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In all living cells phosphatidylserine (PS) is located at the cytosol side of the membrane and becomes exposed at the cell surface only during necrosis or apoptosis. This phenomenon allows measurement of cell death on a cell-by-cell basis, using labeled Annexin V, which has a strong affinity to PS. Two patients with hairy cell leukemia (HCL) who had relapsed after splenectomy and alpha-interferon therapy were treated with 2-chlorodeoxyadenosine (2-CdA) for 7 days. Blood samples were taken from the start of therapy until day 22. Percentages of HCL cells, T cells, B cells, and NK cells were measured with PE-labeled monoclonal antibodies by flow cytometry (FCM). The absolute lymphocyte count dropped rapidly to almost zero in both patients within 7 days. The disappearance rate of lymphocyte subfractions did not show a specific pattern. The percentage of apoptosis in lymphocyte subfractions was measured in freshly prepared cell samples by FCM with FITC-labeled Annexin V in the propidium iodide-negative (non-necrotic) cell fraction. Percentages of PS-positive cells increased gradually till a nadir of Annexin V positivity was reached at 14 and 16 days. Because during the first week the absolute cell counts became almost zero, the absolute numbers of PS-positive cells were still extremely low, i.e., less than 0.1x10(9)/l. Nevertheless, we observed apoptotic cells in circulation after 2-CdA therapy. To our knowledge, this is the first report of the occurrence of apoptosis ex vivo in circulating blood cells after cytotoxic therapy.

Cladribine (2-chlorodeoxyadenosine, 2-CdA) is a new antineoplastic drug which exerts its antilymphoproliferative activity by its resistance to the enzyme adenosine deaminase. Cladribine is mostly administered as a 7-day continuous infusion and in a dose of 0.1 mg/kg/day. However, preliminary data show that the subcutaneous and oral routes of administration might be feasible. The drug is well tolerated, and myelosuppression was found to be the dose-limiting toxicity. Nonhematological toxicity, such as alopecia, nausea, vomiting, stomatitis, diarrhea, and organ toxicity is mild or absent. Cladribine has shown efficacy in phase-II studies in hairy cell leukemia [response rate (RR) = 75-100% and complete response rate (CR) = 46-92%], chronic lymphocytic leukemia (RR = 37-67% and CR = 4-39%), and lymphocytic lymphoma (RR = 43-52% and CR = 14-20%). Furthermore, there is preliminary evidence that cladribine might be effective in the treatment of cutaneous T cell lymphoma (RR = 47% and CR = 20%), acute myeloid leukemia in children (RR = 59% and CR = 47%), acute lymphoid leukemia in children (RR = 14% and CR = 14%) and Waldenström macroglobulinemia (RR = 58% and CR = 3.5%). In multiple myeloma cladribine was not effective. Comparative randomized studies with established first-line and second-line therapeutic regimens are warranted and will define the ultimate place of cladribine in the therapy of malignant hematological disorders.

In this study the hypothesis that inhibition of the de novo pathway results in stimulation of salvage pathway activity was tested. The key enzyme in the balance between these two pathways is ribonucleotide reductase (RR), which can be inhibited by hydroxyurea (HU). The metabolism of 1-beta-D-arabinofuranosylcytosine and 5-Aza-2 deoxycytidine (Aza-dC), which are activated via the salvage pathway, was evaluated in cells from Ara-C-sensitive and -resistant myelocytic leukemia cell line (BNML-Cl/0 and BNML-Cl/Ara-C). The combination of HU and Ara-C caused as much as a threefold increase of Ara-CTP; it significantly increased the incorporation of Ara-C into DNA and induced synergistic cytotoxicity, as evaluated in a colony assay. Even in the deoxycytidine (CdR) kinase-deficient Ara-C-resistant cell line, HU was partially able to restore sensitivity to Ara-C and Aza-dC. dCTP levels are reduced during the first 10 h after incubation with HU, but this effect vanishes at the time when phosphorylation is maximal. Increased CdR kinase activity in cell-free extracts could explain the enhanced synthetic salvage pathway activity, which is likely due to the fact that more enzyme is present (Vmax has increased by Km unchanged). RR inhibition combined with Ara-C might provide a means of eliminating leukemic cells with suboptimal anabolic salvage pathway activity, which otherwise survive Ara-C chemotherapy.

We compared three consolidation regimens in patients with acute myelogenous leukemia in first remission. Thirty-four patients received only intensive consolidation chemotherapy (SIC); 28 patients were scheduled to undergo an autologous bone marrow transplant (auto-BMT) and 44 patients an allogeneic BMT (allo-BMT). Twenty-seven of them were referred in first remission for allo-BMT. Nineteen patients achieved a complete remission after salvage treatment. All other patients obtained a remission after one or two courses of a standard combination of cytosine arabinoside and daunorubicin. Except for the patients who were referred in remission, all patients received intermediate dose cytosine arabinoside and amsacrine as a first consolidation treatment. The median ages of the three groups were 48 (SIC), 39 (auto-BMT) and 33 years (allo-BMT). Two patients relapsed before auto-BMT and 1 before allo-BMT. The median interval from the date of complete remission to the auto- or allo-BMT was 3 months. In total, 80% of the patients of the SIC group relapsed, compared to 50% of the patients belonging to the auto-BMT group and 35% of the 44 patients who were scheduled to receive an allo-BMT. The overall median disease-free survival was 14 months, 30% of the patients being alive and disease-free at 3 years. The disease-free survival rate at three years was 25% for the SIC group, 30% for the allo-BMT group and 40% for the ABMT group (P = 0.45). Our study shows no benefit for bone marrow transplantation over intensive consolidation treatment. However, large randomized trials are required to define the real value of these treatment modalities.