¹Institute of Hematology and Blood Transfusion, Prague, Czech Republic

²2nd Department of Pediatrics, 2nd Medical School, Prague, Czech Republic

Correspondence to: Dr R Formánková, 2nd Department of Pediatrics, 2nd Medical School, Charles University Prague, V Úvalu 84, CX-150 06, Prague 5, Czech Republic

We report a case of a 13-year-old boy who was transplanted for relapse of acute myeloid leukemia (AML). A detailed study of hematopoietic chimerism was performed using polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) at very short time intervals. We used discontinuation of post-transplant immunosuppression and donor lymphocyte infusions (DLI) in order to prevent leukemia relapse that was indicated by a progressive increase in autologous hematopoiesis. Despite the fact that the boy relapsed 10 months after BMT, we could see a significant influence of adoptive immunotherapy on the mixed chimerism status during the post-transplant period. Bone Marrow Transplantation (2000) 25, 453-456.

mixed chimerism; acute myeloid leukemia; relapse; adoptive immunotherapy

Allogeneic bone marrow transplantation (BMT) is a successful treatment for selected pediatric patients with acute and chronic leukemias. Nevertheless, relapse of leukemia is still the most frequent cause of treatment failure. While in patients with chronic myeloid leukemia (CML) a durable complete remission can be achieved using methods of adoptive immunotherapy^1,2,3 in the treatment of post-transplant hematologic relapse, poor results have been reported in acute leukemias. A number of studies have addressed the clinical significance of mixed hematopoietic chimerism^4,5 (MC) in predicting relapse after BMT, mainly in CML. Recently Bader et al⁶ demonstrated a highly significant correlation between increasing MC and relapse of acute leukemia in a group of 55 pediatric patients, and the question whether there is a possibility of relapse prevention using methods of adoptive immunotherapy in the early phase of developing relapse was raised. We report the case of a 13-year-old boy transplanted for AML in whom a detailed study of mixed hematopoietic chimerism was made using PCR of VNTR and who underwent adoptive immunotherapy because of a progressive increase in autologous hematopoiesis.

Case report

A 13-year-old boy was initially diagnosed with AML-M1 in September 1987. He was treated with courses of vincristine, thioguanine, cyclophosphamide, daunorubicin and cytarabine and sustained the first remission for 10 years. He relapsed with AML-M1 in October 1997 and received treatment according to the protocol for AML-REZ-BFM-93. The second remission was achieved and in January 1998 he underwent BMT from his HLA-identical brother. The conditioning regimen consisted of busulphan (16 mg/kg) and cyclophosphamide (120 mg/kg). A combination of cyclosporine (3 mg/kg starting day -1) and a short course of methotrexate (10 mg/m² on days +1, +3 and +6) was used as GVHD prophylaxis. He engrafted neutrophils and thrombocytes on day +17. A BM aspirate on day +21 showed good cellularity with trilineage engraftment. The boy developed graft-versus-host disease (GVHD) grade II (GIT symptoms) on day +26 and treatment with methylprednisolone was started. The patient was discharged home on day +37 on a reducing dose of steroids, in a good clinical condition and with a normal peripheral blood count.

Detailed monitoring of hematopoietic chimerism in the peripheral blood is shown in Figure 1. The results of chimerism in the peripheral blood, the granulocyte and mononuclear cell fractions of bone marrow and the T lymphocyte and granulocyte subpopulations of the peripheral blood are compared in Table 1. Mixed chimerism with only a very low intensity of donor alleles was detected on day +8. On day +14 we could still see approximately 40% of autologous hematopoiesis using densitometric analysis of PCR products. From day +21 until day +49 a stable low level of autologous hematopoiesis was detected in peripheral blood samples (10-12%). The BM aspirate taken on day +21 showed MC in granulocyte and mononuclear fractions. On day +28 we performed separation of the T lymphocyte fraction of the peripheral blood in which we detected approximately 30% autologous hematopoiesis in comparison with only weak signal of autologous hematopoiesis in peripheral granulocytes (about 5%).

