Higher doses of CD34+ peripheral blood stem cells are associated with increased mortality from chronic graft-versus-host disease after allogeneic HLA-identical sibling transplantation

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Abstract

Allogeneic peripheral blood stem cell transplantation (PBSCT) has emerged as an alternative to bone marrow transplantation. PBSCT can be associated with a higher incidence of chronic graft-versus-host disease (cGVHD). In this study, we investigated whether there was a correlation between the composition of PBSC grafts (CD34+ and CD3+ cells) and hematological recovery, GVHD, relapse, and relapse-free survival (RFS) after myeloablative HLA-identical sibling PBSCT. The evolution of 100 acute or chronic leukemia patients was analyzed. Neither hematological recovery, acute or cGVHD, nor relapse, was significantly associated with CD3+ cell dose. Increasing CD34+ stem cells was associated with faster neutrophil (P=0.03) and platelet (P=0.007) recovery. Moreover, 47 of the 78 patients evaluable for cGVHD (60%; 95% CI, 49–71%) developed extensive cGVHD. The probability of extensive cGVHD at 4 years was 34% (95% CI, 21–47%) in patients receiving a ‘low’ CD34+ cell dose (<8.3 × 106/kg), as compared to 62% (95% CI, 48–76%) in patients receiving a ‘high’ CD34+ cell dose (>8.3 × 106/kg) (P=0.01). At a median follow-up of 59 months, this has not translated into a difference in relapse. In patients evaluable for cGVHD, RFS was significantly higher in patients receiving a ‘low’ CD34+ cell dose as compared to those receiving a ‘high’ CD34+ cell dose (P=0.04). This difference was mainly because of a significantly higher cGVHD-associated mortality (P=0.01). Efforts to accelerate engraftment by increasing CD34+ cell dose must be counterbalanced with the risk of detrimental cGVHD.

Introduction

The use of allogeneic peripheral blood stem cells (PBSC) as an alternative to bone marrow (BM) has rapidly grown.1,2,3,4,5,6,7,8,9,10,11,12 After HLA-identical sibling transplantation, PBSC transplantation is associated with quicker platelet and neutrophil recovery than BM.8,9,10,11,12,13 Some data also suggested a better survival among patients with advanced leukemia receiving PBSC.12 This can be because of a decreased incidence of relapse or reduced transplant-related mortality or both.12,14 However, the use of PBSC for allogeneic transplantation is still controversial. This is in part because of unresolved concerns about the long-term effects of growth factors treatment in healthy volunteers and to uncertainties regarding possible increased graft-versus-host disease (GVHD) especially in good-risk patients with a low relapse rate following BM transplantation. Different reports showed a higher incidence of chronic GVHD (cGVHD) among recipients of allogeneic PBSC.9,11,13,15 The initial report from the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC)11 showed that PBSC, when compared to BM, was associated with a similar rate of acute graft-versus-host disease (aGVHD), but an increased incidence of cGVHD. Recently, we performed a detailed analysis of clinical features of PBSC-associated cGVHD over a longer period of time (median follow-up, 4 years). Our results showed a significant effect of stem cell source on the incidence, prevalence, presentation, and therapy of cGVHD.16

When compared with BM, PBSC grafts contain significantly more nucleated cells, more CD34+ hematopoietic stem cells, and more CD3+ lymphocytes.15,17 It has been shown that granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ hematopoietic stem cells not only participate in engraftment, but also have an immunogenic role.18,19,20,21 In the setting of T-cell-depleted allogeneic transplants using CD34+ positive selection as T-cell-depletion method, Urbano-Ispizua et al22 could show that a high CD34+ cell dose not only does not improve the clinical results, but also actually may be associated with a poorer outcome. With this background, we therefore hypothesized that variations in the cell composition of PBSC grafts could lead to differences in graft-versus-host immune reactions, thereby affecting transplant related events also in the context of non-T-cell-depleted allogeneic transplants. To test this hypothesis, we performed a retrospective analysis in 100 patients receiving an HLA-identical sibling PBSC graft for acute or chronic leukemia. We asked whether there was a correlation between the cellular composition of the graft, especially CD34+ and CD3+ cell dose, and the kinetic of hematological recovery, risks of aGHVD and cGVHD, disease relapse, overall survival (OS), and relapse-free survival (RFS).

