Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells.

BACKGROUND: Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting. OBJECTIVE: To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method. METHODS: We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, DeltaNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture. RESULTS: Overall recovery of CD34+ cells after culture was 128.5%; DeltaNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after DeltaNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7-1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and DeltaNGFR selection. CONCLUSION: We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.

HIC gene, a candidate suppressor gene within a minimal region of loss at 7q31.1 in myeloid neoplasms.

We studied monosomy and deletions of chromosome 7 in 170 patients with myeloid disorders and we identified a minimal region of loss in 7q31.1 spanning between the D7S2554 and D7S2460 markers. The closest gene to our most deleted microsatellite, D7S2554, is the human I-mfa domain containing (HIC) gene, alias MyoD family inhibitor domain containing (MDFIC). We investigated the involvement of HIC in myeloid neoplasms by screening for mutations the coding regions and the intron-exon boundaries of this gene in 15 patients who presented chromosome 7 deletions in the region of HIC. No mutations were found in the coding region of this gene.

The achievement of durable complete cytogenetic remission in late chronic and accelerated phase patients with CML treated with Imatinib mesylate predicts for prolonged response at 6 years.

Despite the positive results achieved by Imatinib mesylate (Imatinib) in the treatment of chronic myeloid leukemia (CML), over the past several years, Imatinib does not eradicate the leukemic clone. The long-term duration of response to the drug is not known. Long-term follow-up of CML patients treated with Imatinib will ultimately define the durability of such treatment and the frequency of reemergence of progressive disease. We present the results of a 6-year follow-up of 40 CML patients either in chronic or accelerated phase who obtained a durable (>6 months) complete cytogenetic remission (CCyR) after treatment with Imatinib in a single center. In 34 cases CCyR was obtained at an Imatinib dose of 400-600 mg/day and in 6 cases after a dose increase to 600-800 mg/day. At a median follow-up of 68 months, 6 cytogenetic relapses (15%) were observed. No progressions to more advanced phases of disease have been detected during the follow-up period. Cytogenetic relapse was predicted by either a decrease in the amount of BCR-ABL transcript of less than 2 logs after the achievement of CCyR (p=0.0041) or a time-to-CCyR of more than 12 months (p<0.0001). This 6-year follow-up of the efficacy of Imatinib therapy in CML patients who obtained a durable CCyR indicates that the relapses rate is low over this period of observation and that the rate of relapse does not increase over time.

Immunoglobulin gene rearrangements and in vitro growth of stromal cells are prognostic indexes in B-chronic lymphocytic leukemia.

B-chronic lymphocytic leukemia is a disease characterized by an accumulation of monoclonal B cells that are resistant to apoptosis. In chronic lymphocytic leukemia, the prognosis depends on the stage of the disease, according to the classifications of Rai and Binet. However, in recent years, the number of patients with very early disease (stage 0 of Rai) and without any clinical symptom, has considerably increased because of the extensive use of automatic apparatus for leukocyte counting and immunophenotypic analysis of lymphocytes. It has become, therefore, useful to find new prognostic criteria particularly for these patients. In the present study, 30 patients with B-chronic lymphocytic leukemia were investigated for stage of the disease, survival, immunoglobulin gene rearrangements, presence of nurse like cells in in vitro cultures and spontaneous clinical lymph node regression. We observed that all these criteria are useful prognostic indexes for the disease.

Gynaecomastia in men with chronic myeloid leukaemia after imatinib.

cKit and platelet-derived growth-factor receptor (PDGFR) are receptor tyrosine kinases expressed in the testis, are involved in testosterone production, and are inhibited by imatinib. We measured hormone concentrations in 38 men receiving imatinib for chronic myeloid leukaemia at baseline and during treatment. Mean follow-up was 23.6 months (SD 7.5). We noted seven cases of gynaecomastia (18%, 95% CI 6-30%). A comparison of hormone concentrations in 21 patients before and during treatment showed that patients who developed gynaecomastia had a reduction in free testosterone concentrations of 29.53 pmol/L (95% CI 11.63-47.43), while patients who did not had a decrease of 6.36 pmol/L (-1.02 to 13.74). In most men with chronic myeloid leukaemia studied here, imatinib was associated with a reduction in the production of testicular hormones and in some, with the development of gynaecomastia.

