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Increasing Mixed Chimerism Is an Important Prognostic Factor for Unfavorable Outcome in Children With Acute Lymphoblastic Leukemia After Allogeneic Stem-Cell Transplantation: Possible Role For Pre-Emptive Immunotherapy?

From the University Children's Hospital, and University of Tübingen, Department of Medical Biometry, Tübingen; University Children's Hospital, Essen; University Children's Hospital, Duesseldorf; Hannover Medical School, Children's Hospital, Hannover; University Children's Hospital, Freiburg; University Children's Hospital Charité, Berlin; University Children's Hospital, Jena; University Children's Hospital, Greifswald; University Children's Hospital, Frankfurt, Germany; St Jude Children's Research Hospital, Memphis, TN

Address reprint requests to Peter Bader, PD, MD, University Children's Hospital, Department of Pediatric Hematology and Oncology, Hoppe-Seyler-Strasse 1, D-72070 Tübingen, Germany; e-mail: peter.bader{at}med.uni-tuebingen.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: We recently reported that children with acute leukemias who show increasing mixed chimerism (MC) after allogeneic stem-cell transplantation have a significantly enhanced risk of relapse. Here we present the results of a prospective multicenter study to investigate (1) whether relapse of acute lymphoblastic leukemia (ALL) can be determined in advance by serial analysis of chimerism, and (2) if outcome can be influenced by withdrawal of immunosuppression and/or by low-dose donor lymphocyte infusion when increasing MC is detected.

PATIENTS AND METHODS: Serial and quantitative analysis of chimerism was performed using a fluorescent-based short-tandem-repeat–polymerase chain reaction in 163 children with ALL.

RESULTS: One hundred one patients revealed complete chimerism (CC) or low-level MC (CC/low-level MC); increasing MC was found in 46 patients; and decreasing MC, in 16 patients. Relapse was significantly more frequent in patients with increasing MC (26 of 46) than in patients with CC/low-level MC (eight of 101) or in patients with decreasing MC (0 of 16; P < .0001). The probability of 3-year event-free survival (EFS) was 54% for all patients, 66% for patients with CC/low-level MC (n = 101), 66% for patients with decreasing MC (n = 16), and 23% for patients with increasing MC (n = 46; P < .0001). Of the 46 patients with increasing MC, 31 received immunotherapy. This group had a significantly higher 3-year EFS estimate (37%) than the 15 patients who did not receive immunotherapy (0%; P < .001).

CONCLUSION: Serial analysis of chimerism reliably identifies patients at highest risk to relapse. The 3-year EFS of patients with increasing MC without immunotherapy was 0%, by which overt relapse could be prevented in a considerable group of patients.

	INTRODUCTION

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Allogeneic stem-cell transplantation (alloSCT) is now performed with increasing success for children with particularly high-risk acute leukemia. Disease recurrence remains the most important barrier to the success of this treatment option.¹ After recurrence, only another alloSCT can induce further long-term remission, but it carries a high rate of procedural mortality.^2-5 In recent years, alternative approaches aimed at augmenting or inducing an immune graft-versus-leukemia (GVL) effect have been used to treat leukemia that has relapsed after alloSCT.^6-14 Such approaches include abrupt cessation¹⁵ or rapid tapering of cyslosporine (CSA),¹⁶ administration of cytokines,^9,14,17 and donor lymphocyte infusion (DLI) with or without cytokines.^18,19 Although the benefit of immunotherapy for chronic myelogenous leukemia (CML) is well documented,^6,7,20-23 there are fewer reports of success in patients with acute leukemia.^9-11,18,24

Clinical response to immunotherapy has usually been associated with measurable graft-versus-host disease (GVHD), possibly caused by high doses of donor cells given to patients in frank hematologic relapse. However, there is evidence that low doses of donor T cells may also induce an effective immune response and produce long-term remission in patients whose leukemia burden is small.^18,25-27 Our recent prospective study demonstrated that patients with acute leukemia who show increasing autologous marrow repopulation (increasing mixed chimerism [MC]) after alloSCT are at a significantly greater risk of relapse than those who do not show increasing MC (P < .0001).^28,29 Here, we report the results of a large, prospective, multicenter trial of patients with acute lymphoblastic lymphoma (ALL) showing that (1) serial analysis of chimerism reliably identifies patients with highest risk of relapse, and (2) that overt hematologic relapse of ALL can principally be prevented by withdrawal of CSA and/or administration of low-dose DLI on the basis of chimerism results.

