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In this study, we used a common procedure to assess the potential of mobilized peripheral blood (MPB) and umbilical cord blood (UCB) as sources of mesenchymal stem cells (MSCs) in comparison with bone marrow (BM). We tested three methods: plastic adhesion supplemented with 5% of BM‐MSC conditioned medium, unsupplemented plastic adhesion, and selection of CD133‐positive cells. MSCs derived from MPB or UCB are identified by their positive expression of mesenchymal (SH2, SH3) and negative expression of hematopoietic markers (CD14, CD34, CD45, HLA‐DR). We observed that the CD133‐positive cell fraction contains more MSCs with high proliferative potential. Placed in appropriate conditions, these cells proved their capacity to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal/glial cells. MPB‐ and UCB‐MSCs express Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity. The selection of CD133‐positive cells enabled us to obtain a homogeneous population of MSCs from UCB and MPB. These sources may have a major clinical importance thanks to their easy accessibility.

The effect of 3 anthracene derivatives, mitoxantrone, ametantrone, bisantrene, on 4 normal human bone marrows, was studied using the myeloid stem cell assay developed by Pike and Robinson, in order to define to what extent this test could be used to predict the relative clinical hematologic toxicity of new anticancer agents. For the 3 drugs, an exponential relationship between colony survival and drug concentration was found, but was much steeper for mitoxantrone (slope = -195.2 ± 8.8/µg/ml) than for ametantrone (slope = 5.1 ± 1.0/µg/ml, p?0.001) and bisantrene (slope = 7.1 ± 0.3/µg/ml, p?0.001). The difference of slope between ametantrone and bisantrene was of borderline significance (p?0.05). The ratios of concentrations inducing a 50% growth inhibition for mitoxantrone versus bisantrene and for ametantrone versus bisantrene were close to the corresponding ratios of concentrations inducing a 90% growth inhibition. The relative in vitro toxicities reproduce very well the relative myelosuppression observed in clinical trials with mitoxantrone versus bisantrene but the results were less satisfactory for the comparison of these 2 agents with ametantrone. In addition, our data suggest that, for these 3 compounds, intrinsic myeloid progenitor sensitivity is a major determinant of leukopenia.

We studied the production of cytokines (G-CSF, GM-CSF, IL-6, LIF and IL-10) by bone marrow stromal cells of five untreated patients with B-CLL, in Rai stage 0,1 and II, and of 8 healthy subjects. The production of G-CSF, GM-CSF, LIF and IL-10 did not differ significantly between controls and B-CLL patients. However, the ability of stromal cells to release IL-6 in response to LPS was decreased in all patients: 36 5 ng/ml versus 123 47 ng/ml for normal controls (P < 0.004). Moreover, a soluble activity that inhibited hematopoietic colony formation was detected in B-CLL stromal cell conditioned media. Some potential inhibitors were envisaged and the results indicated an increased production of TGF-P by B-CLL stromal cells compared to normal stromal cells (respectively 53 10 versus 15 4 ng/ml, P < 0.03). The reduced capacity of B-CLL stromal cells to produce IL-6 was associated with this excessive release of TGF-bT; indeed, addition of anti-TGF-bT neutralizing antibody to B-CLL stromal cells, before LPS stimulation, totally normalized the production of IL-6.TGF-bT and IL-6 were also measured in serum samples from normal subjects and B-CLL patients. No significant difference was seen in the production of total TGF-bT (bioactive and latent forms) between normal and B-CLL sera but the mean level of bioactive protein in B-CLL sera was increased in comparison with normal sera (1.74 0.44 versus 0.67 0.2 ng/ml, P < 0.04). Furthermore, low serum levels of IL-6 were found in normal and B-CLL sera but detectable levels of IL-6 were less frequent in B-CLL sera. In conclusion, in B-CLL, decreased IL-6 production by stromal cells and inhibiting activity on hematopoietic progenitors are attributable to increased production of TGF-bT. This increased amount of TGF-bT is also seen in sera of B-CLL patients. Our report supports the hypothesis that overproduction of TGF-bT could play a pathogenic role in some manifestations of this disease such as bone marrow failure, deficiency in platelet and Ig production.

No relevant conflicts of interest to declare.

