Autologous stem cell transplantation for high-risk pediatric solid tumors

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1 Bone Marrow Transplantation, (1999) 24, Stockton Press All rights reserved /99 $ Autologous stem cell transplantation for high-risk pediatric solid tumors JP Perentesis, E Katsanis, TE DeFor, JP Neglia and NKC Ramsay Bone Marrow Transplantation Program, Department of Pediatrics and Cancer Center, University of Minnesota, Minneapolis, MN, USA Summary: Many solid tumors exhibit a steep dose-response to alkylating agents, and autologous stem cell transplantation (ASCT) allows escalation of the chemotherapy dose for treatment of high risk solid tumors. We have transplanted 24 children and young adults with relapsed or metastatic solid tumors on two consecutive ASCT protocols consisting primarily (protocol MT 8911) or exclusively (MT 9408) of alkylating agents. The median time to neutrophil engraftment was 21 days in protocol MT 8911 (no prophylactic use of growth factors) and 14 days in MT 9408 (G-CSF, 5 g/kg, started on day 0). Disease-free survival estimated by the Kaplan Meier method is 39% (95% CI: 19 59%) at 2 years after transplant and 34% (95% CI: 14 54%) at 4 years after transplant. Six of the nine patients with metastatic or relapsed disease that were transplanted while in complete remission (four patients with Ewing s sarcoma family of tumors and two patients with anaplastic Wilms tumor) are alive and disease-free with a median follow-up of 37 months (range months). The estimated 4 year survival for patients receiving a transplant while in high risk remission was 78% (95% CI: %). In contrast, 13/15 patients that were transplanted while in partial remission died because of progressive disease or transplant-related complications. There were three transplant-related deaths (12.5%), including one patient with multiorgan failure, and two patients with complications of hepatic veno-occlusive disease. Our data indicate that autologous stem cell transplantation should be considered for consolidation therapy of high risk and relapsed pediatric patients with solid tumors who have achieved complete remission. Keywords: autotransplantation; pediatric solid tumors The prognosis for children and adolescents with solid tumors has improved during the last two decades because of the use of dose-intensive chemotherapy regimens. However, the majority of patients that have metastatic disease at diagnosis or those that relapse die despite aggressive surgery, radiation and conventional salvage chemotherapy. Correspondence: Dr NKC Ramsay, Room 660, University of Minnesota Cancer Center, 425 East River Road, Minneapolis, MN 55455, USA Received 26 November 1998; accepted 22 April 1999 Autologous stem cell transplantation (ASCT) for pediatric solid tumors allows further escalation of multiagent chemotherapy and potentially exploits the steep alkylating agent dose-response curve that is observed with some solid tumors. 1 3 ASCT is under evaluation as consolidation therapy for patients with metastatic disease and for those who have relapsed after intensive therapies Durable responses have been reported for neuroblastoma, 10,13,14,16 Ewing s sarcoma, 5,9,15 17 rhabdomyosarcoma, 8,15,17 Wilms tumor 15,18 and brain tumors We present our experience with two consecutive studies evaluating the role of myeloablative chemotherapy followed by ASCT for young patients with aggressive solid tumors. We find that high-dose alkylator therapy with hematopoietic stem cell rescue is an effective therapy for patients with high risk pediatric solid tumors. Furthermore, our pilot investigation indicates that disease status at the time of ASCT is the most important predictor of outcome with the majority of patients transplanted in complete remission surviving free of disease. Patients with active disease at the time of transplant generally fare poorly despite the use of an intensive preparative regimen. Patients and methods Patients Between October 1989 and February 1998, 24 children and young adults with high risk solid tumors received ASCT at the University of Minnesota. Demographic characteristics of these patients are listed in Table 1. Transplantation