Search

Current assays of human committed-stem cells are of limited value in predicting the rate of engraftment or in assessing the integrity of the stem cell pool after allogeneic bone marrow (BM) transplantation (BMT). We have used a limiting dilution assay of mafosfamide-resistant progenitors (pre-colony-forming units [CFU]), which are ancestral to committed progenitors such as CFU-granulocyte-macrophage (GM) to analyze the kinetics of myeloid engraftment after BMT and to assess the size of the stem cell pool at intervals up to 66 months thereafter. In 24 patients transplanted for chronic myeloid leukemia in chronic phase (eight with matched unrelated donors and 16 with sibling donors), the rate of neutrophil engraftment correlated strongly with the number of pre-CFU transfused per kilogram recipient body weight (r = .7, P < .005) but not with CFU-GM per kilogram or nucleated cells per kilogram. In 25 patients studied 6 to 66 months after allogeneic BMT, the mean number of pre-CFU in the marrow was 3.1/10(5) mononuclear cells (MNC) (median, 3.47; range, 0.4 to 23.3), compared with 24.7/10(5) MNC (median, 27.3; range, 4.2 to 180) in 25 normal subjects. CFU-GM were also reduced in these patients, but with considerable overlap into the normal range (mean +/- SD: 54 +/- 45.6 per 10(5) MNC; normal, 129 +/- 61.6). Low pre-CFU but not low CFU-GM levels were associated with reduced peripheral blood white blood cell counts in post-BMT patients. Pre-CFU and CFU-GM levels were not related to the interval posttransplant and remained low for up to 66 months. We conclude that the pre-CFU assay measures a population of stem/progenitor cells that are important in the kinetics of engraftment after allogeneic BMT. Our data suggest that pre-CFU levels may remain low for some years after BMT in humans.

The neutropenia-related morbidity and mortality occurring after autologous bone marrow transplantation (ABMT) is increased by marrow purging procedures. While phase I through III clinical trials showed the enhancing activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophil recovery after ABMT with unpurged marrow, controversial results have been reported when purged marrow was used. Therefore, it was the aim of the present study to evaluate the efficacy of rhGM-CSF administration in a group of patients (n = 15) with lymphoid malignancies transplanted in complete remission with mafosfamide-purged (n = 10) or unpurged (n = 5) marrow. Mafosfamide concentrations used for marrow purging were evaluated on an individual basis by means of a recently described technique that destroys the granulocyte-macrophage (granulocyte-macrophage colony- forming units [CFU-GM]) compartment, but spares 50% of the more primitive stroma adherent colony-forming cells (CFU-Blast). rhGM-CSF (10 micrograms/kg/d) was started within 24 hours of ABMT and administered in a 4-hour infusion daily until the absolute neutrophil count (ANC) reached 500 x 10(6)/L and then for 7 more days. Patients receiving mafosfamide-purged or unpurged marrow failed to show any difference in terms of median number of days required to achieve an ANC > or = 500 x 10(6) (13 v 14.0, P > .4) cells/L. As compared with retrospective controls, granulocytic recovery was reduced by a median time of 11 (P < or = .0005) and 5 (P < or = .0005) days for patients grafted with purged and unpurged marrow, respectively. The number of CFU-GM (mean +/- SD) infused per kilogram of body weight was significantly lower in patients who received purged autografts as compared with those receiving unpurged autografts (0.85 +/- 0.79 x 10(4) v 15.7 +/- 9.2 x 10(4), P < or = .0005). The dose of CFU-GM progenitors infused per kilogram of body weight did not correlate (r = .031, P > .05) with the time required to reach an ANC > or = 500 x 10(6) cells/L. The number of CFU-Blast (mean +/- SD) infused per kilogram of body weight was not significantly different between patients who received purged or unpurged autografts (5.05 +/- 2.51 x 10(3)/kg v 6.18 +/- 2.66 x 10(3)/kg, P < or = .375). A statistically significant correlation (r = -.658, P < or = .05) was observed between the number of CFU-Blast infused and the number of days required to reach an ANC > or = 500 x 10(6) cells/L.(ABSTRACT TRUNCATED AT 400 WORDS)

