Marrow purging in autologous bone marrow transplantation for T-lineage acute lymphoblastic leukemia: efficacy of ex vivo treatment with immunotoxins and 4-hydroperoxycyclophosphamide against fresh leukemic marrow progenitor cells

A lymphoblast progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed intact ricin immunotoxins (IT) and 4-hydroperoxycyclophosphamide (4-HC) against clonogenic primary T-lineage marrow blasts freshly obtained from 12 T-lineage acute lymphoblastic leukemia (ALL) patients. Residual T-lineage blast colonies were observed after treatment with 1 micrograms/mL T101 (anti-CD5)-Ricin (R) + G3.7 (anti-CD7)-R in eight of 12 cases and after 100 micrograms/mL 4-HC in six of nine cases. By comparison, a combination of IT and 4-HC proved very effective against T-lineage leukemic progenitor cells, and no residual blast colonies were observed in any of the eight cases studied. We conclude that future trials should consider combined treatment protocols such as IT + 4-HC for more effective purging of autologous marrow grafts.     

Autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide purged marrows for children with acute non-lymphoblastic leukemia in second remission

Although autologous bone marrow transplantation (ABMT) following purging with 4-hydroperoxycyclophosphamide (4HC) has been effective for some adults with acute non-lymphoblastic leukemia (ANLL), there are few data on ABMT for children. We have performed ABMT for 13 patients (median age, 5 years) with ANLL in second remission. Bone marrow was treated ex vivo with 4HC to kill residual leukemic cells. In order to enhance the anti-leukemic effect of 4HC, exogenous red blood cells were eliminated from the marrow/4HC mixture. All patients were conditioned for transplantation with busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg), followed by infusion of the 4HC treated marrow. Eleven of 13 patients had complete hematologic reconstitution; there were no transplant-related deaths. Five patients relapsed at 2, 4, 5, 6 and 6 months post-BMT. Eight patients are disease-free survivors; the median time for survival is 24 months, with a projected disease-free survival of 61%. ABMT is a safe and efficacious treatment modality for children with ANLL in second remission. Our results suggest that the disease-free survival for pediatric patients may be superior to that seen in adults.     

Autologous bone marrow transplantation for acute myelogenous leukemia using 4-hydroperoxycyclophosphamide and VP-16 purged bone marrow.

Thirty adult patients with acute myelogenous leukemia (AML) in remission were treated with hyperfractionated total body irradiation, VP-16, and cyclophosphamide followed by infusion of autologous bone marrow purged with 4-hydroperoxycyclophosphamide and VP-16. Fifteen patients were transplanted in first remission (R1), 13 in second remission (R2), and two in third remission (R3). All patients had hematopoietic engraftment. The median time to achieve a white blood cell count of 1.0 x 10(9)/l and a neutrophil count of 500 x 10(6)/l was 32 days. The median time to achieve an unsupported platelet count of 50 x 10(9)/l was 70 days. There were four transplant-related deaths, all in patients in R2. Ten patients have relapsed, including both patients transplanted in R3. The disease-free survival (DFS) of all patients is 52%, median follow-up not yet reached. Although the number of patients in each group is small and follow-up is limited, there is a trend toward improved actuarial DFS in patients transplanted in R1 compared with patients transplanted in R2 (72 vs 39%; p = 0.081). Use of VP-16 in the conditioning regimen as well as in the purging procedure is feasible in the treatment of patients with AML.     

Autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide purging for acute myeloid leukaemia beyond first remission: a 10-year experience

Between January 1987 and January 1997, 69 eligible patients with acute myeloid leukaemia (AML) in either second (CR2) or third (CR3) complete remission (CR2 = 60, CR3 = 9) underwent 4-hydroperoxycyclophosphamide-purged autologous bone marrow transplantation (BMT) at the Johns Hopkins Oncology Center. The patients' median age was 27 years (range 1-62) and all received busulphan and cyclophosphamide as their preparative regimen. The probability of event-free survival (EFS) at 5 years was 30% [95% Confidence Interval (CI): 19-42%] for CR2 patients and 22% (3-51%) for those in CR3, with a median follow up of 8 years in the surviving group. The median time to an absolute neutrophil count of 0.5 x 109/l was 45 d (range 20-185). Relapse was the major cause of failure with a relapse rate of 55% in CR2 and 44% in CR3, while the non-relapse, transplant-related mortality rate was 15% in CR2 and 33% in CR3. In univariate analysis, patient age, cytogenetics, white blood cell count at presentation, CR1 duration and the sensitivity of clonogeneic leukaemia (CFU-L) in the graft to 4HC were all prognostic for EFS. Using each of these significant variables in multivariate modelling, patient age and sensitivity of CFU-L to 4HC were determined to be predictors of EFS. 4HC-purged autologous BMT produced results similar to allogeneic BMT for AML patients beyond first remission.     

Allogeneic bone marrow transplantation for high-risk acute lymphoblastic leukemia during first complete remission

Fifty-three patients with high-risk acute lymphoblastic leukemia (ALL) under age 50 with a histocompatible sibling donor received high-dose radiochemotherapy followed by allogeneic bone marrow transplantation (BMT). The high-risk factors used to identify the patients were: white blood cell count at initial presentation, cytogenetic abnormalities, age, extramedullary leukemic infiltration, and time from initial therapy to complete remission. Patients with one or more of the above risk factors who received BMT have a disease-free survival of 61% with a median follow-up of 66 months (range 11 months to 10.6 years), and an actuarial relapse rate of 10%. This study demonstrates that patients with high-risk ALL achieve a significant disease-free survival and cure rate with the use of allogeneic fully matched sibling BMT. However, a properly designed prospective study comparing the outcome of BMT with the best currently available chemotherapy data is required to define the ultimate role of BMT in this group of patients.     

The effect of graft purging with 4-hydroperoxycyclophosphamide in autologous bone marrow transplantation for acute myelogenous leukemia.

BACKGROUND: Autologous bone marrow transplantation is an important therapy for patients with acute myelogenous leukemia (AML). However, leukemia in the graft may contribute to posttransplant relapse. Treatment of the graft with 4-hydroperoxycyclophosphamide (4HC) is sometimes used to decrease numbers of infused leukemia cells (4HC purging). No large controlled trials evaluating efficacy and toxicity of 4HC purging are reported. METHODS: We studied 294 patients reported to the Autologous Blood and Marrow Registry receiving either a 4HC-purged (n = 211) or unpurged (n = 83) autograft for AML in first (n = 209) or second (n = 85) remission. Analyses were restricted to patients transplanted less than 6 months after achieving remission. Using Cox proportional hazards regression, we compared time to treatment failure (death or relapse, inverse of leukemia-free survival) after 4HC-purged vs unpurged transplants while controlling for important prognostic factors. RESULTS: Median duration of posttransplant neutropenia was 40 (range, 10-200) days after 4HC-purged transplants and 29 (9-97) days after unpurged transplants (p < 0.01). Transplant-related mortality was similar in the two groups. In multivariate analysis, patients receiving 4HC-purged transplants had lower risks of treatment failure than those receiving unpurged transplants (relative risk, 0.69, p = 0.12 in the first posttransplant year; relative risk, 0.28, p < 0.0001 thereafter). Adjusted three-year probabilities of leukemia-free survival (95% confidence interval) were 56% (47-64%) and 31% (18-45%) after 4HC-purged and unpurged transplants in first remission, respectively. Corresponding probabilities in second remission were 39% (25-53%) and 10% (1-29%). CONCLUSION: Grafts purged with 4HC are associated with higher leukemia-free survival after autologous bone marrow transplants for AML.     

