ TIL levels.

Results: Gene expression analysis revealed significant upregulation of CTL and NK cell-associated genes including GNLY, KLRC2, and GZMB in TIL-high TNBCs. Immunohistochemical validation confirmed that TNBCs with higher TILs had a greater amount of CD56⁺, CD57⁺, GNLY⁺, and GZMB⁺ TILs not only in absolute number but also in proportion relative to CD4⁺ or CD8⁺ TILs. High TIL and its subset (CD4⁺, CD8⁺, CD56⁺, CD57⁺, GNLY⁺, and GZMB⁺ TIL) infiltration correlated with favorable clinicopathological features of tumor. In survival analysis, high CTL-NK score was found to be an independent prognostic factor for better disease-free survival (DFS) of the patients. Furthermore, uniformly high TIL infiltration was linked to better DFS, whereas cases with heterogeneous TIL infiltration showed no difference in survival compared to those with uniformly low TIL infiltration.

Conclusion: Our study showed that CTL and NK cell-associated gene expression and protein levels differ significantly according to TIL levels and that CTL-NK score and distribution of TILs within tumors have a prognostic value. These findings emphasize the importance of CTLs and NK cells as well as the spatial uniformity of TIL infiltration in clinical outcome of TNBC patients, providing valuable insights for refining prognostic assessments and guiding immunotherapeutic strategies.

Keywords: Cytotoxic T lymphocyte; NK cell; Spatial distribution; Triple-negative breast cancer; Tumor infiltrating lymphocyte.

Fig. 1

Heatmap of immune-related genes generated by unsupervised clustering. Samples were grouped into three based on their immune-related gene expression; one cluster had lower expression (cluster 1), another one had higher expression (cluster 2), and the last one showed intermediate expression (cluster 3). The clustering of each group was not determined by recurrence status. However, a potential relationship between immune-related gene expression patterns, TIL levels, and prior neoadjuvant chemotherapy (NAC) was observed. Cluster 1 consistently exhibited tumor-infiltrating lymphocyte infiltration of less than 10%. Additionally, none of the cases in Cluster 2 had undergone NAC. Color keys for sample annotation are displayed on the right

Fig. 2

Representative images of H&E and immunohistochemical staining from one case showing low TIL and the other case showing high TIL. The case with low TIL infiltration also shows a low level of CD4⁺, CD8⁺, CD56⁺, CD57⁺, GNLY⁺, and GZMB⁺ cell infiltration. On the other hand, the case with high TIL infiltration shows abundant CD4⁺, CD8⁺, CD56⁺, CD57⁺, GNLY⁺, and GZMB⁺ cell infiltration. Each circle represents one high power field

Fig. 3

Kaplan-Meier survival curves for disease-free survival according to TIL, TIL subsets, and CTL-NK score. (A-F) High infiltration of TIL and CD4⁺, CD8⁺, CD56⁺, CD57⁺ and GNLY⁺ TIL subset was associated with increased disease-free survival (p = 0.018, 0.004, 0.005, 0.014, 0.046, and 0.037, respectively). (G) GZMB⁺ TIL subset infiltration was not associated with disease-free survival (p = 0.261). (H) CTL-NK score of 2 or higher was associated with increased disease-free survival (p = 0.012)

Fig. 4

Kaplan-Meier survival curves for disease-free survival according to heterogeneity in TIL infiltration and its subset infiltration. (A) A significant difference in disease-free survival was noted between the group with uniformly high infiltration and the group with uniformly low infiltration as well as between the group with heterogeneous infiltration and the group exhibiting uniformly high infiltration (adjusted p = 0.009 for both). (B) Regarding CD4⁺ TIL subset infiltration, better prognosis was observed in groups with heterogeneous levels compared to those with consistently low levels (adjusted p = 0.003). (C) When disease-free survival was compared among the groups with different levels of heterogeneity of CD8⁺ TIL subset infiltration, no significant differences were observed between the groups. (D-F) Although not statistically significant, similar trends for poor survival were noted in both uniformly low and heterogeneous infiltration groups of CD56⁺, CD57⁺, and GNLY⁺ TIL subset. (G) When the disease-free survival was assessed among the groups divided based on the heterogeneity of GZMB⁺ TIL subset infiltration, no significant differences were observed among them