Abstract 3435

Background: MSC are pluripotent cells capable of differentiation into various mesenchymal tissues. These cells can also reconstitute the bone marrow stroma, source of several hematopoietic growth factors. It has been reported that infusion of MSC can enhance hematopoietic recovery. For these reasons, we elaborate a phase II pilot study of MSC infusion in patients (pts) with poor hematopoietic recovery after allogeneic transplantation. Objectives: to evaluate the safety of infusion of ex vivo amplified MSC after allogeneic transplantation. Secondly, to assess the improvement of hematopoietic recovery after MSC infusion in pts with poor engraftment at the day 30 or more after an allogeneic HSCT after informed consent. Methods: MSC were amplified from the bone marrow (BM)-mononuclear cells of the HSC donors. MSC expansion was made in a commercial serum-free medium (UltraCulture, Cambrex, Walkersville, MD) supplemented with a serum substitute (Ultroser, Pall Biosepra, Cergy-Saint-Christophe, France) as previously reported by our group (Eur J Haematol, 2006). Ex vivo expanded allogeneic MSC were intravenously infused at the dose of 1×106 MSC/kg of recipient body weight. Results: six patients (pts) were enrolled in this study. Three were fully related and 3 had haploidentical sibling donors. Median time from transplantation to MSC infusion was 112 (92–186). In two of these pts we observed a normalisation of the platelet level, the white blood cell count and an increase in the reticulocytosis 16 and 21days after MSC infusions. Four were unevaluable due to many interfering patches. Pts were closely monitored during the MSC infusion and over several hours. No immediate side effects were observed. Patients were monitored by PCR for viral reactivation. In one pt the PCR CMV became positive on the day 12 of the MSC infusion. This pt had an haploidentical HSCT (CMV+/+) and at the time of infusion had no GVHD, no corticoid treatment or previous CMV reactivation. A preemptive treatment with gancyclovir was given. However 15 days after the start of the treatment, we observed an increase of the level of the PCR, treatment was switched for foscavir and the PCR became negative. At the day 53 positive EBV PCR was efficiently treated by four injections of rituximab. 77 and 102 days after MSC infusion the pt needed new preemptive treatments with foscavir for new CMV reactivation. Unfortunately the pt developed encephalitis and died from CNS CMV infection. No other complications linked to the MSC infusion were observed in this pilot study. Conclusions: we did not observed immediate side effects linked to the infusion of ex vivo expanded allogeneic MSC. However one of our pts died from CMV infection. It is difficult to assess that the immunosuppressive effects of MSC and not those of the transplantation were responsible of this fatal infection. Nevertheless future studies have to closely monitor infectious complications and pts with active infection or viral reactivation should not receive MSC infusion.

The purpose of our study was to evaluate the capacities of cord blood (CB) CD34+ cells to proliferate and differentiate ex vivo into myeloid lineage in response to cytokines and to compare them with mobilized peripheral blood (MPB) cells. Briefly, 2.5 × 104 CD34+ cells, isolated from CB (n=10) and MPB (n= 9), were cultured in 5 ml MacoBiotech HP01 (Macopharma) with SCF, Flt3-L, IL-3 and G-CSF. At day 9, 106 cultured cells were replated for 5 additional days. Cells were counted and evaluated for their CD34, CD13 and CD15 expression. Differentiation into myeloid compartment was assessed by CD11b and CD16 coexpression on CD15+ cells. We observed that leucocyte expansion was significantly higher in CB than in MPB at day 9 (24.3±3.8 vs 15.2±1.9) and at day 14 (224.7±54.2 vs 72.9±20.0). A similar difference was observed for CD34+ cell expansion (8.7±1.4 vs 3.4±0.5 at day 9 and 31.3±4.6 vs 7.6±2.4 at day 14). at day 9, despite superior CB leucocyte expansion, CD13+ and CD15+ cell number produced per CD34+ cell seeded at day 0 were similar in CB and in MPB (18.5±2.4 vs 14.4±2.5 for CD13+ and 7.1±1.3 vs 6.0±1.2 for CD15+). Increasing the culture period led to higher numbers of CD13+ and CD15+ cells in CB than in MPB. This increase was due to a total leucocyte expansion rather than to high CD13+ and CD15+ cell percentage. The distribution of CD11b−CD16−, CD11b+CD16− and CD11b+CD16+ subpopulations in CD15+ cells was comparable in CB and in MPB after 9 days of culture, with a majority of relatively immature CD11b−CD16− myeloid progenitor cells. However, after 5 additionnal days of culture, MPB CD15+ cells expressed a more mature phenotype than CB CD15+ cells, with a dramatic increase of CD11b+CD16− cells (promyelocytes and myelocytes). In conclusion, our study suggests that, despite the high CB cell capacity of expansion in our culture conditions, CB CD34+ cell differentiation process into myeloid lineage appears to be slower. This difficulty of CB cells to reach maturation in vitro is likely to be related with the longer delay of neutrophil recovery after CB transplantation.