Autologous bone marrow transplantation after a long first remission for children with recurrent acute lymphoblastic leukemia

Fifty-one children with acute lymphoblastic leukemia (ALL) in second or subsequent remission after a first remission of at least 24 months underwent purged, autologous bone marrow transplantation (ABMT). Bone marrow was harvested in remission and purged in vitro with monoclonal antibodies specific for leukemia-associated antigens. Ablative chemotherapy included cytarabine, teniposide, and cyclophosphamide followed by total body irradiation. Of the 51 patients treated between November 1980 and June 1991, 5 died of treatment-related complications, 18 relapsed, 1 died of a second tumor at 6.7 years, and 27 remained in continuous complete remission for a median of 39 months (range, 9+ to 124+). Event-free survival (EFS) (+/- SE) at 3 years after ABMT was 53% +/- 7%. Leukemia-free survival (LFS) was 58% +/- 8%. In multivariate analysis, the most significant predictors of EFS were duration of longest pre-ABMT remission and remission duration immediately before ABMT. For LFS, the most significant predictors were cell dose per kilogram of marrow reinfused and duration of longest pre-ABMT remission. We conclude that ABMT for this population is an effective therapy available to the majority of children with relapsed ALL.     

Autologous bone marrow transplantation in high-risk remission B-lineage acute lymphoblastic leukemia using a cocktail of three monoclonal antibodies (BA-1/CD24, BA-2/CD9, and BA-3/CD10) plus complement and 4-hydroperoxycyclophosphamide for ex vivo bone marrow purging.

Fourteen patients with high-risk B-lineage acute lymphoblastic leukemia (ALL) in complete remission underwent autologous bone marrow transplantation (BMT) using a combined immunochemopurging protocol. A monoclonal antibody (MoAb) cocktail of BA-1, BA-2, and BA-3 plus rabbit complement (C') plus 4-hydroperoxycyclophosphamide (4-HC) was used to eliminate residual occult leukemia cells from autografts. All patients were conditioned with single-dose total body irradiation (TBI) followed by high-dose Ara-C. All 14 patients engrafted at a median of 24 days (range, 12 to 36 days). Three patients are alive and disease free at 3.5 years, 3.9 years, and 4.1 years post-BMT. The Kaplan-Meiser estimate and standard error of the probability of sustained remission was 23% +/- 12% at 3.5 years post-BMT with a mean relapse-free interval of 1.4 +/- 0.4 years. The disease-free survival (DFS) at 3.5 years was 21% +/- 11%, with a mean DFS time of 1.3 +/- 0.4 years. A novel and quantitative minimal residual disease (MRD) detection assay, which combines fluorescence-activated multiparameter flow cytometry and cell sorting with leukemic progenitor cell (LPC) colony assays, was used to analyze remission BM samples from B-lineage ALL patients for residual LPC, and to evaluate the efficacy of ex vivo BM purging. Notably, the minimal residual leukemia burden before BMT, as measured by the percentage of B-lineage LPC in the pre-BMT remission BM samples, indicated the outcome of the BMT. The median value for the minimal residual leukemia burden before BMT was 0.0035% (35 LPC/10(6) mononuclear cells). The Kaplan-Meier estimates and standard errors of the probability of remaining in remission after BMT were 43% +/- 19% for patients whose BM samples contained less than or equal to 0.0035% LPC and 0% +/- 0% for patients whose BM samples contained greater than 0.0035% B-lineage LPC (P less than .05). In contrast to the minimal residual leukemia burden measured by the described MRD assay system, the percentage of blasts or TdT+ cells in the remission BM samples did not correlate with the probability of relapse. The applied purging protocol showed variable success in destroying target B-lineage LPC populations contaminating the autografts. While in some cases purging was highly effective, eliminating up to greater than or equal to 4 logs of residual B-lineage LPC, in other cases only 0.1 to 0.2 logs of B-lineage LPC were purged.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autologous bone marrow transplantation for acute myelocytic leukemia in first remission: a European survey of the role of marrow purging

