Time-dependent effect of non-Hodgkin's lymphoma grade on disease-free survival of relapsed/refractory patients treated with high-dose chemotherapy plus autotransplantation.

Evaluation of time to event outcomes usually is examined by the Kaplan-Meier method and Cox proportional hazards models. We developed a modified statistical model based on histologic grade and other variables to describe the time-dependent outcome for autologous stem cell transplant (autotransplant) performed for non-Hodgkin's lymphoma (NHL) based on histologic grade and other variables. One hundred and fourteen relapsed or refractory NHL patients were treated using BCNU 600 mg/m2, etoposide 2400 mg/m2, and cisplatin 200 mg/m2 IV followed by autotransplant. Median age was 53.5 (range: 25-70) years, 78 patients had aggressive NHL and 36 indolent NHL. Seventy-five patients received involved-field radiotherapy just prior to transplant. At a median follow-up of 33 (range: 3 to 118) months, the estimated 5-year Kaplan-Meier probabilities of overall survival and disease-free survival were 61% and 51%, respectively. Cox proportional hazards model analysis showed that proportionality did not hold for lymphoma grade, indicating that the relationship between the grade and disease-free survival differed over time. By piece-wise Cox model, the relative risk for experiencing relapse or death after 1 year in patients with indolent compared with patients with aggressive NHL was 2.81 (p=0.019) with 95% confidence interval (1.19, 6.65). The time-dependent effect of lymphoma grade on disease-free survival suggests the need for early (within first year) incorporation of novel therapeutic approaches in management of patients with indolent NHL undergoing autotransplant.

Pre-mobilization therapy blood CD34+ cell count predicts the likelihood of successful hematopoietic stem cell mobilization.

We examined pre-mobilization blood CD34+ cell count to predict ability to mobilize adequate peripheral blood progenitor cells (PBPC) in 106 cancer patients and 36 allogeneic donors. Mean pre-mobilization therapy blood CD34+ cell count was 3.1 cells x 10(6)/l (s.d. = 3.9, r = 0.3-37) and mean CD34+ cells collected were 5.3 x 10(6) cells/kg/leukapheresis procedure (s.d. = 7.0, r = 0.03-53). Yields correlated with pre-mobilization CD34+ cells x 10(6)/l (r = 0.37, P-value < 0.0001); correlation was stronger in allogeneic donors (r = 0.56, P-value = 0.0004) and males (r = 0.46, P-value < 0.0001). Based on classification and regression tree multivariate analysis, the predictive value of pre-mobilization blood CD34+ cell count was confounded by other variables, including age, gender, mobilization regimen and malignancy type. We generated an algorithm to predict a minimum PBPC yield of 1 x 10(6) CD34+ cells/kg/leukapheresis procedure after mobilization. A threshold pre-mobilization blood CD34+ cell count of 2.65 cells x 10(6)/l was the most important decision point in predicting successful mobilization. Only 2% of subjects with pre-mobilization blood CD34+ cell counts > 2.65 cells x 10(6)/l did not achieve the minimum per apheresis, whereas 24% with pre-mobilization values below threshold had inadequate mobilization. Prospectively identifying individuals at risk for mobilization failure would allow for improved treatment planning, resource utilization and time saving.

Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation.

Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and represent potential cellular therapy to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD). We investigated the role of human MSCs in NOD-SCID mice repopulation by unrelated human hematopoietic cells and studied the immune interactions between human MSCs and unrelated donor blood cells in vitro. When hematopoietic stem cell numbers were limited, human engraftment of NOD-SCID mice was observed only after coinfusion of unrelated human MSCs, but not with coinfusion of mouse mesenchymal cell line. Unrelated human MSCs did not elicit T-cell activation in vitro and suppressed T-cell activation by Tuberculin and unrelated allogeneic lymphocytes in a dose-dependent manner. Cell-free MSC culture supernatant, mouse stromal cells and human dermal fibroblasts did not elicit this effect. These preclinical data suggest that unrelated, human bone marrow-derived, culture-expanded MSCs may improve the outcome of allogeneic transplantation by promoting hematopoietic engraftment and limiting GVHD and their therapeutic potential should be tested in clinic.

Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH).

Patients with Hurler syndrome (mucopolysaccharidosis type-IH) and metachromatic leukodystrophy (MLD) develop significant skeletal and neurologic defects that limit their survival. Transplantation of allogeneic hematopoietic stem cells results in partial correction of the clinical manifestations. We postulated that some of these defects may be corrected by infusion of allogeneic, multipotential, bone marrow-derived mesenchymal stem cells (MSC). Patients with Hurler syndrome (n = 5) or MLD (n = 6) who previously underwent successful bone marrow transplantation from an HLA-identical sibling were infused with 2-10 x 10(6)/kg MSCs, isolated and expanded from a bone marrow aspirate of the original donor. There was no infusion-related toxicity. In most recipients culture-purified MSCs at 2 days, 30-60 days and 6-24 months after MSC infusion remained of host type. In two patients the bone marrow-derived MSCs contained 0.4 and 2% donor MSCs by FISH 60 days after MSC infusion. In four patients with MLD there were significant improvements in nerve conduction velocities after MSC infusion. The bone mineral density was either maintained or slightly improved in all patients. There was no clinically apparent change in patients' overall health, mental and physical development after MSC infusion. We conclude that donor allogeneic MSC infusion is safe and may be associated with reversal of disease pathophysiology in some tissues. The role of MSCs in the management of Hurler syndrome and MLD should be further evaluated.

G156A MGMT-transduced human mesenchymal stem cells can be selectively enriched by O6-benzylguanine and BCNU.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are being investigated for a potential therapeutic role as hematopoietic support cells following chemo-radiotherapy and as vehicles of gene delivery. Although hMSCs can be safely infused into humans and experimental animals, there is limited evidence regarding their engraftment and proliferation in vivo. We developed a drug resistance gene transfer strategy to mark and selectively enrich marked hMSCs using chemotherapy. We have determined that hMSCs are markedly sensitized to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro when pretreated with O(6)-benzylguanine (BG) resulting in a more than four-fold decrease in BCNU IC(90). The MFG retroviral vector encoding a bicistronic transcript for green fluorescent protein (GFP) and mutant (G156A)-methylguanine methyltransferase (G156A-MGMT), which encodes O(6)-alkylguanine-DNA alkyltransferase (AGT), conferring, BG plus BCNU resistance, transduced a high percentage of hMSCs. Transduced hMSCs had high expression of GFP and AGT and became significantly resistant to BG and BCNU. Furthermore, the proportion of GFP expressing transduced hMSCs increased from 32 +/- 14% to 70 +/- 14% following BG and BCNU treatment in vitro. Intravenously infused hMSCs were detected in NOD-SCID mice 8 weeks later by PCR analysis but could not be recultured from the bone marrow. GFP-expressing hMSCs inoculated into subcutaneous wounds in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mouse could be recultured at a low frequency, but enriched by BG and BCNU treatment from 0.05 +/- 0.03% to 0.55 +/- 0.4 (p = 0.028, Welch t-test). Our results indicate that hMSCs are sensitive to BG and BCNU, predicting significant toxicity to the hematopoietic microenvironment with this therapy. G156A-MGMT is a powerful selectable gene for a second marker gene in hMSCs. Drug resistance gene transfer into hMSCs may allow in vivo enrichment of hMSCs when MSC homing and engraftment into target tissues is optimized.

Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors.