Background : Recent studies have demonstrated the transmigration capacity of mesenchymal stem cells (MSC) over the endothelial cell barrier into various tissues including brain, bone marrow and many others. Nevertheless, little is know about the agents that promote and control their migration. Methods : The MSC (5.105 cells) migration potential was evaluated using matrigel invasion chamber assay in the presence/absence of SDF-1 (100ng/mL), PDGFbb (100ng/mL), IGF (500ng/mL), IL-6 (100ng/mL) and 10% of conditioned medium in the bottom chamber. The chemotactic properties of these cytokines/chemokines were compared to the spontaneous migration of MSC in DMEM medium without FCS. After 24 hours, cells that migrated through the membrane were counted. The presence of specific receptors was analysed using Raybio Assay, Western Blot and semi-quantitative RT-PCR. Results : We observed that PDGFbb has the highest chemoattractive property on the MSC and correlates with the strongest expression of PDGF-bb receptor. We also noted that the migratory response to IL-6, IGF and SDF-1 is correlated with the expression of their respective receptors. However, a weak migration was observed in response to SDF-1 due to the low extracellular expression of CXCR4. Nevertheless, a high intracellular expression of CXCR4 was demonstrated with western blots and FACS analysis. In addition, using Raybio assay, we demonstrated the expression of various receptors and metalloproteinases involved in the migration of MSC including ALCAM, L-selectin, ICAM-1, MMP2 and MMP9. Conclusions : Our observations suggest that chemokines or cytokines involved in the regulation of immunity or the inflammatory process promotes the migration of MSC into bone marrow or damaged tissues.

Background: Patients (pts) with MLD have neurological and musculo-skeletal defects with a limited survival. HSCT has been reported as an effective treatment to stabilize or improve defects associated with this disease. Koç and colleagues reported that donor allogeneic MSC infusion is safe and may be associated with reversal of the disease pathophysiology in some tissues (Bone Marrow transplant, 2002). We decide thus to perform a non myeloablative familial HSCT in adult pt with a symptomatic MLD in order to evaluate the safety and the benefit allogeneic HSCT with MSC infusion in a patient with MLD. An informed consent was obtained from the pt. Patient: a 23 years old women who presented an adult form of MLD for three years was admitted in our department. The most important symptoms associated with the disease were dizziness, proximal weakness of the lower limbs, difficulty to walk, disorder of the memory and urinary incontinence. The reduced intensity conditioning was preferred to decrease the morbidity and mortality of the procedure. MSC were isolated and amplified from the BM-mononuclear cells of the HSC donor. MSC expansion was made in a commercial serum-free medium (UltraCulture, Cambrex, Walkersville, MD) supplemented with a serum substitute (Ultroser, Pall Biosepra, Cergy-Saint-Christophe, France) as previously reported by our group (Eur J Haematol, 2006). Ex vivo expanded allogeneic MSC were intravenously infused at the dose of 1×106 MSC/kg of recipient body weight. The conditioning regimen of the HSCT consisted in Fludarabine and ATG combination and 5× 106 CD34 cells were infused concomitantly with 1.106 MSC cells. We did not observed any immediate or delayed side effects after the MSC infusion. The patient did not presented any complications after the HSCT. At day 8 of the transplantation she had an normal hematological recovery. The platelet nadir was 72.000/mm3 and she did not need any transfusion. With regards of her neurological status, since 22 months the patient had no new deterioration and we observed a stabilisation of the clinical manifestations. Conclusions: This case report suggests the feasibility and the potential efficacy of reduced intensity conditioning allogeneic HSCT with MSC infusion for patient with MLD. A larger trial is required to confirm this observation.

Background:The anti-leukemic in vitro activity of valproic acid (VPA), a commonly used antiepileptic agent, was tested on lymphocytes derived from 40 patients with chronic lymphocytic leukemia (CLL) (Binet stage A=34, B=3, C=3). These patients had not been previously treated or remained untreated for the previous 6 months. Combined analysis of ZAP-70, CD38 and IgVHmutational status was performed for each patient.

Background : Recent studies have demonstrated the transmigration capacity of mesenchymal stem cells (MSC) over the endothelial cell barrier into various tissues including brain, bone marrow and many others. Nevertheless, little is know about the agents that promote and control their migration.

