In normal thymus and bone marrow, a large number of unwanted lymphocytes (cells which failed to rearrange antigen receptor genes or which acquired autoreactivity) are rapidly eliminated by programmed cell death (apoptosis) in situ. Since leukemic cells are derived from these organs, knowledge of the regulation of apoptosis is important to understand leukemogenesis and also to establish a new treatment of leukemia. When leukemic cells are cultured in vitro, most of the cells die by apoptosis unless these cells are placed on bone marrow stromal cells. Although cell-to-cell contact is needed, the bone marrow microenvironment may provide some essential factors to support cell survival like colony stimulating factors for cytokine-dependent cell lines. The role of bcl-2 is to block apoptosis in peripheral lymphoid organs and also in B-cell diseases such as follicular lymphoma. Recently, other bcl-2 related proteins, such as bcl-x and bax were discovered. Virtually all the cytotoxic agents and irradiation kill leukemic cells by induction of apoptosis. Some molecules such as p53, c-myc, bcl-2 and c-jun are known to modulate the process of apoptosis, however, the exact cellular mechanism is still unknown. Biologic response modifiers such as interleukins and some ligands to the cellular antigens like Fas are also involved in the process of apoptosis. The future direction of leukemia research should focus on the molecular events of apoptosis. The method of detecting apoptosis is also mentioned.

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Most cases of adult T-cell leukemia (ATL) are thought to result from vertical transmission, with few clear-cut cases linked to horizontal transmission. We analyzed the percentage of flower-like cells, which included both flower cells (ATL cells morphologically) and abnormal lymphocytes, per 400 lymphocytes in carriers and noncarriers alike, and postulated that this percentage might be a risk factor for ATL in HTLV-I carriers. Accordingly, we analyzed for (1) any difference in the percentage of flower-like cellsbetween symptomless carriers of HTLV-I and noncarriers ; (2) any difference in the incidence of flower-like cells between the vertical and sexual transmission groups of pregnant women who were positive for HTLV-I ; and (3) any difference in the incidence of flower-like cells between pregnant women and their mothers who were positive for HTLV-I. The percentage of flower-like cells in the symptomless carriers was significantly higher than that in the noncarriers. The mean percentage of flower-like cells in the symptomless carriers was between one and two percent and was stable, unaffected by generation or by transmission route. Even though the flower-like cells are most likely transformed by HTLV-I, the percentage of flower-like cells does not appear to be a risk factor for ATL. It is not known why so few cases of ATL developed in our sexual transmission group, which had the same percentage of flower-like cells as did the vertical transmission group.

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To investigate the incidence and antibody production of hepatitis C virus (HCV) in children with hematologic diseases, patients were divided into four groups as follows : (1) patients with hematologic diseases (acute lymphoblastic leukemia and aplastic anemia) with a past history of receiving several blood transfusions, (2) hemophilic patients during treatment with anti-hemophilic products, (3) those with idiopathic throm bocytopenic purpura or congenital immunodeficiency treatment with gamma-globulin and (4) those with bone marrow transplantation. Anti-HCV antibodies were assayed by 1st and 2nd generation enzyme immunoassays (1st and 2nd EIA), and recombinant immunoblot assay-II (RIBA-II). In addition, HCV-RNA detection by polymerase chain reaction and the HCV genotyping method developed by Dr. Okamoto were also used. The detection of antibodies for the c33c and c22-3 antigens by RIBA-II was reliable for diagnosing HCV hepatitis. A good correlation was seen between the positivity rate for the above described antigens and that for HCV-RNA. Analyses of HCV genotyping revealed that the type II HCV was seen with high frequency in the group of patients with hematological diseases receiving blood transfusion and type I HCV in hemophilic patients. From these findings it is considered that the combined use of RIBA-II with HCV genotyping is a rapid and reliable means of identifying HCV infection and to distinguish the HCV-genotype in children with hematological diseases.

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We investigated serum thymidine kinase (s-TK) activity sequentially in five children with non-Hodgkin's lymphoma, neuroblastoma and acute lymphoblastic leukemia, who had been treated with high-dose chemotherapy before harvesting peripheral blood stem cells. s-TK activity showed low levels after chemotherapy, then increased rapidly. A significant correlation was found between s-TK activity and CD34 positive cell count. s-TK activity and CD34-positive cells increased to a maximum level when the white blood cell count increased to 5, 000-15, 000/μl. It was suggested that s-TK levels reflect proliferation of hematopoietic stem cells, and s-TK may be an index of peripheral blood stem cell harvest.