	PATIENTS AND METHODS

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Patients
Between January 1996 and December 2001, 163 children who had received alloSCT for ALL underwent transplantation in 13 pediatric transplantation centers in Germany. Posttransplantation samples for evaluation of hematopoietic chimerism were sent to the study center in Tübingen, Germany. The study protocol was approved by the clinical ethics committee of the University of Tübingen, and the study was conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from the patients and parents according to institutional guidelines. Data were obtained for analysis until May 2002. Well-defined, high-risk patients in complete remission 1 were recommended to undergo alloSCT according to the guidelines of the German ALL-BFM-95 multicenter trial for primary ALL, and patients in complete remission 2, according to the German ALL-REZ-BFM-96 multicenter trial for relapsed-state ALL. Patient characteristics are summarized in Table 1. The median age was 9.3 years, and the median duration of follow-up was 1.37 years. Table 1 summarizes the types of stem-cell donors, the sources of stem cells, and the conditioning regimens. GVHD prophylaxis was performed uniformly in patients who received their grafts either from a matched family donor (MFD) or from a matched unrelated donor (MUD). GVHD prophylaxis for MFD consisted of 3 mg/kg CSA starting on day 1. GVHD prophylaxis for MUD consisted of methotrexate administered additionally on days 1, 3, and 6. GVHD prophylaxis was performed variably in patients who received T-cell–depleted stem cells from a mismatched unrelated donor or mismatched family donor.

Immunotherapy for patients receiving CSA consisted of immediate discontinuation of the immunosuppressive agent. Chimerism was then assayed weekly until complete chimerism (CC) status was restored. If MC continued to increase after cessation of CSA, a DLI was given.

Immunotherapy for patients not receiving CSA consisted of DLI as frontline treatment. The cell dose administered was based on the number and potential severity of human leukocyte antigen mismatches between the donor and recipient, and ranged from 2.5 x 10⁴ to 1 x 10⁶ per kilogram of body weight. After DLI, chimerism status was assayed weekly until CC status was restored. Patients who showed a further increase in MC were given an additional DLI after at least 3 weeks had elapsed.

Definition of Chimerism Status and Response
The patients were subdivided exclusively on the basis of serial analysis by STR—polymerase chain reaction, within the limits of sensitivity (approximately 1%). Patients who showed no evidence of autologous cells at any time posttransplantation were considered to have CC. Additionally, patients who showed weak autologous signals or signals that were not confirmed by a repeat analysis 1 week later were defined as having low-level MC. Patients who showed a marked increase (5% or more) in the proportion of autologous cells between two consecutive assessments were defined as having increasing MC. Patients who showed autologous signals immediately posttransplantation that decreased during follow-up were categorized as having decreasing MC.

Response was defined as a return to CC. GVHD was graded according to clinical criteria, as previously described.^34,35

Statistical Methods
The probability of survival and of event-free (EFS) and relapse-free survival was estimated by the Kaplan-Meier method.³⁶ Univariate analyses of prognostic factors were performed by using the log-rank test and Fisher's exact test. Multivariate analyses have been performed using Cox proportional hazards model to analyze simultaneously different risk factors.

	RESULTS

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Patients
Of the 163 patients studied, 101 showed either CC (n = 59) or low-level MC (approximately 1% autologous cells; n = 42), 46 patients developed increasing MC, and 16 showed decreasing MC. All patients were in complete hematological remission at the time at which MC was detected the first time. Of the 46 patients with increasing MC, pre-emptive immunotherapy was offered to 31. Fifteen patients with increasing MC received no additional treatment. Although the study protocol assigned all patients with increasing MC to be treated as described above, each transplantation center made the final decision on whether to start immunotherapy. Four patients' physicians decided against treatment on the basis of the chimerism results. Two patients did not achieve stable engraftment. One patient was not treated because she had a severe viral infection. In two patients, relapse occurred before treatment could be initiated. In six children, their MUDs were not available when increasing MC was detected, and they all went on to overt relapse. Characteristics of patients with increasing MC are shown in Tables 2 and 3. Univariate analyses revealed that none of the parameters of sex, other category of donor, number of remissions, or conditioning regimen, showed any significant association to increasing MC. Patients who received T-cell—depleted stem cells developed increasing MC more frequently compared with the remaining patients (42% [28 of 67] v 19% [18 of 96]; P < .005). In the mismatched family donor subgroup, the frequency of increasing MC was 43% (13 of 30) compared with 19% (nine of 48) in the group of patients who received their transplant from an MFD (P < .05).

Survival
The probability of 3-year EFS was 54% for all 163 patients (Fig 1). Patients who showed CC or low-level MC (n = 101), or who showed decreasing MC (n = 16), had significantly higher 3-year EFS estimates (66% and 66%, respectively) than did patients with increasing MC (23%; P < .0001; Fig 2). By contrast, patients with increasing MC who received additional immunotherapy (n = 31) had an estimated 3-year EFS of 37%, while that of similar patients who did not receive additional treatment (n = 15) was 0% (P < .005; Fig 3).