We analyzed data from 263 patients with acute myelocytic leukemia (AML) autografted in first remission (CR) during the period from January, 1982 to January, 1987 at one of 34 centers in the European Bone Marrow Transplant Group. The median age of patients was 30 years (range, 1 to 65). The median interval between achieving CR and autografting was 5 months (range, 1 to 23). Of the 263 patients, 131 patients received cytoreductive regimens that included total body irradiation (TBI); the remainder received various combinations of cytotoxic drugs. Sixty-nine patients received autologous marrow purged in vitro with mafosfamide, and 194 received unpurged marrow. The median follow-up was 28 months (range, 12 to 97). For patients with standard risk AML in CR1 autografted after TBI (n = 107), the leukemia-free survival (LFS) was higher, and the probability of relapse was lower in recipients of purged than of unpurged marrow (63% versus 34%, P = .05 and 23% versus 55%, relative risk 0.34, P = .005, respectively). The superior results of purging were most obvious in patients autografted within 6 months of achieving CR (probability of relapse, 20% versus 61%, P = .01). Patients with longer intervals between CR and autografting had higher LFS and lower probability of relapse than those autografted early in CR (intervals greater than 9 months, 7 to 9 months, 4 to 7 months, and less than or equal to 3 months: LFS = 56%, 40%, 35%, 27%, P = .007, probability of relapse = 25%, 56%, 59%, 67%, P = .005; respectively). We conclude that marrow purging with mafosfamide may be valuable for patients autografted early in first CR.     

Autologous bone marrow transplantation for acute leukaemia in remission

Between 1980 and 1985, 175 patients with acute leukaemia in first or subsequent complete remission (CR) were treated by chemotherapy or chemoradiotherapy followed by transfusion of autologous bone marrow cells that had been collected days or months previously. In 85 cases, autologous marrow cells were treated ex vivo with cytotoxic drugs or monoclonal antibodies with the intention of removing residual leukaemic cells. The actuarial relapse-free rate was 52% at 2 years. Of 89 patients autografted for acute non-lymphocytic (myeloid) leukaemia (ANLL), 60 were treated in first remission and 18 in second CR; their relapse-free rates at 2 years were 67% and 41% respectively (P less than 0.001). In contrast, of 77 patients autografted for acute lymphoblastic leukaemia (ALL), 32 were treated in first CR and 28 in second CR and their actuarial relapse free rates at 2 years were 56% and 55% respectively (P = NS). There was no significant difference in leukaemia relapse rates between patients autografted with purged and those autografted with non-purged marrow cells. These preliminary results suggest that autologous bone marrow transplantation may be valuable if offered to patients with ANLL in first CR or to patients with ALL in first or second CR but the need for marrow purging remains uncertain.     

Variability in 4-hydroperoxycyclophosphamide activity during clinical purging for autologous bone marrow transplantation

We examined the effects of varying incubation conditions on the in vitro activity of 4-hydroperoxycyclophosphamide (4HC). 4HC activity against CFU-GM and against the K562 tumor cell line decreased with increasing the RBC concentration of the incubation mixture. Increasing the concentration of nucleated bone marrow cells in the incubation mixture also decreased the 4HC activity. Evaluation of 53 consecutive patients undergoing autologous bone marrow transplantation (BMT) revealed that the incubation RBC concentration during clinical purging showed a similar effect on CFU-GM recovery. Aldehyde dehydrogenase content of RBCs and nucleated marrow cells appears to be the cause of the inhibition of 4HC activity. Although there was no difference in individual CFU-GM sensitivity to 4HC among normals, previously treated patients undergoing autologous BMT showed significant variability in CFU-GM sensitivity to 4HC. The combined effects of incubation RBC concentration and individual patient 4HC sensitivity appear to account for most of the variability in CFU-GM recovery and speed of hematologic recovery after clinical purging with 4HC.     