was conducted at a median of 16 months (range months) after initial diagnosis. Patients enrolled on these ASCT studies possessed high risk disease characteristics that included widely metastatic disease at diagnosis, single or multiple disseminated relapses, and/or therapy-refractory disease (Table 2). Signed informed consent approved by the University of Minnesota Committee on the Use of Human Subjects in Research was obtained from parents, legal guardians, or patients. Clinical and laboratory data were retrieved from patient charts and the University of Minnesota Bone Marrow Transplant Program Data Base that prospectively collects data on all patients. Transplant procedure The two ASCT protocol conditioning regimens used are outlined in Table 3. All patients had a double lumen central

2 610 Table 1 Patient characteristics Protocol MT 8911 MT 9408 Total Dates 10/89 to 5/94 5/95 to 2/98 10/89 to 2/98 No. of patients Male/Female 7/6 6/5 13/11 Age years/median (range) 7 ( ) 10.3 ( ) Diagnosis Ewing s sarcoma tumor family a Neuroblastoma Rhabdomyosarcoma Wilms tumor Osteosarcoma Yolk sac tumor Time from diagnosis to transplant, median months (range) 18 (6 133) 11 (6 46) 16 (6 133) Follow-up of survivors, median (range) 52 (46 69) 17 (8 31) 30 (8 69) a Includes one desmoplastic small round cell tumor. Table 2 Disease characteristics UPN Age Sex Disease a Status at Outcome Diagnosis Diagnosis Diagnosis Pre-SCT SCT b after SCT c stage d primary site metastatic sites relapse sites F Wilms Anaplastic CR1 Alive CR 4 Left Kidney/Spillage Lung, IVC, Right Atrium, Liver, Adrenal M EWSFT DSRCT CR1 Alive CR Diffuse Intra- Peritoneal/Omental Abdominal Nodes, Colonic abdominal Wall (transmural) F EWSFT Ewing s CR1 Alive CR Metastatic Right Forearm Lung, Marrow M EWSFT Ewing s CR2 Alive CR Localized Right Chest Wall None Lung F EWSFT Ewing s CR2 Alive CR Metastatic Left Ilium Lung Lung F Wilms Anaplastic CR2 Alive CR 2 Right Kidney None Lung M RMS-Embryonal CR2 Died w disease 4 Right Calf Lung Pancreas, Mesentery, Abdominal Nodes M EWSFT Ewing s CR3 Died w disease Localized L4 Vertebrae None Lungs F Osteosarcoma CR5 Died regimen Localized Right Femur None Lungs, Diaphragm, Ribs (VOD) F Neuroblastoma PR1 Alive CR 4 Right Adrenal Retroperit/Spinal, Bone, Marrow F EWSFT Ewings PR1 Alive SD Metastatic Pelvic/Ilium Lung, Bone, Marrow F EWSFT Ewings PR1 Died w disease Metastatic Pelvic Lung, Marrow M Neuroblastoma PR1 Died w disease 4 Adrenal Abdominal Nodes, Marrow M Neuroblastoma PR1 Died w disease 4 Adrenal Marrow, Liver, Abdominal Nodes M RMS Alveolar PR1 Died w disease 4 Left Foot Marrow, Testes, Bone, Abdominal Nodes, Chest Wall M EWSFT Ewing s PR1 Died regimen Metastatic Right Femur Lung, Marrow, Bone (VOD) M EWSFT Ewing s PR2 Died w disease Localized Ulna None Bone, Thoracic Nodes F Malignant yolk sac tumor PR2 Died w disease Localized Presacral None Pelvis (local), Liver, Lung F Neuroblastoma PR2 Died w disease 4 Adrenal Marrow, Abdominal Nodes, Marrow, Mediastinal Bone Adenopathy M Neuroblastoma PR2 Died w disease 4 Right Adrenal Marrow Bone, Marrow M Wilms Anaplastic PR2 Died w disease 4 Right Kidney Lung Lung F Neuroblastoma PR2 Died regimen 4 Adrenal Marrow, Brain, Bone Bone, Abdominal (MOF) Nodes, Lung M RMS Alveolar PR3 Died w disease 3 Face/Masticator None Pterygoid (local), Thorax/Pleural, Hilar M EWSFT Ewing s PR4 Died w disease Localized Left Fibula None Bone (local), Lungs a EWSFT = Ewing s sarcoma family of tumors; DSRCT = desmoplastic small round cell tumor; RMS = rhabdomyosarcoma. b CR = complete remission; PR = partial remission. c VOD = veno-occlusive disease of the liver; MOF = multiorgansystem failure. d National Wilms Tumor Study Group Staging System for Wilms tumor; International Neuroblastoma Staging System for Neuroblastoma; Intergroup Rhabdomyosarcoma Clinical Group Staging System for Rhabdomyosarcoma. venous catheter in place before conditioning treatment and were hospitalized in single rooms with high-efficiency particulate air filtration. Patients enrolled on protocol MT 9408 received phenytoin 24 h prior to the first dose of busulfan and until 24 h after the final dose. The majority of patients received autologous bone marrow cells, with two patients in protocol MT 8911 and four patients in protocol MT 9408 receiving G-CSF-mobilized peripheral blood stem cells

