Immune Signatures Following Single Dose Trastuzumab Predict Pathologic Response to PreoperativeTrastuzumab and Chemotherapy in HER2-Positive Early Breast Cancer

Abstract

Purpose: Recent data suggest that intrinsic subtype and immune cell infiltration may predict response to trastuzumab-based therapy. We studied the interaction between these factors, changes in immune signatures following brief exposure to trastuzumab, and achievement of pathologic complete response (pCR) to subsequent preoperative trastuzumab and chemotherapy in HER2-positive breast cancer.

Experimental design: In patients enrolled on two multicenter trials (03-311 and 211B), tumor core biopsies were obtained at baseline and after brief exposure to single-agent trastuzumab or nab-paclitaxel. Gene expression profiles were assessed to assign PAM50 subtypes, measure immune cell activation, and were correlated with response.

Results: The pCR rate was significantly higher in HER2-enriched tumors in the Discovery, 03-311 (36%, P = 0.043) dataset, as compared with other subtypes, which validated in 211B (50%, P = 0.048). Significant increases in a signature of immune cell admixture (Immune Index) were observed only following brief exposure to trastuzumab in HER2-enriched tumors (Discovery/03-311, P = 0.05; Validation/211B, P = 0.02). Increased Immune Index was predictive of response after brief exposure (03-311, P = 0.03; 211B, P = 0.04), but not at baseline, in addition to increased expression of a CD4(+) follicular helper T-cell signature (03-311, P = 0.05; 211B, P = 0.04). Brief exposure to trastuzumab significantly increased gene expression of the T-cell marker PD-1 in HER2-enriched tumors (Discovery/03-311, P = 0.045) and PD-1 positivity by IHC (Validation/211B, P = 0.035).

Conclusions: Correlations between pCR rates, increases in Immune Index and markers of T-cell activity following brief exposure to trastuzumab in HER2-enriched tumors provide novel insights into the interaction between tumor biology, antitumor immunity, and response to treatment, and suggest potential clinically useful biomarkers in HER2(+) breast cancers. Clin Cancer Res; 22(13); 3249-59. ©2016 AACR.

Figure 1

PAM50 subtyping. A, hierarchical clustering of microarray samples from the 03-311 cohort using the PAM50 genes. Three major clusters were identified, including Luminal, HER2-Basal, and HER2-enriched subtypes that correspond well with ER/PR IHC and the expression levels of proliferation genes. B, hierarchical clustering of samples from the 211B trial using targeted RNA sequencing of the PAM50 genes. Samples that showed poor expression of a majority of genes were considered to have low tumor content. C, rates of pCR across PAM50 subtypes in 03-311 cohort. Shown are the percentages of pCR within each subtype. Significance of the rate of pCR in the HER2-enriched subtype as compared with the remaining tumors was assessed using a two-sided Fisher exact test. D, rates of pCR across PAM50 subtypes in 211B cohort. Shown are the percentages of pCR within each subtype. The hypothesis that the HER2-enriched subtype exhibited the highest rate of pCR as compared with the rest was evaluated using a one-sided Fisher exact test.

Figure 2

Comparisons of Immune Index following brief exposure to therapy across PAM50 subtypes. A, plotted are the Immune Index (log2) values in individual patient biopsies at baseline and after a single dose of trastuzumab in the 03-311 cohort across the HER2-enriched, luminal, and HER2-basal subtypes. Significance was calculated using a one-sided nonparametric Wilcoxon test. B, Immune Index values at baseline and following brief exposure to therapy (either trastuzumab or nab-paclitaxel) in the 211B cohort across the three subtypes.

Figure 3

Association of immune signatures with response. A, shown are the Immune Index (log2) values at baseline and postexposure to trastuzumab across response groups in the 03-311 trial. B, Immune Index at baseline and post-exposure to trastuzumab across response groups in the 211B trial. Significance was assessed using a one-sided Wilcoxon test. C, plotted is the 8-gene signature index associated with CD4⁺ Tfh activity in the baseline and postexposure timepoint across response groups in the 03-311 trial. D, the Tfh signature index is plotted across response groups at baseline and postexposure to trastuzumab in the BrUOG 211B cohort.

Figure 4

Modulation of PD-1 expression upon brief exposure to trastuzumab. A, plotted are the normalized log2 expression levels of PD1 in the 03-311 trial at baseline and post brief exposure to trastuzumab across the three PAM50 subtypes. The ladder plots indicate changes in expression in matched baseline and postexposure biopsy samples. Significance was assessed using a paired one-sided Wilcoxon test evaluating for increase in PD1 expression upon brief exposure to trastuzumab. B, the bar graphs plotted on top represent the number of tumors with low/medium (blue) or high (red) PD-1 protein expression by IHC at baseline and post brief exposure to trastuzumab across subtypes in the 211B trial. Significance was assessed using a Fisher exact test. Representative IHC stains of PD1 in the 211B trial in the low/negative, medium, and high categories at shown at 400× magnification in the bottom.