BACKGROUND: Umbilical-cord blood from unrelated donors who are not HLA-identical with the recipients can restore hematopoiesis after myeloablative therapy in children. We studied the use of transplantation of umbilical-cord blood to restore hematopoiesis in adults. METHODS: Sixty-eight adults with life-threatening hematologic disorders received intensive chemotherapy or total-body irradiation and then transplants of HLA-mismatched umbilical-cord blood. We evaluated the outcomes in terms of hematologic reconstitution, the occurrence of acute and chronic graft-versus-host disease (GVHD), relapses, and event-free survival. RESULTS: Of the 68 patients, 48 (71 percent) received grafts of umbilical-cord blood that were mismatched for two or more HLA antigens. Of the 60 patients who survived 28 days or more after transplantation, 55 had neutrophil engraftment at a median of 27 days (range, 13 to 59). The estimated probability of neutrophil recovery in the 68 patients was 0.90 (95 percent confidence interval, 0.85 to 1.0). The presence of a relatively high number of nucleated cells in the umbilical-cord blood before it was frozen was associated with faster recovery of neutrophils. Severe acute GVHD (of grade III or IV) occurred in 11 of 55 patients who could be evaluated within the first 100 days after transplantation. Chronic GVHD developed in 12 of 33 patients who survived for more than 100 days after transplantation. The median follow-up for survivors was 22 months (range, 11 to 51). Of the 68 patients, 19 were alive and 18 of these (26 percent) were disease-free 40 months after transplantation. The presence of a high number of CD34+ cells in the graft was associated with improved event-free survival (P=0.05). CONCLUSIONS: Umbilical-cord blood from unrelated donors can restore hematopoiesis in adults who receive myeloablative therapy and is associated with acceptable rates of severe acute and chronic GVHD.

Mesenchymal stem cells: heading into the clinic.

In recent years, there has been an increasing interest in non-hematopoietic pluripotent progenitor cells that are found in the bone marrow. Mesenchymal stem cells (MSCs) are the first non-hematopoietic progenitors to be isolated from the bone marrow and extensively characterized. In addition to their ability to support hematopoiesis, MSCs can differentiate into osteocytes, chondrocytes, tenocytes, adipocytes and smooth muscle cells. This article will review our current understanding of bone marrow stroma and MSCs and their potential therapeutic role in the setting of hematopoietic stem cell transplantation.

Autologous CD34+ enriched peripheral blood progenitor cell (PBPC) transplantation is associated with higher morbidity in patients with lymphoma when compared to unmanipulated PBPC transplantation.

High-dose chemotherapy followed by CD34+ enriched peripheral blood progenitor cell (PBPC) transplantation is used for the treatment of primary refractory or relapsed Hodgkin's and non-Hodgkin's lymphomas. The CD34+ enrichment procedure, while reducing tumor burden, may compromise immunological reconstitution in the transplanted patient and result in increased rates of post-transplant infection. We compared infectious complications in patients with lymphoma who were treated with high-dose chemotherapy and supported either with CD34+ enriched PBPC (n = 19) or unmanipulated PBPCs (n = 24). Analysis was limited to patients discharged from initial hospitalization for transplantation with a minimum of 1 year followup and free of lymphoma recurrence. We found a statistically significant increase in the number of patients with one or more infectious events in the CD34+ transplant group (14/19) compared with the unmanipulated PBPC group (9/24, P < 0.01). Greater numbers of patients with two or more infectious events were observed in the CD34+ group (7/19 vs 2/24, P < 0.03) and an increased incidence of bacterial infections was observed in the CD34+ group (10/19 vs 5/24, P < 0.05). Two deaths due to infectious complications were observed in the CD34+ group. There was no significant difference in blood lymphocyte or monocyte recovery between the groups. These data demonstrate a significant increase in the long-term incidence of infectious events in lymphoma patients transplanted with autologous CD34+ enriched PBPCs compared to unmanipulated PBPCs. Thus, patients who undergo CD34+ enriched PBPC transplantation should be followed closely for infectious complications and prolonged infectious prophylaxis should be considered.

Limiting numbers of G156A O(6)-methylguanine-DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection.

The limited efficacy of hematopoietic gene therapy can be improved by in vivo selection for transduced long-term repopulating cells (LTRC). We selected for G156A MGMT (triangle upMGMT) transduced LTRC present in 5 x 10(4) to 100 x 10(4) marrow cells infused into nonmyeloablated mice by the administration of O(6)-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engraftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, triangle upMGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, triangle upMGMT(+) CFU were frequently detected, and their proportions increased with each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% triangle upMGMT(+) CFU for at least 6 months, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell toxicity allows triangle upMGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than that observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduced hematopoietic cells, potentially decreasing cumulative drug-induced myelosuppression in patients with cancer. These data also suggest that triangle upMGMT may be a potent, dominant, selectable marker for use in dual gene therapy.