Autologous bone marrow transplantation for acute myeloid leukemia using monoclonal antibody-purged bone marrow

We report our experience from a clinical trial of autologous bone marrow transplantation (ABMT) in the treatment of 30 patients with acute myeloid leukemia (AML) using monoclonal antibody (MoAb) and complement-treated bone marrow. All patients were in complete remission (CR) at the time of transplant: 6 patients were in first CR, 18 in second CR, and 6 in third CR. The median age of all patients was 42 years (range 11 to 57 years). For marrow ablation, 28 patients were treated with cyclophosphamide and total body irradiation. One patient was treated with busulfan and cyclophosphamide and one was treated with busulfan and VP-16. Each patient was then transfused with autologous bone marrow that had been harvested previously and treated with two MoAbs, PM-81 and AML-2-23, and rabbit complement. Median time to recovery of neutrophils (500/microL) was 30 days, and platelets (20,000/microL) was 45 days. Median time for initial erythrocyte engraftment, assessed by a flow cytometric reticulocyte assay, was 13 days. Median overall and relapse-free survival of first CR patients was at least 17.4 months post-ABMT and the 2- and 3-year actuarial overall and relapse-free survival was 67% (+/- 19%). Median survival for the 24 patients in second or third CR was 6.8 months post-ABMT and 9.3 months since CR; however, six patients survived disease-free from 16 to 61 months post-ABMT. For the second and third CR group it was observed that six patients (5 of the 6 survivors) showed "inversions," when their post-ABMT remission lasted longer than any previous one. Actuarial 2- and 3-year disease-free and overall survival of patients in second and third CR was 25% (+/- 9%) and 18% (+/- 9%), and 29% (+/- 9%) and 23% (+/- 9%), respectively. ABMT avoids the problems of graft-versus-host disease and of finding suitable donors for allogeneic marrow transplantation.     

Autologous bone marrow transplantation for childhood acute lymphoblastic leukaemia in second remission - long-term follow-up

From 1984 to 1996, 31 consecutive children without sibling donors, aged 5-19 years (median 8) with acute lymphoblastic leukaemia (ALL) in second complete remission (CR), received unpurged autologous bone marrow transplantation (ABMT) after melphalan and single fraction total body irradiation (TBI). ABMT was performed using fresh unmanipulated marrow harvested after standard reinduction and consolidation therapy 2-11 months (median 5) after relapse. With a median survival of 2.9 years the probability of survival for all patients in continuing second CR was 45.1% (95% CI, 24%-62%) after 5 years. Regimen-related and non-leukaemia mortality was 7% (95% CI, 2%-26%). The longest time to second relapse from ABMT was 3.1 years. Pituitary and gonadal dysfunction requiring hormonal replacement therapy occurred in the majority of long-term survivors. Twelve patients developed cataracts. ABMT with melphalan/single fraction TBI has proved an effective anti-leukaemia treatment with low regimen-related mortality but significant long-term morbidity. The current approach of allogeneic BMT from an unrelated donor when no sibling donor is available, following conditioning with cyclophosphamide/ fractionated TBI has resulted in a reduced relapse rate and improved short-term overall survival in the treatment of relapsed childhood ALL. However, long-term results are awaited.     

Allogeneic or autologous bone marrow transplantation for acute lymphoblastic leukemia in first complete remission

Forty-seven patients with high risk acute lymphoblastic leukemia (ALL) received an allogeneic (allo) or autologous (auto) bone marrow transplant (BMT). Patients in both groups were comparable in terms of age, initial presentation of ALL and induction chemotherapy. Allo patients were transplanted earlier (median 3 months after CR) than auto patients (median 6.5 months after CR). Auto patients received more consolidation chemotherapy before BMT. All patients received total body irradiation 2.2 Gy/day x 5 days after cyclophosphamide 60 mg/kg x 2 (18 allo and five auto) or melphalan 140 mg/m2 (seven allo and 17 auto). Prevention of graft-versus-host disease (GVHD) was by conventional immunosuppression in 17 patients and T cell depletion in eight. Seven patients (28%) developed moderate to severe acute GVHD. Auto marrow was treated in vitro in each case. Seven patients died in CR from BMT complications (five allo and two auto). The probability of relapse was 9% for patients receiving allo BMT and 52% for patients receiving auto BMT (p less than 0.01). The disease-free survival was 71% for allo BMT and 40% for auto BMT (p = NS). Early BMT is an effective form of consolidation for high risk patients with ALL in first CR. An allogeneic anti-leukemia effect was demonstrated in this study.     