View larger version (11K): [in this window] [in a new window]   Fig 1. Kaplan-Meier analysis of event-free survival (EFS) for all study patients (N = 163). The 3-year EFS estimate was 54%.

View larger version (17K): [in this window] [in a new window]   Fig 2. Kaplan-Meier analysis of event free survival (EFS) according to chimerism status. CC/LL-MC, complete chimerism/low-level mixed chimerism; de-MC, decreasing mixed chimerism; in-MC, increasing mixed chimerism.

View larger version (13K): [in this window] [in a new window]   Fig 3. Kaplan-Meier analysis of event-free survival (EFS) in patients with increasing mixed chimerism (MC). Patients with increasing MC (in-MC) who received additional therapy (A, n= 31) and patients with increasing MC who did not receive additional treatment (B, n = 15).

Toxicity and GVHD
GVHD showed higher frequencies in patients with CC/low-level MC (69% [70 of 101]) or decreasing MC (69% [11 of 16]) than in patients with increasing MC before pre-emptive immunotherapy (17% [8 of 46]; P < .0001; Table 6).

In the group of 31 children with immunotherapy, three died of transplant-related toxicity (infection, n = 2; acute GVHD, n = 1), one patient died of multiple organ failure during the course of an overwhelming virus infection, and one patient developed grade 2 acute GVHD of the intestines that required additional immunosuppression (this patient responded to immunotherapy but died of severe infection with multiple-organ involvement). The third patient did not respond to low-dose DLI and progressed to frank hematological relapse. Two additional DLIs with increasing cell doses (up to 5 x 10⁶) were given. The patient's leukemic blast cells disappeared, and complete donor chimerism was restored; however, the patient died on day +323 of severe acute GVHD of the skin, liver, and intestines.

	DISCUSSION

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Despite the completion of several studies, the efficacy of immunotherapy after allogeneic transplantation for acute leukemia is still the subject of debate.^6-9,18,37

The results reported here demonstrate that serial characterization of posttransplantation chimerism offers the possibility of identifying those patients who are at highest risk to develop relapse. Moreover, these results show that many patients with increasing MC, who otherwise face almost certain relapse and death, can be rescued by additional immunotherapy. The probability of 3-year EFS in patients with increasing MC was 0% in the untreated group, but 37% in patients who received early treatment. We acknowledge that because this study was not randomized, we cannot rule out selection bias as a factor in our results. However, the striking difference between the outcomes of treated and untreated patients is likely to reflect the effect of therapy, as the two groups of patients with increasing MC with or without prophylactic therapy did not differ with regard to relevant transplant factors such as donor, type of graft, conditioning regimen, or T-cell depletion of the graft.

A GVL reaction in patients with ALL is suggested by the higher incidence of relapse in the absence of GVHD^38-40 or with the use of T-cell–depleted allografts.⁴¹ These experimental results impressively mirror our finding that the highest frequency of increasing MC was detectable in those cohorts that received T-cell–depleted stem cells. This confirms that a T-cell depletion substantially reduces the alloreactivity of a graft, facilitates the recurrence of autologous hematopoiesis, and allows the underlying disease to newly expand.^42-44 Additional support for the GVL effect in patients with ALL can be found in a study from Locatelli et al in which it was reported that low-dose CSA reduces the risk of relapse in children with acute leukemia receiving grafts from human leukocyte antigen–identical siblings.⁴⁵ Slavin et al^18,25 reported the first successful implementation of DLI in a patient whose ALL relapsed after alloSCT. Since that time, experience with immunotherapy^15,46-48 has shown that DLI initiated during frank hematologic relapse induces complete remission in 8% of patients with ALL and in 22% of patients with acute myeloid leukemia. If tumor burden is reduced by chemotherapy before DLI, the rate of complete response is significantly improved (to 33% in ALL and 37% in acute myeloid leukemia).^49,50 These results suggest that immunotherapy offers the greatest benefit to patients with acute leukemia when it is administered before hematologic relapse occurs.^18,25,27 Therefore, it is desirable to identify patients who are at greatest risk of relapse early, so that additional immunotherapy can be delivered as prophylaxis.