3 Table 3 Conditioning regimens 611 Day Drug Dose MT Etoposide 1800 mg/m 2 i.v. 8 Thiotepa 300 mg/m 2 i.v. 7 Thiotepa 300 mg/m 2 i.v. 6 Thiotepa 300 mg/m 2 i.v. 5 Cyclophosphamide 300 mg/m 2 i.v. 4 Cyclophosphamide 300 mg/m 2 i.v. 3 Cyclophosphamide 300 mg/m 2 i.v. 2 Cyclophosphamide 300 mg/m 2 i.v. 1 0 PBSC or marrow MT Busulfan 1 mg/kg q 6 h p.o. 7 Busulfan 1 mg/kg q 6 h p.o. 6 Busulfan 1 mg/kg q 6 h p.o. 5 Melphalan 50 mg/m 2 i.v. 4 Melphalan 50 mg/m 2 i.v. 3 Thiotepa 250 mg/m 2 i.v. 2 Thiotepa 250 mg/m 2 i.v. 1 0 PBSC or marrow (PBSC). Two patients in each protocol received bone marrow in addition to PBSC because an insufficient number of CD34 cells were present in the PBSC collection. Supportive care All patients received prophylaxis for Pneumocystis carinii with trimethoprim-sulfamethoxazole, and all fevers were treated empirically with broad-spectrum antibiotics. Cytomegalovirus (CMV) seronegative patients received blood products from seronegative donors or filtered blood products to remove CMV infected leukocytes. CMV and herpes simplex seropositive patients received acyclovir 10 mg/kg or 5 mg/kg, respectively, intravenously every 8 h as prophylaxis against acute infection. Standard blood product support guidelines were used, including irradiated packed red cell transfusions to maintain hemoglobin 8.0 gm/dl, and in the absence of bleeding, irradiated platelet transfusions to maintain platelet counts /l. Patients on MT 8911 received growth factors only for delayed engraftment while all patients on protocol MT 9408 received granulocyte colony-stimulating factor (G- CSF, 5 g/kg/day) beginning on day 0 and continuing until the absolute neutrophil count was greater than /l on each of 2 successive days. The time to neutrophil engraftment was defined as the first of 3 consecutive days on which the absolute neutrophil count (ANC) was /l. Statistical analysis The Kaplan Meier product-limit method was used to assess survival 27 and the log-rank statistic was used to test differences between groups. 28 Patients were censored at the time of death in the analysis of nonfatal events such as engraftment and time to platelet transfusion independence. Probability MT 8911 MT 9408 P = Days Figure 1 Time to neutrophil engraftment (ANC /l) for patients transplanted on protocols MT 8911 and MT Results Engraftment The median time to engraftment, defined as the first of 3 consecutive days of an ANC /l, was 21 days (range days) in protocol MT 8911 and 14 days (range days) in MT 9408 (P = 6; Figure 1). The overall estimate for ANC engraftment by day 45 was 78% (95% confidence interval (CI): 61 95%), and 100% by day 80. In protocol MT 9408, 56% of patients achieved platelet transfusion independence by day 100 following ASCT. Data on time to platelet transfusion independence were not available for protocol MT Toxicity and post-transplant complications Regimen-related toxicity and post-transplant complications are summarized in Table 4. There were three early deaths that were in part attributable to regimen-related toxicity. One patient enrolled on protocol MT 8911 developed multiorgan failure and died on day 5, with severe mucositis and skin toxicity, congestive heart failure, renal failure and respiratory distress syndrome. In protocol MT 9408, there were two deaths, on days 19 and 36, from complications of hepatic veno-occlusive disease. One of the patients that died with veno-occlusive disease was also receiving treatment for Candida kruseii septicemia. Fourteen of the other 21 patients had at least one docu- Table 4 Post-transplant complications Protocol MT 8911 MT 9408 Regimen-related mortality 1 2 Deaths due to infection 0 0 Number of patients with 9 5 bacteremias Number of patients with fungemia 0 1 Viral infections Varicella zoster 6 2

