Immune Phenotype and Response to Neoadjuvant Therapy in Triple-Negative Breast Cancer

Abstract

Purpose: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR.

Experimental design: We obtained pretreatment core-needle biopsies from 105 patients with stage I-III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT.

Results: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage.

Conclusions: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

©2021 American Association for Cancer Research.

Figures

FIGURE 1.. A more clonal T-cell population in pre-treatment tumor tissue is associated with response to neoadjuvant therapy, an immunologically active infiltrate and PD-L1 positivity, which is also associated with response to neoadjuvant therapy.

(A) Pre-treatment T-cell clonality scores in tumor tissue from patients with NAT-sensitive disease (pCR, n=40) and NAT-resistant disease (RCB I-III, n=58). (B) Associations between T-cell clonality and cell densities of CD3+, CD3+CD8+, CD3+PD-1+, and CD3+CD8+PD-1+ cells. (C) PD-L1+ tumors (n=24) have higher in T-cell clonality scores (n=27) compared with PD-L1- tumors (n=78). (D) PD-L1+ tumors are more likely to respond to neoadjuvant therapy compared to PD-L1- tumors.

FIGURE 2.. Response to neoadjuvant therapy is associated with a higher ratio of CD3+:CD68+ and CD3+CD8+:CD68+ cells as well as increased spatial proximity of T-cell subsets to tumor cells in the tumor-immune microenvironment.

(A) Ratio of CD3+:CD68+ and CD3+CD8+:CD68+ cells in the tumor-immune microenvironment in patients with NAT-sensitive disease (pCR, n=42) and NAT-resistant disease (RCB I-III, n=60). (B) Representative multiplexed images (Cyan: CK, Red: CD3, Yellow: CD68) of the tumor-immune microenvironment from a patient with NAT-sensitive disease (pCR) and another with NAT-resistant disease (RCB-III). (C) Increased proximity (reduced distance) between tumor cells and CD3+ and CD3+CD8+ T-cells. (D) Schematic diagram illustrating that increased spatial proximity of T-cell subsets to tumor cells in the tumor-immune microenvironment is associated with response to neoadjuvant therapy.

FIGURE 3.. Spatial proximity of T-cell subsets to tumor cells in the tumor-immune microenvironment and response to neoadjuvant therapy (NAT).

Tumor cells were further away from CD3+PD-1+ and CD3+CD8+PD-1+ T-cells in NAT-sensitive (pCR) tumors (p=NS). Spatial proximity of tumor cells and CD3+(CD8+)PD-L1+ T-cells were similar in NAT-sensitive (pCR) and NAT-resistant (RCB I-III) tumors.

FIGURE 4.. A higher ratio of CD3+:CD68+ cells and increased proximity of T-cells to tumor cells in the tumor-immune microenvironment is associated with increased expression of interferon-γ pathway associated genes, increased transcriptional activity in immune related pathways and reduced transcriptional activity in tumor-associated pathways.

(A) A higher ratio of CD3+:CD68+ cells in the tumor-immune microenvironment is correlated positively with increased expression of interferon-γ pathway associated genes. (B) A higher ratio of CD3+:CD68+ cells in the tumor-immune microenvironment is associated with increased transcriptional activity in immune related pathways but reduced transcriptional activity in tumor-associated pathways. (C) Increased proximity (reduced distance) of T-cells to tumor cells in the tumor-immune microenvironment is associated with increased expression of interferon-γ pathway associated genes. (D) Increased proximity (reduced distance) of T-cells to tumor cells in the tumor-immune microenvironment is associated with increased transcriptional activity in immune related pathways but reduced transcriptional activity in tumor-associated pathways.

FIGURE 5.. Immune parameters enhance response prediction in patients receiving neoadjuvant therapy when considered in parallel with traditional clinical predictors.

(A) Receiver operating characteristic (ROC) curve demonstrating predictive accuracy of clinical stage alone (Area Under the Curve [AUC]=63%). (B) ROC curve demonstrating predictive accuracy of a multivariable model based on clinical stage and PD-L1 (AUC=68%). (C) ROC curve demonstrating predictive accuracy of a multivariable model based on clinical stage, and tumor-CD3+ cell distance (AUC=72%). (D) ROC curve demonstrating predictive accuracy of a multivariable model based on clinical stage, PD-L1, and tumor-CD3+ cell distance (AUC=74%).