Following the increasing MC post-transplant immunosuppression was stopped on day +63, but the patient responded only transiently and chimerism studies showed increasing recipient allele intensity again until day +100. Therefore donor lymphocyte infusion was indicated. The boy received the first course of DLI (1 ´ 10⁵/kg CD3⁺ cells) on day +127 and the second course (5 ´ 10⁵/kg CD3⁺ cells) on day +169. Within 3 weeks after both the first and the second DLI we could observe a trend to a decrease but not disappearance of specific recipients alleles. Decreasing MC was seen also in the T lymphocyte fraction of the peripheral blood. Although the bone marrow aspirate taken on day +180 confirmed hematological remission, MC was detected in the granulocyte and mononuclear fractions of BM. Starting from day +252, the patient showed an increasing MC in peripheral blood samples. An increasing intensity of autologous alleles was seen also in the T lymphocyte fraction of peripheral blood in contrast with only weak signal of MC in peripheral granulocytes. Since microscopic examination of BM smear on day +273 revealed sporadic cells containing Auer rods, more detailed immunophenotyping was performed at this point. It documented the presence of 2% of immature (CD117⁺, CD34⁺) myeloid cells with an aberrant expression of CD7 and/or asynchronous expression of CD15. From this point on, the level of minimal residual disease (MRD) was also followed by three-color flow cytometry. The boy received the third DLI (1 ´ 10⁸/kg CD3⁺) on day +276 with only a very small and transient subsequent decrease of MC. Full hematological relapse of AML-M1 in the bone marrow aspirate was diagnosed on day +295 after BMT. Both the aberrant expression of CD7 and/or the asynchronous expression of CD15 were confirmed on a subset of CD34⁺, CD117⁺, CD33⁺ cells. Treatment with intensive chemotherapy was immediately started and he achieved remission after the first cycle of chemotherapy. For the second allogeneic transplantation he was prepared with a conditioning regimen consisting of total body irradiation (TBI) (12Gy) and melphalan (140 mg/m²) with the same GVHD prophylaxis. The second transplant with peripheral blood stem cells from the same donor was performed in January 1999, 12 months after the first BMT. From day +8 after the second BMT, complete chimerism (CC) was detected in all peripheral blood samples taken weekly until day +100 and at least one a month in the subsequent period. CC was seen repeatedly also in the T lymphocyte fraction of peripheral blood. The frequency of CD7⁺, CD117⁺, CD33⁺ cells was less than 1 in 10^-4. Cells with an asynchronous CD15 expression (CD15⁺, CD117⁺, CD33⁺) were detectable at 0.1%. Bone marrow aspirates on day +100 and day +180 confirmed hematological remission and the boy remains well with no evidence of GVHD 8 months after second allogeneic BMT.

Methods

Genomic DNA was isolated from peripheral blood using the modified salting out method,⁷ from Ficoll separated mononuclear and granulocyte fractions of bone marrow or from granulocyte, mononuclear and T lymphocyte fractions of peripheral blood. Pre-transplant recipient and donor DNA samples were amplified with six different pairs of primers to choose the best informative VNTR systems. The loci MCT118⁸ and YNZ22⁹ were selected with a maximum sensitivity of 0.5%. PCR products were separated by electrophoresis in 1.5% agarose gel, stained with ethidium bromide. DNA fragments were visualized on an ultraviolet transilluminator and photographed. Signals were analyzed densitometrically⁶ and results were analyzed using the software GelBase (Ultraviolet Products Ltd, Cambridge, UK). Peripheral blood samples were taken weekly from day +8 until day +100 after BMT and then at least once a month. If MC was detected, we intensified follow-up.

Standard direct three-color fluorescence flow cytometry on FACSCalibur (San Jose, CA, USA) was performed. For MRD analysis the live-gating procedure for CD33⁺ cells was used. Prior to each tube one blank tube with distilled water was run for 2 min to ensure the tubing was clear; if more than 30 events were acquired, cleansing of the cytometer was repeated.

Discussion

It is widely recognized that patients suffering from CML in whom MC is detected after BMT have an increased risk of developing relapse and it was demonstrated that MC has prognostic value to predict relapse also in patients with acute leukemias.^6,10 In contrast to patients with relapse of CML after BMT, in whom a durable complete remission can be achieved using DLI, the question of using adoptive immunotherapy in patients with acute leukemia is still unclear. It results from earlier studies^1,2 that starting the treatment with adoptive immunotherapy in frank hematologic relapse has no or only a very limited effect in acute leukemias. However we assume that also in patients with acute leukemia it is possible to use successful methods of adoptive immunotherapy when the treatment is started early in leukemia recurrence.

Relapses of acute leukemias usually develop within a very short time in comparison to chronic leukemias. We consider PCR of VNTR sequences as a simple and rapid method which allows a very intensive monitoring scheme. Monitoring of hematopoietic chimerism at short time intervals allows early prediction of relapse in children with acute leukemia and allows the use of different forms of adoptive immunotherapy (discontinuation of post-transplant immunosuppression or DLI) in the period of minimal disease burden.

Bader et al¹¹ demonstrated two cases of children with AML and MDS, respectively, in whom a long-lasting remission was achieved using adoptive immunotherapy. Unfortunately, in our case withdrawal of post-transplant immunosuppression together with DLI did not prevent relapse in a boy with acute leukemia. However, we could see a significant decrease of mixed chimerism in relation to discontinuation of post-transplant immunosuppression and with application of DLI. We suppose that recurring even if short-term effects of adoptive immunotherapy probably set back the relapse and extended continuation of remission after BMT that allowed us to perform the second transplant.

Our observations confirm the fact that the antileukemic effect of allogeneic lymphocytes and discontinuation of post-transplant immunotherapy can efficiently enhance the GVL effect, deficiency of which is one of the factors responsible for relapse of leukemia.

Relapses in children transplanted for acute leukemia remain the most important cause of treatment failure. Detailed monitoring of hematopoietic chimerism in the post-transplant period allows identification of patients with high risk of developing relapse who are indicated for treatment with adoptive immunotherapy. Further studies must confirm the efficiency of post-transplant adoptive immunotherapy in children with acute leukemia and find the best strategy for its timing and dosage in different stages of myeloid and lymphoid leukemia.

Acknowledgements

We would like to acknowledge Dr Ond&rcaron:ej Hrušák for MRD analysis. We thank Dr Jan Trka for his advice in the preparing of the manuscript. This work was supported in part by grants IGA-4918-3 and IGA-4554-3 from Czech Ministry of Health.

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Figure 1 Monitoring of MC in peripheral blood.

Table 1  Comparison of MC results in peripheral blood, bone marrow and T cell and granulocyte subpopulation of peripheral blood