Alterations in immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients receiving HER2-targeted neo-adjuvant therapy

Abstract

Background: The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from pre- (n = 41) and post- (n = 25) neo-adjuvant treatment blood samples. Direct/trastuzumab-ADCC cytotoxicity of patient-derived PBMCs against K562/SKBR3 cell lines was determined ex vivo. Pembrolizumab was interrogated in 21 pre-treatment PBMC ADCC assays. Thirty-nine pre-treatment and 21 post-treatment PBMC samples were immunophenotyped. Fc receptor genotype, tumour infiltrating lymphocyte (TIL) levels and oestrogen receptor (ER) status were quantified.

Results: Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours.

Conclusions: PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response.

Fig. 1. Breakdown of available samples.

Consort diagram (a) for patient samples used in immune cytotoxicity (IC) assays, immunophenotyping (IMP) experiments and FCGR SNP genotyping. Sample overlap (b) between IC and IMP experiments.

Fig. 2. Pre- and post-treatment direct cytotoxicity, ADCC and SNP status.

a Direct cytotoxicity levels elicited against K562 cells by patient PBMCs pre- (n = 40) and post-treatment (n = 25). The K562 dataset lost a pre-treatment sample due to a technical issue acquiring the sample. b Direct cytotoxicity and trastuzumab-mediated ADCC levels elicited against SKBR3 cells by patient PBMCs pre- (n = 41) and post-treatment (n = 25). An optimal pooled t test was used to determine statistical significance; all p values were adjusted for multiple testing, *p < 0.05, **p < 0.01, NS not significant. c FCGR SNP frequency and association with pCR in ICORG 10-05 patients. Data covers 37 pre-treatment samples and 24 post-treatment samples from (a) and (b). H histidine, R arginine, I isoleucine, T threonine, Q glutamine, D aspartic acid, N asparagine, V valine, F phenylalanine. Fisher’s Exact test, significant if p < 0.05, NA not available.

Fig. 3. Pre- and post-treatment immune cell subsets.

Proportion of CD45+ cells staining positive for a CD3, b CD4, c CD8, d CD56, e CD19, f CD14, and g PD-1 in paired pre- and post-treatment samples. A paired Student’s t test was used to determine statistical significance, all p values were corrected for multiple testing. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant.

Fig. 4. Direct cytotoxicity and ADCC by pCR status.

Paired pre- and post-treatment direct cytotoxicity levels of patient PBMCs against a K562 and b SKBR3 by pCR. The K562 dataset lost a paired pCR sample due to a technical issue acquiring the sample. c Pre- and post-treatment trastuzumab-mediated ADCC against SKBR3 by patient PBMCs by pCR. A paired Student’s t test was used to determine statistical significance. All p values were adjusted for multiple testing. NS not significant.

Fig. 5. Immune cell subsets by pCR status.

Proportion of CD45+ cells which stained positive for a CD3, b CD4, c CD8, d CD56, e CD19, f CD14, and g PD-1 in paired pre- and post-treatment samples based on pCR vs No pCR. A paired Student’s t test was used to determine statistical significance. All p values were corrected for multiple testing, *p < 0.05, **p < 0.01.

Fig. 6. Pembrolizumab-responsive PBMCs in the No pCR cohort.

Trastuzumab ADCC levels with and without pembrolizumab against SKBR3 cells using pre-treatment patient PBMCs from a pCR (n = 7) and b No pCR (n = 14) cohorts. Bars represent the average of three technical replicates +/− Std. Dev. c Comparison of pre- and post-treatment CD56+CD16+ levels by treatment response. d Comparison of pre- and post-treatment CD14+CD16+ levels by treatment response. e PD-1+CD56 +CD16+/− and PD-1+CD14+CD16+/− as a percentage of CD45+ immune cells or subset total (CD56+ or CD14+) +/− Std. Dev. based on assay response in (a) and (b). f Trastuzumab ADCC levels with and without pembrolizumab against SKBR3 cells using post-treatment patient PBMCs from the No pCR patient cohort. Average of three technical replicates +/− Std. Dev. g Average pre-treatment infiltrating lymphocyte levels in tumour, stroma and combined for patients whose pre-treatment PBMC samples responded to pembrolizumab in (b) (n = 6) versus patient samples that did not respond to pembrolizumab in (a) (n = 7) +/− Std. Dev. Unpaired or paired (c, d) Student’s t tests were used to determine statistical significance, *p < 0.05.