In recent pediatric collaborative studies of acute myeloid leukemia (AML), patients with Down's syndrome (DS) have better outcome than other patients when they were treated according to their intensive AML protocols. This may be attributed to enhanced sensitivity of DS AML cells to selected chemotherapeutic agents. We evaluated a less intensive chemotherapeutic regimen which was specifically designed for children with AML-DS. Remission induction chemotherapy consisted of daunorubicin (25 mg/m2/day for 2 days), cytosine arabinoside (100 mg/m2/day for 7 days), and etoposide (150 mg/m2/day for 3 days). Patients received one to seven courses of consolidation therapy of the same regimen. Thirty-three patients were enrolled on the study and their clinical, hematologic and immunophenotypic features were analyzed. Of the 33 patients, all were younger than 4 years and diagnosed as having acute megakaryoblastic leukemia or myelodysplastic syndrome. All patients achieved a complete remission and estimated 8 year event-free survival rate was 80 ± 7%. Three patients relapsed and two died due to cardiac toxicity and one due to septic shock. The results of our study showed that patients with AML-DS constitute a unique biologic subgroup and should be treated according to a less intensive protocol designed for AML-DS.
In children with Down's syndrome (DS), the clinical and biologic features of acute myeloid leukemia (AML) are quite different from those in children without DS.12 We previously reported the clinical, hematologic, and immunophenotypic features in 20 children with DS.3 All 14 children, 3 years old or younger, had acute megakaryoblastic leukemia (AMKL). These children were characterized as having a history of myelodysplastic syndrome (MDS) and a poor response to chemotherapy. Only one child has remained in continuous complete remission (CR) for more than 1 year. These children were treated by various types of chemotherapy, including acute lymphocytic leukemia (ALL)-oriented chemotherapy, low-dose cytosine arabinoside (AraC) or high-dose AraC.
Based on the results of the retrospective survey, we initiated a multi-center trial to evaluate a chemotherapeutic regimen which was specifically designed for AML-DS. On the other hand, several large collaborative groups that included children with DS in their recent AML protocol reported that children with AML-DS have a superior event-free survival (EFS) rate compared to AML patients without DS.45678 This report describes the clinical and laboratory features as well as the outcome in 33 children with DS included in our AML-Down study.
Patients and methods
Between September 1987 and August 1997, 33 children with newly diagnosed AML/MDS and DS were treated according to the AML-Down protocol in 12 institutions. This analysis was performed in May 1998. The median follow-up period was 41 months, ranging from 9 to 114 months. Patient 1 has been reported previously.3 Informed consent was obtained according to each institution's guidelines before the initiation of therapy. The patients were classified according to the French–American–British (FAB) Cooperative Group Criteria, which defined acute leukemia as the presence of over 30% blasts in the bone marrow.9 The patients with an inspirable bone marrow and more than 30% blasts in their peripheral blood were defined as having suspected acute leukemia.
The bone marrow and peripheral blood samples were stained with Wright–Giemsa stain and cytochemical methods for peroxides, periodic acid-Schiff (PAS), and α-naphthyl butyrate esterase with and without sodium fluoride inhibition. Ultrastructural and ultracytochemical studies were performed on the blasts from 19 patients according to a previously reported method.10 The buffy coat layer of the peripheral blood or bone marrow was fixed in a mixture of paraformaldehyde, glutaraldehyde and tannic acid for the detection of platelet peroxidase (PPO) activity. All specimens were post-fixed with osmium tetroxide, dehydrated, and embedded in epoxy resin for transmission electron microscopy.
For cell marker studies, mononuclear cells were isolated by Ficoll–Hypaque density gradient centrifugation from the bone marrow and/or peripheral blood. In patients who did not have an adequate bone marrow aspirate, the immunologic studies were performed on their peripheral blood mononuclear cells. The cells were analyzed by flow cytometry with a panel of monoclonal antibodies (MoAbs). For the assessment of megakaryocytic lineage, at least 10% of the blast cells need to be positive for the PPO reaction or one or more of the platelet-specific MoAbs. For the scoring of the other immunologic markers, the samples were defined as positive if >20% of the cells were stained.
Remission induction chemotherapy consisted of daunorubicin (25 mg/m2/day on days 1 and 2), AraC (100 mg/m2/day on days 1–7), and etoposide (150 mg/m2/day on days 3–5). Each drug was administered over a 1-h infusion. Pirarubicin (25 mg/m2/day) or mitoxantrone (25 mg/m2/day) was used in 10 patients and in three patients, respectively, instead of daunorubicin. Patients who achieved a CR received one to seven courses of consolidation therapy of the same regimen (five courses, 16; seven courses, four; three courses, three; two courses, two; one course, one). Prophylactic therapy for central nervous system (CNS) involvement was not included in the protocol.
Random-donor platelet transfusions were given to all patients with evidence of bleeding and were given prophylactically whenever the platelet count was below 20 × 109/l. A hemoglobin level of greater than 8 g/dl was generally maintained. Prophylactic oral antibiotics were prescribed for the duration of therapy. Broad-spectrum antibiotics were commenced empirically after appropriate culture in patients who became pyrexial. The antibiotics were altered on the basis of bacteriological isolates when appropriate.
