Early clearance of hairy cell leukaemia in the bone marrow after first-line treatment with cladribine predicts a favourable outcome.

First-line purine nucleoside analogues (PNAs) in hairy cell leukaemia (HCL) allow deep and long-lasting responses. We retrospectively analysed 53 HCL patients treated frontline with cladribine and assessed for response at 2 and 6 months after treatment to evaluate the kinetics of response. The estimated median progression-free survival was significantly different according to the degree of residual HCL infiltrate detected by immunohistochemistry at the bone marrow biopsy at 2 months (≤5% vs. >5%, 247 vs. 132 months, respectively, p = 0.033), but not at 6 months (p = 0.79). Our data suggest a favourable prognostic impact of early marrow HCL clearance in patients treated with cladribine.

Occurrence of thrombotic events in acute promyelocytic leukemia correlates with consistent immunophenotypic and molecular features.

Although the occurrence of thrombosis in acute promyelocytic leukemia (APL) has been reported during retinoic acid treatment, no studies carried out in large clinical cohorts have specifically addressed this issue. We analyzed 124 APL patients treated with the all-trans retinoic acid and idarubicin protocol and compared clinico-biologic characteristics of 11 patients who developed thrombosis with those of 113 patients who had no thrombosis. In seven patients, the events were recorded during induction, whereas in four patients deep vein thrombosis occurred in the post-induction phase. Comparison of clinico-biological characteristics of patients with and without thrombosis revealed in the former group higher median white blood cell (WBC) count (17 x 10(9)/l, range 1.2-56, P=0.002), prevalence of the bcr3 transcript type (72 vs 48%, P=0.01), of FLT3-ITD (64 vs 28%, P=0.02), CD2 (P=0.0001) and CD15 (P=0.01) expression. No correlation was found with sex, age, French-American-British subtype, all-trans-retinoic acid syndrome or with thrombophilic state that was investigated in 5/11 patients. Our findings suggest that, in APL patients consistent biologic features of leukemia cells may predict increased risk of developing thrombosis.

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.

Monitoring of CD34+ cells during leukapheresis allows a single, successful collection of hemopoietic progenitors in patients with low numbers of circulating stem cells.

We have studied a total of 188 patients with hematological malignancies, submitted to mobilization therapy with G-CSF associated or not with chemotherapy in order to: (1) establish the lower limit of circulating progenitor cells that allows the collection of 2 x 10(6) CD34+ cells/kg by a single leukapheresis, utilizing the instrument set on standard parameters; (2) evaluate whether the number and quality of CD34+ cells collected remain stable during leukapheresis; and (3) collect a sufficient number of circulating CD34+ cells by a single procedure in patients in whom such an approach would have been insufficient to reach the target with the instrument set on standard parameters. The retrospective analysis conducted in 85 patients showed that 19 circulating CD34+ cells/microl represented the cut-off number capable of discriminating between patients who will require one or more apheresis to collect 2 x 10(6) CD34+ cells/kg. The validity of this value was prospectively confirmed in 70 subsequent patients. Based on in vitro results that showed the stability in the number of CD34+ cells, the proportion of different CD34+ cell subpopulations and the clonogenic capacity of the stem cell compartment during leukapheresis both in the blood of the patients and in samples taken directly from the instrument, we have adapted the blood volume to be processed in 33 patients with <19 PB CD34+ cells/microl. Stem cell collection was monitored during the leukapheresis and the procedure was prolonged for a time period estimated to be sufficient to reach the target number of CD34+ cells with a single procedure. The median increment of the total blood volume processed, calculated from the volume set automatically by the instrument was 25.2%, with a median of 3.3-fold total blood volume processed. In all cases, a sufficient CD34+ cell collection was completed in a single procedure. After autograft, the pattern of blood reconstitution was similar to that of all the other patients.

Modulation of VLA-4 and L-selectin expression on normal CD34+ cells during mobilization with G-CSF.

