Introduction

Graft-versus-leukemia (GVL) effect plays an important role in curing children with acute lymphoblastic leukemia (ALL) treated with hematopoietic stem cell transplantation (HSCT).^{1, 2, 3, 4} Seventy percent of children with ALL who are candidates for HSCT lack a human leukocyte antigen (HLA)-matched sibling donor (MSD). For these children, HSCT from a matched unrelated donor (MUD) represents an alternative option. Matched sibling stem cells are always preferred compared to unrelated stem cells mainly due to the reduced likelihood of graft-versus-host disease (GVHD) and decrease in treatment-related mortality (TRM). Nonetheless, with recent advances in HSCT in terms of better molecular HLA typing, excellent supportive care, better diagnostic tools, new antifungal medications and new agents for management of GVHD, the outcome of MUD HSCT in pediatric ALL has improved dramatically so that it is comparable or even superior to that of MSD HSCT.^{5, 6} With the high incidence of GVHD in the MUD transplants, children who receive MUD HSCT are likely to benefit from GVL effect.

The intensity of the GVHD prophylactic regimen inversely correlates with the incidence of acute GVHD.⁷ In adult studies, the probability of developing moderate to severe acute GVHD with HLA identical MSD may be less than 20–40%, but with unrelated transplants it is 60–90%.⁸ In pediatrics, the overall incidence of GVHD may be less than in adults, however, children receiving unrelated donor marrow grafts experience a much higher incidence of GVHD compared to those receiving MSD marrow grafts. This difference probably justifies different GVHD prophylactic strategies between MSD and MUD transplantation. Furthermore, Nordlander et al.⁹ reported that combination prophylaxis with methotrexate (MTX) and cyclosporine (CSA) was an independent risk factor for relapse after HSCT (P=0.01) in ALL. Although we aim for lower levels of CSA in MSD transplantations, both MSD and MUD transplant recipients received double prophylaxis with CSA and MTX for GVHD, and this practice is standard in many other transplant institutions. Since there is no known measure for GVL, we hypothesize that when treating the same disease with comparable disease biology, remission status, using the same conditioning regimen and GVHD prophylaxis and using the same supportive care measures, outcome should be somewhat similar. However, relapse rate is different between MSD and MUD recipients and outcome, therefore, is different. We hypothesize that this difference in relapse rate is an indirect measure of GVL effect, and to compare the GVL effect between MSD and MUD recipients, we compared the relapse rate between the two groups and estimated the event-free survival (EFS) based on the occurrence of acute GVHD.

Patients and methods

This study was approved by our institutional review board. The health records of consecutive children who received HSCT for a diagnosis of ALL (B- and T-cell lineage) in The Hospital for Sick Children, Toronto from July 1998 to June 2006 were reviewed in a retrospective analysis. The criteria for inclusion of patients were the following: (1) recipients of a T-cell-replete fully matched sibling (MSD) or unrelated (MUD) bone marrow grafts. Recipients of a mismatched bone marrow donor, peripheral and cord progenitor stem cells and children with Down's syndrome were excluded. HLA matching consisted of serologic or low-resolution molecular typing for class I and molecular typing using high-resolution techniques for class II; (2) a myeloablative conditioning regimen of cyclophosphamide 50 mg/kg intravenously over 1 h daily for 4 days, followed by fractionated total-body irradiation (1200 cGy, in six fractions over 3 days); (3) GVHD prophylaxis with CSA at 3 mg/kg per day starting at day -1 and a short course of MTX at a dose of 10 mg/m² intravenously at day +3, +6, +11 and +18; (4) patients were to be in complete morphological remission at time of HSCT. Unmanipulated bone marrow was collected according to established methods.¹⁰ All patients had indwelling central venous catheters, and the majority received nutritional support with total parental nutrition. Infection prophylaxis included fluconazole for fungal prophylaxis,¹¹ ganciclovir for cytomegalovirus (CMV) prophylaxis,¹² growth factors and intravenous immunoglobulin, as well as the use of laminar airflow rooms from day 0 onwards. Pneumocystis jirovecii pneumonia prophylaxis was given for at least 6 months post HSCT or longer, if patients were still on CSA, and pneumococcal prophylaxis with penicillin continued for at least 1 year or until the administration of pneumococcal vaccine. Patient characteristics are summarized in Table 1. Taking into account a significant P-value of 0.1, there were no statistically significant differences in any of the patients' characteristics between the MSD and the MUD groups and, therefore, no adjustments were required.

Table 1 - Baseline demographics.