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Seven patients with hematologic malignancies were treated with granulocyte transfusions (GTX) for severe infection due to prolonged neutropenia. In the course of treatment, we performed 29 GTX. G-CSF was administered subcutaneously at a dose of 1, μug/kg to each GTX donor, 12 hours prior to leukapheresis in an attempt to increase the yield of granulocytes after informed consent was obtained. Leukapheresis techniques including hydroxyethyl starch (HES) was performed using Hemonetics model 50. Hydrocortisone was not administered. The mean volume of processed blood was lower (1.6-1.8 l) than that by conventional methods (2.4-3.7 l). The collecting time was also shortened. No side effects were seen in donors. The mean number of granulocytes collected was 2.54+1.2 × 10¹⁰ which is about 3 times higher than that of the conventional method using hydrocortisone stimulation. In conclusion, G-CSF was safe to administer to normal individuals, and significantly improved the quantity of neutrophils collected. This study indicates that GTX donors and patients benefit from G-CSF administration to the donor prior to leukapheresis.

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Cytomegalovirus (CMV) pneumonia is one of the serious complications known to occur after allogeneic bone marrow transplantation (BMT). The diagnosis is usually made by viral culture technique using the specimens obtained from blood, urine, throat swab, and bronchoalveolar lavage fluid or from lung biopsy specimens in marrow recipients with interstitial pneumonia. However, the specificity and sensitivity of viral culture is occasionally insufficient to make a correct diagnosis, and biopsy is invasive. In addition, it takes several days to obtain results. HRP-C7, originally reported by Eizuru et al. in 1991, is a human monoclonal antibody to immediate early antigen of CMV, and the immunoperoxidase method with HRP-C7 is reported to be reliable for the detection of CMV antigenemia. Furthermore, it takes only 24 hours to obtain the results. We experienced four cases of CMV infection after allogeneic BMT. Two of these four cases were diagnosed in their early phase of infection utilizing the HRP-C7 method, and treated with combination of ganciclovir and high-dose immunoglobulin before detection of abnormalities on chest X-ray film. Antigenemia resolved within 10 days after the initiation of the treatment. As a result, the patients did not develop typical symptoms and signs of CMV pneumonia. Three of four cases including these two are alive without disease recurrence.

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To a 7-year-old boy with AML during 1st complete remission we performed the bone marrow transplantation in 1988 from his HLA identical younger sister. In bone marrow harvesting we diagnosed her as malignant hyperthermia under general anesthesia with succinylcholine. We continued bone marrow harvesting after changing anesthesia into intravenous anesthesia with ketamine, but sequela did not remain. Although he had been in complete remission for 7 months, AML relapsed. After chemotherapy 2nd complete remission did not occur and bone marrow suppression was severe. We decided 2nd BMT to attain remission and harvested her bone marrow again before 2nd BMT using neuroleptanesthesia. In 2nd bone marrow harvesting, there was no trouble. Her bone marrow was cryopreserved and he received bone marrow transplantation after 114 days of her bone marrow harvesting. But he died of sepsis after 43 days of 2nd BMT. Anesthesia for BMT donor is necessary to be safe. This is a valuable case suggesting important problems we must consider in safety of BMT donor, especially in bone marrow bank system that started in Japan in 1992.

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We report a 20-month-old boy with hemolytic-uremic syndrome (HUS) who had unusual findings. A bone marrow aspirate on day 4 of the disease revealed dyshematopoiesis and phagocytosis by the monocytes. The second specimem showed a hyperplastic marrow, but neither dyshematopoiesis nor phagocytosis on day 30. The patient was treated with anticoagulants, and both renal and hematological abnormalities returned to normal on day 21. The relationship between the abnormal findings and HUS was discussed. We think that bone marrow studies and measurement of cytokines may give a clue to the pathogenesis of HUS.

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We report a case of rhinocerebral mucormycosis that developed after multi-drug chemotherapy for relapsed acute lymphoblastic leukemia (ALL). A4-year-old boy with common ALL relapsed hematologically and received a chemotherapy consisting of prednisolone, etoposide, cytosine arabinoside and mitoxantrone. Several days after persistent high fever unresponsive to antibiotics, his left cheek became reddened and swollen, and massive destruction of his hard palate was noted. Pathological examination of the palate revealed the infiltration of fungi which showed characteristics of Mucorales. We started intravenous amphotericin-B therapy, but due to its severe toxicity, had to change it to 5-fluorocytosine and fluconazole. The granulocyte-colony stimulating factor therapy was also adopted for persistent severe neutropenia. However, despite all efforts, he died 5 months after diagnosis of mucormycosis. An intensive prophylaxis is required to prevent lethal fungal infections including mucormycosis during severe neutropenia after combined chemotherapy.