Patients and methods

Patients and donors

This analysis included 100 patients who received a PBSC transplantation between April 1995 and July 2001 from HLA-identical donors for acute or chronic leukemia. These patients were included in two consecutive trials by the SFGM-TC teams and were previously reported for other purposes.11,23 The majority of the patients included in this analysis (n=59) were treated at the Marseille, Montpellier, and Lyon transplantation centers. Written informed consent was obtained from each patient and donor. The protocols were approved by the scientific committee of the SFGM-TC and the local ethical committee of Marseille II (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale). All donors were HLA-A-, HLA-B-, and HLA-DR-matched siblings. Donors were treated with G-CSF (Lenograstim, Aventis, Montrouge, France) at a dose of 10 μg/kg per day. Cytapheresis was performed starting from day 5 of G-CSF treatment. Patients' and donors' characteristics are shown in Table 1. According to current practice in France, at the time of transplantation, none of these patients was scheduled to receive prophylactic G-CSF after transplantation. The type of myeloablative preparative regimen that was used in each case depended on the disease status of the patient and on each participating center's program.24,25,26,27 The day of cell infusion was designated as day 0. Patient management was performed according to standard procedures of each participating center and was expected to be the same for all patients for a given center. The graft was analyzed in terms of hematopoietic progenitors (CD34+ cells) and CD3+ lymphoid cells using standard flow cytometry procedures at each center. Cyclosporine and methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3 and 6) regimen was the usual GVHD prophylaxis regimen.28 Cyclosporine was started intravenously on day −1, usually at the dosage of 2–3 mg/kg, and switched to oral formulation as soon as the patient was able to take medication postengraftment. The dosage was adjusted to blood levels and renal function according to each center's practice.

Table 1 Patients' and donors' characteristics

Clinical outcomes and GVHD assessment

Clinical outcomes after transplantation that were considered included kinetic of neutrophil and platelet engraftment, aGVHD, cGVHD, disease relapse, and RFS. Time to neutrophil engraftment was defined as the first of three consecutive days in which the absolute neutrophil count (ANC) exceeded 500/μl. Time to platelet engraftment was defined as the first of 3 days with 25 000/μl without platelet transfusion during a 5-day period. aGVHD was evaluated according to standard criteria.29 The diagnosis of cGVHD was made based on both clinical and/or histology criteria of skin and other affected sites as previously described.30,31 cGVHD was defined as any GVHD present after day 100. Extensive cGVHD was defined according to standard criteria.32 Data concerning GVHD were carefully assessed in a central way through review of report forms and medical charts in all cases by M Mohty and DB. For comparison of ‘low’ vs ‘high’ CD34+ doses, the limit of 8.3 × 106/kg was defined as the median of CD34+ dose received by patients surviving beyond day 100 and evaluable for cGVHD.

Statistics

All data were computed using SPSS for Windows (SPSS, Inc, Chicago, IL, USA). The Mann–Whitney test was used for comparison of continuous variables. Categorical variables were compared using the χ2 test corrected with the Yates method if necessary.33 Spearman's rank correlation coefficient was used to estimate the correlation between cell types and to associate cell dose with time to engraftment. The probability of developing cGVHD was depicted by calculating the cumulative incidence34 with relapse and death without relapse or cGVHD as competing risks.35 Cumulative incidence estimate was also used to measure the probability of relapse.34 RFS was defined as survival in continuous complete remission; relapse and death in remission were events, and patients surviving in continuous complete remission were censored at last contact. OS and RFS were analyzed using the Kaplan–Meier product-limit estimates.36,37 Differences between groups were tested using the log-rank test when Kaplan–Meier analysis was performed.38 Since cGVHD begins after day 100 following transplantation, OS and RFS were also analyzed by the landmark method using day 100 after transplantation as a ‘landmark’ time.39 The association of time to cGVHD or RFS, with the transplanted CD34+ and CD3+ cell doses and other noncell-type variables (age, sex, cytomegalovirus (CMV) serologic status, risk of disease (standard risk vs advanced disease; advanced disease was defined as chronic myeloid leukemia in accelerated phase or blast crisis, or acute leukemias beyond first complete remission, or refractory anemia with excess of blasts), conditioning regimen, major ABO mismatch, and GVHD prophylaxis) was evaluated in multivariate analysis, with the use of Cox's proportional hazards regression model.40

Results

Patients' characteristics

Patients' and donors' characteristics are shown in Table 1. The majority of the patients included in this study (n=69) received a preparative regimen including cyclophosphamide (120 mg/kg) and total-body irradiation (TBI).24 In all, 16 patients were not treated with TBI but received instead busulfan (16 mg/kg) and cyclophosphamide (200 mg/kg).25 The remaining 15 patients received other conditioning regimens including TBI associated with etoposide, cytarabine, or melphalan.26,27 In total, 81 patients received cyclosporine and methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3 and 6) as GVHD prophylaxis.28 The remaining 19 patients received cyclosporine in association with steroids.

