Rescue from failed growth factor and/or chemotherapy HSC mobilization with G-CSF and plerixafor (AMD3100): an institutional experience.

Auto-SCT has been shown to be a potentially curative treatment for a variety of hematological malignancies. Auto-SCT is dependent on the successful mobilization and collection of hematopoietic stem cells to ensure engraftment. The inability to mobilize sufficient number of hematopoietic stem cells using standard cytokine-assisted mobilization strategies excludes eligible patients from potentially curative auto-SCT. Plerixafor (AMD3100; Mozobil), a novel bicyclam antagonist of the SDF-1alpha/CXCR4 complex, has been reported previously to augment PBSC mobilization in patients undergoing their first planned stem cell mobilization and collection attempt. In our experience, 17 of 20 patients otherwise eligible for auto-SCT who failed previous mobilization attempts had successful mobilization of CD34(+) hematopoietic stem cells with one apheresis procedure, and an additional patient required two aphereses procedures, when treated with the combination of plerixafor and G-CSF on a compassionate use protocol available at our institution.

Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA-94 trial.

Fifty-four percent of adults with acute lymphoblastic leukemia (ALL) who entered the LALA-94 trial experienced a first relapse. We examined the outcome of these 421 adult patients. One hundred and eighty-seven patients (44%) achieved a second complete remission (CR). The median disease-free survival (DFS) was 5.2 months with a 5-year DFS at 12%. Factors predicting a better outcome after relapse were any transplant performed in second CR (P<0.0001), a first CR duration >1 year (P=0.04) and platelet level >100 x 10(9)/l at relapse (P=0.04). Risk groups defined at diagnosis and treatment received in first CR did not influence the outcome after relapse. The best results were obtained in a subset of patients who were eligible for allogeneic stem cell transplantation (SCT). Geno-identical allogeneic SCT was performed in 55 patients, and 3 patients received donor lymphocyte infusions. Forty-four transplantations were performed from an unrelated donor (of which four were cord blood). We conclude that most adult patients with recurring ALL could not be rescued using current available therapies, although allogeneic SCT remains the best therapeutic option.

Long-term complete responses after 131I-tositumomab therapy for relapsed or refractory indolent non-Hodgkin's lymphoma.

We present the long-term results of 18 chemotherapy relapsed indolent (N = 12) or transformed (N = 6) NHL patients of a phase II anti-CD20 (131)I-tositumomab (Bexxar) therapy study. The biphasic therapy included two injections of 450 mg unlabelled antibody combined with (131)I-tositumomab once as dosimetric and once as therapeutic activity delivering 75 or 65 cGy whole-body radiation dose to patients with normal or reduced platelet counts, respectively. Two patients were not treated due to disease progression during dosimetry. The overall response rate was 81% in the 16 patients treated, including 50% CR/CRu and 31% PR. Median progression free survival of the 16 patients was 22.5 months. Median overall survival has not been reached after a median observation of 48 months. Median PFS of complete responders (CR/CRu) has not been reached and will be greater than 51 months. Short-term side effects were mainly haematological and transient. Among the relevant long-term side effects, one patient previously treated with CHOP chemotherapy died from secondary myelodysplasia. Four patients developed HAMA. In conclusion, (131)I-tositumomab RIT demonstrated durable responses especially in those patients who achieved a complete response. Six of eight CR/CRu are ongoing after 46-70 months.

Autologous stem cell transplantation in adults with acute lymphoblastic leukemia in first complete remission: analysis of the LALA-85, -87 and -94 trials.

To evaluate the results of autologous stem cell transplantation (ASCT) in a large population of adults with acute lymphoblastic leukemia (ALL) in first complete remission (CR), we performed an individual data-based overview of the last three trials from the LALA group. Overall, 349 patients with ALL prospectively randomized in the consecutive LALA-85, -87, and -94 trials to receive either ASCT or chemotherapy as post-CR treatment were analyzed. Eligibility criteria were 15-50-year-old patients without sibling donors in both LALA-85/87 trials and 15-55-year-old patients with high-risk ALL and no sibling donors in the LALA-94 trial. Intent-to-treat analysis, which compared 175 patients from the ASCT arm to 174 patients from the chemotherapy arm, showed that ASCT was associated with a lower cumulative incidence of relapse (66 vs 78% at 10 years; P=0.05), without significant gain in disease-free or overall survival. Despite a possible lack of statistical power, a nested case-control analysis performed in 85 patient pairs adjusted for time to transplant and prognostic covariates confirmed these intent-to-treat results in patients actually transplanted. Of interest, the reduced relapse risk after ASCT translated in better disease-free survival in the 300 rapid responders who reached CR after the first induction course.