4 612 mented episode of bacteremia, but all were successfully treated with antibiotic therapy. Organisms isolated from these episodes included Staphylococcus epidermidis (Seven patients), Enterobacter cloacae (two patients), Achromobacter xyloxida (two patients), Streptococcus mitis (two patients), Escherichia coli (one patient), Staphylococcus aureus (one patient), Pseudomonas maltophilia (one patient), Pseudomonas aeruginosa (one patient), Neisseria spp. (one patient), Citrobacter freundi (one patient), Acinetobacter (one patient), and alpha hemolytic Streptococcus (one patient). Four patients were successfully treated for bacteremia with more than one organism. Eight patients developed varicella zoster at a median of 4 months after ASCT, with a range of months after transplant. Two patients developed hemorrhagic cystitis that responded to intravenous fluid flushes and correction of coagulation abnormalities. All patients (on both protocols) developed grade IV stomatitis requiring parenteral nutrition and analgesics. Outcome As of September 1998, eight patients are surviving free of disease with median follow-up among survivors of 30 months and a range of 8 69 months. The actuarial diseasefree survival is 39% (95% CI: 19 59%) at 2 years after transplant and 34% (95% CI: 14 54%) at 4 years after transplant. As shown in Figure 2, the 2 year disease-free survival was similar for both protocols and was 38% for MT 8911 (95% CI: 12 64%) and 42% for MT 9408 (95% CI: 11 73%; P ). The majority (6/9) of patients who were transplanted while in complete remission are alive and disease-free with a median follow-up of 37 months (range months). The other three patients who were transplanted while in remission have died after relapse (two patients) or as a consequence of regimen-related toxicity (one patient with hepatic veno-occlusive disease). The estimated overall survival for patients receiving a transplant while in high risk remission was 78% (95% CI: %) at 2 and 4 years after transplant (Figure 3). The corresponding disease-free survival for these patients was 78% (95% CI: %) at 2 years after transplant and 62% (95% CI: 27 97%) at 4 years after transplant. This group of responders included four patients with Ewing s sarcoma family tumors (three Ewing s sarcoma and one desmoplastic small cell tumor) and two patients with relapsed or metastatic anaplastic Wilms tumor. In contrast, the estimated overall survival for patients receiving transplants while only in partial remission was 15% (95% CI: 0 34%) at 2 years and 8% (95% CI: 0 22%) at 4 years (P 1 when compared to remission patients; Figure 3). Only two of 13 patients who were transplanted while in partial remission are surviving free of disease, and include a patient transplanted with advanced neuroblastoma and a patient with Ewing s sarcoma. These patients remain in remission at 52 months and 8 months after transplant, respectively. We also assessed the risk of disease-related treatment failure, measured by relapse in the patients who were in CR at the time of ASCT and by disease progression in the patients with residual disease at the time of ASCT. The 2 year relapse/disease progression rate was similar for both protocols and was 69% for MT 8911 (95% CI: 43 95%) and 45% for MT 9408 (95% CI: 11 79%; P = 0.16). The overall incidence of relapse and disease progression was 59% (95% CI: 38 80%) at 2 years and 66% (95% CI: 44 88%) at 4 years after transplantation (Figure 4). Discussion Autologous stem cell transplantation is currently being evaluated for virtually all types of pediatric solid tumors. 15,29 For many tumors, however, it has been difficult to draw firm conclusions on the advantages of this approach when compared to non-myeloablative treatment regimens. This is due to the relatively small number of pediatric patients that are transplant candidates, the heterogenous group of solid tumors in most series, the type and dose intensity of previous chemotherapy, and the disease status at the time of myeloablation. Alkylating agents are ideal for stem cell transplantation Probability MT 8911 MT 9408 P > 0 Probability Remission Relapse P < Years Figure 2 Disease-free survival following autotransplantation. Thirteen patients were treated on protocol MT 8911 and 11 on MT 9408 (P 0). Tick marks indicate patients surviving disease-free Years Figure 3 Impact of disease status at the time of transplant on overall survival. Tick marks indicate surviving patients.

