In vitro and in vivo correlation of cyclosporin A‐induced apoptosis in acute lymphoblastic leukemia

To the Editor: Cyclosporin A (CsA) is a cyclic endecapeptide immunosuppressive agent which inhibits T-cell responses and is used in the prevention of graftversus-host disease, in the treatment of allograft rejection and some autoimmune disorders (1). CsA has been shown to bind to an intracellular protein, cyclophilin, and this binary complex inhibits the activity of calcineurin, a calciumand calmodulindependent serinekhreonine phosphatase (2). This results in an impairment of transcriptional activity of lymphoid cells (3). Furthermore, CsA has been shown to be an uncompetitive inhibitor of proteasome activity and prevents NF-KB activation (4). CsA exhibits toxicity against leukemic T-cell lines (5) . More recently, CsA (5 pM) was found to produce a median cell kill of 69% in seven B-lineage acute lymphoblastic leukemia (ALL) cell lines. Against lymphoblasts from patients with B-lineage ALL, the compound (at 5 pM) reduced the leukemic cell number by a median of 87% (6). These results stimulated us to administer CsA to a patient with refractory B-lineage ALL in an attempt to treat the leukemia, since CsA produced significant apoptosis of his ALL cells in vitro. Mononuclear cells were collected from heparinized venous blood by density-gradient centrifugation (Ficoll; Pharmacia, Uppsala, Sweden). The cells were resuspended in RPMI 1640 tissue culture medium supplemented with 10% heat inactivated fetal calf serum (Biochrom, Berlin, Germany). A CsA preparation for intravenous administration (Sandimmun; Novartis, Basel, Switzerland) was used. The cells were incubated with or without CsA (but with 0.1% ethanol) in different concentrations at 37 "C with 5% C02 and 95% humidity for 24 or 48 h. Apoptosis was quantified by flow cytometry analysis of FITC-conjugated annexin-V bindings to plasma membrane externalization of phosphatidylserine as an early event of apoptosis (7). Cell membrane permeabilization was detected by propidium iodide (PI) labeling. CsA blood concentrations were determined in the Institute of Clinical Pharmacology, CharitC, Berlin. Quantitative measurements of CsA were routinely carried out with the Abbott Tdx polyclonal antibody flourescence polarization immunoassay (FPIA, parent compound and metabolites, limit of quantification <65 yglL). When the Syva enzyme multiplied immunoassay technique (EMIT, specific measurement of the parent compound, limit of quantification 6 4 0 pg/L) is used, this difference in the methodology is emphasized when the results are given. Mean accuracy for both assays was <7% over the complete working range. In November 1995 the diagnosis of an ALL was made in a 16-year-old patient who was treated according to the protocol of the German ALL Study Group. Immunotyping revealed a commonALL, and cytogenetics were normal (46, XU). A complete response was obtained, but during maintenance therapy the patient relapsed in January 1997. A second remission was obtained with intensive therapy including high-dose cytarabine. A bone marrow transplantation could not be performed because of hepatolienal candidiasis. A second relapse occurred in May 1997, and the leukemia has been refractory to different chemotherapy protocols since then. In March 1998, the leukocyte count increased to 47 OOO/mm3, consisting of 98% blasts and 2% lymphocytes. CsA-induced apoptosis and cell kill were quantified by flow cytometry analysis. The effect of different concentrations of CsA and the impact of incubation periods of 24 and 48 h, respectively, are shown in Fig. 1. After an incubation period of 48 h, the cytotoxicity of CsA on ALL blasts was more pronounced than after 24 h. An attempt of therapy with CsA was made after informed consent was given by the patient. CsA was administered as a 2-h loading dose of 5 mg/kg followed by a continuous infusion of 15 mg/kg for 60 h as shown to be feasible in a phase I study with CsA used as a multidrug resistance modifier (8). The patient was monitored closely. Blood CsA levels