Allogeneic stem cell transplantation (allo-SCT) is a well-established treatment modality for children with severe aplastic anemia (SAA). Treatment failures are rare and mostly caused by graft rejection. Increasing mixed chimerism represents a stage at the very beginning of graft rejection, where immunological intervention might be an effective prophylactic approach. To substantiate this, we: (1) monitored peripheral blood cells from children with SAA after allo-SCT and performed pre-emptive immunotherapy in patients with increasing MC. In all, 23/34 courses of 32 children with SAA after allo-SCT showed a complete chimerism (CC) throughout and 10/34 developed different types of mixed chimerism (MC). Altogether, 4/10 with MC spontaneously developed decreasing MC, 2/10 courses persisted with low proportions of autologous cells below 30% (stable-MC), 4/10 developed increasing MC and one patient showed an autologous recovery. All patients with CC, decreasing MC or stable MC remained in continuous complete remission (CCR). In all, 2/4 patients with increasing MC developed graft rejection. Based on these observations, 2/4 new patients with increasing MC received low-dose DLIs prophylactically, and remained in CCR without any GVHD. These results substantiate that low-dose DLI in children with SAA and increasing MC can prevent graft rejection with a calculable risk to induce severe GVHD.
Allogeneic stem cell transplantation (allo-SCT) is a convincing treatment modality in children and young adults with severe aplastic anemia (SAA).1 Best results can be achieved with stem cells from a matched family donor (MFD), but in the last few years other allo-SCT procedures using stem cells from matched unrelated, mismatched unrelated or haploidentical donors (MUD, MMUD or haplo) were applied with good success, as well.2,3,4,5 Treatment failures in SAA are rare and are often caused by graft rejection.5,6 Divers studies focused on individual chimerism development showed that the mixed chimerism (MC) phenotype is linked to graft rejection.7,8,9,10 In addition, we recently demonstrated that patients with different types of acute leukemias and increasing MC after allo-SCT are at the highest risk to relapse, and that early immunotherapeutical intervention in this stage significantly improves the outcome.11,12,13 In this study, we asked whether the phenotype of increasing MC might also be a reliable indicator for threatening graft rejection in SAA patients after allo-SCT, and whether graft rejection can be prevented by additional immunotherapy, as well.
Patients, materials and methods
In total, peripheral blood samples from 32 children, who received allo-SCT for SAA, were referred from nine pediatric transplant centers in Germany to the study center in Tübingen. The study period lasted from January 1993 to June 2002. The study protocol was approved by the Clinical Ethics Committee of the University of Tübingen, and conducted according to the principles of the Declaration of Helsinki. Informed assent and consent were obtained from the patients and parents, respectively, according to institutional guidelines. Data were obtained for analysis until December 2002. The median follow-up was 42 months (2.5–108 months). Patient characteristics, types of stem cell donors, sources of stem cells and conditioning regimens are summarized in Table 1.
All patients were diagnosed and treated according to the guidelines of the actual SAA protocol of the German and Austrian Paediatric Aplastic Anaemia Working Group.14
Quantitative chimerism analyses were performed on peripheral blood weekly until day 100 and then monthly, which is described in detail elsewhere.15,16 In brief, DNA of peripheral blood samples was extracted using the ‘Qiagen Mini Kit’ (Qiagen, Hilden, Germany). Individual hematopoietic chimerism was determined by a quantitative PCR approach based on amplification of short tandem repeat (STR) markers. Amplified fragments were separated by capillary electrophoresis using the ABI prism 300 sequencer (Applied Biosystems). For detailed evaluation, the corresponding peak area values were exported to the GenotyperTM software. The percentage of donor and recipient DNA was calculated from individual proportions of donor and recipient peak areas in relation to the summation of all signals. The sensitivity to detect minor alleles was found in the range of 1%. As a quality control, a follow-up probe was analysed together with the former sample throughout.
