Effect of Cyclin-Dependent Kinase 4/6 Inhibitors on Circulating Cells in Patients with Metastatic Breast Cancer

Abstract

The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard-of-care for estrogen receptor (ER)-positive, HER2-negative (ER+/HER2- advanced/metastatic breast cancer (mBC). However, the impact of CDK4/6i on circulating immune cells and circulating tumor cells (CTCs) in patients receiving CDK4/6i and ET (CDK4/6i+ET) remains poorly understood. This was a prospective cohort study including 44 patients with ER+/HER2- mBC treated with CDK4/6i+ET in either first or second line. Peripheral blood samples were collected before (baseline) and 3 months (t2) after therapy. Immune cell's subsets were quantified by flow cytometry, and microfluidic-captured CTCs were counted and classified according to the expression of cytokeratin and/or vimentin. Patients were categorized according to response as responders (progression-free survival [PFS] ≥ 6.0 months; 79.1%) and non-responders (PFS < 6.0 months; 20.9%). CDK4/6i+ET resulted in significant changes in the hematological parameters, including decreased hemoglobin levels and increased mean corpuscular volume, as well as reductions in neutrophil, eosinophil, and basophil counts. Specific immune cell subsets, such as early-stage myeloid-derived suppressor cells, central memory CD4+ T cells, and Vδ2+ T cells expressing NKG2D, decreased 3 months after CDK4/6i+ET. Additionally, correlations between the presence of CTCs and immune cell populations were observed, highlighting the interplay between immune dysfunction and tumor dissemination. This study provides insights into the immunomodulatory effects of CDK4/6i+ET, underscoring the importance of considering immune dynamics in the management of ER+/HER2- mBC.

Keywords: CDK4/6 inhibitor; ER+/HER2− breast cancer; advanced breast cancer; circulating tumor cell; immunomodulation; metastatic breast cancer; myeloid-derived suppressor cell; peripheral immune cell.

Figure 1

Significant differences at baseline between responders (Resp, blue) and non-responders (NResp, red) patients for (A) carcinoembryonic antigen (CEA); (B) basophil counts; (C) effector Vδ2 T cells. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times the IQR from the first and third quartiles. p-values were determined using the Mann–Whitney–Wilcoxon test and p < 0.05 was considered significant.

Figure 2

Significant correlations between CTCs (presence or absence at baseline with immune subsets and serum IFN−δ. (A) effector CD8+ T cells; (B) Vδ1 T cells; (C) effector Vδ1 T cells; (D) Vδ1 T cells expressing NKG2D; (E) ratio CD8Eff/CD8Mem; (F) ratio CD8Eff / CD8CM; (G) central memory CD8+ T cells; (H) naïve Vδ1 T cells; (I) central memory Vδ1 T cells; (J) serum IFN−δ. The absence of CTCs at baseline is represented as an orange boxplot and the presence of CTCs at baseline (≥1) is represented as a green boxplot. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times the IQR from the first and third quartiles. p-values and r-coefficients were determined using point-biserial correlation and p < 0.05 was considered significant.

Figure 3

Impact of 3 months (t2) of CDK4/6i+ET on (A) hemoglobin levels (g/dL); (B) mean corpuscular volume (MCV; fL); (C) leucocyte counts (×10⁹/L); (D) neutrophil counts (×10⁹/L); (E) eosinophil counts (×10⁹/L); and (F) monocyte counts (×10⁹/L). Baseline is represented as a blue boxplot and t2 is represented as a green boxplot. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times the IQR from the first and third quartiles. p-values were determined using the Wilcoxon test and p < 0.05 was considered significant.

Figure 4

Impact of 3 months (t2) of CDK4/6i+ET on circulating immune cell subsets: (A) early-stage myeloid-derived suppressor cells (eMDSCs); (B) central memory CD4+ T cells; and (C) Vδ2 T cells expressing NKG2D. Baseline is represented as blue boxplot and t2 is represented as green boxplot. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times the IQR from the first and third quartiles. p-values were determined using Wilcoxon test and p < 0.05 was considered significant.

Figure 5

Comparison between Resp and NResp patients 3 months after the start of CDK4/6i+ET (t2) for (A) early-stage myeloid-derived suppressor cells (eMDSCs); (B) leucocyte counts (×10⁹/L); and (C) basophil counts. NResp patients are shown as red box plots and Resp patients are shown as blue box plots. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times the IQR from the first and third quartiles. p-values were determined using the Mann–Whitney–Wilcoxon test and p < 0.05 was considered significant.

Figure 6

Variation in (A) Treg III; (B) Treg I; and (C) CD8+ T cells expressing NKG2D, between baseline and t2 in Resp and NResp patients. NResp patients are shown as red box plots and Resp patients are shown as blue box plots. The lines within each box represent the median values, the boxes’ limits indicate the first and third quartiles, and the whiskers represent the smallest and largest values within 1.5 times the IQR from the first and third quartiles. p-values were determined using the Mann-Whitney-Wilcoxon test and p < 0.05 was considered significant.