Background: The anti-leukemic in vitro activity of valproic acid (VPA), a commonly used antiepileptic agent, was tested on lymphocytes derived from 40 patients with chronic lymphocytic leukemia (CLL) (Binet stage A=34, B=3, C=3). These patients had not been previously treated or remained untreated for the previous 6 months. Combined analysis of ZAP-70, CD38 and IgVH mutational status was performed for each patient. Methods: Mononuclear cells were incubated with VPA at 1, 5 and 10 mM for 24 hours. Cell viability was assessed by trypan blue exclusion assay, apoptosis by annexin V/propidium iodide(PI) labelling and PI staining after cell permeabilisation. Caspase activation was studied by flow cytometry analysis after cell treatment with selective caspase inhibitors. Results: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in all CLL patients tested. VPA-treatment induced apoptotic changes in CLL cells including phosphatidylserine (PS) externalisation and DNA fragmentation. The mean apoptotic rate was similar between IgVH mutated and unmutated patients or ZAP-70+/ZAP-70- cases. VPA induced apoptosis by the extrinsic pathway involving engagement of the caspase-8 dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA increased significantly the sensitivity of leukemic cells to TRAIL (tumor necrosis factor a-related apoptosis-inducing ligand). In addition, VPA overcomed the prosurvival effects of bone marrow stromal cells. Conclusions: These data indicate that VPA, at the pharmacological concentration of 1 mM, is a potent inducer of apoptosis in CLL and should be further explored as a single agent. Also the combination of VPA and TRAIL may be a promising approach in the treatment of CLL.

Background: Patients (pts) with MLD have neurological and musculo-skeletal defects with a limited survival. HSCT has been reported as an effective treatment to stabilize or improve defects associated with this disease. Koç and colleagues reported that donor allogeneic MSC infusion is safe and may be associated with reversal of the disease pathophysiology in some tissues (Bone Marrow transplant, 2002). We decide thus to perform a non myeloablative familial HSCT in adult pt with a symptomatic MLD in order to evaluate the safety and the benefit allogeneic HSCT with MSC infusion in a patient with MLD. An informed consent was obtained from the pt.

The purpose of our study was to evaluate the capacities of cord blood (CB) CD34+cells to proliferate and differentiate ex vivo into myeloid lineage in response to cytokines and to compare them with mobilized peripheral blood (MPB) cells. Briefly, 2.5 × 104CD34+cells, isolated from CB (n=10) and MPB (n= 9), were cultured in 5 ml MacoBiotech HP01 (Macopharma) with SCF, Flt3-L, IL-3 and G-CSF. At day 9, 106cultured cells were replated for 5 additional days. Cells were counted and evaluated for their CD34, CD13 and CD15 expression. Differentiation into myeloid compartment was assessed by CD11b and CD16 coexpression on CD15+cells. We observed that

Background: MSC are pluripotent cells capable of differentiation into various mesenchymal tissues. These cells can also reconstitute the bone marrow stroma, source of several hematopoietic growth factors. It has been reported that infusion of MSC can enhance hematopoietic recovery. For these reasons, we elaborate a phase II pilot study of MSC infusion in patients (pts) with poor hematopoietic recovery after allogeneic transplantation.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation of CD5+ B cells with significant resistance to apoptosis and therefore prolonged survival. CLL remains an incurable disease requiring innovative new approaches to improve overall patient outcome. OBJECTIVE: Histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) have shown antitumoral activity at micromolar concentrations in a variety of human cancers in vitro and in vivo. These different studies suggested extrinsic, intrinsic and caspase-independent apoptosis as relevant death pathway depending upon cell types. RESULTS: In this study, we examined the effects of SAHA on CLL cells in vitro. SAHA induced apoptosis in a dose-dependent manner in all (n=25) CLL samples tested, including previously untreated and chemo-resistant CLL patients. The level of apoptosis, as measured by annexin binding to exposed PS residues, increased after 48 hours of SAHA treatment and was significant in the treated cells at concentrations of 10 and 20 μM (respectively 45±5 and 52±4% of apoptotic cells versus 29.5±4 for untreated cells, p<0.0001). The pre-treatment of cells with the pan-caspase inhibitor Z-VAD before SAHA-treatment had no effect on PS externalization but inhibited DNA degradation demonstrating that caspases are critical for inducing DNA fragmentation. Using specific caspase inhibitors (DEVD, VEID and IETD) we demonstrated the participation of caspases-3, -6 and -8 in cell apoptosis. In addition, inhibition of the initiator caspase in the intrinsic/mitochondrial pathway, caspase-9, did not influence cell apoptosis. Thus, extrinsic pathway seems activated during SAHA-induced apoptosis. We have next investigated the expression of FAS and TRAIL-R1 (DR4) by CLL cells after SAHA treatment. Only a small proportion of CLL cells displayed detectable expression of CD95 (12.5±2% CD19+CD95+). 24h treatment with SAHA resulted in increase in FAS expression compared to control (33±5.6%, p<0.02). However, a FAS-blocking antibody (ZB4) did not inhibit SAHA-induced apoptosis arguing against a role of FAS/FAS-L signaling pathway in the induction of apoptosis by SAHA. The expression of TRAIL-R1 was very low and not upregulated by SAHA treatment. To explore the mechanism by which SAHA triggers the extrinsic pathway in CLL cells, the effects of SAHA on the level of various apoptosis-regulatory proteins (FLIP, FADD…) are now evaluated. CONCLUSIONS: SAHA induces apoptosis in CLL cells via the extrinsic pathway involving caspase-8 activation. Since the majority of cytotoxic agents operate via the intrinsic pathway and since defects in the mitochondrial pathway exist in chemoresistant CLL patients, it is important to identify agents that exert their cytotoxic effect via the extrinsic pathway. Moreover, the combination of SAHA with conventional drugs could be of therapeutic effect in CLL.