Randomized cross-over trial of progenitor-cell mobilization: high-dose cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) versus granulocyte-macrophage colony-stimulating factor plus G-CSF.

PURPOSE: Patient response to hematopoietic progenitor-cell mobilizing regimens seems to vary considerably, making comparison between regimens difficult. To eliminate this inter-patient variability, we designed a cross-over trial and prospectively compared the number of progenitors mobilized into blood after granulocyte-macrophage colony-stimulating factor (GM-CSF) days 1 to 12 plus granulocyte colony-stimulating factor (G-CSF) days 7 to 12 (regimen G) with the number of progenitors after cyclophosphamide plus G-CSF days 3 to 14 (regimen C) in the same patient. PATIENTS AND METHODS: Twenty-nine patients were randomized to receive either regimen G or C first (G1 and C1, respectively) and underwent two leukaphereses. After a washout period, patients were then crossed over to the alternate regimen (C2 and G2, respectively) and underwent two additional leukaphereses. The hematopoietic progenitor-cell content of each collection was determined. In addition, toxicity and charges were tracked. RESULTS: Regimen C (n = 50) resulted in mobilization of more CD34(+) cells (2.7-fold/kg/apheresis), erythroid burst-forming units (1.8-fold/kg/apheresis), and colony-forming units-granulocyte-macrophage (2.2-fold/kg/apheresis) compared with regimen G given to the same patients (n = 46; paired t test, P<.01 for all comparisons). Compared with regimen G, regimen C resulted in better mobilization, whether it was given first (P =.025) or second (P =.02). The ability to achieve a target collection of > or =2x10(6) CD34(+) cells/kg using two leukaphereses was 50% after G1 and 90% after C1. Three of the seven patients in whom mobilization was poor after G1 had > or =2x10(6) CD34(+) cells/kg with two leukaphereses after C2. In contrast, when regimen G was given second (G2), seven out of 10 patients failed to achieve the target CD34(+) cell dose despite adequate collections after C1. Thirty percent of the patients (nine of 29) given regimen C were admitted to the hospital because of neutropenic fever for a median duration of 4 days (range, 2 to 10 days). The higher cost of regimen C was balanced by higher CD34(+) cell yield, resulting in equivalent charges based on cost per CD34(+) cell collected. CONCLUSION: We report the first clinical trial that used a cross-over design showing that high-dose cyclophosphamide plus G-CSF results in mobilization of more progenitors then GM-CSF plus G-CSF when tested in the same patient regardless of sequence of administration, although the regimen is associated with greater morbidity. Patients who fail to achieve adequate mobilization after regimen G can be treated with regimen C as an effective salvage regimen, whereas patients who fail regimen C are unlikely to benefit from subsequent treatment with regimen G. The cross-over design allowed detection of significant differences between regimens in a small cohort of patients and should be considered in design of future comparisons of mobilization regimens.

CD34+ selection of hematopoietic blood cell collections and autotransplantation in lymphoma: overnight storage of cells at 4 degrees C does not affect outcome.

The purpose of this study was to investigate whether storing mobilized peripheral blood progenitor cell (PBPC) collections overnight before CD34+ selection may delay platelet count recovery after high-dose chemotherapy and CD34+-enriched PBPC re-infusion. Lymphoma patients underwent PBPC mobilization with cyclophosphamide 4 g/m2 i.v. and G-CSF 10 microg/kg/day subcutaneously. Patients were prospectively randomized to have each PBPC collection enriched for CD34+ cells with the CellPro CEPRATE SC System either immediately or after overnight storage at 4 degrees C. Thirty-four patients were randomized to overnight storage and 34 to immediate processing of PBPC; 15 were excluded from analysis due to tumor progression or inadequate CD34+ cell mobilization. PBPC from 23 patients were stored overnight, while 30 subjects underwent immediate CD34+ selection and cryopreservation. Median yield of CD34+ enrichment was 43.6% in the immediate processing group compared to 39.1% in the overnight storage group (P = 0.339). Neutrophil recovery >500 x 10(9)/l occurred a median of 11 days (range 9-16 days) in the overnight storage group compared to 10.5 days (range 9-21 days) in the immediate processing group (P = 0.421). Median day to platelet transfusion independence was 13 (range 7-43) days in the overnight storage group vs 13.5 (range 8-35) days in those assigned to immediate processing (P = 0.933). We conclude that storage of PBPC overnight at 4 degrees C allows pooling of consecutive-day collections resulting in decreased costs and processing time without compromising neutrophil and platelet engraftment after infusion of CD34+-selected progenitor cells. Bone Marrow Transplantation(2000) 25, 559-566.

Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.

PURPOSE: Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We hypothesized that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by hematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of culture-expanded MSCs in breast cancer patients receiving autologous peripheral-blood progenitor-cell (PBPC) infusion. PATIENTS AND METHODS: We developed an efficient method of isolating and culture-expanding a homogenous population of MSCs from a small marrow-aspirate sample obtained from 32 breast cancer patients. Twenty-eight patients were given high-dose chemotherapy and autologous PBPCs plus culture-expanded MSC infusion and daily granulocyte colony-stimulating factor. RESULTS: Human MSCs were successfully isolated from a mean +/- SD of 23.4 +/- 5.9 mL of bone marrow aspirate from all patients. Expansion cultures generated greater than 1 x 10(6) MSCs/kg for all patients over 20 to 50 days with a mean potential of 5.6 to 36.3 x 10(6) MSCs/kg after two to six passages, respectively. Twenty-eight patients were infused with 1 to 2.2 x 10(6) expanded autologous MSCs/kg intravenously over 15 minutes. There were no toxicities related to the infusion of MSCs. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion in 13 of 21 patients (62%). Median time to achieve a neutrophil count greater than 500/microL and platelet count >/= 20,000/microL untransfused was 8 days (range, 6 to 11 days) and 8.5 days (range, 4 to 19 days), respectively. CONCLUSION: This report is the first describing infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed rapid hematopoietic recovery suggests that MSC infusion after myeloablative therapy may have a positive impact on hematopoiesis and should be tested in randomized trials.

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

Human long-term culture initiating cells are sensitive to benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and protected after mutant (G156A) methylguanine methyltransferase gene transfer.

Human hematopoietic progenitors express low levels of O6-alkylguanine-DNA alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), particularly following O6-benzylguanine (BG)-mediated O6-alkylguanine-DNA alkyltransferase inhibition. Expression of the BG-resistant mutant (G156A) methylguanine methyltransferase (deltaMGMT) gene in hematopoietic cells confers resistance to BG and BCNU. Because BCNU targets both early and late human hematopoietic cells and results in prolonged and cumulative myelosuppression, we attempted to protect early hematopoietic progenitors (long-term culture initiating cells (LTC-ICs)) by retroviral-mediated transfer of the deltaMGMTgene. A total of 33-56% of LTC-ICs were transduced with MFG-deltaMGMT retrovirus as determined by evidence of provirus in secondary colony-forming units at 5 weeks of culture under conditions optimal for the survival and proliferation of early hematopoietic progenitors. The addition of flt-3 ligand to cultures increased the transduction rate of LTC-ICs. Furthermore, 17.8 +/- 8.1% of deltaMGMT-transduced LTC-ICs survived doses of BG and BCNU; these doses allowed the survival of only 0-1% of untransduced LTC-ICs. This finding compares favorably with the 8-12% of CD34+ cell-derived colony-forming units that we previously showed became resistant to BG and BCNU after deltaMGMTgene transfer. Thus, deltaMGMT transduction of human early hematopoietic progenitor LTC-ICs confers resistance to BG and BCNU and may allow transduced LTC-ICs selective survival and enrichment over untransduced cells in patients undergoing BG and BCNU chemotherapy.

DeltaMGMT-transduced bone marrow infusion increases tolerance to O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and allows intensive therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea-resistant human colon cancer xenografts.