Correlation between hematological recovery and cellular composition of PBSC grafts

The number of cell subtypes in PBSC grafts from this study was highly variable. The collection of at least 4 × 106/kg of recipient weight CD34+ cells was possible from 87 donors. The majority of patients (38%) received a CD34+ cell dose between 4 and 8 × 106/kg. In all, 10 patients (10%) received a CD34+ cell dose between 2 and 4 × 106/kg, whereas three patients received less than 2 × 106/kg despite performing two or more apheresis in donors. In all, 20 patients (20%) and 16 patients (16%) received, respectively a CD34+ cell dose between 8 and 12 × 106/kg and between 12 and 16 × 106/kg. A total of 13 patients (13%) received more than 16 × 106/kg. The distribution of CD3+ lymphoid cells in PBSC grafts was also highly heterogeneous. No predominant subgroup could be detected. Moreover, there was no correlation between CD34+ and CD3+ cells contained in the graft (P=NS). All patients included in this study reached a sustained neutrophil count of more than 500/μl at a median of 15 days (range, 10–32). Two patients failed to reach a sustained platelet count of more than 25 000/μl. Platelet engraftment occurred at a median of 14 days (range, 8–188). An increased CD34+ cell dose was associated with a shorter time to neutrophil recovery (P=0.03). Among the 98 patients who achieved a platelet engraftment, an increased CD34+ cell dose was also associated with a faster time to platelet recovery (P=0.007). In contrast, CD3+ cell dose was not statistically significantly associated with the kinetic of neutrophil or platelet recovery (data not shown).

Correlation between clinical outcomes and cellular composition of PBSC grafts

Table 2 summarizes patients' characteristics according to CD34+ cell dose. All patients in this series were evaluable for aGVHD. In all, 18 patients (18%; 95% confidence interval (CI), 10.5–25.5%) developed grade I aGVHD. A total of 39 patients (30%; 95% CI, 21–39%) developed grade II aGVHD, and 25 patients (25%; 95% CI, 16.5–33.5%) developed grades III–IV aGVHD. Neither the CD34+ cell dose nor CD3+ cell dose was statistically significantly associated with the probability of aGVHD whatever the grade (Table 3). In total, 78 patients (78%; 95% CI, 70–86%) survived relapse-free beyond day 100 and were evaluable for cGVHD. cGVHD developed in 61 patients (78%; 95% CI, 50–72%) at a median time of 5 months (range, 3.2–21.1 months) after transplantation (Table 3). The cumulative incidence of cGVHD among all 100 patients at 4 years was 61% (95% CI, 51–71%) (Figure 1a). In the 78 patients evaluable for cGVHD, 14 (18%; 95% CI, 9.5–26.5%) developed limited cGVHD, whereas 47 (60%; 95% CI, 49–71%) developed clinical extensive cGVHD (Table 3). The probability estimate of clinical extensive cGVHD at 4 years was 34% (95% CI, 21–47%) in patients who received a ‘low’ CD34+ cell dose, as compared to 62% (95% CI, 48–76%) in patients receiving a ‘high’ CD34+ cell dose (P=0.01) (Figure 1b). After controlling for noncell-type variables that can be associated with the development of cGVHD (patient's age, sex mismatch, CMV serologic status, disease stage, presence of major ABO mismatch, use of TBI for conditioning, GVHD prophylaxis using methotrexate vs others), in the multivariate analysis, only the CD34+ cell dose was associated with the risk of cGVHD (P=0.0093) (Table 4). The CD3+ cell dose did not show any suggestion of an association with the hazard of cGVHD.

Table 2 Patients' characteristics by CD34+ cell dose
Table 3 Transplant−related events by CD34+ cell dose
Figure 1
figure1

cGVHD and CD34+ cell dose. (a) Cumulative incidence of cGVHD in the whole study population. (b) Cumulative incidence of extensive cGVHD according to CD34+ cell dose.