Definition of chimerism status and response
A lack of any signal from autologous cells was defined as complete chimerism (CC). Probes of patients that constantly showed autologous signals that did not exceed 30% were specified as stable MC. Follow-ups of patients with MC and declining autologous signals were defined as decreasing MC. A significant increase (5% or more) of autologous cells, which, furthermore, exceeded 30%, was defined as increasing MC.
Strategy for additional immunotherapy
We defined a limit for immunological intervention when we experienced two relapses in patients with increasing MC, exceeding 30% of autologous cells. This cutoff level was then chosen as a result of our experience in the pre-emptive treatment of children with advanced hematological malignancies and increasing MC.
In these patients, we found a low risk to induce severe GVHD by pre-emptive immunotherapy, when treatment was initiated in a stage where patients had high levels of autologous cells. In addition, different previous studies in patients with SAA indicate that lower levels of stable MC were not correlated with an enhanced risk for graft rejection and vice versa.8,9
Taken together, we planed a stratified immunological intervention for such patients:
(1) Immunotherapy for patients receiving CSA should consist of immediate discontinuation of the immunosuppressive agent. Chimerism should then be assayed weekly until CC status would be restored. If MC would continue to increase after cessation of CSA, a DLI should be given. (2) Immunotherapy for patients not receiving CSA should consist of DLI as frontline treatment. Patients who nonetheless would show a further increasing MC should receive additional DLI after at least 3 weeks had elapsed. The cell dose administered should be based on the number and potential severity of HLA mismatches between the donor and recipient, and ranged from 2.5 × 104 to 1 × 105/kg BW. However, the final decision was up to the treating physician.
The CD3-positive cells were collected out of the donor's peripheral blood by positive selection using the magnetic activated cell sorting (MACS) technique provided by Milteny Biotec (Bergisch-Gladbach, Germany). The CD3-positive cells were counted by flow cytometry and frozen in aliquots with 2.5 × 104 cell/kg BW of the patient.17,18
However, immunotherapy was performed only in two patients with increasing MC, where CD34 enrichment instead of CSA was taken for GVHD prophylaxis. Therefore, only DLI could be taken as frontline treatment here.
In all, 32 patients with SAA received allo-SCT and two of them experienced a second allo-SCT after graft rejection. Altogether, 23 of these 34 courses permanently showed CC and 22 of the patients with CC remained in continuous complete remission (CCR). One patient with CC died from infectious disease when severe GVHD had to be treated with an intensive immunosuppressive regimen. Four patients with a decreasing MC and two patients with a stable MC remained in CCR. Despite great efforts, one patient (UPN 32) unfortunately was not engrafted, showed autologous cells throughout and died from infectious disease (Table 1).
Four patients developed an increasing MC with more than 30% autologous cells. The first two of them (UPN 29 and UPN 30, Table 1) rejected their graft, received a second allo-SCT, revealed a CC then and remained in CCR. When two further patients (UPN 27 and UPN 28, Table 1) with increasing MC exceeded 30% of autologous proportions, immunological intervention was initiated. As both patients got CD34 enrichment and not CSA as GVHD prophylaxis, they received immunotherapy with repetitive low-dose DLI as frontline therapy consisting of 2.5 × 104 cell/kg BW CD3-positive T-cells.
One patient (UPN 28) received two DLI series on days +60 and +189. Hematopoiesis showed a shift after the first DLI, which was seen in a decrease of autologous cells from 30% at day +60 to 5% at day +87. Nonetheless, this patient showed a second increase of autologous cells exceeding even 80% at day +170, and received a second DLI series starting at day +189. Autologous proportions again decreased below 30% and persisted in this range (Figure 1). The patient remained in CCR and has been in an excellent clinical condition for 40 months now after allo-SCT.