Immunotherapy after alloSCT may therefore be useful in increasing the alloreactive potential of the graft and augmenting a possible GVL effect. Additionally, the implementation of treatment in an early phase of impending relapse offers the possibility of using cessation of CSA or low-dose DLI, which may be less likely to cause severe acute GVHD.⁵¹

Patients whose frontline treatment was withdrawal of CSA had a higher rate of response (restoration of CC) than patients who received DLI as frontline treatment (P < .05). Response to cessation of CSA was consistently prompt, with a decrease in autologous cells as soon as 1 week afterward. We hypothesize that the increase of donor chimerism after withdrawal of CSA cleared residual disease by reinducing or augmenting the GVL effect. DLI as frontline treatment was less successful. The GVL response is thought to be a complex, multistep process that involves activation of donor T-cells by antigens, clonal expansion of the activated T-cells, and differentiation of these cells into helper or cytotoxic effectors. Therefore, clonal expansion of transfused T-cells is not likely to occur before 3 to 4 four weeks after transfusion. This delay could allow rapidly evolving leukemia cells to proliferate and progress to overt relapse before the GVL effect is augmented by the donor graft. In the majority of patients who had no response to DLI, leukemia progressed to frank hematologic relapse. In such cases, the administered cell doses would have been too small to treat disease recurrence by induction of a GVL effect. Therefore, coadministration of cytokines may be an option to improve the immunogenicity of DLI given as pre-emptive therapy, as it has been shown that costimulation with interleukin-2 to promote the in vivo clonal expansion of the transfused T-cells has induced remission in patients who had no response to DLI alone.¹⁸

Severe acute or chronic GVHD may result from higher cell doses and/or from nonspecific cytokine-mediated stimulation of the alloreactive capacity of infused donor lymphocytes. According to Collins et al, approximately 60% of patients who receive DLI experience acute or chronic GVHD.^15,48 In adult patients, cell doses below 10⁷ per kilogram of body weight are believed not to induce GVHD. In children, however, a cell dose of 10⁵/kg from an unrelated donor can result in uncontrollable and fatal GVHD, despite CC status (T. Klingebiel, unpublished data). In the present study, the administration of low-dose donor T-cells to patients with MC caused no cases of fatal GVHD, except for one patient in whom a dose escalation has been performed because she did not respond to initial low-dose DLI.

Relapse of acute leukemia, especially ALL, is different from relapse of CML. In most patients with CML whose chimerism status has been investigated, a gradual increase in recipient hematopoietic cells was found before relapse occurred. In contrast, relapse of acute leukemia is usually rapid. In a few such patients, bone marrow relapse has occurred without prior detection of recipient cells in the peripheral blood²⁹ or has occurred 1 week after testing showed CC in the peripheral blood.⁴² Most patients at the highest risk of relapse can be identified by frequent monitoring of chimerism status by the semiquantitative method we have described. As shown in this study, virtually all relapses can be detected in advance. However, this technique has limited sensitivity—its lower limit of detection is one recipient cell in 100 donor cells.^52-54 More sensitive techniques, such as real-time quantitative polymerase chain reaction approaches for the characterization of single nucleotide polymorphisms might help to improve chimerism surveillance. Nonetheless, in our study, in contrast to others,^55,56 all patients who remained free of adverse events had immunotherapy that converted increasing MC to CC, whereas no patient with ALL and increasing MC survived without additional treatment. This approach therefore allows the administration of additional treatment according to the individual risk profile of the patient.

In conclusion, our study clearly demonstrates that semiquantitative serial analysis of hematopoietic chimerism is a powerful tool for predicting relapse in children after alloSCT for ALL. Children who developed increasing MC did not survive in this cohort of patients unless pre-emptive immunotherapy could convert increasing MC back to CC. Therefore, adjuvant immunotherapy on the basis of increasing MC seems to be a rational and effective treatment option to treat impending relapse in these children. However, further studies (eg, using dose escalation strategies combined with coadministration of cytokines) are needed to improve adjuvant immunotherapy for the prevention of relapse.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We thank all other participating centers and all colleagues who included fewer than 10 patients into the study: Professor Dr W. Holter, University Children's Hospital Erlangen; Professor Dr A. Reiter, University Children's Hospital Gießen; Professor Dr S. Müller-Weihrich, University Children's Hospital München-Schwabing; Dr I. Schmidt, München v. Haunersches Kinderspital; Dr A. Schulz, University Children's Hospital, Ulm; Professor Dr C. Bender-Götze, München-Poliklinik; PD Dr J. Vormoor, University Children's Hospital Münster. We are grateful to Sharon Naron for excellent editorial assistance. We thank all colleagues who contributed fewer than 10 patients: Professor Holter, Erlangen; Professor Reiter, Giessen, Professor Müller-Weihrich, München-Schwabing; Dr Schmid, München, v. Haunersches Kinderspital; Dr Schulz, Ulm; Professor Bender-Götze, München; PD Vormoor, Münster; and Sharon Naron for excellent editorial assistance.

	NOTES

 
Supported by the Deutsche Krebshilfe (70-2178-Kl I), Bonn, Germany; the Fortüne Program of the University of Tübingen; and by the Förderverein für Krebskranke Kinder Tübingen e.V., Tübingen, Germany.

James F. Beck and Thomas Klingebiel contributed equally to this manuscript.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted May 30, 2003; accepted February 23, 2004.

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