Marrow-ablative chemo-radiotherapy followed by hematopoietic stem cell rescue from an allogeneic source improves outcomes for children with high-risk acute leukemia. The first effective pre-transplant preparative regimens consisted of high-dose cyclophosphamide (CY) and total body irradiation (TBI). Subsequent attempts have been made to improve leukemia-free survival, by adding other chemotherapy agents to these agents. In previous clinical studies of total body irradiation, etoposide, cyclophosphamide (TBI-VP-16-Cy) in adult allogeneic bone marrow transplantation, there has been a high incidence of severe regimen-related toxicity. In this study, we investigated the safety and efficacy of this combination in 41 children who received TBI (12–14 Gy), VP-16 (30 mg/kg), and CY (60 mg/kg × 2) and then either matched sibling or alternative donor transplants for acute leukemia. There was only one case of fatal regimen-related toxicity. The estimated 3-year event-free survival for patients with early or intermediate stage disease was 68% (53–88%). The estimated event-free survival of patients with advanced disease was 17% (5–59%). TBI-VP16-CY is safe in pediatric transplantation, and it has good efficacy for transplant recipients with less advanced disease. Bone Marrow Transplantation (2000) 25, 489–494.
Investigators in the 1980s demonstrated that allogeneic bone marrow transplantation, preceded by high-dose cyclophosphamide (CY) and total body irradiation (TBI),12 improved survival for children with high-risk acute leukemia. Subsequently, attempts were made to further enhance outcomes for both adults and children by augmenting the anti-leukemic activity of this combination. Increasing the TBI dose led to decreased relapse rates, but this was offset by increased treatment-related mortality.3 Alternatively, many investigators sought improved preparative regimen efficacy, by incorporating supplemental chemotherapy agents such as busulphan,4 cytarabine,5 or etoposide.678
Previous clinical studies of one of these novel regimens, total body irradiation, etoposide and cyclophosphamide combined with allogeneic bone marrow transplantation for adults with acute leukemia have yielded mixed results.678 To assess the safety and efficacy of this combination in pediatric patients, we evaluated the outcomes of 41 children and adolescents who received allogeneic bone marrow transplants for acute leukemia after total body irradiation, etoposide and cyclophosphamide.
Patients and methods
The pediatric bone marrow recipients included in this analysis were treated between September 1990 and October 1998 at the University of Rochester Medical Center. Patients were followed until 1 April 1999. During this time, all patients between the ages of 1 and 19 years who were receiving their first sibling or unrelated donor bone marrow transplant, and had acute lymphoblastic leukemia (FAB classification 1 or 2) or acute undifferentiated leukemia, were treated with this regimen. Patients with AML in undelayed first remission with matched sibling donors were treated on Pediatric Oncology Group protocols and not included in this analysis. All other pediatric patients who received allogeneic bone marrow transplants for AML were treated with this regimen. Two patients receiving second transplants received this preparative regimen; different preparative regimens were used in all other second transplants. Written consent, approved by the Institutional Review Board of the University of Rochester Medical Center, was obtained after review of the potential risks and benefits of the therapies used, with the patients and their families.
For sibling transplants, HLA compatibility was determined by serology. In unrelated donor transplants, HLA compatibility was determined by serology for class I and class II loci to 1994. High resolution Genotyping for HLA DRB1 was started thereafter.
All patients were hospitalized in HEPA-filtered rooms on a combined adult/pediatric unit. Fractionated TBI was administered days −8 to −4, either from lateral-opposed fields (1200 cGy as 150 cGy fractions twice daily, n = 8) or from AP–PA fields (1400 cGy as 175 cGy fractions twice daily, n = 33) with lung-shielding to limit the dose to the lungs to 800 cGy. A small mouth shield was used to limit the dose to 900 cGy to this region. Patients treated with the AP–PA approach, also received an electron boost (300 cGy in three fractions) to the rib cage. Lateral opposed fields were chosen for patients unable to lie still for the AP–PA approach. Etoposide was administered at a dosage of 30 mg/kg i.v. as a 4 h infusion on day −4. Hydration (3 l/m2/day) was begun prior to infusion of etoposide and continued for 24 h following completion of CY. CY was administered at a dosage of 60 mg/kg i.v. as a 1 h infusion daily for 2 days (days −3 and −2). Mercaptoethane sulfonate sodium (Mesna) (1 mg/1 mg CY) was administered by continuous i.v. infusion, beginning 3 h before CY and ending 24 h after the second dose. Patients receiving marrow from mismatched relatives or unrelated donors received additional immunosuppression with methylprednisolone, 1 g/m2 i.v. twice daily for 3 days (days −3, −2 and −1).