The second patient (UPN 27) received repetitive DLIs with 2.5 × 104 cell/kg BW CD3-positive T-cells on days +38, +54, +82, +104 and +118, after autologous cells exceeded 30% at day +38. This patient responded to treatment as well, showed an impressive decrease of autologous proportions, a stable MC with autologous proportions in the range of 10% and remained in CCR for now 56 months after allo-SCT (Figure 2).
Acute GVHD grade II–IV was observed in six patients, but not in those who received DLI. However, severe GVHD was more frequent in the patients with CC compared to MC. In all, 5/23 patients with CC developed GVHD grade II–IV (22%), compared to 1/10 patients with MC (10%) (Table 2). Severe GVHD could be controlled in all but one patient. None of the two patients who received additional immunotherapy developed acute or chronic GVHD.
The introduction of fluorescence-based STR–PCR techniques allowed high throughput serial and quantitative assessments of hematopoietic chimerism in short time intervals.15,16 Most of those studies on children after allo-SCT were either focused on more or less homogeneous groups of malignant diseases or on heterogeneous groups where different malignant and nonmalignant diseases were combined. Thus, there exist only a few studies exclusively focused on children with SAA. These studies already give evidence that an SAA patient with a stable MC and low proportions of autologous cells generally remain in CCR, and that an SAA patient with a higher level of autologous cells more likely rejects his graft. Therefore, lower levels of stable MC seem to be not accomplished with an enhanced risk for graft rejection and vice versa.7,9,10,2,21,22
At the beginning of our study, there was also growing evidence that patients after allo-SCT profit from an early and restrained immunological intervention under specific circumstances. This was shown in recent studies on children with acute leukemias after allo-SCT performed by us, where pre-emptive immunotherapy in the stage of increasing MC could effectively prevent relapse in patients with acute leukemias and MDS.12,13,23,24 Either cessation of CSA or low-dose DLI seems to offer a disease-independent potential (1) to shift the proportions of donor to recipient cells, (2) to increase the alloreactive potential of the graft and (3) to achieve a stable engraftment with a low risk to induce severe GVHD. This appears to be of great advantage compared to late interventional strategies, when autologous cells already dominate and transfusion of higher lymphocyte quantities might induce a profound marrow aplasia together with an enhanced risk to induce a severe GVHD.25 To identify the high-risk SAA patients and the optimal timing for immunological intervention, we studied numerous courses of SAA patients after allo-SCT, until we would experience at least two patients with graft rejection. As, in both patients, autologous cells exceeded 30% before graft rejection invariably happened, we considered this as a limit where an intervention is justified. Graft rejection could then be prevented in two patients with this constellation in advance, and this was achieved with only 2.5 × 104 cells/kg BW. A severe GVHD was not seen by this approach, but the theoretical risk to induce GVHD should be kept in mind.
Although the study group of patients with SAA presented here is small and only four patients could be assigned to the high-risk group, it, nonetheless, represents a relatively large study on this rare entity in children. Two patients with a graft rejection compared to two patients with immunological intervention followed by remission are statistical, not calculable, but reflect impressive results on the background of other studies, and possibly an evidence-based standard for future interventions.
In summary, we could show that SAA patients with increasing MC and the highest risk to develop graft rejection can be identified by serial and quantitative analysis of hematopoietic chimerism so early, that a restrained immunological intervention can still prevent graft rejection with a calculable risk to induce severe GVHD.
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We thank all the participating centers and all colleagues who also included less than three patients into the study: Professor Dr B Kremens, University Children's Hospital Essen; Professor Dr C Niemeyer, University Children's Hospital Freiburg; Professor Dr A Reiter, University Children's Hospital Gießen; PD Dr KW Sykora, University Children's Hospital Hannover.
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Hoelle, W., Beck, J., Dueckers, G. et al. Clinical relevance of serial quantitative analysis of hematopoietic chimerism after allogeneic stem cell transplantation in children for severe aplastic anemia. Bone Marrow Transplant 33, 219–223 (2004). https://doi.org/10.1038/sj.bmt.1704337
- increasing mixed chimerism
- graft rejection
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