Background: Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting.

Methods: We assessed multiple immunologic biomarkers in a series of 99 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E-based TILs analysis, standard immunohistochemistry (IHC; CD8, CD20, CD56, FOXP3, LAG-3, B7-H4, HLA-A) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK, PD1, CD3). Association among parameters were assessed (up to n=98) including clinical outcome after surgery (n=94).

Results: As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS (p=0.0093 and p=0.0376, respectively). H&E-scored TILs were highly correlated to CD3, CD4 and CD8 positive T cells (Spearman r range 0.34-0.4850; all p<0.001), while CD20 and CD56 positive cells only make up a small and non-significant proportion of the TILs composition. Likewise, infiltration by overall T cell populations were associated with significantly improved recurrence free survival (RFS; HR range 0.34-0.54) and overall survival (OS; HR range 0.38-0.43), whereas CD20+ B cells were not. Additionally, infiltration by cells expressing LAG3 (HR 0.49) and CD56 (HR 0.44), were each associated with significantly improved OS. Interestingly, colocalizing GZMB+ and CD8+ T cells by mIF yielded a paradoxical finding that cytotoxic CD8+ T cells (CD8+GZMB+ T cells) were associated with worse RFS and OS (HR 1.8 p=0.0338 and HR=2.8 p=0.0013, respectively). In a multivariate model, only CD8+GZMB+ T cell infiltration remained significant for OS (p=0.0224), suggesting independence. CD8+GZMB+ T cell infiltration inversely correlated with TILs (p=0.0293) and CD8/mm2 counts in the tumor core (p=0.0020) and showed a positive correlation with B7H4 tumor expression (p=0.0183). Additionally, high CD8+GZMB+ cases showed low HLA-A (p=0.0189) expression in tumor cells. In accordance, high CD8+GZMB+ tumors mainly correspond to stroma-restricted, margin-restricted and immune-desert tumor immune microenvironment landscapes.

Conclusions: We have found a paradoxical association of GZMB+ CD8+ T cells with a negative prognosis in NAC-treated TNBC. We hypothesize that while these T cells are poised for cytotoxic activity, they remain restricted through sub-localization outside the tumor core, downregulation of HLA-A on tumor cells preventing interaction with the T cell receptor, and upregulation of B7H4 expression, which has been shown to inhibit cytotoxic T cell activity. Patients with high CD8+GZMB+ tumors in the post-NAC setting may benefit from adjuvant immunotherapy, particularly in combination with therapies that enhance MHC-I (e.g. HLA-A) antigen presentation.

Citation Format: Paula I Gonzalez-Ericsson, Violeta Sanchez, Roberto Salgado, Jennifer Bordeaux, Ju Y Kim, Christine Vaupel, Henry Gomez, Melinda E Sanders, Justin M Balko. The immune landscape of residual triple-negative breast cancers after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-07.