A phase 1 clinical-laboratory study of clofarabine followed by cyclophosphamide for adults with refractory acute leukemias

Abstract

Clofarabine has shown impressive response rates in patients with acute leukemias. In vitro investigations with clofarabine in combination with cyclophosphamide in primary cells have demonstrated synergistic cytotoxicity and inhibition of DNA repair. Based on these clinical and laboratory observations, we designed a mechanism-based combination protocol with clofarabine and cyclophosphamide for patients with relapsed acute leukemias. Eighteen patients were treated with cyclophosphamide (200 mg/m(2)) alone on day 0 and with clofarabine plus cyclophosphamide on day 1. Clinical responses, toxicity, DNA damage measured as H2AX phosphorylation, and accumulation of clofarabine triphosphate (TP) were analyzed. At dose level 1 (20 mg/m(2) clofarabine + cyclophosphamide, 6 patients) and dose level 0 (10 mg/m(2) clofarabine + cyclophosphamide, 12 patients) overall response rates were 50% and 30%, respectively, with responses in 4 (67%) of 6 patients with refractory acute lymphoblastic leukemia. Dose-limiting toxicity occurred at dose level 1 with prolonged marrow aplasia. Four (22%) patients died from prolonged aplasia (1), fungal pneumonia (1), or multiorgan failure (2). In 12 of 13 patient samples, increased DNA damage (gammaH2AX) was observed with clofarabine and cyclophosphamide compared with cyclophosphamide alone. In conclusion, pharmacodynamic end points along with clinical results suggest usefulness of this combination strategy, whereas toxicity data suggest reduction in chemotherapeutic intensity. This clinical trial is registered with the National Cancer Institute's PDQ at www.clinicaltrials.gov as no. JHOC-J0561.

Trial registration: ClinicalTrials.gov NCT00293410.

Figures

Figure 1

Changes in γH2AX induced by CY and clofarabine-CY. (A) Increase in H2AX phosphorylation in circulating leukemia cells from peripheral blood. The peripheral blood blasts were obtained from 13 patients and the presence and magnitude of DNA damage was measured by flow-cytometric analysis prior to initial CY (D0 pre), at the end of CY infusion (D0 End CY), 2 hours after EOI of day 0 CY (D0 After CY), and on day 1 following clofarabine-CY (2 hours after CY; D1 After CY). Cellular debris was gated out and nonspecific staining controls were used to mark the lower limit of the positive γH2AX-staining region (SR). Control SR was subtracted from SR of samples to determine the percentage of cells staining positive for γH2AX (expressed as % γH2AX). Pretreatment value was expressed as zero and all other values were percentage change. An expanded y-axis (−100% to 250%) is presented in panel B to highlight the differences in γH2AX at the measured time points.

Figure 2

Increase in γH2AX and sub-2N DNA with clofarabine-CY. Measurements of DNA damage (γH2AX; ○) and apoptosis (sub2N DNA; △) were made on peripheral blood blasts obtained from 13 patients before treatment, after CY on day 0, and after clofarabine-CY on day 1. Values obtained from day 1 after clofarabine-CY blasts were compared with values obtained from pretreatment blasts and day 0 after CY blasts. The relative changes are expressed as fold increase. ● indicates median fold change in γH2AX; and ▴, median fold change in sub-2N DNA.

Figure 3

Increase in H2AX phosphorylation in leukemia cells obtained from bone marrow. The bone marrow blasts were obtained longitudinally from 4 patients and the presence and magnitude of damaged DNA was measured by flow-cytometric analysis on day 0 (Pre-Study), EOI CY (D0 End CY), and day 3 clofarabine followed by CY (D3 End). Cellular debris was gated out and nonspecific staining controls were used to mark the lower limit of the positive γH2AX-staining region (SR). Control SR was subtracted from sample SR to determine the percentage of cells staining positive for γH2AX (expressed as % γH2AX). Pretreatment value was expressed as 0 and all other values were percentage change.

Figure 4

Pharmacokinetic and pharmacodynamic end points during therapy in leukemia blasts obtained from 1 representative patient. Leukemic blasts were obtained before treatment (D0-Pre), after end of infusion clofarabine (D1-EOI), and on day 1 following clofarabine followed by CY 2 hours after CY EOI (D1 After), and clofarabine-TP was extracted and analyzed (▩) using high-pressure liquid chromatography. Samples were obtained prior to pretreatment of clofarabine (D1-Before), after end of infusion clofarabine (D1-EOI), after end of infusion cyclophosphamide (D1-EOI), and on day 1 following clofarabine followed by CY 2 hours after CY EOI (D1 After) for dATP quantitation (■). Additional samples were obtained for γH2AX (□) and analyzed as described in Figure 1.