Graft-versus-host disease prophylaxis and supportive care
In all instances T cell-replete marrow was used. Prophylaxis against GVHD for patients receiving marrow from matched siblings, consisted of i.v. cyclosporin A (1.5 mg/kg twice daily) beginning day −1 and continuing until oral intake was established. Trough whole blood levels were maintained at 150–250 ng/ml. Methotrexate was administered as ‘short-course methotrexate’, 15 mg/m2 on day 1, and 10 mg/m2 on days 3, 6 and 11 post transplant. Cyclosporin A was administered to patients receiving marrow from mismatched relatives or unrelated donors by continuous infusion (3 mg/kg/day) and steady-state levels were maintained at 300–450 ng/ml for the first 50 days, 250–350 ng/ml days 50–100 post transplant. Portable infusion pumps were used for ambulatory patients. Cyclosporin A was changed to oral therapy at day 100 post transplant if there was no significant GVHD at that time. The ‘short-course methotrexate’ was modified for these patients as 10 mg/m2 on day 1, and 7.5 mg/m2 on days 3, 6 and 11 post transplant. In addition, methylprednisolone was started day 7 at a dosage of 1.5 mg/kg i.v. (or an equivalent oral dose of prednisone) daily to day 40. In the absence of GVHD, the dose was then tapered weekly by 10%. Supportive care for patients with histocompatible sibling donors included i.v. acyclovir (250 or 500 mg/m2 three times daily) for prophylaxis of HSV and/or CMV, and intravenous immunoglobulin (0.4 g/kg) every 2 (if at risk for CMV) or 4 weeks. Ceftazidime with gentamicin or imipenem were used for empiric treatment of neutropenic fever. CMV prophylaxis was modified for patients with mismatched related or unrelated donors. All CMV sero-positive patients received gancyclovir (5 mg/kg i.v. twice daily) during cytoreduction. High-dose acyclovir, as described above, was initiated on day 0 for sero-positive patients and sero-negative patients with sero-positive donors. All of these patients were then switched to gancyclovir when the ANC was >1000/μl post nadir. Similarly, low-dose amphotericin-B (0.1 mg/kg/day) was started empirically on day 0 for recipients of unrelated donor or mismatched sibling donor transplants. The dosage was increased to 0.5–1 mg/kg/day if fever persisted for 72 h while receiving antibacterial therapy.
Definitions and statistical analysis
Engraftment was defined as the first of 2 consecutive days post transplant in which the absolute neutrophil count was equal to or greater than 500. Acute GVHD was graded according to the Seattle criteria.9 Chronic GVHD was graded as limited or extensive with standard criteria.10 Regimen-related bladder, cardiac, renal, pulmonary, CNS, hepatic, oral and gastrointestinal toxicity was graded on a scale of 0 to IV according to the Seattle criteria. According to this system, organ toxicity leading to death by day 28 (by day 100 for pulmonary) is grade IV. Toxicity causing death after day 28 is graded according to the highest toxicity score achieved by day 28.11 Insufficient data were collected to determine the incidence of grade I or II cardiac or CNS toxicity. Using IBMTR criteria, leukemia was staged at the time of transplant as early (first remission), intermediate (second or greater remission) or advanced (relapse).12 Using IBMTR definitions, the type of transplant was classified as matched sibling or alternative donor (mismatched sibling, matched or mismatched unrelated).12 Causes of treatment failure were classified as relapse or transplant-related mortality. Transplant-related mortality was defined as death in continuous complete remission.12 Transplant-related mortality was further categorized into death associated with severe GVHD (grade III–IV AGVHD or extensive chronic GVHD), regimen-related toxicity or infection unrelated to severe GVHD.
Disease-free survival was estimated by the Kaplan–Meier method. To assess the effect of transplant type on survival, patients were divided into matched sibling transplant and alternative donor transplant groups. Similarly, to evaluate the effect of disease stage, patients were divided into early/intermediate stage disease and advanced stage disease groups. Comparisons of survival outcomes for these groups were performed, using the log-rank test. Sub-grouping that accounted for both transplant type and disease stage concurrently was not feasible given the limited size of the study. Comparisons between groups were also made for baseline characteristics, incidence of GVHD and cause of treatment failure. For quantitative variables, Student's t-test was used to test for significant differences. For proportions, Fisher's exact test was used. A P value of 0.05 was used as the cutoff for statistical significance.