The Kaplan–Meier method was used to estimate the survival curves.11 Hematologic complete remission (CR) was defined by criteria established by the National Cancer Institute (NCI) conference on AML.12 EFS was defined as the time a patient entered the study to induction failure, marrow relapse, or death from any cause. Toxicity was also graded according to NCI criteria. The χ2 test was used to compare the toxicities between induction therapy and the last consolidation therapy.
Clinical and laboratory findings
The clinical and laboratory findings in the 33 patients are summarized in Table 1. All patients were younger than 4 years. Twenty patients were male and 13 were female. Fifteen of the 33 patients had a history of a transient myeloproliferative disorder in neonate. Congenital heart anomalies were present in 18 patients. Nine of these 22 patients with AML or suspected AML had a history of MDS. MDS in children with DS is associated with isolated thrombocytopenia as a common presenting feature. These patients have clinical findings such as pancytopenia, fewer than 30% leukemic blasts in the bone marrow, and dysplastic features in three cell lineages, particularly the megakaryocytes. Transformation to overt leukemia occurred within 12 months from the initial presentation. Their white blood cell counts ranged from 2.1 to 47.3 × 109/l (median: 6.2 × 109/l), their hemoglobin levels from 3.7 to 12.8 g/dl (median: 8.3 g/dl), and their platelet counts from 9 to 184 × 109/l (median: 25 × 109/l).
The immunologic and ultrastructural studies showed that at least one of the megakaryocytic markers (CD41, CD42 or PPO activity) was expressed on the leukemic blasts in all the patients with AML or suspected AML. Based on these findings, all these patients were diagnosed as having acute megakaryoblastic leukemia (AMKL). Expression of glycophorin A on the leukemic blasts was detected in four of the 16 AMKL patients studied. Strikingly, clear coexpression of the CD7 antigen was observed in 20 of the 21 AMKL patients studied.
Cytogenetic results were available in 29 patients. In addition to the constitutional aberrations, other acquired chromosomal abnormalities could be detected in 24 of 29 patients. The modal chromosomal number ranged from 47 to 57. An additional chromosome was found, involving chromosomes No. 8 (n = 8), No. 21 (n = 6) and No. 19 (n = 5). Four patients had monosomy 7 with additional aberrations. Rare translocations, such as t(3;3)(p25;p10), t(2;12)(p13;p11), and t(1;3)(p36;?), were found in some patients. According to the FAB classification, 11 patients had MDS, 18 had AML, and four had suspected AML.
All patients achieved a CR; 30 patients after one cycle of induction therapy and three patients after two cycles. Two patients who relapsed during therapy failed to respond to re-induction therapy and died 6 and 7 months following their relapse. One patient who relapsed after cessation of chemotherapy received re-induction therapy containing high-dose AraC, and achieved a second CR. This patient has remained in a CR for more than 5 years. During CR, two patients died due to cardiac toxicity and one due to septic shock. Except for one patient, all patients have been off therapy for 3 to 105 months. The EFS rate at 8 years is 80 ± 7% (Figure 1).
The toxicity of the regimen was relatively tolerable. However, two patients with congenital heart anomalies developed congestive heart failure and died. One patient died during consolidation therapy and one patient died 34 months after completing therapy. We compared the nonhematologic toxicity of the induction therapy and the last consolidation therapy (Table 2). Although the patients were treated with the same regimen, the incidence of severe infections was more frequent during the induction therapy compared with during the last consolidation therapy.
The present study confirmed our previous report,3 that almost all children with AML-DS can be classified as having FAB M7 by immunophenotyping and electron microscopic studies. Several collaborative study groups recently reported the distribution of FAB subtypes in AML-DS.468 The presence of myelofibrosis and a low percentage of blasts often makes it difficult to classify subtypes according to the FAB criteria. However, every report has confirmed that M7 is the most common form in AML-DS, followed by M0 and M6. The percentage of M7 ranged from 42 to 100% in these studies. The blasts in patients with FAB M0, M6 and M7 are very similar in morphology and it is only possible to discriminate among them using panel of MoAbs and ultrastructural studies. In reports from the Berlin–Frankfurt–Munster (BFM) Group6 and the Pediatric Oncology Group (POG),4 laboratory tests using anti-platelet MoAbs were not performed in all patients with AML-DS. Only our study used an ultrastructural PPO reaction routinely to determine the cell lineage of the leukemic blasts. In light of this, the incidence of FAB M7 might have been underestimated in other studies.
As previously reported, the blasts in patients with DS reacted not only with anti-platelet MoAbs but also with MoAbs expressed on other cell lineages.1314 In our study, the expression of glycophorin A was detected in four of 16 patients. In the BFM Group study, glycophorin A positivity was observed in five of 24 patients.6 Since glycophorin A is expressed only in mature erythroid cells,15 a higher percentage of blasts from patients with AML-DS might be positive for erythroid characteristics if a sensitive method to detect erythroid markers were used. Ito et al16 showed that erythroid-specific mRNAs encoding γ-globin and erythroid δ-amino levulinate synthase were expressed in blasts from all of their patients with M7-DS. Co-expression of the CD7 and CD34 antigens was also frequently noted in our study. In addition to mature T cells, the CD7 antigen is expressed on immature hematopoietic cells.1718 Cells with the potential to form both megakaryocytes and erythroid precursors have been isolated from normal bone marrow cells.19 These findings suggest that leukemic blasts arise from bipotent erythro–megakaryocyte progenitors in some patients with AML-DS.