Alpha1 acid glycoprotein binds to imatinib (STI571) and substantially alters its pharmacokinetics in chronic myeloid leukemia patients.

PURPOSE: Imatinib (Glivec) is a potent inhibitor of bcr/abl, an oncogenic fusion protein that causes chronic myelogenous leukemia (CML). alpha1 acid glycoprotein (AGP) binds to imatinib with high affinity and inhibits imatinib activity in vitro and in vivo in an animal model. A pharmacokinetics analysis of imatinib was undertaken in CML patients. EXPERIMENTAL DESIGN: Imatinib plasma concentrations were measured in 19 CML patients treated with imatinib (400 or 600 mg/day). Five patients received a concomitant short-term course of clindamycin (CLI). RESULTS: A positive correlation between AGP and imatinib plasma levels was observed. CLI administration decreased imatinib plasma concentrations, evaluated as area under the curve (AUC) and peak concentrations (C(max)). The effects of a bolus of CLI was studied in three patients on imatinib 23 h after the last imatinib dose. Within 5-10 min in three of three cases, CLI caused a decrease in imatinib plasma concentrations of 2.6-, 2.7-, and 4.7-fold, respectively. In vitro experiments using fresh blasts from CML patients showed that AGP, at concentrations observed in the patients, decreased imatinib intracellular concentrations up to 10 times and blocked imatinib activity. The incubation with CLI restored imatinib intracellular concentrations and biological activity. CONCLUSION: AGP exerts significant effects of the pharmacokinetics, plasma concentrations, and intracellular distribution of imatinib in CML patients; these data indicate that plasma imatinib levels represent unreliable indicators of the cellular concentrations of this molecule.

Differences between in vivo and in vitro sensitivity to imatinib of Bcr/Abl+ cells obtained from leukemic patients.

Imatinib mesylate (imatinib) inhibits Bcr/Abl, an oncogenic fusion protein. The in vitro effects of imatinib on BCR/ABL+ leukemic cells include inhibition of Bcr/Abl tyrosine phosphorylation, block of proliferation, and induction of apoptosis. The in vivo effects of imatinib were evaluated in 12 CML (chronic myeloid leukemia) patients in blast crisis or accelerated phase who were treated with imatinib. Treatment caused a decrease in spontaneous proliferation of leukemic cells in 10 of 12 evaluable patients and the development of apoptosis in 9 of 11 cases. Imatinib also caused an inhibition of Bcr/Abl autophosphorylation; however, the degree of inhibition obtained in vivo was substantially lower than that achieved in vitro with similar concentrations of imatinib. In seven patients cells could be evaluated at relapse: spontaneous proliferation was no longer inhibited and Bcr/Abl phosphorylation was comparable or superior to that present at the beginning of treatment, before imatinib administration. Plasma imatinib concentrations were not reduced. Leukemic cells obtained at relapse maintained in vitro sensitivity (Bcr/Abl autophosphorylation and proliferation inhibition) to imatinib concentration measured in vivo (3 microM or higher), although a partial resistance to the antiproliferative effects of imatinib was present at low (0.01-0.3 microM) concentrations. In four patients, addition of erythromycin to blood samples obtained at relapse restored imatinib sensitivity in terms of phosphorylation inhibition, indicating that the majority of plasma imatinib was not available to cells and probably bound to alpha1 acid glycoprotein. These data suggest that measurements of Bcr/Abl kinase activity in peripheral blood samples may represent a more reliable indicator of active concentrations than the measurement of imatinib plasma levels.