Tumor Immune Microenvironment Changes by Multiplex Immunofluorescence Staining in a Pilot Study of Neoadjuvant Talazoparib for Early-Stage Breast Cancer Patients with a Hereditary BRCA Mutation

Abstract

Purpose: The immunological profile of early-stage breast cancer treated with neoadjuvant PARP inhibitors has not been described. The aim of this study was to delineate the changes in the tumor immune microenvironment (TiME) induced by talazoparib.

Patients and methods: Patients with operable germline BRCA1/2 pathogenic variant (gBRCA1/2+) breast cancer were enrolled in a feasibility study of neoadjuvant talazoparib. Thirteen patients who received 8 weeks of neoadjuvant talazoparib were available for analysis, including 11 paired pre- and post-talazoparib core biopsies. Treatment-related changes in tumor-infiltrating lymphocytes were examined and immune cell phenotypes and their spatial distribution in the TiME were identified and quantified by multiplex immunofluorescence using a panel of 6 biomarkers (CD3, CD8, CD68, PD-1, PD-L1, and CK).

Results: Neoadjuvant talazoparib significantly increased infiltrating intratumoral and stromal T-cell and cytotoxic T-cell density. There was no difference in PD-1 or PD-L1 immune cell phenotypes in the pre- and post-talazoparib specimens and PD-L1 expression in tumor cells was rare in this cohort. Spatial analysis demonstrated that pre-talazoparib interactions between macrophages and T cells may correlate with pathologic complete response.

Conclusions: This is the first study with phenotyping to characterize the immune response to neoadjuvant talazoparib in patients with gBRCA1/2+ breast cancer. These findings support an emerging role for PARP inhibitors in enhancing tumor immunogenicity. Further investigation of combinatorial strategies is warranted with agents that exploit the immunomodulatory effects of PARP inhibitors on the TiME.

Trial registration: ClinicalTrials.gov NCT02282345.

Conflict of interest statement

Conflicts of interest:

E. A. Mittendorf reports personal financial interests: sponsored research agreement from Glaxo SmithKline; honoraria from Physician Education Resource; compensated service on Scientific Advisory Boards for Exact Sciences (formerly Genomic Health), Merck, and Roche/Genentech; and uncompensated service on Steering Committees for BMS, Lilly, Roche/Genentech. E. A. Mittendorf reports institutional financial interests: Clinical trial funding from Roche/Genentech (via SU2C grant). G. J. Whitman is a shareholder of Pfizer and editor for UpToDate. JLitton reports personal financial interests: sponsored research agreements from Pfizer/Medivation, Genentech, BMS, Novartis, GSK, Zenith, Merck, EMD-Serono and Astra-Zenica. And has had uncompensated service on advisory Boards and Steering Committees for Pfizer, Astra-Zeneca and Ayala Pharmaceuticals. The other authors declare no potential conflicts of interest.

©2022 American Association for Cancer Research.

Figures

Figure 1:

Tumor-infiltrating lymphocyte variation before and after neoadjuvant talazoparib. A) Schema of study analysis. B) Representative TILs by H&E in pre- and post-talazoparib specimens. Arrowhead depicts TILs surrounded by invasive breast ductal carcinoma. Scale bar, 100 μM. C) Percentage of TILs in paired pre- and post-talazoparib specimens (n=6). Bars, boxes, and whiskers represent median, interquartile range, and range, respectively. Individual data points are shown and color encodes underlying biology with endocrine receptor (ER+) (red) or triple negative breast cancer (TNBC) (black). Data points with a white asterix in the center represents two paired specimens with overlapping values.

Figure 2:

Neoadjuvant talazoparib increases infiltration of T cell lymphocyte populations by multiplex immunofluorescence. A) Representative 7-color multiplex immunofluorescence microscopy. Staining of pre- and post-talazoparib biopsies with lymphoid biomarker panel by pseudo-coloring (orange, PD-L1; green, PD-1; red, CD3; lavender, CD8; yellow, CD68; cyan, CK; blue, DAPI). Scale bar, 100 μM. Enlarged subsection of core to the right shows individual markers for CD3 and CD8 single channel and composite images, with DAPI nuclear marker (pseudocolored blue). B) Immune cell phenotypes by intratumor (T) and intrastromal (S) compartments grouped by patient and by pre- (upper panel) and post-talazoparib (lower panel) specimens. Patient characteristics are described on the bottom. Stacked bar graphs are shown as proportion of total immune cells evaluated (CD3+ and CD63+). Immune cell subsets are indicated by different colors. C) Pre- and post-talazoparib immune cell densities (cells/mm2) in paired samples (n=11). Bars, boxes, and whiskers represent median, interquartile range, and range, respectively.

Figure 3.

Multiplex immunofluorescence spatial analysis. A) Representative spatial analysis in a paired pre-talazoparib specimens with low T cell infiltration (upper panel, low AUC) and a post-talazoparib specimen with high T cell infiltration (lower panel, high AUC). Spatial analysis of mIF data report of all pseudocolored cell types (left); tumor cells and total T cell X- and Y-spatial coordinate plots (middle); and G(r) function versus r (μm) plots with Kaplan Meier (km) estimate of AUC with radius 0-20μm (shaded blue) of tumor cell and cytotoxic T cell interaction (right). B) Pre-talazoparib G-function analysis probability of interaction between macrophages onto CD8− T cells (upper panel) and macrophages onto cytotoxic T cells (lower panel) stratified by pCR (pCR Yes, n=6, pCR No, n=6). C) Heatmap of probability of pre-talazoparib spatial interactions between each cell phenotype, grouped by pCR. Km: Kaplan Meier estimate of AUC with darker shading representing higher probability of interaction between two cell phenotypes.

Figure 4.

Immune checkpoint expression and composition of immune cells in TiME by CIBERSORT. A) Heatmap analysis of immune checkpoints. Red denotes highly expressed genes, and blue denotes lower expression levels by bulk RNA sequencing. B) Heatmap showing absolute abundance of each immune cell type predicted by CIBERSORT. Red denotes higher cell abundance and blue denotes lower cell abundance. Heatmaps grouped by pre- and post-talazoparib specimens. C) Scatterplots showing enrichment of memory resting CD4+ T cells and M2 macrophages post-talazoparib treatment.