In our previous study, we reported that the patients with AML-DS had a poor prognosis when treated with various types of chemotherapy.3 In 1992, Ravindranath et al4 reported an EFS rate of 100% in 12 children with AML-DS treated using the POG 8498 protocol. Since then, several large collaborative studies have reported their results using standard AML protocols568 (Table 3). The EFS rates ranged from 48 to 100%, which were much better than those in non-DS children.
Unlike other collaborative study groups, we designed a protocol specifically designed for AML-DS, which was much less dose-intensive compared to other protocols. For example, other protocols employed intensive post-remission therapy with high-dose AraC, the total dose of AraC ranging from 25 to 50 g/m2, while we administered conventional doses of AraC for 1 h (median total dose of 4.2 g/m2, range, 2.8–5.6 g/m2). Because AraC has a brief half-life and is active against proliferating cells during the S phase, its anti-leukemia effect is dose and schedule-dependent.20 Although 1 h infusion of conventional-dose Arac is not an effective way of administration, our protocol was quite effective for AML-DS.
In vitro cytotoxicity assays showed that DS myeloblasts were approximately 10-fold more sensitive to AraC than non-DS myeloblasts.21 This is attributed to the enhanced intracellular metabolism of AraC to its active metabolite, 1-β-D-arabinofuranosylcytosine 5′-triphosphate (ara-CTP). The good response to chemotherapy of AML-DS may be due to the altered metabolism of chemotherapeutic agents, which is possibly the result of a gene dosage effect of one or more genes on chromosome 21.
The Children's Cancer Group (CCG) compared intensive timing with standard timing of induction therapy in children with AML.8 Among children without DS, intensive timing achieved a significantly higher CR rate and better EFS rate than the standard timing of induction. In contrast, intensive timing was associated with a higher mortality and reduced EFS rate in children with DS. In our series, one patient died of septic shock during CR. Impairment in humoral and cellular immunity in DS may lead to a lower tolerance of intensive chemotherapy and a high mortality rate from infections.22
Another problem associated with their treatment of AML-DS is the high frequency of congenital heart disease in patients with DS. This study observed that congenital heart anomalies were present in more than half of the patients with AML-DS. One child with a severe heart anomaly died of congestive heart failure during the second round of consolidation therapy. The other child with an atrial septal defect, who was treated with a cumulative daunorubicin dose of 400 mg/m2, died of congestive heart failure after 38 months in CR. Anthracycline therapy in childhood impairs myocardial growth in a dose-related fashion and results in clinically significant heart disease.2324 According to Lipshultz et al,25 cumulative doses of more than 228 mg/m2 of doxorubicin and an age of less than 4 years at treatment were risk factors for the subsequent development of cardiotoxicity in patients with ALL. The median cumulative dose of anthracycline in the present study was 300 mg/m2, and ranged from 100 to 400 mg/m2. In the light of the cardiotoxicity of anthracyclines, the number of cycles of therapy was reduced to four in the last four patients. Although the follow-up period for these patients has been short, all four patients remain in continuous CR. Furthermore, although our protocol did not utilize prophylaxis against CNS leukemia, no patient suffered a CNS relapse, suggesting that prophylactic therapy against CNS leukemia may be unnecessary in patients with AML-DS when treated with effective systemic chemotherapy.
Another frequent finding in patients with AML-DS is a history of MDS.26 Eleven MDS patients were included and seven AML patients in this study had transformed from an antecedent MDS. MDS in childhood is considered to be rare, and the outcome following intensive chemotherapy is very poor.27 However, unlike patients without DS, all the patients with MDS and AML transformed from MDS achieved a CR and remained in CR in our series. Owing to the low blast count in the bone marrow, it is often difficult to determine the cell lineage involved with leukemic clones in patients with MDS. However, the expression of at least one megakaryocytic marker was found in eight of 11 MDS patients. Considering the excellent outcome, MDS in patients with DS may represent an early stage of M7.
Cytogenetic studies revealed that several numerical and structural aberrations are common in patients with AML-DS. Monosomy 7 is generally associated with a dismal prognosis in AML.2829 Of the 24 patients with acquired chromosomal abnormalities, four had monosomy 7. It is noteworthy that two of three patients with relapse had monosomy 7. Further studies are necessary to confirm whether monosomy 7 is a poor prognostic factor even in patients with AML-DS.
In conclusion, children with AML-DS constitute a unique biologic subgroup and should be treated according to a protocol specifically designed for AML-DS. Considering the low relapse rate and high incidence of therapy-related mortality, over treatment should be avoided. Based on our experience, a chemotherapeutic regimen using conventional-dose AraC and less than 250 mg/m2 of an anthracycline is recommended, which offers a reasonable prognosis in patients with AML-DS.
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