Autologous bone marrow transplantation in acute lymphoblastic leukemia: biological and clinical aspects

The purpose of this review is to assess the current status of autologous bone marrow transplantation in acute lymphoblastic leukemia. In the first part of the review the biological aspects of minimal residual disease are discussed and in the second part the most recent clinical results analyzed. Only the studies that contained adequate response and survival data were selected for analysis, including peer-reviewed articles, book chapters and proceedings of international meetings. The most current or complete references were used for series reported more than once.     

Autologous bone marrow transplantation for acute myeloid leukemia using busulfan plus etoposide as a preparative regimen

We have studied the use of a new preparative regimen for the treatment of patients in remission of acute myeloid leukemia (AML) with autologous bone marrow transplantation. Chemotherapy consisted of busulfan 1 mg/kg every 6 hours for 4 days (total dose, 16 mg/kg) on days -7 through -4 followed by an intravenous infusion over 6 to 10 hours of etoposide 60 mg/kg on day -3. Autologous bone marrow, treated in vitro with 100 micrograms/mL of 4-hydroperoxycyclophosphamide, was infused on day 0. We have treated 58 patients up to the age of 60 years, 32 in first remission, 21 in second or third remission, and 5 with primary refractory AML unresponsive to high-dose Ara-C, but achieving remission with aggressive salvage regimens. Of the first remission patients, there has been 1 treatment related death and 5 relapses. With median follow-up of 22 months, the actuarial relapse rate is 22% +/- 9% and disease-free survival is 76% +/- 9% at 3 years. Patients with favorable French-American-British (FAB) subtypes (M3 or M4 EO) did especially well, with no relapses seen in 15 patients observed for a median of 30 months. Actuarial relapse rate at 3 years was 48% for first remission patients with less favorable FAB subtypes. Of patients in second or third remission, there were 5 treatment related deaths and 4 relapses. With median follow-up of 22 months, the actuarial relapse rate is 25% +/- 11% and disease-free survival is 56% +/- 11% at 3 years. Four of five primary refractory patients died during treatment and 1 remains in remission with short follow-up. These preliminary data are very encouraging and, if confirmed, support the use of autologous purged bone marrow transplantation using aggressive preparative regimens as one approach to improve the outcome of adults with AML.     

Monoclonal antibody purging and autologous bone marrow transplantation in acute myelogenous leukemia in complete remission

A mouse monoclonal antibody (S4-7) reacting with human myelomonocytic cells has been previously shown to be suitable for bone marrow purging in selected acute myelogenous leukemia (AML) patients with S4-7 positive leukemic clonogenic cells at diagnosis. The results obtained in seven AML patients who underwent such a treatment, followed by autologous bone marrow transplantation (ABMT), are now reported. Six patients underwent ABMT in first complete remission (CR), one in second CR, after BAVC conditioning regimen. One patient died of infection 1 month after ABMT; in the other six a complete recovery of hemopoiesis was observed. In spite of S4-7 reactivity with normal myelomonocytic cells, a prompt recovery of granulopoiesis was however observed both in in vitro liquid culture and in vivo with a median time of 20 days to reach granulocyte values of 500 x 10(6)/l. The patient transplanted in 2nd CR relapsed 3 months after ABMT. Of the five evaluable patients transplanted in 1st CR, two relapsed 8 and 9 months post-ABMT while three remain in continuous CR at 35, 47, 57 months. Leukemic cells of two of the three patients with recurrent disease were studied at relapse and in both could be detected a significant percentage of S4-7 negative cells, detectable neither at diagnosis nor (one patient) at the time of first relapse after standard chemotherapy.