Severe pustular eruption associated with imatinib and voriconazole in a patient with chronic myeloid leukemia.

Imatinib is a specific and potent inhibitor of the BCR-ABL tyrosine kinase. Several clinical trials have demonstrated the efficacy of imatinib in chronic myeloid leukemia. Adverse cutaneous reactions induced by imatinib are frequent and may be dose related. We report a case of an unusual pustular eruption in a patient with chronic myeloid leukemia, who received high doses imatinib for blast crisis and later voriconazole for invasive pulmonary aspergillosis. At the time of his skin eruption, elevated plasma levels of imatinib were recorded. Imatinib is primarily metabolized by the cytochrome CYP3A4. Voriconazole is a cytochrome CYP3A4 inhibitor and can lead to high plasma levels of imatinib. This case suggests that severe drug reactions to imatinib may be related not only to imatinib doses, but also to elevated plasma drug levels resulting from pharmacokinetic interactions. The monitoring of imatinib plasma levels may be of help for identifying patients at risk for severe toxicity.

Determination of imatinib (Gleevec) in human plasma by solid-phase extraction-liquid chromatography-ultraviolet absorbance detection.

A sensitive HPLC method has been developed for the assay of imatinib in human plasma, by off-line solid-phase extraction followed by HPLC coupled with UV-Diode Array Detection. Plasma (750 microl), with clozapine added as internal standard, is diluted 3 + 1 with water and subjected to a solid-phase extraction on a C18 cartridge. After matrix components elimination with 2000 microl of water (in two aliquots of 1000 microl), imatinib is eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 180 microl 50% methanol. A 50 microl volume is injected onto a Nucleosil 100-5 microm C18 AB column. Imatinib is analyzed using a gradient elution program with solvent mixture constituted of methanol and water containing both 0.05% ammonium acetate. Imatinib is detected by UV at 261 nm. The calibration curves are linear between 0.1 and 10 microg/ml. The limit of quantification and detection are 0.05 and 0.01 microg/ml, respectively. The mean absolute recovery of imatinib is 96%. The method is precise with mean inter-day CVs within 1.1-2.4%, and accurate (range of inter-day deviations -0.6 to +0.7%). The method has been validated and is currently being applied in a clinical study assessing the imatinib plasma concentration variability in a population of chronic myeloid leukemia- and gastro-intestinal stromal tumor-patients.

Campath-1H treatment of T-cell prolymphocytic leukemia in patients for whom at least one prior chemotherapy regimen has failed.

PURPOSE: We conducted a retrospective analysis to evaluate the safety and efficacy of Campath-1H, an anti-CD52 humanized monoclonal antibody, in previously treated T-prolymphocytic leukemia (T-PLL) patients in a compassionate-use program. PATIENTS AND METHODS: Seventy-six patients with T-PLL (including four chemotherapy-naive patients) received 3, 10, and 30 mg of Campath-1H on sequential days, followed by 30 mg three times weekly, as 2-hour intravenous infusions, for 4 to 12 weeks. RESULTS: Median patient age was 60 years (range, 35 to 84). Spleen liver, lymph node, and skin involvement were present in 64%, 40%, 54%, and 18% of patients, respectively. All tested patients had CD2, CD7, CD4, and/or CD8 positivity, whereas CD5 and CD3 were positive in 98% and 96% of tested patients, respectively. The objective response rate was 51% (95% confidence interval [CI], 40% to 63%), with a 39.5% complete response (CR) rate (95% CI, 28% to 51%). The median duration of CR was 8.7 months (range, 0.13+ to 44.4), and median time to progression was 4.5 months (range, 0.1 to 45.4) compared with 2.3 months (range, 0.2 to 28.1) after first-line chemotherapy. The median overall survival was 7.5 months (14.8 months for CR patients). The most common Campath-1H-related adverse events were acute reactions during or immediately after infusions. Fifteen infectious episodes occurred during treatment in 10 patients (13%), leading to treatment discontinuation in three. Eight patients experienced possibly related, late-onset infections. Severe thrombocytopenia and/or neutropenia occurred in six patients (8%), leading to treatment discontinuation in four. Two treatment-related deaths occurred. CONCLUSION: Campath-1H is an active drug in T-PLL patients for whom first-line therapy has failed. It has a favorable risk/benefit ratio and should be prospectively investigated in chemotherapy-naive patients.

[Current treatment of AL amyloidosis]. / Approches actuelles du traitement de l'amyloïdose AL.