Haplo-identical transplant is now established as a procedure of choice for patients who lack a compatible donor. It might even be the best choice for AML, provided there is a GvH NK allo-reactivity. However, patients are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity: : G-CSF + DLI, : GM-CSF + DLI, : patient and disease adapted strategy. Thirty-six consecutive leukemia patients, aged 18–55, were investigated (20 very poor risk, 12 poor risk and 4 better risk). GvH type NK alloreactivity was chosen when possible (21/36) and balanced across the 3 groups. In the first 9 patients, G-CSF was used pot-transplant and prophylactic DLI were given at month 1, 2 and 3. The use of G-CSF and 1 to 3 DLI (10exp4 CD3/kg) was found safe. It resulted in faster CD4 recovery and a low rate of infections. However, it was insufficient to induce a protective GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI (104 CD3/kg) at day 30 unless aGVHD (3 pts). The comparison between the 2 first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD:0. GM-CSF + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD grade 2 or more: 9/12; price to pay: GVHD resulting in 5 deaths in total. Median time to relapse in the 21 first patients was 6 months range (4 – 9). Step 3 (17 patients) consists of a patient adapted strategy: no more aspecific DLI (selected anti-CMV and aspergillus DLI planned in all patients); in myeloid disorders with NK allo-reactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mcg) is given from day 5 to day 9. The follow-up of patients alive in CCR (12), although promising (3 relapses), is currently short (median 8 months), compared to the median of relapse in the 2 first groups (6 months). Overall, TRM at day 100 is 2/29, reflecting the good tolerance of the conditioning in a heavily pre-treated population (median age: 43). Overall relapse rate for all patients treated with GM-CSF, without the benefit of NK-alloreactivity, is 6/16 (median FU: 15 months). We conclude that the third strategy might improve the outcome and the relapse rate without exposing patients to unnecessary severe GVHD. These data will be updated with 6 months more follow-up and 3 more patients.

Background: Chronic lymphocytic leukemia (CLL) is a disease with unpredictable natural history in stage A patients (pts). Recently, It has been demonstrated that ZAP-70 and IgVH mutational status are powerful biological prognostic factors and we previously reported that the doubling time of soluble CD23 (sCD23DT) is a prognostic factor for progression of disease in untreated stage A CLL. Therefore we prospectively evaluated ZAP-70 protein expression and IgVH mutational status in untreated stage A B-CLL pts and correlated these markers with sCD23DT. Methods: sCD23 level was evaluated by commercially available enzyme-linked immunosorbent assay (ELISA). ZAP-70 expression is determined in leukemic cells by flow cytometry, and positive expression was defined as >20% positive cells. When tests became available, ZAP-70 gene expression and IgVH mutational status were also evaluated in all pts included in the sCD23 study. Results: 47 pts with untreated stage A B-CLL are evaluable for the sCD23DT. ZAP-70 and mutational status were evaluated in 34pts. Median age was 62 (42–82) years. The median follow-up was 58 (16–129) months. The sCD23DT was clearly associated with progression to upper stages requiring a treatment: median time to progression was 25 Mos in pts with sCD23 DT inferior to one year comparing to 141 months in pts with sCD23 DT superior to one year (p<0.0001). Time to progression in the group of pts with positive ZAP-70 expression was 19.1 mos comparing to 86.1 mos in ZAP-70 negative group (p=0.0001) Time to progression to treatment for pts with unmutated IgVH was 15,9 mos and 86,1 mos for pts with mutated status (p<0.001). In the group of pts with ZAP-70 positive expression 6pts (67%) needed a treatment. Among ZAP-70+ and sCD23DT< 1 year pts, 100% (6/6) required a treatment. For pts with positive ZAP-70 and a sCD23DT superior to one year, 100% (3/3) of pts do not need a treatment (p=0.0188). In the group of good prognosis pts with negative ZAP-70, 24% (6/25) of the pts required a treatment, median time to progression for these pts was 66,6 mos. in the ZAP-70 negative pts population, 3/25 (12%) have a sCD23DT<1 year and 2 of these pts required a treatment for disease progression. Among the pts with indolent progression, negative ZAP-70 and who do not need a treatment, 96 % of the pts have a sCD23DT superior to one year. In the group of pts with unmutated status (n=8), 88% of the pts needed a treatment and all the pts had a sCD23DT inferior to one year, the only pts who did not required a treatment had a sCD23DT superior to one year. Among patients with mutated IgVH 73% did not require a treatment and 95% of those pts had a sCD23DT superior to one year. Among the mutated pts who progressed to treatment 42% had a sCD23DT inferior to one year, the 58% of treated pts with sCD23DT superior to one year and unmutated status had a median time to progression of 68 mos. In conclusion, CLL patients can be divided into different risk categories according to sCD23 serial determinations. sCD23DT is an additional important prognostic marker for disease progression even in ZAP-70 positive patients.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation of CD5+ B cells with significant resistance to apoptosis and therefore prolonged survival. CLL remains an incurable disease requiring innovative new approaches to improve overall patient outcome.