The knowledge about drug resistance in childhood leukemias and acute lymphoblastic leukemia (ALL) in general is limited. This is because of the lack of a suitable in vitro drug sensitivity assay, which is in part due to low in vitro ALL cell survival. We recently adapted the highly efficient 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay to test cells from ALL patients and showed that its results were comparable with those of the DiSC assay, up to now the most valid but laborious assay. In this study, in vitro drug sensitivity was assessed in cells from 82 children with leukemia, 79 of whom had ALL, with the MTT assay. Dose response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG), prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine arabinoside (Ara-C), L- asparaginase (L-Asp), mafosfamide, and mustine. A cytotoxic effect of methotrexate could be detected in only a few cases. Large interindividual differences in drug sensitivity were detected. Compared with leukemia cells from newly diagnosed patients, leukemia cells from relapsed patients were significantly more in vitro resistant to 6-TG, Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp. Improvements of culture medium and methods to increase MTT reduction were studied. From 10 components tested, addition of insulin and bovine serum albumin to serum-containing medium improved ALL cell survival. Addition of succinate did not increase the amount of MTT reduction. We conclude that the in vitro MTT assay highly facilitates large-scale studies on drug resistance of ALL patients that can lead to rational improvements in existing treatment protocols.

Abstract 1815 AICAR (5-aminoimidazole-4-carboxamide riboside or acadesine) induces apoptosis in chronic lymphocytic leukemia (CLL) cells, without affecting primary T lymphocytes (Campàs et al, Bood 2003). Available treatments for this disease generally induce remission, although nearly all patients relapse and CLL remains incurable. Thus, AICAR is a promising drug for the treatment of this B-cell neoplasm. It has been recently published that AICAR induces apoptosis by a p53- and AMPK-independent mechanism through upregulation of BIM and NOXA in CLL cells (Santidrián&González-Gironès et al, Blood in press). A clinical phase I/II study of AICAR is currently being conducted in CLL patients (http://clinicaltrials.gov/ct2/NCT00559624 ). This clinical study has shown that AICAR plasmatic levels in the micro molar range are achievable and safe when CLL patients are treated with the drug. In vivo assays were performed in mice to analyze the effects of AICAR on the peripheral lymphocyte population. Thus, 0.5 mg/g AICAR was administered intraperitoneally to Balb/c mice every 12 hours and every day blood was collected from the tail of treated (n = 3) and untreated (n = 3) mice. Significant differences (p< .05) were observed between control and treated groups in the number of lymphocytes from day 3 to 6 while the number of total leucocytes did not change. From day 4 the peripheral lymphocyte population started to recover and mice were sacrificed at day 8. In addition, AICAR induced a significant reduction (p< .05) on the percentage of B cells from day 3 to 5. Therefore, AICAR is effective in vivo decreasing the number of peripheral B lymphocytes. From the therapeutic point of view, it is interesting to analyze whether AICAR could synergize with the cytotoxic activity of the current chemotherapy used in CLL patients. Thus, cells from CLL patients (n = 4) were treated with AICAR (0.125, 0.25, 0.5 and 1 mM) and/or dexamethasone (1, 2.5, 5 and 10 μM), fludarabine (0.3, 0.6, 1.5 and 3 μM), chlorambucil (1.25, 2.5, 5 and 10 μM), mafosfamide (the active metabolite of cyclophosphamide) (0.25, 0.5, 1 and 2 μg/mL), rituximab (10, 25, 50 and 100 μg/mL) or alemtuzumab (1.25, 2.5, 5 and 10 μg/mL). The cytotoxic effect of the alkylating agents chlorambucil and mafosfamide was synergic or additive with the effect of AICAR in CLL cells depending on the concentration used of both drugs. The glucocorticoid dexamethasone synergized with AICAR in half of the samples analyzed. As for the nucleoside analogue fludarabine and the monoclonal antibodies rituximab and alemtuzumab, their apoptotic effect was additive with AICAR at some concentrations. Together, AICAR induces apoptosis in B lymphocytes from Balb/c mice when administered intraperitoneally and its cytotoxic effect in CLL cells is synergic or additive with the most common chemotherapy used in CLL treatment. Disclosures: de Frias: Advancell: Employment. Campàs:Advancell: Employment.