AL amyloidosis is a plasma cell disorder characterized by the tissue accumulation of immunoglobulin light chains. The prognosis of AL amyloidosis is poor, with a median survival inferior to two years. Amyloid deposits, responsible for the clinical manifestations of this disease, can regress at least partially after the suppression of the production of amyloid precursors. Therefore, current treatment strategies in AL amyloidosis aim at reducing the plasma cell clones, using chemotherapy regimens applied in multiple myeloma. A combination of melphalan and prednisone is the standard therapy of AL amyloidosis, but its results remain disappointing. Intensive chemotherapy regimens, with high-dose melphalan followed by peripheral blood stem cell transplantation, lead to increased response rates and improved survival, but are complicated by severe short term morbidity and mortality. These regimens offer the best clinical perspectives for selected patients, until the introduction of innovative agents capable of interfering with amyloid fibril formation.

[Autologous transplantation of peripheral blood hematopoietic stem cells in the treatment of hematological malignancies. I: patients]. / Autogreffe de cellules souches hématopoïétiques périphériques dans le cadre du traitement d'hémopathies malignes. Partie I: patients.

61 autologous transplantations for haematological malignancies have been performed with peripheral blood stem cells in 60 patients. 26 grafts were performed for non-Hodgkin's lymphoma (18 patients were transplanted in first remission, 8 patients after progression or relapse), 13 for multiple myeloma, 7 for Hodgkin's disease and 10 for acute myeloid leukaemia. One patient died from thrombosis of the portal vein. 38 patients were in complete remission 29 months (extremes: 14-44) after transplantation. 21 of 60 patients progressed or relapsed after transplantation, and 14 died. No death was attributed to graft failure, infection or haemorrhage. In conclusion, transplantation with peripheral blood stem cells is well tolerated, has a low toxicity rate, and can be used safely for patients with haematological malignancies.

Acute myeloid leukemia in the elderly: results of an individualized approach in two centres.

We retrospectively assessed seventy-four consecutive patients with AML over 65 years of age (median 71; range 65-88) treated with an individualized approach in two specialized cancer centers. Patients were managed according to their performance status (PS) and associated diseases in both institutions. The proportion of patients with poor PS (3-4) was higher in center 1 (37%) than in center 2 (10%) and in center 1 palliative treatment was given more frequently (16/32 patients) than in center 2 (7/42 patients). Fifty-one patients received intensive combination chemotherapy including an anthracycline and ara-C or VP16 (2 patients) and 36 patients received a second intensive course as reinduction or as consolidation treatment after complete remission. Patients not eligible for myelosuppressive chemotherapy were treated with palliative measures (23 patients). With intensive chemotherapy, complete remission (CR) was achieved in 29 of 51 patients (57%), after first (20 patients) or second course (9 patients) and the rate of deaths during induction was 14% (7 patients). The CR rate was lower for patients with performance status >or= 2 (48%) as compared to patients with performance status or= 2) was associated with reduced survival (hazard ratio: 3.29, 95% confidence interval: 1.75-6.17). Overall 2-years and 5-years survival were 20% and 11% for the patients treated intensively. From this study it appears that an individualized approach of treatment with intensive chemotherapy for selected patients offers a substantial CR rate and an improvement in survival. This analysis also suggests that differences in outcome between single institutions can be explained mainly by referral and selection biases

Surface grafting onto template-assembled synthetic protein scaffolds in molecular recognition.

Creating functional biological molecules de novo requires a detailed understanding of the intimate relationship between primary sequence, folding mechanism, and packing topology, and remains up to now a most challenging goal in protein design and mimicry. As a consequence, the use of well-defined robust macromolecules as scaffolds for the introduction of function by grafting surface residues has become a major objective in protein engineering and de novo design. In this article, the concept of scaffolds is demonstrated on some selected examples, illustrating that novel types of functional molecules can be generated. Reengineered proteins and, most notably, de novo designed peptide scaffolds exhibiting molecular function, are ideal tools for structure-function studies and as leads in drug design.

Dose-finding study of valspodar (PSC 833) with daunorubicin and cytarabine to reverse multidrug resistance in elderly patients with previously untreated acute myeloid leukemia.