Haplo-identical transplant is now established as a procedure of choice for patients who lack a compatible donor. It might even be the best choice for AML, provided there is a GvH NK allo-reactivity. However, patients are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity:

Background: Chronic lymphocytic leukemia (CLL) is a disease with unpredictable natural history in stage A patients (pts). Recently, It has been demonstrated that ZAP-70 and IgVH mutational status are powerful biological prognostic factors and we previously reported that the doubling time of soluble CD23 (sCD23DT) is a prognostic factor for progression of disease in untreated stage A CLL. Therefore we prospectively evaluated ZAP-70 protein expression and IgVH mutational status in untreated stage A B-CLL pts and correlated these markers with sCD23DT.

BACKGROUND: Bone Marrow mesenchymal stem cells (MSC) are pluripotent cells that have the capacity to differentiate into several tissue lineages and also the ability to support hematopoiesis. Their use in gene and cell therapy requires their in vitro expansion. Maintenance and proliferation of MSC are strongly dependent on the culture conditions such as properties of the bovine serum added in the culture medium. However, duration and culture conditions are critical for the successful clinical use of MSC. OBJECTIVE: We have evaluated the efficiency of a commercial serum-free medium (UltraCulture, Bio-Whittaker, Walkersville, MD) supplemented with a serum substitute (Ultroser, BioSepra, Cergy-Saint-Christophe, France) in order to work without FBS and to have a more constant composition. This medium (UC) was compared to the classical medium a-MEM containing 10% FBS (Invitrogen, Merelbeke, Belgium). METHODS: BM-mononuclear cells collected from 11 healthy donors were plated in petri dishes at a concentration of 105/cm2. After 3 days, non adherent cells were removed and culture media were added to adherent cells which were maintained at 37°C until they reached confluence. MSC expansion was analysed after the primoculture (PM) and after the first passage (P1). CFU-F (colony forming units-fibroblastic) number, phenotypic analysis and differentiation potential were also evaluated. RESULTS::The mean culture duration was 13±2 and 8±2 days respectively for PM and P1 but the confluence was reached more rapidly for cells cultured in UC. After PM, 0.35x106 and 1.21x106 cells were obtained for MEM and UC respectively (p<0.005). Moreover, around 20% of cells cultured in MEM were CD45+ while the level of CD45+ cells was frequently < 5% in UC indicating that UC medium favored the rapid elimination of hematopoietic cells. After P1, the expansion rate was significantly higher in UC than MEM: respectively 5.13 ± 1.2 and 22.6 ± 3 (p<0.0005). The numbers of CFU-F were always higher in UC demonstrating the enhanced proliferation in serum-free medium. The phenotype of MSC was similar in the both media: SH2+, SH3+, CD44+, CD45−, CD34−, HLA-DR−. More importantly, these cells remained able to differentiate into osteocytes, chondrocytes, adipocytes and neuron-like cells in the both media confirming their multipotentiality. CONCLUSIONS: Our data strongly support UltraCulture medium supplemented with a serum substitute as an optimal medium for MSC culture. Indeed, it allows a better cell expansion, better proliferation and preserves multipotentiality. This medium, reducing culture period and containing low concentration of serum substitute, is of major interest for clinical production of MSC.