Sixteen patients received matched sibling transplants. Alternative donors were used in the other 25 transplants. There were four mismatched sibling transplants (three 5/6, one 4/6), 14 matched unrelated transplants and seven mismatched unrelated transplants (all 5/6). Six patients had early stage disease, 23 patients had intermediate stage disease and 12 had advanced stage disease. Two patients had had previous transplants (one autologous, one allogeneic). The median age at time of transplant was 8.3 years (1.8–18.9 years). There were no statistically significant differences in age or in the proportion of patients receiving alternative donor transplants between the patients with early/intermediate stage disease and the patients with advanced stage disease (Table 1). Similarly, there were no statistically significant differences in age or in the proportion of patients with advanced stage disease between the matched sibling transplant recipients and the alternative donor transplant recipients (not shown).
Engraftment, toxicity and graft-versus-host disease
Two patients died prior to day 30 and before engrafting. Engraftment occurred in all of the other 39 recipients. The median time to engraftment was 20 days (12–41 days). All 41 patients developed either grade I or II stomatitis. Grade I or II bladder, renal, pulmonary, hepatic, oral or gastrointestinal toxicity each occurred in less than 25% of the patients. There was a single case of grade III toxicity. This patient died from VOD and hepatorenal syndrome on day +29. There were no cases of grade IV toxicity.
Of the 39 patients who engrafted, 23 (59%) developed acute GVHD. Eight of these patients had severe disease (grade 3 or 4). Thirty-two patients survived to day +100, and seven of these (22%) developed chronic GVHD. Six patients had extensive disease. In the alternative donor group, the incidence of mild acute or limited chronic GVHD was 46% while the incidence of severe acute or extensive chronic disease was 33%. In the matched sibling donor group the respective figures were 20% and 13%. Statistically, the proportions in the two groups were significantly different (P = 0.02) (Table 2).
Disease-free survival and causes of treatment failure
The median follow-up time for event-free survival was 36 months. In univariate analysis, both donor type and disease stage were important determinants of disease-free survival. The estimated 3-year DFS for patients with matched sibling donors and alternative donors was 75% (95% CI, 56–100%) and 39% (95% CI, 24–64%), respectively. This difference was statistically significant (P = 0.03) (Figure 1). The estimated 3-year DFS for patients with early/intermediate disease and advanced disease was 68% (95% CI, 53–88%) and 17% (95% CI, 5–59%), respectively (Figure 2). This difference was also statistically significant (P < 0.001). Relapse was a more common cause of treatment failure than transplant-related mortality. Twelve patients relapsed, accounting for 60% of the events in the entire cohort. Only seven of the 29 patients with early or intermediate stage disease experienced a relapse (24%). In the advanced stage disease group, on the other hand, five of 12 patients had a relapse of their leukemia (42%). There were five deaths related to severe GVHD, making this the most frequent cause of transplant-related mortality. All five of these events occurred in recipients of marrow from alternative donors. There were two deaths from infection unrelated to GVHD and as previously noted, one death from regimen-related toxicity (Table 3).
Although there are no data from randomized studies regarding the relative efficacy of preparative regimens combining one or more chemotherapy agents with TBI and cyclophosphamide, as a group they are the most commonly used regimens for allogeneic bone marrow transplantation in leukemia. They are employed more frequently in alternative donor transplants than in matched sibling transplants. These regimens were used in 61% of all alternative donor transplants for leukemia reported to the IBMTR between 1985 and 1991.12
In this study, we examined the safety and efficacy of one such regimen, TBI-VP-16-CY, in children. There is a strong basis for investigating the clinical efficacy of TBI, etoposide and cyclophosphamide as a conditioning regimen for allogeneic bone marrow transplantation in acute leukemia. Etoposide combined with TBI, is effective in allogeneic transplants for acute leukemia in adults13 and children.14 In vitro, etoposide exhibits synergy with oxazophorines15 and the non-hematologic toxicity profiles of etoposide and cyclophosphamide do not overlap significantly. Finally, TBI-VP-16-CY has been effective for autologous stem cell transplantation for pediatric ALL.16
However, Brown et al,6 Giralt et al7 and Yau et al,8 in non-randomized clinical studies of adults with acute leukemia undergoing allogeneic bone marrow transplantation, all observed relatively high rates of regimen-related toxicity and no apparent advantage of TBI-VP16-Cy over TBI-Cy or other regimens employed in allogeneic transplantation for acute leukemia. In contrast, the results of this study demonstrate that in children, TBI-VP16-Cy is a safe and effective regimen.
