Children with Down's syndrome have an approximately 20-fold higher incidence of leukemia than unaffected children. The majority of leukemia cases associated with Down's syndrome are acute megakaryocytic leukemia (AMKL). Recently, we and others have demonstrated that mutagenesis of the GATA-1 gene, encoding the erythroid/megakaryocytic transcription factor GATA-1, is a very early event in the development of Down's syndrome-related AMKL (DS-AMKL) in the process of multi-step leukemogenesis. Acquired mutations of the GATA-1 gene have been detected in almost all cases in DS-AMKL and transient myeloproliferative disorder (TMD), “a preleukemia” that may be present in as many as 10% of newborn infants with Down's syndrome. In each, the mutation resulted in the introduction of a premature stop codon in the gene sequence that encodes the N-terminal activation domain and a complete lack of expression of the 50-kD full length GATA-1 protein. Instead, an alternative 40-1W translation product of GATA-1 was expressed from a downstream initiation site. Human tumors have been shown to progress by the accumulation of genetic abnormalities. DS-AMKL is an excellent model of cancer pathogenesis. To understand the mechanisms of a lineage-specific malignant conversion further, it is very important to define the mechanism that results in the high frequency of GATA-1 mutations in Down's syndrome, and to identify the gene or genes on chromosome 21 which cooperate with mutated GATA-1, as well as additional genetic changes in TMD blast cells during the development of DS-AMKL.

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Unrelated cord blood transplantation (UR-CBT) has been established as an alternative for hematopoietic stem cell transplantation. Japan Cord Blood Bank Network offer over 17, 000 cord blood units and more than 1, 500 transplants were done by the beginning of 2004. The advantages of UR-CBT include transplantability between HLA mismatched pairs and immediate availability for urgent transplantation, but delayed hematological recovery and low incidence of engraftment are recognized as major disadvantages. Outcome of UR-CBT for children with acute leukemia revealed no correlation between HLA disparity and acute GVHD, and high relapse rate in patients with advanced disease or who received HLA matched graft. By multivariate analysis, the only prognostic factor for disease-free survival was disease status at transplantation. From now on, effort should be made to improve the outcome of transplant by overcoming disadvantages of CBT concomitant with decreasing the risks of late toxicities.

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Hereditary spherocytosis (HS) is a disease based on abnormalities of the red cell membrane that shows clinical heterogeneity. To assess the relationship between the severity of red cell membrane abnormalities and clinical features, 28 HS patients underwent red cell membrane analysis by SDS polyacrylamide gel electrophoresis (PAGE), as well as hematology tests and clinical examination. A good correlation was found between the hemoglobin level and the amount of membrane protein band 3 or the size of the spleen. Patients with low levels of band 3 tended to have a low hemoglobin and a large spleen was also associated with a low hemoglobin. These data suggest that the severity of anemia is related to the amount of membrane band 3 protein in HS patients. Splenectomized patients had low levels of band 3 before splenectomy, except for 3 patients with near normal band 3 levels. From these data, patients with a low amount of band 3 should receive splenectomy because these patients also have severe anemia.

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We report a 10-year-old girl with Ph-negative chronic myeloid leukemia (CML) and fusion signal bcr/abl on chromosome 9. She complained of right femoral swelling. She was diagnosed as having CML in accelerated phase by laboratory investigations and bone marrow. The biopsy of the femoral swelling showed granulocytes at all stages of development. Cytogenetic studies (G-banding) revealed a 46, XX normal female karyotype in bone marrow cells. Fluorescence in situ hybridization studies revealed the bcr/abl fusion signal on chromosome 9, two bcr signals both on chromosomes 22 and an abl signal on chromosome 9. Molecular studies (RT-PCR) revealed the presence of a b3a2 rearrangement. An allogeneic bone marrow transplantation (BMT) from her HLA-identical sister was performed 6 months after diagnosis. She developed a lymphoid blast crisis 3 months after BMT. Despite attempts at chemotherapy and donor lymphocyte infusion twice, the disease progressed and she died 14 months after transplant. In past reports, the prognosis of the patients with both fused bcr/abl gene located on chromosome 9 and extramedullary disease is poor. The correlation between fused bcr/abl gene located on chromosome 9 and extramedullary disease is unknown.

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We report a two-year-old girl with acute lymphoblastic leukemia (ALL) complicated with lung abscess under induction chemotherapy. Her leukemic cells showed L 1 morphology in a FAB-classification, CD10 and CD19 positive in surface markers, and 47, XX, + X, t (2; 11) (q11; q23) with MLL-LAF4 fusion protein. Under induction chemotherapy using the TCCSG L99-15 standard risk protocol, the patient showed persistent fever with an elevated level of CRP and infiltration in the right-middle lobe at a chest X-ray film. We failed to obtain the origin in several bacterial examinations. Anti-bacterial and anti-fungal agents did not induce the complete improvement of infiltration. Four months after diagnosis of lung abscess, the right-middle lobe was surgically resected and unrelated bone marrow stem cell transplantation was successfully performed. She is now well, in complete remission without any pulmonary complications.