5 Probability Years Figure 4 Relapse/disease progression following autotransplantation. Thirteen patients were treated on protocol MT 8911 and 11 were treated on MT For patients that were in remission at the time of ASCT, tick marks indicate relapse. For patients that had residual disease at the time of ASCT, tick marks indicate disease progression. conditioning since their dose-limiting toxicity is predominantly myelosuppression. Their dose can be escalated to non-hematologic dose-limiting toxicity and the patients rescued with stem cell infusion. Previous reports have documented a 3 20% incidence of transplant-related mortality following autotransplantation for pediatric solid tumors The first regimen that we evaluated, protocol MT 8911, consisting of etoposide, thiotepa and cyclophosphamide was associated with one patient experiencing peri-transplant mortality. The second regimen that we evaluated, MT 9408, consisted exclusively of alkylating drugs (busulfan, melphalan and thiotepa) and was associated with two toxic deaths, both from complications of hepatic veno-occlusive disease. In addition, the majority of patients in both protocols suffered from severe mucositis and episodes of the septicemia. The regimen-related toxicity was similar to that reported by the Seattle group using the same conditioning in an adult patient population undergoing transplantation for breast, lymphoma, ovarian and other cancers. 30 Nonetheless, these results highlight the need for ancilliary measures to decrease the risk of veno-occlusive disease and other conditioning regimen-associated toxicities to the gastrointestinal tract. Chemoprotectants such as amifostine may hold promise of reducing the risk of regimen-related toxicity while preserving the efficacy of high-dose chemotherapy with ASCT. 31,32 A striking finding in both of our preparative regimens was that the majority of patients transplanted in complete remission survive disease-free while the majority of those with measurable disease progressed in spite of the intensive multiagent chemotherapy. The European Blood and Marrow Transplant Registry (EBMT) has accumulated data from a large number of patients treated at different institutions with different conditioning regimens. 15 These data are in accord with our findings and show that pediatric patients with chemotherapy-sensitive solid tumors and minimal residual disease at transplantation have superior outcomes when compared with those with bulky chemoresistant tumors. Our favorable survival data in high risk Ewing s sarcoma patients are comparable to those reported by others. Four of 10 patients (40%) are alive and disease-free with a follow-up of 20, 23, 35 and 37 months. Hartmann et al 33,34 reported 11 complete responses in 27 Ewing s sarcoma patients (41%) receiving melphalan alone as the preparative regimen. Burdach et al 5 and Ladenstein et al 9 found the estimated disease-free survival following transplantation for Ewing s sarcoma to be 46% and 34%, respectively, compared to only 2% in historical controls receiving conventional chemotherapy regimens. Taken together, these studies indicate that autologous stem cell transplantation should be considered in pediatric patients with high risk Ewing s sarcoma. In addition, these results suggest that the combination of high-dose busulfan, melphalan and thiotepa are active in patients with Ewing s sarcoma who have failed intensive regimens containing other chemotherapy agents. As with Ewing s sarcoma, ASCT is currently under active investigation for the treatment of patients with high risk rhabdomyosarcoma. Children with relapse of rhabdomyosarcoma have a poor prognosis when treatment includes only non-marrow ablative chemotherapy, radiation therapy, or surgery. These treatments may produce some regression of the tumors, but responses are usually shortlived and the majority of patients relapse and ultimately succumb to their disease. 35 Overall, less than 5 10% of these patients are long-term survivors from recurrence if conventional chemotherapy alone is employed for treatment. 36 High-dose chemotherapy with autologous stem cell rescue has been proposed for the consolidation of second complete remission of rhabdomyosarcoma after relapse, 15 and initial promising results using this approach have been recently reported. Employing a conditioning regimen of melphalan and total body irradiation, Hartmann and colleagues 33 reported a 67% disease-free survival in patients with metastatic disease. A separate study of children with high risk rhabdomyosarcoma (relapses or metastatic disease at diagnosis) employing high-dose chemotherapy and total body irradiation revealed 45% long-term survival. 37 Pinkerton et al 12 treated pediatric patients diagnosed with stage IV rhabdomyosarcoma with high-dose melphalan and autologous transplant, and observed a 3 year relapse-free survival of 25%. Cumulative data from the EBMT Registry showed an overall survival at 3 years of 12% for rhabdomyosarcoma patients transplanted in second complete or partial remission and 28% for those treated as consolidation of first remission. 15 Refinements of conditioning regimens have improved these results, and a recent study of 26 patients with high risk rhabdomyosarcomas and undifferentiated sarcomas employing high-dose induction chemoradiotherapy followed by consolidation with high-dose melphalan and etoposide with autologous bone marrow transplantation revealed a 2 year progression-free survival of 53%. 17 Significantly, Koscielniak and coworkers 8 found that patients with localized tumor at relapse demonstrated extremely good outcome (four of nine alive with no evaluable disease) when they were treated with high-dose chemotherapy with stem cell rescue. Our protocol employs alkylating agents, including melphalan, which are among the agents of choice for the treat- 613

