Low-Dose Cyclophosphamide Induces Antitumor T-Cell Responses, which Associate with Survival in Metastatic Colorectal Cancer

Abstract

Purpose: Anticancer T-cell responses can control tumors, but immunosuppressive mechanisms in vivo prevent their function. The role of regulatory T cells (Tregs) in metastatic colorectal cancer is unclear. We have previously shown depletion of Tregs enhances colorectal cancer-specific effector T-cell responses. Low-dose cyclophosphamide targets Tregs in animal models and some human studies; however, the effect of cyclophosphamide in metastatic colorectal cancer is unknown.Experimental Design: Fifty-five patients with metastatic colorectal cancer were enrolled in a phase I/II trial and randomly assigned to receive 2-week-long courses of low-dose (50 mg twice a day) cyclophosphamide or not. The absolute number, phenotype, and antitumor function of peripheral blood-derived lymphocyte subsets were monitored throughout treatment, as well as during 18-month follow-up.Results: Initially, cyclophosphamide reduced proliferation in all lymphocyte subsets; however, a rapid mobilization of effector T cells overcame this decrease, leading to increased absolute T-cell numbers. In contrast, a reduction in proportional and absolute Treg, B-cell, and NK-cell numbers occurred. The expansion and subsequent activation of effector T cells was focused on tumor-specific T cells, producing both granzyme B and IFNγ. Cyclophosphamide-treated patients demonstrating the most enhanced IFNγ+ tumor-specific T-cell responses exhibited a significant delay in tumor progression [HR = 0.29; 95% confidence interval (CI), 0.12-0.69; P = 0.0047), compared with nonresponders and no-treatment controls.Conclusions: Cyclophosphamide-induced Treg depletion is mirrored by a striking boost in antitumor immunity. This study provides the first direct evidence of the benefit of naturally primed T cells in patients with metastatic colorectal cancer. Our results also support the concept that nonmutated self-antigens may act as useful targets for immunotherapies. Clin Cancer Res; 23(22); 6771-80. ©2017 AACR.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

R. Harrop holds ownership interest (including patents) in Oxford BioMedica. No potential conflicts of interest were disclosed by the other authors.

©2017 American Association for Cancer Research.

Figures

Figure 1

The effect of metronomic, low-dose cyclophosphamide on CD4+CD25hiFoxp3+ Treg. A, 50 mg cyclophosphamide was administered twice daily to 27 colorectal cancer patients on treatment days 1–7 and 15–21, with blood samples being taken at regular intervals, as shown in this schematic. B, Example FACS plots of CD25hi- and Foxp3-expressing CD4+ T cells at TD1 and TD4, and the gating used to denote the Treg percentage (top right quadrant). C, The percentage of Tregs was measured at the indicated time points throughout treatment. D, Peripheral Treg numbers were derived from the number of CD3+CD4+ T cells from whole blood samples and the proportion of CD4+ T cells expressing CD25hiFoxp3 in subsequent phenotypic analysis. The percentage of Tregs expressing Helios (E) and Ki67 (F) was measured at the indicated time points throughout treatment. G, The percent depletion of Treg numbers at treatment day 18 in comparison with treatment day 1 was correlated with the percent reduction in Ki67-expressing Tregs at treatment day 8 (TD8) in comparison with treatment day 1 (TD1). Black triangles/lines indicate patients taking cyclophosphamide (n = 27); clear triangles/dashed lines indicate control patients at the same stage of tumor progression (n = 25). Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 2

T- and NK-cell number and proliferation in response to cyclophosphamide. Absolute numbers of CD3+CD4+(Foxp3−) T-cells (A), CD3+CD8+ T cells (B), and CD3−CD56+ NK-cells (C) per microliter of whole blood were analyzed at indicated time points. The expression of Ki67 in CD4+(Foxp3−) T cells (D), CD8+ T cells (E), and CD3−CD56+ NK cells (F) was monitored throughout cyclophosphamide treatment. Black triangles/lines indicate patients taking cyclophosphamide (n = 27); clear triangles/dashed lines indicate control patients at the same stage of tumor progression (n = 25). Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3

Enhanced CD8+ T-cell function during cyclophosphamide treatment. Example FACS plots of perforin (A) and granzyme B (B) expression, and % expression of perforin (C) and granzyme B (Grz-B; D) in CD3+CD8+ T cells throughout cyclophosphamide treatment. Black triangles/lines indicate patients taking cyclophosphamide (n = 27); clear triangles/dashed lines indicate control patients at the same stage of tumor progression (n = 25). Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Dual-color FluoroSpots analyzing the production of IFNγ (green spots), and granzyme B (red spots) were performed, and representative well images are shown from two patients before treatment (TD1) and during cyclophosphamide treatment (TD4), with PBMCs stimulated with 5T4 peptide pools (E). The average SFC/105 PBMCs from duplicated wells of single- (IFNγ/granzyme B) and dual-cytokine– producing T cells in response to 5T4 peptide pool stimulus in five cyclophosphamide-treated patients, is shown (F).

Figure 4

Augmented antitumor (5T4)-specific responses during cyclophosphamide treatment associate with prolonged survival. PBMCs were cultured with 5T4 peptide pools (see Supplementary Fig. S2 for sequences), and IFNγ+ T-cell responses were measured by ELISpot. Representative examples from three patients at TD1 and TD8 are shown (A). The total number of spots to all positive identified 5T4 epitopes (B), tuberculin PPD (C), and HA (D) were enumerated and normalized to SFC/105 cultured PBMCs. Black triangles/lines indicate patients taking cyclophosphamide (n = 27); clear triangles/dashed lines indicate control patients at the same stage of tumor progression (n = 25). Significant differences are indicated (*, P < 0.05; **, P < 0.01). E, The mean change in 5T4-specific T-cell response at TD8–22 in comparison with TD1 was measured for all patients, with patients being separated into responders (white circles, n = 19) and nonresponders (black triangles, n = 8) below the 95% CI of responses at +105 SFC/105 cultured PBMCs. F, This change in 5T4 T-cell response was correlated with the patient's mean change in absolute peripheral CD3+ T-cell numbers during cyclophosphamide treatment. G, High anti-5T4 T-cell responses were associated with PFS. In addition, control patients who did not take any medication during the trial were also included for analysis (dashed lines; n = 8). 5T4 T-cell responders versus nonresponders and controls: HR, 0.29; 95% CI, 0.12–0.69; P = 0.0047.