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We report a 14-year old boy with an acute unclassified leukemia (AUL) with CD7 and CD13 expression without other lineage-specific markers. He was diagnosed with T-cell acute lymphoblastic leukemia (ALL) at initial presentation because of the presence of a mediastinal mass and a negative cytochemical reaction for myeloperoxidase (MPO). He entered complete remission with standard chemotherapy for ALL and underwent allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling at the 8th month. The post transplantation course was uneventful. The disease recurred in the bone marrow (BM) and in the skin 23 months after BMT. We characterized the blast cells again and made the diagnosis of AUL because of negative cytoplasmic expression for lineage-specific antigens : MPO, CD3 and CD79a. After unsuccessful induction treatment, he presented multiple bone metastases and hypercytokinemia-like symptoms. Finally, he entered hematological remission with cyclophosphamide and prednisolone 4 months after relapse. With a local irradiation to the right tibia, he received allogeneic peripheral blood stem cell transplantation (PBSCT) from the same donor as in the initial BMT. Chronic graft-versus-host disease in the oral mucosa and liver required intensive immunosuppressive therapy for 6 months and he relapsed in the BM 12 months after PBSCT. Although it is not well recognized in the literature, AUL with CD7 and CD13 positivity should be categorized as a unique entity with a dismal prognosis.

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More than 20 years have passed since the first allogeneic bone marrow transplantation (BMT) was performed for the treatment of childhood leukemia in Tokai University and BMT from an HLA-matched related donor has been established as a curative therapy for hematologic malignancies and other hematologic or hereditary disorders. During the second decade, unrelated donor BMT through the Japan Marrow Donor Program started, and as another approach of searching for available donor in patients without HLA-matched related donor, CD34-positive cell transplantation from HLA-two or three-loci mismatched parental donors has been tried. Such trials did not realize sufficient results readily because serious complications including severe graft-versus-host disease (GVHD) or serious infection often developed as posttransplant complications. Though CD34-positive cell transplantation is difficult to establish as a first-line transplant, unrelated BMT has become a mode of curative transplant after introducing the combination of methotrexate and tacrolimus for the prophylaxis of GVHD. In addition, the developments of new preconditioning regimens facilitated improved results of UBMT. The future problems in BMT for childhood disease are accomplishing a well-designed multicenter study and maintaining quality of life.

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The number of patients undergoing allogeneic PBSCT in children is not increasing compared with that in adults, probably because donor safety of the procedure including administration of G-CSF has not been confirmed. The controversy about donor safety among physicians is always focused on the comparison of safety between bone marrow transplantation and PBSCT. However, both have similar potential problems for the donor, and offer tremendous benefits to the recipients in comparison with unrelated donors. One cannot draw a conclusion easily. In the present state, both alternatives should be explained in detail to the potential donors and their family.

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The number of unrelated cord blood transplantation (CBT) is increasing because of its immediate availability and transplantability across the HLA barriers. The multivariate analysis of unrelated CBT for children with acute leukemia in Japan showed that the favourable factor for neutrophil engraftment was larger cell dose, and risk factors of leukemic relapse were advanced stage at transplant, HLA 6/6 match, and grade 0-I acute GVHD. The risk factor for event-free survival was advanced stage at transplant and it was not associated with either cell dose or HLA disparity. In patients with non-malignant diseases, the incidence of graft failure was high and survival was poor especially in patients with aplastic anemia and metabolic diseases. It is important to increase the engraftment rate and reduce the transplant-related complications such as posttransplant infections to improve the outcome of unrelated CBT.

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Based on the hypothesis that long-term microchimerism of fetal or maternal cells in the donor circulation might be an indicator of acquired immunological hyporesponsiveness to non-inherited maternal HLA antigens (NIMA) or inherited paternal HLA antigens (IPA), several groups have recently reported the feasibility of non-T-cell-depleted blood and marrow transplantation between HLA-haploidentical and NIMA or IPA mismatched family members. To evaluate the safety and efficacy of mother donor superiority over father donor, we performed a nationwide retrospective analysis on the clinical outcomes of 452 patients who received the first allogeneic stem cell transplantation from their mother or father, registered when the bone marrow transplantation committee of the Japanese Society of Pediatric Hematology since 1983 to 2001. Mother versus father donor was not a significant factor in the over-all survival of pediatric allo-SCT patients. The retrospective study revealed NIMA mismatched sibling donors' efficacy and safety; the NIMA concept could be applied to select HLA mismatched relatives at better compatibility.

