Systemic immune effects of adjuvant chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide and/or radiotherapy in breast cancer: a longitudinal study

Abstract

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n = 21) and another group with radiotherapy (RT) (n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher (P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of T(reg) cells (CD4(+)CD25(high)) were low in the RT + CT group during follow-up, as well as expression of TCRxi, Zap70, p56(lck), P59(fyn) and PI3 k in CD4(+) cells. In contrast, expression of intracellular cytokines (IFN-gamma, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy.

Fig. 1

Absolute numbers and cytotoxic activity of NK cells in patients and healthy volunteers. NK cytotoxicity against K562 (expressed as LU₃₀) (a); percentage of IFN-γ producing NK cells (b), absolute numbers of CD3⁻CD56⁺ NK cells (c) in breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05

Fig. 2

Absolute numbers of T cell subsets in patients and healthy volunteers. a CD3⁺ T cells, b CD4⁺ T cells and c CD8⁺ T cells in breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05

Fig. 3

Regulatory T cells and CD28 expression on patients and healthy controls. Absolute numbers of CD8⁺CD28⁺ T cells (a), CD4⁺CD28⁺ (b), CD4⁺CD25⁺ (c) breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05

Fig. 4

Expression of ZAP70 in T cells of patients and healthy controls. Absolute numbers of CD4 T cells expressing signalling molecule ZAP70 in breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05

Fig. 5

Expression of PI3-k in T cells of patients and healthy controls. Mean fluorescent intensity of PI3-k expressing CD4 (a) and CD8 (b) T cells in breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05

Fig. 6

Cytokine production by T cells of patients and healthy controls. Percentage of IFN-γ (a), IL-4 (b), IL-2 (c) producing CD4 T cells in breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05

Fig. 7

PPD-induced T cell proliferative response in patients and healthy controls. T cell proliferative response to PPD in breast cancer patients who had received prior adjuvant radiotherapy (RT) (n = 20) (filled square), radio-chemotherapy (RT + CT) (n = 21) (filled diamond) and in normal healthy controls (NC) (n = 11) (filled triangle). Data at each time point is shown as mean ± SEM. † Significant P value for repeated measurements ANOVA, comparing two groups of patients at four different time points. * Significant P value for ANOVA with Post Hoc test, comparing healthy controls and patients at three different time points. Differences were considered to be statistically significant if P was less than or equal to 0.05