Background: Mesenchymal Stem Cells (MSC) can be isolated from bone marrow, adipose and fetal tissues, but their presence in Mobilized Peripheral Blood (MPB) and in Umbilical Cord Blood (UCB) remains controversial. Methods: In this study we evaluated whether MPB (n=6) and UCB (n=8) could be two other sources of MSC beside Bone Marrow (BM). CD133 positive cell fraction was isolated through immunomagnetic system and MNC were seeded in medium supplemented or not with 5% of conditioned medium durong the first 48 hours of adhesion. MSC derived from MPB or UCB were identified by their expression of mesenchymal (SH2, SH3) and hematopoietic markers (CD14, CD34, CD45 and CD62-E). MPB and CB-MSC were also tested for their capacity to generate CFU-F (Fibroblast colony-forming units) and to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells after specific induction. Finally we tested through RT-PCR the gene expression of Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity. Results: Through CFU-F, we observed that the selection of CD133 positive cells allows to obtain a great amount of MSC in comparison with the MNC fraction seeded (MPB-MSC, 139 versus 72 and UCB-MSC 165 versus 8 after the primoculture). After four passages, more than 1.108 cells were obtained by the cell expansion assays from 1.106 cells seeded in primoculture. During the first culture, whatever the medium used or cell fraction seeded, the cell population was composed of osteoblasts, osteoclasts, marcophages and hematopoietic cells with less than 5%SH3 positive cells. After two passages, cells derived from MPB or UCB expressed mesenchymal markers (SH3 and SH2) and Oct4. Under appropriate conditions these cells were able to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells. Conclusion: These results indicate that MSC are present in MPB and UCB, with similar characteristics to bone marrow (self-renewal capacity and multi-diffentiation potential), and may have a major clinical importance due to their accessibility. They could be an attractive target for cellular and gene therapies Mesenchymal marker expression (%) T0 (%) second passage (%) MPB SH2 2,7±1,1 96,1±2,9 SH3 4,9±1,5 95,7±3,3 UCB SH2 1,4±0,6 98±0,7 SH3 5,2±1,8 96,5±2,5

Haplo-identical transplant is now established as a procedure of choice for patients who lack a compatible donor. It might even be the best choice for AML, provided there is a GvH NK alloreactivity. However, patients are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity: (1): G-CSF + DLI, (2): GM-CSF + DLI, (3): patient and disease adapted strategy. Thirty-four consecutive leukemia patients, aged 18–55, were investigated (20 very poor risk, 11 poor risk and 3 better risk). GvH type NK alloreactivity was chosen when possible (19/34) and balanced across the 3 groups. In the first 9 patients, G-CSF was used pot-transplant and prophylactic DLI were given at month 1, 2 and 3. The use of G-CSF and 1 to 3 DLI (104 CD3/kg) was found safe. It resulted in faster CD4 recovery and a low rate of infections. However, it was insufficient to induce a protective GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI (104 CD3/kg) at day 30 unless aGVHD (3 pts). The comparison between the 2 first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD:0. GM-CSF + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD grade 2 or more: 9/12; price to pay: GVHD resulting in 5 deaths in total. Median time to relapse in the 21 first patients was 6 months (range 4 to 9). Step 3 (14 patients) consists of a patient adapted strategy: no more aspecific DLI (selected anti-CMV and aspergillus DLI planned in all patients); in myeloid disorders with NK alloreactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mcg) is given from day 5 to day 9. The follow-up of these 14 patients, although promising (2 relapses), is currently short (median 5 months), compared to the median of relapse in the 2 first groups (6 months) but will be updated for the meeting (10 months and 17 patients). Overall, TRM at day 100 is 3/29, reflecting the good tolerance of the conditioning in a heavily pre-treated population (median age : 43). Overall NR-mortality at one year is 8/26, but was greater in the GM-CSF + DLI group, reflecting the impact of severe aGVHD. In the current group, its is 2/14 at 3 months. We conclude that the third strategy might improve the outcome and the relapse rate without exposing patients to unnecessary severe GVHD.

