Changes in Peripheral and Local Tumor Immunity after Neoadjuvant Chemotherapy Reshape Clinical Outcomes in Patients with Breast Cancer

Abstract

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).

Experimental design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1-high (PD-1^HI) CD8⁺ peripheral T cells and examination of a cytolytic gene signature in whole blood.

Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.

Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Figure 1:. Immunologic changes in breast tumors after neoadjuvant chemotherapy.

A) High levels of sTILs are associated with RFS (left; n=41) and OS (right; n=42) after surgery in TNBC. Patients are stratified based on post-NAC sTILs ≤ 30% or > 30%, scored as recommended by the International TILs Working Group^,, according to the predefined cut point. B) Heatmap demonstrating gene expression patterns for 770 immune-related genes (NanoString Pan-Cancer Immune Panel) across all patients (TNBC and non-TNBC; n=83 total patients, 166 samples). C) Heatmap of gene expression patterns as detailed in panel B, instead depicting the change in expression of each gene in matched paired (pre- and post-NAC; n=83) samples. Red data points represent an upregulation, while blue data points represent a downregulation in the post-NAC residual disease compared to the pre-treatment diagnostic biopsy.

Figure 2:. Identification of immune-associated genes associated with RFS and OS in TNBC after chemotherapy.

A) Individual genes (changes pre- to post-NAC) were tested iteratively in a univariate cox-proportional hazards model for their association with RFS (left) or OS (right) after chemotherapy and surgery. Individual genes are colored for their statistical significance (red: nominal p-value <0.05; green: q-value (FDR) <0.10; black: not significant). Selected top genes are labeled but are limited in number for clarity. Genes with negative coefficients (left of the center line) are associated with better outcome, while genes with positive coefficients (right of the center line) are associated with worse outcome. B) Representative Kaplan-Meier plots for selected detrimental (CDH1; e-cadherin) and beneficial (CD70) genes are shown. Strata are defined by tertiles, and generally represent upregulation during NAC (blue), no change/equivocal (green), and downregulation (red). P-values represent the log-rank test for trend.

Figure 3:. Upregulation of immune-associated gene sets after chemotherapy are associated with improved RFS and OS in TNBC.

A) Gene set scores were calculated by summing expression levels of all gene set member genes across each candidate gene set (n=70). Changes pre- to post-NAC was then calculated for each TNBC patient (n=44) and each gene set score was tested iteratively in a univariate cox-proportional hazards model for association with RFS (left) or OS (right) after chemotherapy and surgery. Individual gene sets are colored for their statistical significance (red: nominal p-value <0.05; green: q-value (FDR) <0.10; black: not significant). Selected top gene sets are labeled but are limited for clarity. Gene sets with negative coefficients are associated with better outcome, while gene sets with positive coefficients are associated with worse outcome. B) Representative Kaplan-Meier plots for selected gene set changes with beneficial associations are shown (left: T cell activation; right: NK cell functions). Strata are defined by tertiles, and generally represent upregulation during NAC (blue), no change/equivocal (green), and downregulated (red). P-values represent the log-rank test for trend.

Figure 4:. Evidence of enhanced T cell functionality in the CD8+ PD-1^HIperipheral compartment

A) Clinical details of 4 patients analyzed prospectively for changes in peripheral blood T cell functionality. NST indicates no special type. B) Polyfunctionality of PD-1^HICD4+ and PD-1^HICD8+ T cells isolated from PBMCs in 4 patients prior and after NAC (>1000 individual cells/sample/timepoint) was determined by Isoplexis single-cell cytokine profiling. Polyfunctionality is defined as the percentage of cells capable of producing ≥ 2 cytokines following CD3/CD28 stimulation. The percentage of cells in each sample capable of secreting 2, 3, 4, or 5+ cytokines are depicted in stacked bars. Characteristics of each of the 4 patients are shown above the bars. Patients with TNBC (Pt. 1 and Pt. 4) had greater increases in polyfunctionality in the CD8+ compartment with NAC. C) Heatmap representation of log cytokine signal intensity of each cell in each patient sample, pre and post NAC. Each row represents one PD-1^HiCD8+ T cell. White indicates no cytokine secreted. D) TCRβ chain repertoire analysis in CD8+ peripheral blood T cells. Upper plots indicate the number of individual T cells sequenced plotted by sample on the left Y axis; number of clonotypes (unique CDR3 amino acid sequences) plotted by sample on the right Y axis. In the lower graph, each sample is divided into the number of clonotypes comprising expanded (hyper-expanded, large, medium, small, and rare) compositions of the detected repertoire (categories divided by orders of magnitude of fraction of total repertoire). E) The fraction of repertoire clonotypes identified in PD-1^HI versus PD-1^NEG CD8+ T cells (before or after NAC) classified as ‘hyperexpanded’ or ‘large’ (comprising >0.1% of repertoire). P value represents a 2-sample 2-tailed t-test.

Figure 5:. Single-cell RNA sequencing of CD8+ PD-1^HI peripheral T cells from 2 patients with TNBC after NAC demonstrate high expression of cytolytic markers.

A) UMAP plots of 1,964 PD-1^HICD8+ peripheral T cells across 2 patients (672 and 1,292 respectively) are shown. Five (5) clusters (0–4) were defined. B) Percent of cells sequenced comprising each cluster are plotted. C) Heatmap identifying top 10 most differentially expressed transcripts across clusters. D) A selection of genes defining cluster 0 are highlighted. Data depicted include combined cells from both Pt.1 and Pt.4. E) UMAP plots of 7,062 PBMCs from 2 TNBC patients (3,525 cells from patient 4 and 3,537 cells from patient 5). Cell type annotations are defined by SingleR. 8 gene score is defined by expression of FGFBP2 + GNLY + GZMB + GZMH + NKG7 + LAG3 + PDCD1 – HLA-G. F) Violin plots showing expression of the 8 gene score by cell type. Overlaid box plots show mean and interquartile range for each cell type.

Figure 6:. An 8-gene activated T cell signature derived from whole blood at surgery is associated with pCR and prognosticates recurrence in RD patients.

A) Individual gene plots of 8 analyzed genes by nanoString from RNA derived from whole blood sampled within 14 days leading up to definitive surgery. Datapoints are stratified by untreated patients (No NAC), those experiencing pCR (pCR), those with RD that did not recur (RD not recur) and those with RD that recurred (RD recur) within 3 years after surgery. Box plots represent the interquartile range. P values represent Kruskal-Wallis tests. * indicates p<0.05 by post-hoc Dunn test. B) A composite gene signature derived as PDCD1 + NKG7 + LAG3+ GZMH + GZMB + GNLY + FGFBP2 – HLA-G (sum of Z-scores), stratified by outcome, as in (A). C) Heatmap showing row-standardized (Z-score) gene expression for genes assayed across all patients.