INTRODUCTION: This trial was designed to determine the maximum tolerated dose of intravenous daunorubicin (DNR) in combination with valspodar and to test the feasibility of P-glycoprotein modulation using valspodar in elderly patients with previously untreated acute myelogenous leukemia receiving standard induction chemotherapy. METHODS: Patients > or =60 years of age with previously untreated AML received valspodar (10 mg/kg/24 h by continuous intravenous infusion [CIV] on days 1-4 with a 2-mg/kg loading dose on day 1) in conjunction with two cycles of induction chemotherapy consisting of cytarabine (200 mg/m(2) CIV on days 1-7), and DNR (35 mg/m(2) [cohort 1] or 45 mg/m(2) [cohort 2] on days 1-3, intravenous bolus). Patients were assessed for dose-limiting toxicities (DLT), response rate, event-free and overall survival, and pharmacokinetics of valspodar and DNR. RESULTS: Valspodar was well tolerated at the lower DNR dose level (ie, 35 mg/m(2)) resulting in a 21% rate of DLT and only three toxic deaths. Treatment-related mortality was unacceptably high at the 45 mg/m(2) DNR dose level. The complete response rate was 49% overall and similar in both cohorts. The median overall survival of patients was 333 days in cohort 1 compared to 98 days in cohort 2. At baseline, 70% of assessable patients were P-glycoprotein positive. CONCLUSION: Substantial inhibition of P-glycoprotein activity can be achieved in this patient population at clinically tolerable doses of valspodar and DNR. The maximum tolerated dose of DNR was established as 35 mg/m(2). This regimen is being further evaluated in phase III trials.

Intensive treatment strategies in multiple myeloma.

Intensive therapy regimens, followed by autologous (ABMT) or allogeneic bone marrow transplantation, have improved the prognosis of multiple myeloma (MM) over the last decade. However, although these regimens increase the overall response rate and survival, they are not curative, since most patients relapse after 1.5 to 3 years. The most active drug in high-dose therapy of MM is melphalan at doses of 140 mg/m2 or higher. Allogeneic bone marrow transplantation appears to be more effective than ABMT, but is not applicable in the majority of patients and is complicated by a very high toxicity rate. New clinical strategies aimed at improving the results of ABMT have been tested in phase I-II trials. The use of peripheral blood stem cell (PBSC) transplantations has allowed a reduction in the toxicity of high-dose regimens, but has not led to an increase in the overall response rate or survival. Likewise, the application of double transplants appears to improve the response rate in some patients, but has not resulted in an increase in survival. Studies testing the role of positive- or negative- selection procedures to deplete the graft of contaminating myeloma cells are still in early phases. Whether these novel clinical approaches will improve the response to high-dose therapy in MM will be defined by several ongoing phase III studies.

D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by >/=5 microM thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 microM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.

Thrombin-induced GPIb-IX centralization on the platelet surface requires actin assembly and myosin II activation.

In resting platelets, the GPIb-IX complex, the receptor for the von Willebrand factor (vWF), is linked to underlying actin filaments by actin-binding protein (ABP-280). Thrombin stimulation of human platelets leads to a decrease in the surface expression of the GPIb-IX complex, which is redistributed from the platelet surface into the open canalicular system (OCS). Because the centralization of GPIb-IX is inhibited by cytochalasin, it is believed to be linked to actin cytoskeletal rearrangements that take place during platelet activation. We have further characterized the mechanism of GPIb-IX centralization in platelets in suspension. Following thrombin stimulation, GPIb-IX shifts from the membrane skeleton of the resting cell to the cytoskeleton of the activated cell in a reaction sensitive to cytochalasin B. The cytoskeletal association of GPIb-IX involves ABP-280, as it correlates with the incorporation of ABP-280 into the activated cytoskeleton and because no dissociation of the ABP-280/GPIb-IX complexes is detected after thrombin activation. However, the incorporation of GPIb-IX into the cytoskeleton is complete within 1 minute, whereas GPIb-IX centralization requires 5 to 10 minutes for completion. The movement of GPIb-IX to the cytoskeleton of activated platelets is therefore necessary, but not sufficient for GPIb-IX centralization. Blockage of cytosolic calcium increases induced by thrombin by loading with the cell permeant calcium chelator Quin-2 AM inhibited GPIb-IX centralization by 70%, but did not prevent its association with the activated cytoskeleton. Quin-2 loading did, however, decrease the incorporation of myosin II into the activated cytoskeleton. The role of myosin II was further probed using the myosin light chain kinase (MLCK) inhibitor wortmannin. Wortmannin prevents myosin II association to the activated cytoskeleton and inhibits GPIb-IX centralization by 50%, without affecting actin assembly or the association of GPIb-IX to the cytoskeleton. Only micromolar concentrations of wortmannin, high enough to inhibit MLCK, prevent GPIb-IX centralization. These results indicate that thrombin-induced GPIb-IX centralization requires a minimum of two steps, one associating GPIb-IX to the activated cytoskeleton and the second requiring myosin II activation. The involvement of myosin II implies that GPIb-IX/ABP-280 complexes, linked to actin filaments, are pulled into the cell center, and that platelets may exert contractile tension on vWF bound to its receptor.

Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation.

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.