Background: Additional infusion of ex vivo differentiated myeloid progenitor may reduce or abrogate severe neutropenia following high-dose chemotherapy and peripheral blood (PB) transplantation. Normal HPC as well as HPC from patients in clinical remission can be used as source of cells. The aims of our study were to compare the ex vivo expansion of myeloid progenitor cells starting from pretreated patients (PP) and normal donors (ND) and to evaluate if the cells collected from poor mobilizers -for which the ex vivo expansion is particularly interesting- present similar ex vivo expansion capacities than cells from good mobilizers. Methods: In this study, 11 normal donors (ND) and 17 patients (PP) were evaluated (5 NHL, 2 HK, 3 MM, 3 lymphoma, 1AML, 1 CLL and 2 solid tumor). The CD34+ cells were plated for 7 days in serum-free medium with SCF, IL-3, G-CSF and FLT3- ligand. The expression of CD34, CD13, CD15 and CD16 Ags and the presence of primary (myeloperoxydase, MPO) and secondary granules (lactoferrin, LF) were used to evaluate maturation into myeloid lineage. Functional capacities of the ex vivo generated cells were also analysed using oxydative burst activity and clonogenic capacities (CFU-GM). Results: (1) the proliferation capacities of total leucocytes were higher in ND than in PP (28.8±5.6 vs 17.2±4.0 fold, p<0.04) with a comparable CD34+ cell expansion (8.7±3.0 vs 7.8±2.7 fold, NS); (2) in patients, 65.3±5.8% of the expanded cells were CD13+ and 23.2±4.1% remained CD34+ with 7.8±2.5 % of immature CD34+CD13− cells; these results were similar to that observed in ND (72.7±7.5% CD13+, 23.0±5.3% CD34+, 9.0±3.2% CD34+CD13−); (3) the number of myeloid progenitors produced per seeded CD34+ cell consisted of 19.0±3.7 CD13+, 11.8±3.7 CD15+, 10.2±3.2 CD16+ and 15.4±4.4 MPO+/LF+cells in PP and 25.3±5.4 CD13+, 12.5±3.8 CD15+, 7.6±2.1 CD16+ and 21.7±7.2 MPO+/LF+ cells in ND (NS); (4) these cells, essentially promyelocytes and metamyelocytes, were able to proliferate in clonogenic assay and presented oxydative burst activity; (5) we failed to observe a correlation between the cell expansions (total leucocytes, CD34+ cells and myeloid progenitor cells) and the diagnosis; (6) no correlation was observed between the mobilization of CD34+ cells into PB and the capacities of cells to be expanded in vitro. Conclusions: in our culture system, the generation of cells committed to the myeloid lineage from CD34+ cells is feasible from ND as well as from PP, whatever is the diagnosis. The ex vivo expanded cell population consisted of cells at all stages of differentiation including immature hematopoietic progenitors. The mobilisation rate doesn’t correlate with the proliferative capacities of cells, suggesting that ex vivo expansion can be performed even in subjects with poor mobilization.

Haplo-identical transplant is now established as a procedure of choice for patients who lack a compatible donor. It might even be the best choice for AML, provided there is a GvH NK alloreactivity. However, patients are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity: (1): G-CSF + DLI, (2): GM-CSF + DLI, (3): patient and disease adapted strategy. Thirty-four consecutive leukemia patients, aged 18–55, were investigated (20 very poor risk, 11 poor risk and 3 better risk). GvH type NK alloreactivity was chosen when possible (19/34) and balanced across the 3 groups. In the first 9 patients, G-CSF was used pot-transplant and prophylactic DLI were given at month 1, 2 and 3. The use of G-CSF and 1 to 3 DLI (104CD3/kg) was found safe. It resulted in faster CD4 recovery and a low rate of infections. However, it was insufficient to induce a protective GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI (104CD3/kg) at day 30 unless aGVHD (3 pts). The comparison between the 2 first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD:0. GM-CSF + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD grade 2 or more: 9/12; price to pay: GVHD resulting in 5 deaths in total. Median time to relapse in the 21 first patients was 6 months (range 4 to 9). Step 3 (14 patients) consists of a patient adapted strategy: no more aspecific DLI (selected anti-CMV and aspergillus DLI planned in all patients); in myeloid disorders with NK alloreactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mcg) is given from day 5 to day 9. The follow-up of these 14 patients, although promising (2 relapses), is currently short (median 5 months), compared to the median of relapse in the 2 first groups (6 months) but will be updated for the meeting (10 months and 17 patients). Overall, TRM at day 100 is 3/29, reflecting the good tolerance of the conditioning in a heavily pre-treated population (median age : 43). Overall NR-mortality at one year is 8/26, but was greater in the GM-CSF + DLI group, reflecting the impact of severe aGVHD. In the current group, its is 2/14 at 3 months. We conclude that the third strategy might improve the outcome and the relapse rate without exposing patients to unnecessary severe GVHD.

Background: Mesenchymal Stem Cells (MSC) can be isolated from bone marrow, adipose and fetal tissues, but their presence in Mobilized Peripheral Blood (MPB) and in Umbilical Cord Blood (UCB) remains controversial.

BACKGROUND: Bone Marrow mesenchymal stem cells (MSC) are pluripotent cells that have the capacity to differentiate into several tissue lineages and also the ability to support hematopoiesis. Their use in gene and cell therapy requires their in vitro expansion. Maintenance and proliferation of MSC are strongly dependent on the culture conditions such as properties of the bovine serum added in the culture medium. However, duration and culture conditions are critical for the successful clinical use of MSC.

Background:Additional infusion of ex vivo differentiated myeloid progenitor may reduce or abrogate severe neutropenia following high-dose chemotherapy and peripheral blood (PB) transplantation. Normal HPC as well as HPC from patients in clinical remission can be used as source of cells. The aims of our study were to compare the ex vivo expansion of myeloid progenitor cells starting from pretreated patients (PP) and normal donors (ND) and to evaluate if the cells collected from poor mobilizers -for which the ex vivo expansion is particularly interesting- present similar ex vivo expansion capacities than cells from good mobilizers.