Serum cytokine levels in breast cancer patients during neoadjuvant treatment with bevacizumab

Abstract

A high concentration of circulating vascular endothelial growth factor (VEGF) in cancer patients is associated with an aggressive tumor phenotype. Here, serum levels of 27 cytokines and blood cell counts were assessed in breast cancer patients receiving neoadjuvant chemotherapy with or without bevacizumab (Bev) in a randomized cohort of 132 patients with non-metastatic HER2-negative tumors. Cytokine levels were determined prior to treatment and at various time-points. The cytotoxic chemotherapy regimen of fluorouracil, epirubicin, and cyclophosphamide (FEC) had a profound impact on both circulating white blood cells and circulating cytokine levels. At the end of FEC treatment, the global decrease in cytokine levels correlated with the drop in white blood cell counts and was significantly greater in the patients of the Bev arm for cytokines, such as VEGF-A, IL-12, IP-10 and IL-10. Among patients who received Bev, those with pathological complete response (pCR) exhibited significantly lower levels of VEGF-A, IFN-γ, TNF-α and IL-4 than patients without pCR. This effect was not observed in the chemotherapy-only arm. Certain circulating cytokine profiles were found to correlate with different immune cell types at the tumor site. For the Bev arm patients, the serum cytokine levels correlated with higher levels of cytotoxic T cells at the end of the therapy regimen, which was indicative of treatment response. The higher response rate for Bev-treated patients and stronger correlations between serum cytokine levels and infiltrating CD8T cells merits further investigation.

Keywords: Bevacizumab; VEGF-A; cytokines; immunity; neoadjuvant.

Figures

Figure 1.

Co-expression of 27 cytokines in the serum of breast cancer patients during therapy. Correlation heatmaps assess the strength of correlation between the 27 cytokines measured by the Luminex assay. Correlations are assessed by Spearman rank test, only significant correlations (Bonferroni corrected P < 0.05) are shown. An initial correlation heatmap included cytokine levels is shown for all samples at screening (A, n = 88). Both arms of the randomized control trial are represented: left panel: chemotherapy, right panel: Combination (+ Bev) arm at the different time points of measurement, (A, week zero) pre-treatment; (B & C, week 12, n = 41 and 44) after FEC therapy; (D & E, week 25, n = 40 and 40) after taxane; and (F & G, week 31, n = 45 and 45) six weeks after surgery.

Figure 2.

Changes in serum cytokine levels between two consecutive time points. Differences in cytokine levels between two successive time points are presented as barplots in both treatment arms, (A) Differences between 0 and 12 weeks, n = 85, (B) between 12 and 25 weeks n = 80, (C) between 25 and 31 weeks, n = 80. Error bars represent ± 1 standard deviation. * P

Figure 3.

Effects of treatments on blood cell counts and correlation with cytokines levels. Comparison of the blood cell counts (109 per liter): white blood cells (WBC, A), neutrophils (Neutro, B) and platelets (C), at the different time points of treatment. * indicates t-test P < 0.01 between two successive time points, error bars represent standard deviation. Heatmap showing Spearman rank correlations between cytokine levels and blood cell counts at screening n = 98, (D), 12 weeks, (n = 44, Bev; n = 41, Chemo) (E) and 25 weeks (n = 40, Bev; n = 40, Chemo) (F). All correlations are shown independently of p values. Heatmaps were obtained using the pheatmap R package.

Figure 4.

Effect of Bevacizumab on serum cytokines levels. Cytokine serum levels were assessed in the two treatment arms: chemotherapy (blue), chemotherapy + Bev (green), and at four different time points, 0, 12, 25 and 31 weeks. To find the most relevant and significant differences between the two arms, we first tested for global differences using ANOVA (Table 2), and only for the significant values showing a significant result in the global three-way ANOVA (FDR-adjusted P < 0.1) (and TNF-alpha) further tested for significant difference at each time point using t-test. For (A) VEGF-A, (B) IL-12, (C) IP-10, and (D) IL-10, we found a global difference between their levels when comparing the two arms (ANOVA P < 0.05) and further identified significant differences at the indicated time points: * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent ± 1 standard deviation.

Figure 5.

Cytokine levels associated with pathological complete response. Cytokine levels were assessed in the patients with pathological complete response in both treatment arms: chemotherapy (left panel) and combination arm (right panel). To find the most relevant and significant differences between responders and non-responders, we first tested for global differences using ANOVA (Table 3) and only for the significant values (ANOVA P

Figure 6.

Levels of CD8T and T follicular helper cells according to response and treatment. Levels of infiltrating CD8T cells and T follicular helper cells were inferred using CIBERSORT and expression levels. In both treatment arms levels of CD8T cells and T follicular helper are plotted at the different time points (screening, 12 and 25 weeks). The global levels of CD8 and follicular T cells appeared significantly different only in the combination (+Bev) arm (ANOVA P

Figure 7.

Levels of CD8T cells and T follicular helper cells according to levels of INF-γ and IL-17A. Levels of infiltrating CD8T cells and T follicular helper cells inferred using CIBERSORT were analyzed in regard to the circulating levels of INF-γ and IL-17A respectively. In each treatment arm, we divided patients for having low or high levels of circulating cytokines according to the median level of cytokine and further assessed for differences in infiltrating CD8T or T follicular helper cells. Only significant Students t-test p-values (P