It is clear from the results of our study, that adding etoposide to TBI-CY does not increase regimen-related toxicity in children. There was only one case of fatal regimen-related toxicity (VOD) in our cohort. Similarly, Horstmann et al17 did not observe any severe regimen-related toxicity in nine children with acute leukemia who received allogeneic bone marrow transplants after TBI-VP16-CY. On the other hand, in all three aforementioned adult studies, toxicity and in particular, pulmonary toxicity, was a major problem. Giralt et al7 and Yau et al8 reported incidences of diffuse alveolar hemorrhage causing death of 8% and 7%, respectively. The lower toxicity rates observed in the present study and in the Horstmann et al17 study are likely partly attributable to the younger age of the patients. Although in some large single-center studies of regimen-related toxicity, older age was not identified as a risk factor,1118 in multivariate analyses of IBMTR data older age was a strong predictor of interstitial pneumonitis1920 and hepatic veno-occlusive disease.21 The dissimilarity in the incidence of toxicity between our study and the previous studies may also be due in part to dosing differences. In the adult studies, the TBI dose ranged from 1020 cGy to 1200 cGy and only Giralt et al appeared to have used lung shielding. We employed a higher TBI dose (1400 cGy) for most patients, but the lungs were shielded to a dose of 800 cGy. In the adult studies, the total doses of cyclophosphamide ranged from 100 mg/kg to 180 mg/kg and the total doses of etoposide ranged from 1500 to 1800 mg/m2 (equivalent to 50–60 mg/kg). In this study, the patients received a comparable cyclophosphamide dose (120 mg/kg), but a lower total dose of etoposide (30 mg/kg).
The event-free survival rate was very good for the favorable risk groups, whether the data are analyzed by disease stage or transplant type. The 3-year survival rates for the matched sibling transplant recipients and the patients with early or intermediate stage disease were 75% and 68%, respectively. These results compare quite favorably to outcomes reported by the IBMTR for pediatric matched sibling transplants performed from 1984 to 1990. In that study, the 2-year disease-free survival rates for patients with ALL and AML in second remission were 45% and 39%, respectively.22
An accurate assessment of the comparative efficacy of TBI-VP16-CY, TBI-CY and other regimens is more difficult to make. Brochstein et al2 reported results similar to ours with TBI-CY alone. In their cohort, the estimated disease-free survival rates for matched sibling transplant recipients with ALL and AML in second remission were 64% and 75%, respectively. Our cohort was not large enough to perform a subgroup analysis that concurrently accounted for disease stage, type of leukemia and transplant type (matched sibling vs alternative donor). This would permit a more precise subgroup comparison of the two studies. In addition to differences in subgrouping, it has been over a decade since Brochstein et al reported their results. Certainly, supportive care has improved since that time. On the other hand, because of improvements in non-transplant therapy for acute leukemias, especially ALL, patients needing hematopoietic stem cell transplants may now have more treatment-refractory disease. These and other differences underscore the need for multi-institutional, registry or randomized-controlled studies to assess the comparative efficacy of preparative regimens.
Unfortunately, this intensified regimen does not appear to significantly alter the outlook for children with advanced stage disease. The 3-year disease-free survival for these children was only 17%. Both disease relapse and transplant-related mortality contributed to the poor outcomes. Two-year disease-free survival rates for children reported to the IBMTR with AML and ALL in relapse were 26% and 25%, respectively.
In chronic myeloid leukemia much of the benefit of allogeneic hematopoietic stem cell transplantation is derived from the graft-versus-leukemia effect and gains will certainly be realized in the future through a better understanding of this phenomenon. On the other hand, in acute leukemia and ALL in particular,23 the graft-versus-leukemia effect is far less pronounced, and improved outcomes may have to await more effective preparative regimens. With this in mind, and given the age-related differences in susceptibility to regimen-related toxicity, registry or randomized controlled studies of preparative regimens are badly needed for children with acute leukemia.
Future phase I–II studies of intensified regimens are also warranted to determine optimal pediatric dosing. Given the very low toxicity rates observed in this study, the anti- leukemic effect of TBI-VP16-CY might be safely enhanced through dose escalation of etoposide, which was used at a relatively low dose in our regimen.
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Cite this article
Duerst, R., Horan, J., Liesveld, J. et al. Allogeneic bone marrow transplantation for children with acute leukemia: cytoreduction with fractionated total body irradiation, high-dose etoposide and cyclophosphamide. Bone Marrow Transplant 25, 489–494 (2000). https://doi.org/10.1038/sj.bmt.1702181
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