Table 4 Multivariate analysis of risk factors for time to chronic GVHD and RFS

Outcome

In all, 13 patients had a recurrence of their underlying disease at a median time of 7 months (range, 2–36). The cumulative incidence of relapse at 4 years was 13% (95% CI, 6–20%) with no significant difference between patients receiving a ‘low’ or a ‘high’ CD34+ dose (Table 5). Of the 100 patients included in this study, 48 (48%) died during the follow-up period. In total, 11 deaths among patients receiving a ‘high’ CD34+ dose were directly attributed to cGVHD, as compared with only three patients among patients receiving a ‘low’ CD34+ dose (P=0.01) (Table 5). Deaths attributed to aGVHD, relapse, and infections were comparable in both groups (Table 5). The Kaplan–Meier estimate of RFS among all 100 patients at 5 years was 50% (95% CI, 40–60%). When we considered the whole population, the rate of RFS was not statistically significantly different between patients receiving a ‘low’ CD34+ cell dose and patients receiving a ‘high’ CD34+ cell dose (P=NS) (Figure 2a). However, because cGVHD begins by day 100 following transplantation and this may impact the patient clinical management, we also attempted to detect a survival difference exclusively in patients who survived beyond day 100 using a landmark analysis. Among patients surviving relapse-free beyond day 100 and evaluable for cGVHD (n=78), there was a trend toward a better OS in patients receiving a ‘low’ CD34+ cell dose as compared to those receiving a ‘high’ CD34+ cell dose (P=0.07). More importantly, in this group of patients surviving beyond day 100, those receiving a ‘low’ CD34+ cell dose had a significantly better RFS as compared to those receiving a ‘high’ CD34+ cell dose (P=0.04) (Figure 2b). The Kaplan–Meier estimate of RFS at 5 years was 76% (95% CI, 61–87%) in patients receiving a ‘low’ CD34+ cell dose and evaluable for cGVHD as compared to 50% (95% CI, 34–66%) in those receiving a ‘high’ CD34+ cell dose and evaluable for cGVHD.

Table 5 Outcome by CD34+ cell dose
Figure 2
figure2

RFS and CD34+ cell dose. (a) RFS according to CD34+ cell dose in the whole study population. (b) Landmark analysis of RFS according to CD34+ cell dose among patients surviving beyond day 100 and evaluable for cGVHD.

In multivariate analysis in the group of 78 patients surviving relapse-free beyond day 100 and evaluable for cGVHD, disease status (standard risk vs advanced disease) and CD34+ cell dose were the only variables statistically significantly associated with the hazard of RFS (Table 4).

Discussion

In the current study, we have analyzed the impact of the number of CD34+ and CD3+ cells given in HLA-identical PBSC grafts on various clinical outcomes. We have confirmed that the number of cell subtypes in PBSC grafts can be highly variable. We found that CD34+ cell dose in PBSC grafts can affect both hematological recovery and the development of clinical extensive cGVHD. Moreover, in patients surviving beyond day 100, RFS was significantly higher in patients receiving a ‘low’ CD34+ cell dose as compared to those receiving a ‘high’ CD34+ cell dose. This difference was mainly because of a significantly higher cGVHD-associated mortality.

The importance of the number of CD34+ stem cells was first shown in the autologous setting, where higher numbers were associated with a faster hematological recovery and an economic advantage.41,42 Moreover, a minimal CD34+ cell dose was shown necessary for engraftment in autologous PBSC transplantation.43 In the current study, as has been suggested by our previous work,23 an increased allogeneic CD34+ cell dose leads to a faster neutrophil and platelet engraftment. This observation is supported by other studies.7,44,45 However, increasing the stem cell dose above a certain threshold may not necessarily translate toward a continuous faster neutrophil recovery.46

Different studies established the role of CD3+ lymphoid cells in the induction of aGVHD and cGVHD.47 In the current study, we did not find a significant association between CD3+ cell dose and the risk of aGVHD. This is consistent with all previous studies, but one,48 suggesting that the risk of aGVHD was not increased after allogeneic PBSC transplantation when compared to bone marrow.15 The lack of correlation between the high CD3+ lymphoid cells infused in PBSC grafts and the risk of aGVHD might be explained by the diminished alloresponsiveness of G-CSF-mobilized T cells.49,50 Recently, in a haplo-mismatch setting, postgrafting G-CSF has been shown to impair immune recovery.51 G-CSF is also known to reduce the production of inflammatory cytokines, to mobilize greater numbers of CD14+ monocytes with suppressor-cell function,52 and to mobilize large numbers of plasmacytoid dendritic cells that can induce a type 2 helper T-cell response.53 Altogether, these effects may lead to downregulation of the inflammatory response involved in aGVHD.54,55

We did not find a significant correlation between CD34+ cell dose and the risk of aGVHD. This stands in contrast to the results reported by Przepiorka et al56 These discrepancies may be explained by the differences in GVHD prophylaxis regimens used in these studies since the GVHD prophylaxis regimen can significantly influence the risk of aGVHD.57 In our study, methotrexate was omitted on day 11. The omission of the dose of methotrexate on day 11 can increase the risk of aGVHD57 that predisposes patients to the development of cGVHD. The majority of our patients (81%) received a homogeneous GVHD prophylactic regimen consisting of short-course methotrexate and cyclosporine.28,58 Thus, it is not likely that this dose regimen will have a different effect in the presence of low doses of CD34+ cells than it would have in the presence of high doses.