Abstract 3749 Regulatory T cells (Tregs) contribute to the maintenance of self-tolerance and have been shown to ameliorate acute GVHD both in murine models and in the clinical setting. However, translation of results and mechanistic insights from animal models to the clinic is difficult due to inherent incongruities in the use of post-grafting immunosuppression for prevention of GVHD. Administration of high-dose cyclophosphamide (Cy) in mouse models of allogeneic bone marrow transplantation (BMT) and its identical use as a short course single agent prophylaxis for GVHD in a HLA-matched setting provides an ideal model for studying the interaction between postgrafting immunosuppression and Tregs. We explored the hypothesis that Tregs play a critical role in prevention of GVHD in a MHC-matched mouse model of alloBMT using C57Bl/6-Foxp3-DTR mice (H-2b, CD45.2+) as donors, in which Foxp3+ Tregs can be selectively depleted in vivo by administration of diptheria toxin (DT; 2 doses on days 0 and +1). Donor mice were further crossed with those expressing luciferase (luc+) so that the in vivo expansion and engraftment of transplanted T cells could be noninvasively tracked by bioluminescent imaging (BLI) in addition to standard metrics of GVHD severity. We transplanted 1.2×107 splenocytes (or purified conventional T cells) from C57Bl/6-luc+-Foxp3-DTR mice (H-2b, CD45.2+) plus 1×107 bone marrow (BM) cells from B6-Ly5.2 (H-2b, CD45.1+) donors into myeloablatively conditioned allogeneic Balb/b recipients (H-2b, CD45.2+, 775 cGy) to induce acute GVHD. BLI on day 4 post-BMT revealed a significantly higher bioluminescent signal (photons/second/mouse) in animals treated with DT compared to PBS recipients (p < 0.001). Treatment of chimeras with Cy on day+3 led to a significantly reduced expansion of alloreactive luc+ T cells on days 4, 7, 14 and 21 (p < 0.05; Cy vs DT-treated chimeras and controls). Conversely, BLI of chimeras that received DT on days 0 and +1 followed by Cy on day +3 demonstrated a moderate increase in proliferation of donor derived luc+ T-cells in comparison to Cy recipients suggesting that the depletion of CD4+Foxp3+ Tregs early after transplant was detrimental to the protective action of the drug. Clinical severity of GVHD was significantly increased in chimeras with depleted Foxp3+ Tregs (P < 0.05, compared to PBS-treated chimeras), while Cy administration completely prevented manifestation of GVHD (P< 0.01, Cy vs PBS- and DT-treated chimeras). Consistent with BLI findings, depletion of Foxp3+ Tregs early after transplant abrogated the protective effect of Cy on GVHD, reflected in increased morbidity and mortality. To determine the effect of Cy on Foxp3 expression, in vitro experiments were conducted with CD4+Foxp3-GFPneg T cells co-cultured with allogeneic DCs and treated with mafosfamide. While exposure to increasing doses of the drug (2.5 and 12.5 μg/ml) caused a proportionally higher cell kill, the surviving T cells showed a marked induction of GFP expression in comparison to cells in the untreated culture. Taken together, these results suggest that Tregs are critical for prevention of GVHD in the context of GVHD prophylaxis with single agent high-dose Cy and that this approach besides limiting alloreactive T cell expansion may also foster conversion of naïve peripheral CD4+ T cells into CD4+Foxp3+ Tregs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4635 Objective: Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia among elderly people in Europe and North America. CLL is considered as a cancer involving mainly deregulated apoptosis. A strong difference between patients in the disease progression, response to anti-cancer therapy, and outcome is a major challenge in elaboration of efficacious treatment. Therefore, it is important to develop personalized therapeutic protocols based on the individual patient's sensitivity to medication. The aim of our ex vivo studies was to monitor the changes in responses of leukemic cells to anti-cancer agent(s) and correlate results obtained by DSC and other supplementary techniques with patients clinical response. Methods: Leukemic cells were obtained from peripheral blood of 14 patients prior to the onset of CC (cladribine + cyclophosphamide) therapy. Primary tumour cell samples were exposed ex vivo to CM or FM, as well as to ROSC alone for 48 hrs. We monitored the induction and progress of apoptosis in CLL cells exposed ex vivo to purine analogs combined with mafosfamide i.e. CM (cladribine + mafosfamide), FM (fludarabine + mafosfamide), and additionally to R-roscovitine (ROSC), an inhibitor of cyclin-dependent kinases, by a few techniques, i.e. cell viability test, apoptosis assay, differential scanning calorimetry (DSC), immunoblotting and determination of caspase-3/7 activity in the culture medium. The characteristic changes in chromatin complex induced upon the above treatments were detected by DSC, a simple thermal technique. This study received approval of the Medical University of Lodz Ethical Committee (RNN/196/07/KE). Informed consent was obtained from all the patients. Results: Marked differences in the individual patients’ sensitivity to the agents used were registered. A decrease or even loss of transition at 95±5°C in thermal scans of nuclear fraction preparations occurred in the treated cells in which apoptosis was induced. Remarkably, the changes in thermal profiles coincided with an accumulation of apoptotic cells, a decrease of the number of viable cells, and the changes in cellular levels as well as functional status of apoptosis-related proteins (caspase-9, Mcl-1) and CLL prognostic factor – kinase Zap-70. However, no significant changes were observed in thermal profiles of nuclei originating from CLL cells resistant to the treatment. Our studies revealed that the ex vivo exposure of CLL cells to CM combination, equivalent to clinically used CC regimen, was of prognostic value. Among the DSC profiles of nuclear preparations isolated from leukemic cells treated ex vivo with CM 70% of the cases examined indicated the decrease or loss of transition at about 95 ± 5°C. Moreover, we noticed a statistically significant dependence between the patient's response to CC treatment and thermal transition reduction at 95 ± 5°C (p=0.034). Interestingly, the results of the examined group did not show any correlation between disease advance (Rai stage) or cytogenetic abnormalities versus the clinical patient's response to the used treatment. Conclusion: Our results demonstrate the advantage of DSC in the evaluation of the treatment efficacy and indicate that its application could facilitate the choice of highly effective therapy for individual patients. Disclosures: No relevant conflicts of interest to declare.