We have evaluated the immunophenotype, functional activity and clonogenic potential of CD34+ cells from peripheral blood (PB) of normal donors before and after 4 and 6 days of G-CSF administration. The percentage and absolute number of CD34+ cells significantly increased at days 4 and 6 of G-CSF administration, compared to the steady-state level (P < 0.0001). Two-colour fluorescence analysis showed, at days 4 and 6, a lower proportion of CD34+/c-kit+ compared to the steady-state level (P < 0.0001), but a similar expression of CD13, CD33, CD38, HLA-DR and Thy-1 antigens on CD34+ cells. The expression of adhesion molecules on CD34+ cells revealed a significant reduction of CD11a (P = 0.009), CD18, CD49d and CD62L (P < 0.0001) at days 4 and 6, compared to the baseline level. Three-colour staining showed a reduction of the more immature compartment (34+/DR-/13-) and an increase of the more differentiated compartment (34+/DR+/13+). Downregulation of VLA-4 during mobilisation was seen almost exclusively on more committed cells (34+/13+); downregulation of CD62L, on the contrary, was observed on both early progenitors (34+/13-) and more committed cells (34+/13+). The expression of 34+/VLA-4+ decreased on both c-kit+ and c-kit- cells, while the expression of 34+/62L+ decreased on the c-kit+ cells only. In vivo administration of G-CSF reduced the adherence capacity of CD34+ cells to normal BM stroma; in vitro incubation with SCF or IL-3 enhanced the expression of CD49d on CD34+ cells, while GM-CSF reduced the expression of CD62L. SCF was the only cytokine able to induce a significant increase of CD34+ cell adherence to preformed stroma. Pre-incubation with the blocking beta2 integrin monoclonal antibody caused a reduction of CD34+ cell adherence. In conclusion, the decrease of CD49d expression on mobilized CD34+ cells correlates with a poor adhesion to BM stroma; CD34+ cells incubated in vitro with SCF showed, conversely, a higher expression of CD49d and a greater adherence capacity on normal preformed stroma.

Interferon-alpha and bcr-abl antisense oligodeoxynucleotides in combination enhance the antileukemic effect and the adherence of CML progenitors to preformed stroma.

We have studied the in vitro effect of IFN-alpha and bcr-abl antisense oligodeoxynucleotides (As ODN) alone and in combination with the aim of enhancing the antileukemic activity of the two single agents and evaluating whether the two agents in combination might restore the adherence capacity of chronic myeloid leukemia (CML) progenitors to preformed stroma. We have also correlated the increased adhesion found after in vitro treatment with the expression of adhesion molecules on leukemic progenitors. Incubation of the BV173 cell line with escalating doses of IFN-alpha (100-10000 U/ml) showed a colony growth inhibition between 10 and 30%. IFN-alpha and junction-specific As ODN in combination showed a greater antiproliferative effect compared to that observed with the two agents used alone. In particular, As ODN at a concentration of 40 microg/ml in combination with IFN-alpha at 100 and 1000 U/ml showed a greater inhibitory effect compared to that obtained with IFN-alpha only. Addition of As ODN to IFN-alpha at 10000 U/ml did not result in a greater BV173 inhibition. In a further set of experiments, primary cells from 16 CML patients at diagnosis were incubated with 40 microg/ml of J-spec As ODN, several control ODNs and IFN-alpha at 1000 U/ml alone and in combination. A significantly greater elimination of CML progenitors was found after treatment with the combination of IFN-alpha and J-spec As ODN, compared to any other treatment group, confirmed also by a more marked effect on p210 expression. The deficient adhesion of CML progenitors on human preformed stroma was restored at levels similar to that of normal bone marrow cells after treatment with IFN-alpha and/or J-spec As ODN, while the phenotypic analysis showed that the combined treatment increased significantly the expression of CD49b and CD62L on CML CD34+ cells. However, when the expression of adhesion molecules was blocked with specific monoclonal antibodies, only CD49d (expressed on more than 90% of CML CD34+ cells) appeared to influence the functional activity of adhesion molecules. In conclusion, IFN-alpha and bcr-abl As ODN in combination exert a marked in vitro antileukemic activity and could be a useful approach for in vitro purging of CML cells prior to autologous transplantation.

Evaluation of marrow and blood haemopoietic progenitors in chronic lymphocytic leukaemia before and after chemotherapy.

We have evaluated the number and differentiation pattern of CD34+ cells, as well as the CFU-GM, BFU-E and CFU-GEMM progenitors from the blood (PB) and marrow (BM) of 53 chronic lymphocytic leukaemia (CLL) patients. Twenty-four patients were untreated and 29 were studied at 2 months from the last course of fludarabine or chlorambucil; 6 patients, studied after fludarabine therapy, were further evaluated after mobilization with cyclophosphamide and G-CSF. PB of untreated patients showed a median number of CD34+ cells, CFU-GM, BFU-E and CFU-GEMM/10(5) seeded cells and per litre of PB similar to those of normal controls. No differences were also found in the number of clonogenic progenitors/10(5) cells in patients studied before and after therapy, while significantly fewer BFU-E/l of PB were found after fludarabine. The number of circulating CD34+ cells/l of PB was significantly lower in patients treated with fludarabine or chlorambucil compared to untreated patients. BM growth was significantly reduced in untreated CLL patients compared to healthy donors. Treatment with fludarabine or chlorambucil restored BM progenitors at levels similar to those of normal controls; this effect did not occur for CFU-GM in patients treated with fludarabine. Three-colour fluorescence analysis demonstrated a differentiation pattern of CD34+ cells, with a greater expression of CD13 and CD33 after treatment with fludarabine compared to untreated patients and normal controls. In 4 patients previously treated with fludarabine who underwent a successful cyclophosphamide and G-CSF mobilization therapy, 4x10(6) CD34+ cells/kg were collected. These 4 patients showed a notable increase of CD34+ cells and of clonogenic cells in the PB, but a marked decrease of BM progenitor cells. The 2 patients who failed CD34+ cell mobilization had a reduced CFU-GM growth both in the PB and in the BM. Taken together, these studies indicate that residual haemopoietic progenitors are present in untreated CLL patients and that stem cell mobilization and collection can be carried out following fludarabine treatment.