Full table

Definitions

Neutrophil engraftment was defined as the first of three consecutive days following the neutrophil nadir of absolute neutrophil count >0.5 10⁹/l and platelet engraftment was defined as the first of three consecutive days of unsupported platelets >20 10⁹/l. Primary graft failure was defined in patients surviving beyond day +28 as failure to attain an absolute neutrophil count >0.5 10⁹/l before death or receipt of a second graft.

Clinical grading of acute graft-versus-host disease (aGVHD) was performed according to previously reported criteria.¹³ Chronic graft-versus-host disease (cGVHD) was diagnosed and classified according to standard criteria.¹⁴ Tissue biopsy samples were obtained to confirm GVHD diagnosis whenever clinically indicated and feasible. TRM was defined as any death in remission.

Statistical analysis

Differences in continuous outcomes between the MSD and MUD groups were compared using the Wilcoxon rank-sum test. Categorical variables were compared using the ² test or Fisher's exact test as appropriate. EFS, overall survival (OS), cumulative incidence of TRM and cumulative incidence of relapse were described according to the Kaplan–Meier method, and groups were compared using the log rank test. In the determination of EFS, death, relapse, graft rejection, and graft failure were considered competing risks. All statistical analysis was performed using the SAS statistical program (SAS-PC, Version 9.1; SAS Institute Inc., Cary, NC, USA).

Results

Seventy-three patients were included in the study, 37 in the MSD group and 36 in the MUD group. Since length of remission in relapsed ALL has prognostic significance, we evaluated the length of remission in CR2 patients by measuring the time from original diagnosis to HSCT, and there was no significant difference between MSD and MUD recipients (Table 1). Length of remission for CR3 patients was not calculated due to the small number of patients, and these patients are high-risk patients regardless of remission time.

Time from last CR to HSCT was expectedly shorter for the MSD recipients compared to MUD recipients, with a median time of 94 days (range 32–296) for the MSD group and 128 (range 72–305) for the MUD group. In the MUD group, one patient died as a result of sepsis before day +28, and was not evaluable for engraftment or GVHD. There was no primary graft failure in either group. Two patients experienced TRM in the MSD group compared to seven in the MUD group. Acute bacterial septic shock caused TRM in the two MSD recipients. Causes of TRM in the MUD group were the following: acute bacterial septic shock in three patients, disseminated fungal infection in two patients, veno-occlusive disease in one patient and idiopathic pneumonia syndrome in one patient. Sixteen patients relapsed in the MSD group versus six in the MUD group. Six children received HSCT in CR3, three from MSD and three from MUD transplantation. All three who received MSD transplantation relapsed and died, while one patient died in the MUD group due to TRM. Expectedly, incidence of aGVHD was higher in MUD recipients (P=0.02). For a median follow-up of 55 months (range 13–104 months), there was a statistically significant difference in the 3-year relapse rate between the MSD group and the MUD group (P=0.03). However, for engraftment, 3-year TRM, 3-year EFS and 3-year OS, there were no significant differences between the two groups. Table 2 summarizes the outcomes of the two groups; EFS and OS are shown in Figures 1 and 2, respectively. In estimating 3-year relapse rate and 3-year EFS based on aGVHD for all patients, there was a trend toward more relapse and less EFS for patients without aGVHD. However, results did not reach statistical significance (Table 3). Although the two groups' characteristics were comparable, the time from CR to HSCT was different, however, the sample size did not permit multiple regression analyses.

Figure 1.

Event-free survival in months of 73 patients with ALL post HSCT comparing recipients of fully matched bone marrow unrelated donors (continuous line) versus fully matched sibling donor (dotted line).

Full figure and legend (31K)

Figure 2.

Overall survival in months of 73 patients with ALL post HSCT comparing recipients of fully matched bone marrow unrelated donors (continuous line) versus fully matched bone marrow sibling donors (dotted line).

Full figure and legend (32K)

Table 2 - Outcome of HSCT in pediatric ALL.

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Table 3 - Relapse and EFS based on aGVHD for all patients.