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A variety of prognostic factors have been known for childhood acute lymphoblastic leukemia (ALL) : leukemia cell biology, host factors, response to therapy, others (age, leukocyte count, sex). Each study group defines its own risk-classification using these prognostic factors; however, uniform criteria are not available yet. Ideally, the EFS should be 100% for a group where the reduction of intensity is planned. Similarly, the EFS should be 0% for a group where a very intensive therapy such as transplantation is incorporated. In the near future, it would be difficult to provide suitable treatments for a substantial number of small risk groups.

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From January 94 to December 99, we prospectively assigned 463 consecutive children with ALL to receive one of four treatment protocols on the stratification by age, white-cell count and certain risk factors at the time of diagnosis. Patients at low-risk and intermediate-risk (55 percent of the total) were treated for two or three years, receiving an escalating dose of anti-metabolites, consisting of MTX, 6-MP and L-Asp, using none or a small amount of alkylating agents, anthracyclines and etoposide, which have adverse late effects. MTX was increased to a 500 mg/m², 24-hour injection without CF rescue, from the 225 mg/m² bolus injection which was used in the previous study (CCLSG 911, 874, 841, 811 protocols). 6-MP was also increased to 250 mg/m² from 175 mg/m² and L-Asp was newly introduced to intermittent injection during the maintenance therapy. After the initial 7 months' chemotherapy, patients at high-risk and high-high-risk were assigned institutionally to 1) uninterrupted chemotherapy for three years or 2) peripheral blood stem cell (PBSC) collection after a mobilization regimen with G-CSF and mega-dose chemotherapy (MCVAC regimen) plus PBSC rescue (PBSC transplantation : PBSCT), with all therapy completed within 9 months. It is noteworthy, that no patients assigned PBSCT were still receiving therapy after the PBSCT, so therapeutic duration was only 25 percent that of chemo-arm patients (9 months versus 36 months). In a combined analysis of all of the patients who underwent PBSCT in their first complete remission (n=44), hematological engraftment was seen in all but 1 patient who died 6 days after PBSCT due to severe infection with resistant Pseudomonous aeruginosa. At a median follow-up of 90 months, the mean event-free survival rates in the low-risk (n=106), intermediate-risk (n=149), high-risk (n=161), high-high-risk (n=47) were 71.5 ± 5.3, 82.3 ±3.7, 70.3 ±4.4 and 30.7 ± 22.0 percent, respectively. As compared with our previous protocols, the CCLSG 941 protocol has resulted in improved event-free survival; excepting low-risk group protocol, without using any dose of alkylating agents, anthracyclines and etoposide. Among high-and high-high-risk group, excluding early toxic death (n=10) and early relapse (n=13) within 7 months, there is currently significant difference (p < 0.05) in the event-free survival between chemo-arm and PBSCT-arm (83.0 versus 63.5%). The further evaluation of long-term efficacy and toxicity of the procedure with a large number of patients is feasible.

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In MLL96/MLL98 studies carried out between 1996 and 2002, the event-free survival for MLL-positive ALL infants improved with the combination of intensive chemotherapy and hematopoietic stem cell transplantation (HSCT). The effect of HSCT for MIL positive infant ALL can be predicted by several reasons as follows; the number of patients who relapsed immediately after induction therapy decreased, and the outcome of patients who received HSCT at first remission was superior, compared with that of those who received HSCT under other conditions. When all patients were classified into four subgroups according to age and white blood cell count at diagnosis, most of the patients under 6 months of age with high white blood cell count relapsed within a couple of months after the achievement of remission, suggesting that this patient group should be treated with HSCT. In contrast, it is possible that other groups can be cured with chemotherapy alone. In several studies of Western countries, the outcome of MLL-positive ALL infants has gradually improved with intensive chemotherapy. In our next study, the true risk factors of MLL-positive infant ALL should be clarified to introduce chemotherapy for this patient group to prevent early and late toxicities.

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With the improvement in the treatment outcome of pediatric acute myeloid leukemia (AML), treatment stratification based on prognostic factors became important for further improvement in treatment results and QOL of children with AML. Children treated in the Japan cooperative study ANLL91 (started August 1991) had a 7-year event-free survival rate of 55.2%. This result was superior to those achieved in other clinical trials reported at that time. Analysis of ANLL91 identified that cytogenetics, response to induction therapy, age at onset, and initial WBC counts were predictive of outcome. Based on these results and reports of other pediatric AML clinical trials, the Japan cooperative study AML99 was planned for testing the concept of risk-stratified treatment and opened in January 2000. The results of ANLL91 study and the interim reports of AML99 study are presented and risk group stratifications in recent pediatric AML clinical trials are discussed.

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