The issue of cGVHD after allogeneic PBSC transplantation is still unclear. A number of studies, including randomized studies, have reported a higher incidence of cGVHD among recipients of allogeneic PBSC from HLA-identical sibling donors.9,11,13 Other studies did not report such an increased incidence.8,10,12 Differences such as the length of follow-up, the number and type of patients, the type of GVHD prophylaxis, the regimen of G-CSF used for the mobilization of PBSC, and the use of postgraft G-CSF have been suggested to explain these different results. The present study provides insights toward understanding factors predicting cGVHD incidence and possibly explaining some of the conflicting data reported so far on the risk of cGVHD following allogeneic PBSC transplantation. An increased CD34+, but not CD3+, cell dose was significantly associated with an increased incidence of cGVHD, especially in its clinical extensive form. Patients receiving CD34+ cell dose in excess of 8 × 106/kg are likely to have a higher risk of developing extensive cGVHD. The latter is a critical issue, since extensive PBSC-associated cGVHD can be more severe, more difficult to treat, and may impact patients' long-term well-being.16,59 This may represent in some situations an acceptable trade-off if the counterpart is a better disease control. This was not the case in this study. Moreover and unexpectedly, in patients surviving relapse-free beyond day 100 and evaluable for cGVHD, a ‘high’ CD34+ cell dose had a negative impact on OS and RFS because of an increased cGVHD-related mortality. The latter further confirms the deleterious effects associated with the infusion of ‘high’ doses of allogeneic stem cells, where the increased severity of cGVHD will not necessarily translate into a reduced risk of relapse.45,60 Although previous studies using allogeneic BM as a source of stem cells showed an inverse association between cGVHD and risk of relapse,61,62 our study supports that allogeneic PBSC and BM do not appear to be simply interchangeable sources of hematopoietic grafts, and well-established dogma in the allogeneic BM transplantation setting need yet to be established in the PBSC setting. The majority of the patients included in this study (76%) were good-risk acute or myeloid leukemia patients. As some studies support that allogeneic PBSC may benefit poor-risk patients,12,14 additional studies will be necessary, addressing specifically disease recurrence and survival in other groups of patients, especially high-risk patients. These issues must be carefully assessed in order to determine the patient or disease subgroup for whom an overall benefit is associated with allogeneic PBSC.

At present, little is known to explain this surprising association between CD34+ cell dose and the risk of detrimental cGVHD. Our basic research work is now focusing on the different differentiation pathways and lymphoid and dendritic cell repopulation potential of G-CSF mobilized CD34+ PBSC. This might bring insights to elucidate the pathophysiology underlying the association between CD34+ cell dose and cGVHD. Cell dose is a continuous variable, and further investigations are warranted to identify the optimal CD34+ cell dose. However, the results of this study, combined to those of other groups,45 suggest that a CD34+ dose between 4 × 106/kg and 8 × 106/kg might be an acceptable trade-off because of a nonincreased risk of deleterious extensive cGVHD and an acceptable kinetic of hematological recovery.

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Acknowledgements

This work was supported in part by a grant from the French Ministry of Health (PHRC 1996) and a grant from the Ligue Nationale de Lutte Contre le Cancer. In addition, Laboratoires Chugai Pharma France (Paris la Défense) provided some support for this study, but did not participate in either the definition or data analysis. Mohamad Mohty was supported by grants from the SFGM-TC, the ‘Fondation de France’ and the ‘Fondation pour la Recherche Médicale’ (Paris, France). We thank D Buckner for critical reading of the manuscript. We thank the clinical research technicians from each participating center for help in data collection. We thank AG Le Coroller (INSERM U379, Marseille) for help with statistical analysis. We also thank the following members of the SFGM-TC for their active participation: JJ Sotto (Grenoble), M Legros (deceased, Clermont-Ferrand), and V Lapierre (Villejuif).

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Correspondence to D Blaise.

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Keywords

  • CD34+ stem cells
  • chronic GVHD
  • leukemia
  • allogeneic myeloablative transplantation

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