O6-Benzylguanine (BG) is a potent inhibitor of the DNA repair protein 06-alkylguanine DNA alkyltransferase (AGT), and sensitizes tumors to BCNU in vitro and in xenografts. The combination of BG and BCNU is now undergoing phase I clinical testing. The maximally tolerated dose of BCNU given after BG is expected to be lower then the doses tolerated as a single agent owing to BG sensitization of hematopoietic progenitors. We have previously shown that retroviral expression of G156A mutant MGMT (deltaMGMT) in mouse and human marrow cells results in significant BG and BCNU resistance. In this study we evaluated the effect of deltaMGMT-transduced marrow infusion on the therapeutic index of multiple BG and BCNU treatments in tumor-bearing nude (nu/nu athymic) mice. Prior to subcutaneous implantation of BCNU-resistant SW480 human colon cancer cells, cohorts of mice were given intraperitoneal injections of nonablative doses of BG (30 mg/kg) and BCNU (10 mg/kg, one-half of the LD10) and then infused with 1-2 x 10(6) isogeneic deltaMGMT (n = 29 mice) or lacZ-transduced (n = 20 mice) marrow cells. The xenograft-bearing mice were treated with multiple cycles of BG (30 mg/kg) and BCNU (10-25 mg/kg). After three cycles, deltaMGMT mouse bone marrow was repopulated with CFU containing the provirus, and demonstrated a 2.7-fold increase in AGT activity and a 5.5-fold increase in BCNU IC90 compared with LacZ mice. After five cycles, the BCNU IC90 of CFU cells increased nine-fold over control cells, indicating selective enrichment of CFU precursor cells expressing high levels of deltaMGMT. Starting with the third cycle of therapy, tolerance to BG and BCNU was significantly improved in deltaMGMT mice compared with LacZ mice, as evidenced by preserved peripheral blood counts, bone marrow cellularity, and CFU content 1 and 2 weeks posttreatment and a significantly higher survival rate. Xenograft growth was significantly delayed in mice tolerating multiple cycles and higher dose intensity of BG and BCNU as compared with mice receiving less intensive therapy. We conclude that deltaMGMT-transduced marrow cells can improve the therapeutic index of BG and BCNU by selectively repopulating the marrow and providing significant marrow tolerance to this combination, allowing intensive therapy of a BCNU-resistant tumor.

Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction.

Human mesenchymal stem cells (hMSC)-nonhematopoietic cells within the bone marrow microenvironment that can be culture expanded to a uniform population of fibroblastic cells-have been shown to support long-term hematopoiesis of CD34+ cells. Because direct contact between stromal elements and CD34+ cells enhances long-term engraftment, we postulated that hMSC would be a good alternative to the more heterogeneous stroma currently used in gene transfer studies. We used hMSC to support retroviral gene transfer of the G156A MGMT (deltaMGMT) gene encoding an alkyltransferase (AGT), which confers drug resistance to a combination of O6-benzylguanine (BG) plus the alkylating agents BCNU and temozolomide (TMZ) in human hematopoietic progenitors. In the presence of IL-3, IL-6, SCF, or leukemia inhibitory factor (LIF) and Flt-3 ligand, hMSC facilitated expansion and retroviral transduction of human peripheral blood-mobilized CD34+ cells. Furthermore, the transduced cells expressed AGT in 29% of hematopoietic cells and were 5-fold more resistant to BCNU and TMZ than were untransduced cells. Unirradiated hMSC present as support cells were simultaneously transduced and expressed AGT in 26% of the cells. Thus, the homogeneous nature of hMSC, and their ability to support gene transfer and be transduced themselves suggest they may be useful in clinical gene transfer protocols and have broad therapeutic applications.

Heterogeneity of O6-alkylguanine-DNA-alkyltransferase measured by flow cytometric analysis in blood and bone marrow mononuclear cells.