Resistance towards the tyrosine kinase inhibitor (TK) imatinib frequently emerges in advanced stages of chronic myelogenous leukemia (CML) and complicates disease treatment. Nilotinib (AMN107) represents a novel BCR-ABL TK inhibitor with reported antiproliferative activity against imatinib-resistant cells in vitro. Since combination chemotherapy is a strategy to increase the antileukemic efficacy, we were interested whether combinations of nilotinib and other established chemotherapeutic agents such as mafosfamide, busulfan, treosulfan, daunorubicin or mitoxantrone display synergistic activity in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. Tetrazolium-based MTT assays (48 h incubation) revealed as expected that substantially higher concentrations of single agent nilotinib are required for growth inhibition of the imatinib-resistant K562-R (IC50=19.76±4.94 μM) and LAMA84-R (IC50=0.14±0.03 μM) cell lines as compared to the imatinib-sensitive counterparts (IC50=0.10±0.03 μM for K562 and IC50=0.03±0.01 μM for LAMA84, respectively). Combination treatment of both imatinib-sensitive LAMA84 and -resistant LAMA84-R cells resulted in variable antiproliferative effects. Combination index (CI) values calculated according to the median-effect method of Chou and Talalay denoted mostly additive (CI=1) and antagonistic (CI>1) effects. In both lines a clear synergistic activity with CI In BaF3 cells expressing imatinib-resistant mutants of BCR-ABL (E255K and T315I) IC50-values of single agent nilotinib were 0.37±0.04 μM and 23.58±1.92 μM, respectively. Importantly, combination treatment of imatinib-resistant BaF3 cells with both nilotinib plus daunorubicin and nilotinib plus mitoxantrone led to enhanced antiproliferative effects with synergism (CI The results demonstrate the ability of established chemotherapeutic agents to enhance the antileukemic efficacy of nilotinib and indicate the potential of the applied combinations for further study in the clinical setting.