6 614 ment of high risk rhabdomyosarcoma. 34 Two of the patients receiving ASCT on our protocol had bulk disease at the time of transplant, and relapsed within a year of the procedure. A third patient was in remission at the time of transplant but also developed recurrent disease and died. These findings suggest that while ASCT is active in the treatment of high risk rhabdomyosarcoma, future protocols should incorporate systemic chemoprotectants to permit additional alkylator dose escalation or novel post-transplant biological or tumor-targeted therapies to eliminate minimal residual disease. Despite advances in conventional intensive chemoradiotherapy and surgery, the prognosis for advanced stage neuroblastoma with these therapies remains dismal with less than 20% 5 year disease-free survival. Autologous stem cell transplantation for high risk neuroblastoma has been used extensively during the last decade with long-term survival rates of 25 40%. 10,13,14 During the period of our study, the majority of patients receiving transplantation for neuroblastoma at the University of Minnesota had multiple poor prognostic disease features, and were not eligible for Children s Cancer Group (CCG) neuroblastoma therapy regimens. Five of the six patients transplanted on protocol MT 8911 had bulky disease at the time of transplantation and progressed. Furthermore, neither marrow nor peripheral blood stem cells were purged in this study. Gene marking studies have confirmed the importance of purging in autotransplantation for neuroblastoma. 38 Therefore, the poor outcome (one of six surviving disease-free) reflects a nonrepresentative sample of neuroblastoma patients undergoing transplantation. No neuroblastoma patients were treated on the more recent MT 9408 protocol since all transplant candidates qualified for CCG treatment regimens. The recently completed CCG 3891 study of 539 high risk (ie 85% stage IV) neuroblastoma patients treated patients with uniform induction chemotherapy followed by randomization to either myeloablative chemoradiotherapy supported by purged autologous bone marrow transplantation (ABMT) or to intensive non-myeloablative chemotherapy. This study revealed significantly improved event-free survival (EFS) in the ABMT group which was additionally increased by the use of 13-cis-retinoic acid to treat minimal residual disease after transplant (55% EFS in patients receiving ABMT retinoic acid vs 16% EFS in patients receiving intensive non-myeloablative chemotherapy). 39 The majority of children with Wilms tumor are cured with chemotherapy. Those presenting with metastatic disease or patients relapsing during initial chemotherapy may be considered for autotransplantation. Two of three patients treated at our institution are disease-free at 59 and 74 months following transplantation. EBMT data confirm the effective role of stem cell transplantation for this group of patients. Of 27 patients with relapsed Wilms tumor who received autologous transplants, 48% were reported to be disease-free. 15 The French Society of Pediatric Oncology has employed a three-drug combination of melphalan, etoposide and carboplatin, followed by ASCT for the treatment of 31 patients with chemotherapy-responsive high risk recurrent or stage IV anaplastic histology Wilms tumor. They observed a disease-free survival of 50% at 3 years in this group of very poor prognosis patients. 18 In summary, our data indicate that autologous stem cell transplantation after a high-dose alkylating agent conditioning regimen can be used to successfully treat a large subset of pediatric patients with high risk solid tumors. These findings are in accord with preclinical studies 1 3 demonstrating that marked escalation of alkylating agent dose is effective in killing diverse histologic types of solid tumors that are otherwise resistant to non-myeloablative doses of chemotherapy. Our results suggest that high risk patients with metastatic or poor prognosis relapsed disease should be aggressively treated to achieve successful remission induction and then considered for this ASCT regimen. However, high risk patients who are unable to achieve remission with contemporary therapies may benefit from the use of new agents or biologicals to induce a state of minimal residual disease prior to myeloablative chemotherapy with ASCT. This investigation also provides the foundation for the incorporation of future studies of chemoprotectant agents such as amifostine (reviewed in Ref. 40) into this effective high-dose alkylating agent chemotherapy conditioning regimen. We hypothesize that the use of chemoprotectants will reduce regimen-related morbidity, and may potentially allow for future investigation of alkylating agent dose escalation. In addition, the development of novel cytokines that possess greater efficacy for stem cell mobilization and the use of pro-thrombopoietic growth factors after ASCT will potentially increase the safety and utility of this regimen by reducing complications from infections and other aspects of regimen-related morbidity observed in our study. 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