Alkyltransferase (AGT) repairs alkylation at O6-guanine in DNA and is a major determinant of susceptibility to alkylating chemotherapeutic agents and carcinogens. Using a newly developed flow cytometry assay with the monoclonal anti-AGT antibody, mT3.1, we compared AGT expression in single-cell suspensions with standard biochemical and Western blot assays to validate the fluorescence-activated cell sorting (FACS) method and develop potential applications. From Chinese hamster ovary cells (CHO) transfected with human O6-methylguanine-DNA methyl-transferase cDNA, 6 CHO-O6-methylguanine-DNA methyl-transferase clones were isolated that expressed 0.3 to 64 fmol/microgram DNA (by biochemical assay) of human AGT. FACS yielded a linear relationship between mean fluorescence intensity and both AGT activity by biochemical assay and AGT protein by Western blot. Using this standard curve, FACS-analyzed AGT protein content in human peripheral blood mononuclear cells (PBMCs) from normal donors ranged from 6.1 to 12.8 fmol/microgram DNA, similar to those obtained by biochemical assay and Western blot. This suggests that the level of immunoreactive protein appears to be an accurate predictor of AGT activity in the steady state. FACS-AGT in PBMCs from normal donors had a low index of heterogeneity within the sample. In contrast, by FACS-AGT analysis of human bone marrow samples and granulocyte-colony-stimulating factor-mobilized PBMCs, AGT was lower and had an 8-fold higher index of heterogeneity than observed in PBMCs from normal donors. After treatment with O6-benzylguanine (O6-bG), Western and FACS-AGT detected significant levels of AGT protein for up to 24 h, whereas biochemical assay showed AGT activity less than 5% of the basal level. Because only the biochemical assay accurately measures net AGT activity, the AGT-FACS assay will not be useful in clinical trials to assess the efficacy of O6-bG or other AGT inhibitors. Thus, AGT-FACS can rapidly assess the heterogeneity of steady-state AGT in single-cell suspensions and may be useful for assay in lymphocytes, bone marrow cells, leukemic myeloma plasma cells, or cells transfected with the AGT gene; Western blot analysis is better for small samples such as tumor biopsies, whereas biochemical assay is best able to measure enzyme activity and its inactivation by O6-bG or other agents.

Autologous CD34+ cell transplantation for patients with advanced lymphoma: effects of overnight storage on peripheral blood progenitor cell enrichment and engraftment.

In order to demonstrate the feasibility of mobilization, enrichment and engraftment of autologous peripheral blood CD34+ cells in patients with relapsed lymphoma, 59 peripheral blood progenitor cell (PBPC) collections from 21 patients were enriched for CD34+ cells using CEPRATE SC (CellPro, Bothell, WA, USA) immunoaffinity column. Following high-dose chemotherapy, a mean of 17 x 10(8) (range, 3-34) nucleated cells/kg containing 8.7 x 10(6) (0.3-26) CD34+ cells/kg were re-infused. Blood cell recovery in these patients was compared to engraftment capacity of unenriched PBPCs in a cohort of lymphoma patients treated with an identical high-dose chemotherapy regimen. Neutrophil and platelet engraftment was rapid in both groups including five patients who received < or = 1 x 10(6) CD34+ cells/kg. After infusion of CD34+ enriched cells, neutrophils exceeded 0.5 x 10(9)/l in 11 (8-14) days and platelets exceeded 20 x 10(9)/l (untransfused) in 15 (9-39) days. In order to optimize the immunoaffinity column utilization we stored the first PBPC collections overnight at 4 degrees C and combined them with the next day's collection prior to the CD34+ enrichment procedure in 11 patients. This maneuver resulted in a significant decrease in the CD34+ cell recovery (resulting in reinfusion of a mean of 42% less CD34+ cells). Although overnight storage did not affect neutrophil engraftment, platelet engraftment was prolonged in this group of patients even when > 2.0 x 10(6) CD34+ cells/kg were re-infused. The overnight storage procedure should be further evaluated for its effects on the CD34+ immunoaffinity enrichment procedure, megakaryocyte progenitors and platelet engraftment. We conclude that CD34+ cells enriched from peripheral blood result in rapid engraftment after high-dose chemotherapy in patients with advanced lymphoma that is comparable to that of patients receiving unenriched PBPCs.