Spatial interactions between immune cells as a predictor of pCR to neoadjuvant chemotherapy (NACT) and nivolumab for ER+/HER2− breast cancer (BC): Analysis of the phase II GIADA trial.

567 Background: Adding an immune checkpoint inhibitor (ICI) to NACT increases pathological complete response (pCR) rates in ER+/HER2− high-risk BC, at the cost of immune-related toxicity. Biomarkers are needed to improve patient (pts) selection. We here assess the association between immune cell spatial interactions and pCR in the GIADA trial (Dieci, CCR 2022). Methods: In the phase II GIADA trial, 43 premenopausal pts with stage II–IIIA ER+/HER2− Luminal B-like BC (Ki67 ≥ 20% and/or G3) received NACT with ECx3 followed by nivolumab (240mg q2w x8) and endocrine treatment. Tumor biopsies at baseline (T0) and after EC (T1) were assessed using multiplex immunofluorescence panels: (1) CD4, CD8, Granzyme B, FoxP3, CD20, pan-cytokeratin (CK), and DAPI; (2) CD3, CD68, CD163, PD-1, PD-L1, CK, and DAPI. Interactions were quantified using a count-within approach. The number of reference immune cells located in a ≤20 µm radius of cells with a different phenotype (high probability of cell-cell contact) was calculated and normalized to number of reference cells. Association with pCR was assessed by binomial generalized linear models. Results: At baseline (T0), spatial interaction of tumor cells (CK+) with multiple immune cells (CD4+, CD8+, FOXP3+, CD68+), including cytotoxic, immunosuppressive and macrophagic subpopulations, was associated with pCR. Moreover, interaction of cytotoxic CD8+GranzymeB+ T-cells with regulatory FOXP3+ cells was associated with pCR. Interactions between PD-1+ and PD-L1+ cells were associated with pCR, mainly CK+PD-L1+/CD3+PD-1+ in tumor area and CD3+PD-1+/CD68+PD-L1+ in stroma. After chemotherapy (T1), only spatial interactions between tumor cells and CD8+ and CD4+ T-cells were associated with pCR. The role of cytotoxic CD8+GranzymeB+ T-cells appeared stronger as interactions with multiple immune subpopulations (CD4+, CD8+, CD8+GranzymeB+, FOXP3+, CD20+) were associated with pCR, overall (OR 1.06-1.73) and in stroma (OR 1.06-1.40). Interaction of CK+ tumor cells with PD-1+ cells in the tumor area and with CD68+ PD-L1+ macrophages in the stromal area was associated with pCR. Conclusions: Spatial immune profiling identifies distinct tumor-immune interaction patterns associated with pCR in high-risk ER+/HER2− BC treated with NACT and ICI. Clinical trial information: NCT04659551 . Selected spatial interactions significantly associated with pCR. T0 overall T0 tumor T0 stroma T1 overall T1 tumor T1 stroma CK+ / CD4+ OR 1.08, p=0.034 OR 1.13, p=0.017 OR 1.07, p=0.038 CK+ / CD8+ OR 1.21, p=0.005 OR 1.34, p=0.005 OR 1.06, p=0.049 OR 1.12, p=0.008 OR 1.16, p=0.021 OR 1.08, p=0.011 CK+ / FOXP3+ OR 1.21, p=0.017 OR 1.28, p=0.024 CK+ / CD68+ OR 1.07, p=0.016 OR 1.09, p=0.007 CK+PD-L1+ / CD3+PD-1+ OR 1.22, p=0.025 CD3+PD-1+ / CD68+PD-L1+ OR 1.08, p=0.047 CK+ / PD-1+ cells OR 1.13, p=0.027 OR 1.27, p=0.015 OR 1.29, p=0.027 CK+ / CD68+PD-L1+ OR 1.21, p=0.036 OR 1.44, p=0.030