B-type chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease. Unmutated immunoglobulin (Ig) VH genes, i.e. a post-germinal center genotype, are associated with a more aggressive natural course of disease. To explore the impact of Ig variable region hypermutation on response to cytotoxic therapies we analyzed the sensitivities to a panel of cytotoxic modalities by use of the DiSC assay that measures cell death. These data were compared to the Ig VH mutation status in 113 B-CLL patients. 29 patients exhibited IgHV hypermutation (age 66.3±1.8 years, 18 male, 11 female) whereas 84 patients (age 62.2±1.1, 66 male, 18 female) were of pre-germinal center B-cell origin. Hypermutated samples were from Binet stage A in 11 cases (unmutated 12), Binet stage B in 5 cases (14), Binet stage C in 10 cases (44), and in stage A/B in 2 cases (10 cases), Chi-square test p=0.02. 11/29 patients with Ig VH hypermutation and 54 of the 84 of the patients with unmutated B-CLL had received prior chemotherapy. Ig VH hypermutation had no impact in the percentage of surviving cells after 92 hours for ionizing irradiation (IR, 2 Gy by a 137Cs source) (hypermutated, 35.3%±8.2, unmutated 34.2%±4.5). Furthermore, there was no significant difference for the log LC90 values for chlorambucil (Mann-Whitney U test, p=0.22), doxorubicin (p=0.86), fludarabine (p=0.36), and mafosfamide (p=0.49). Interestingly, the log LC90 value for methylprednisolone showed a significant difference and was lower in the unmutated (n=83, mean±SEM 0.8±0.13) as compared to the hypermutated samples(n=29, 1.36±0.2; p=0.038). Conclusions: The mutation status of the IgHV gene segment does not appear to be responsible for differences in the response to various drug treatments in B-CLL. Vice versa, differences in gene expression that were described in hypermutated versus unmutated B-CLL do not seem to impact on drug sensitivity profiles. Thus, both DiSC assay result and VH Gene hypermutation are valuable independent prognostic markers.

Autologous bone marrow transplantation (ABMT) makes it possible to escalate the dose of cytotoxic treatment to a lethal range. Disease- free survival (DFS) following myeloablative therapy and ABMT has been shown to be superior to conventional treatment in high risk patients with acute myelogenous leukemia (AML). It was the purpose of the present study to compare hematopoietic reconstitution, actuarial DFS, and relapse rate of patients transplanted in first complete remission (CR) of AML with those in second or subsequent CR, and to evaluate transplant related mortality. Fifty-two patients with AML, 22 in first CR (low risk) and 30 in second or subsequent CR (high risk), underwent total body irradiation (12.1 to 16.7 Gy) and cyclophosphamide (CY) treatment (200 mg/kg) followed by ABMT. The autograft was incubated with the active CY derivative Mafosfamide (ASTA Werke, Bielefeld, Federal Republic of Germany) to reduce the number of possibly contaminating clonogenic tumor cells. All patients showed three lineage engraftments with platelet recovery observed as being the slowest. The transplant related death rate was low at 5.8%. There was no significant difference in the kinetics of polymorphonuclear (PMN) cell or platelet reconstitution between the low and high risk patient subgroups. The estimated probability of DFS (relapse) after ABMT in first CR was 61% (36%) compared with 34% (65%) in second or subsequent CR, the longest follow-up being 55 months and 57 months, respectively (median follow-up 31 months and 19 months, respectively). ABMT offers a stable long-term DFS when performed in first CR with no relapses occurring in over a year after transplantation. Six later relapses, however, were seen after ABMT in second or subsequent CR, although DFS was not statistically different from that of first remission patients (P = .72).

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6- contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.