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Discussion

In this single-institution experience of HSCT for pediatric ALL patients receiving dual GVHD prophylaxis, we found that relapse rate post HSCT was higher in recipients of bone marrow stem cells from MSD transplantation compared to MUD transplantation, suggesting different GVL effects. Patient characteristics, including ALL biology, in both groups were comparable. Stem cell transplantation technology, HLA typing, supportive care, conditioning regimens and GVHD prophylaxis were identical in both groups. The choice of MSD or MUD was based only on availability and all patients entered remission in a timely manner and maintained remission prior to HSCT. Time from entering remission to HSCT was expected to be longer for the unrelated group, since the search process for an unrelated donor could take anywhere between 6 and 20 weeks. This might have lead to MUD recipients receiving extra chemotherapy prior to HSCT. In the absence of a very sensitive method to measure the quality of remission beyond morphology such as minimal residual disease, the effect of more chemotherapy on the quality of remission prior to HSCT is unknown. We acknowledge this limitation in our study. However, timing for HSCT was mainly related to donor availability and there was no evidence in selecting better or worse patients sooner or later.

In allogeneic HSCT, the immune recognition of host antigens by donor T lymphocytes leads to a beneficial GVL effect as well as to life-threatening GVHD. Effective GVHD prophylaxis is extremely important in reducing TRM following HSCT and has dramatically improved HSCT outcome.¹⁵ However, the role of MTX, given with CSA after HLA-identical sibling bone marrow transplantation in pediatric ALL, needs to be defined. In a randomized prospective study of 80 patients with hematological malignancies, Lee et al.¹⁶ reported a significantly lower relapse rate in the CSA-only group compared to the CSA plus MTX group, with a similar TRM in both groups, suggesting a beneficial GVL effect with the reduction in GVHD prophylaxis. Several other randomized trials have investigated immunosuppression regimens after HLA-identical sibling bone marrow transplantations. Earlier trials showed that patients who received post transplant immunosuppression with CSA plus MTX had a lower frequency of grade II–IV aGVHD than patients who received CSA alone or MTX alone.^{17, 18} However, in these early studies, CMV interstitial pneumonitis contributed to almost 50% of the deaths. In contrast to clinical trials performed in early 1980s, lower TRM rates have been obtained in recent clinical trials when patients were given GVHD prophylaxis with CSA only, and CMV-related deaths have markedly reduced with effective anti-CMV agents such as ganciclovir.¹⁹ In the current study, children who were at risk for CMV disease because of the recipient's or the donor's prior CMV status received empirical prophylaxis with ganciclovir, and there was no CMV-related death in any patient.

Several reports have demonstrated lower relapse rate associated with GVHD, presumably through a better GVL effect.^{1, 4, 9, 20, 21} Although great variations exist in the prophylaxis of GVHD in children undergoing HSCT, the European Group of Blood and Marrow Transplantation (EBMT) Working Party for Paediatric Diseases and the International BFM Study Group subcommittee Bone Marrow Transplantation (IBFM-SG) aimed at evaluating current standards for GVHD prophylaxis, and 75% agreed to CSA-only prophylaxis at 3 mg/kg/day for children less than 16 years old and receiving HSCT from an MSD for a malignant diagnosis.²² Furthermore, in a recently published report of individual patient data meta-analysis of allogeneic peripheral blood stem cell transplant versus bone marrow transplant, in the management of hematological malignancies that included nine randomized control trials with various hematological malignancies, relapse rate was 36.6% among recipients of BM when four doses of MTX were used along with CSA for GVHD prophylaxis, compared to 26% relapse rate in the three-dose studies. The author suggested that there is a disadvantage for BM recipients when given the 4th dose of MTX because the fourth dose of MTX has probably provided an extra immunosuppression among BM recipients resulting in a reduced anti-leukemic effect. This difference in relapse was not seen in recipients of peripheral blood stem cells, probably due the much higher T-cell counts in peripheral blood stem cells.²³ In our study, EFS and OS were higher in MUD recipients; however, results did not reach statistical significance. With the state-of-the-art HLA molecular typing, excellent diagnostic tools and new agents to treat infections and GVHD, unrelated transplantation results may further improve in future. However, better HLA typing could, theoretically, reduce GVHD incidence and may increase relapse, and the balance continues to be very fine between the unwanted complications of GVHD and the benefits of GVL.

In conclusion, transplant-related mortality post HSCT has been significantly reduced in recent years; however, relapse continues to be a major problem in MSD HSCT for pediatric ALL, when dual GVHD prophylaxis with CSA and MTX is used. In this study, there was less relapse when unrelated donors were utilized, presumably through a better GVL effect. In MSD transplantation, attempts for less GVHD prophylaxis, active early manipulation of the immunosuppressive therapy or other immune interventions post HSCT need to be further explored.

References

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Acknowledgements

We are indebted to the patients, patients' families, nursing and medical staff of the 8B unit and the oncology units at our partner centers for the provision of excellent patient care, which has been crucial for the achievement of the results reported here.