CD34+ cell mobilization for allogeneic progenitor cell transplantation: efficacy of a short course of G-CSF.

BACKGROUND: G-CSF-mobilized PBPCs are considered the richest source of HPCs for both autologous and allogeneic transplantation, but, despite their wide use, the best dose and schedule for G-CSF administration have not been definitively established. STUDY DESIGN AND METHODS: With a target of collecting from the peripheral blood > or = 4 x 10(6) CD34+ cells per kg of body weight of the recipient, the short-course administration of glycosylated G-CSF (gly-G-CSF) in 30 healthy donors for an allogeneic transplantation was investigated. Gly-G-CSF was given subcutaneously at a dose of 10 microg per kg per day in two divided doses over 3 days and was followed by a leukapheresis (on the 4th day) 12 hours after the last dose. RESULTS: A median of 53.5 circulating CD34+ cells per microL (range, 19-190) was found in the 30 donors on the day of first leukapheresis, which allowed a median CD34+ cell collection of 6.0 x 10(6) per kg of body weight of the donor and 6.5 x 10(6) per kg of body weight of the recipient. In 25 (83%) of 30 donors, a single procedure was sufficient to collect the target CD34+ cells, while in the other 5, two leukapheresis procedures were required. Hematologic reconstitution was observed in all patients at a median of 14 days (range, 10-23) for neutrophils and 14.5 days (range, 11-46) for platelets. With a median infusion of 3.9 x 10(8) CD3+ T-lymphocytes per kg of body weight of the recipient (range, 1.3-7.8), acute and chronic GVHD occurred in 13 (43%) of 30 and 15 (60%) of 25 evaluable patients, respectively. After a median follow-up of 337 days from transplant, 22 (73%) of 30 patients are alive in complete remission. CONCLUSION: A schedule consisting of 3-day administration of gly-G-CSF followed by a single leukapheresis can be proposed and widely accepted by healthy donors, as 84 percent of them reach the target in the estimated time with a reduced drug exposure. The cost of the procedure is reduced, in terms of both the growth factor administration and the number of leukapheresis procedures. The search for the optimum methods of donor management may improve the acceptability of this procedure and increase the number of allogeneic transplantations from PBPCs.

BCR-ABL antisense oligodeoxynucleotide in vitro purging and autologous bone marrow transplantation for patients with chronic myelogenous leukemia in advanced phase.

BCR-ABL antisense oligodeoxynucleotides (ODN) have provided evidence of antileukemia effect when tested in vitro against Philadelphia-positive (Ph-pos) cells and in vivo when injected into leukemic mice. On the basis of the results obtained in vitro at diagnosis, eight patients with chronic myelogenous leukemia (CML) were selected and submitted to autologous bone marrow transplantation (ABMT) with bone marrow (BM) cells purged in vitro with junction-specific (J-sp) BCR-ABL antisense ODN at the time of transformation in accelerated phase or during second chronic phase. Mononuclear BM cells were treated in vitro for 24 or 72 hours with 150 micro/mL of antisense ODN yielding a median recovery of 47.6% mononuclear cells, 48.8% CD34(+) cells, and 20.3% clonogenic cells. After a conditioning regimen including busulphan and etoposide, the reinfused treated cells allowed engraftment and hematologic reconstitution in all patients. Evaluation of the antileukemic effect by standard cytogenetic analysis and fluorescence in situ hybridization showed a complete karyotypic response in two cases and a minimal or no response in the other six. The patient autografted in second chronic phase died in blast crisis 7 months after ABMT; of the seven patients autografted in transformation, three developed blast crisis 21 to 39 months after reinfusion, one died from unrelated BMT complications 30 months after ABMT, and three are in persistent second chronic phase 14 to 26 months after autograft. The low toxicity of the protocol and the hemopoietic reconstitution observed in all patients make this approach feasible; the marked karyotypic response observed in some patients and the duration of the second chronic phase show that ODN-mediated BM purging and autograft is a promising treatment for this high-risk group of CML.