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A five-year-old boy with common ALL was complicated by pulmonary mycosis and a large fungal brain abscess during the first remission induction therapy. Treatment by systemic AMPH-B administration was effective to the lung lesion but not to the brain abscess, and then brain lobectomy was performed to resect the abscess lesion. Aspergillus species was identified in the resected specimen by microscopic examination and enzyme immunostain. Simultaneously, treatment of ALL had been capable to be continued with some modification to the scheduled protocol, and completely finished in three years and nine months. Since then, no sign of recurrence of ALL nor mycosis has been observed. This case suggests that an advanced deep-seated mycosis intractable to the systemic administration of antifungal agents should be considered the indication of surgical resection.

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The mechanism that attributes to EDTA-dependent pseudothrombocytopenia in a 10-years-old boy was immunologically studied by using flow cytometry. Platelets aggregates were brought by incubating with patient serum in the presence of EDTA and was stained with FITC-conjugated goat anti-human immunoglobulin G (IgG) polyclonal antibodies. Moreover, the potential of mouse monoclonal antibody to react with the determinant specific for glycoprotein IIb/IIIa complex on platelets was totally abrogated when platelets were pre-incubated with the patient serum together with EDTA. These data indicate the presence of EDTA-dependent IgG antibodies in the patient serum which provoke platelet aggregation to react specifically with glycoprotein IIb/ IIIa complex on the surface of platelets.

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Two children with acute lymphoblastic leukemia that were complicated by CNS hemorrhage or peripheral thrombosis associated with chemotherapy of multiple drugs including L-asparaginase (L-Asp) were reported. Case 1 is a 13-year-old boy with common ALL. He was treated with dexamethasone, VCR, THP-ADR and L-Asp. On the day following four times of L-Asp injection, he developed generalized tonic and chronic seizure. At that time CT scan of the head revealed a left parietal intracranial hemorrhage. He was given fresh frozen plasma and antithrqmbin III concentrates. His outcome was good and he could receive subsequent therapy without further complications. Case 2 is a 9-year-old girl with common ALL. She was treated with prednisolone, VCR, THP-ADR and L-Asp. On the day following nine times of L-Asp injection, her left leg became painfully swollen. Peripheral venous thrombosis was diagnosed. We studied changes of blood coagulation factors and fibrinolytic proteins during the therapy of Tokyo Children Cancer Study Group 12th and 13th protocols. Plasma level of factors VII, IX, X, XI, XII and AT III was decreased and also fibrinogen concentration became low during the therapy with L-Asp. We also noticed that plasma level of blood coagula-tion factors was decreased even though during the chemotherapy without L-Asp.

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We report a 2-year-old boy who was admitted to our hospital because of large anterior mediastinal mass. Initial Symptoms were swelling of right maxilla and oral tumor. Biopsy showed infiltration of his-tiocytes. Thoracotomy revealed white colored tumor extending over the heart and lungs to such an extent that only partial excision was performed. Histological examination showed destruction of thymic architecture with infiltrating large histiocytes which were strongly positive for S-100 protein, and Birbeck's granules were detected by electron microscopic examination. Flow cytometric analysis revealed a marked decrease of CD1⁺ and CD4⁺ 8⁺ cells. Diagnosis of Langerhans' cell histiocytosis was made and chemotherapy with VP16 and AraC had been performed for one year. One year later, he showed relapse at the left jaw joint. The same chemotherapy was effective and was continued for an additional year. He has been free from disease for three years since the last therapy.

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We report herein a 4-month-old girl with acute megakaryoblastic leukemia, who presented with hypercalcemia at diagnosis and showed infiltration to the lung, heart and brain. Her serum calcium level was elevated at 16.8 mg/di on admission. Bone X-ray revealed marked osteolytic change of the whole skeleton. Chest roentgenogram and computed tomography revealed diffuse infiltration in the bilateral lung fields, and a mass lesion in the left atrium was shown by echocardiogram. After chemotherapy, serum calcium level normalized quickly. Although brain involvement appeared during the course of treatment, the lesions of organ involvements were improved by more intensive chemotherapy. She is alive 36 months after the onset, and has remained disease-free 16 months after cessation of chemotherapy. Only two cases who were acute megakaryoblastic leukemia with hypercalcemia were reported, while no case with hypercalcemia at the onset was reported. This is the first report of a pediatric case of affected megakaryoblastic leukemia with hypercalcemia. Since her organ involvements were observed with roentgenogram, computed tomography and echocardiogram, without pathological examinations, this case may provide useful information.

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Here we report a 1-year-old boy with hemophagocytic syndrome presenting with high grade fever, severe pancytopenia, elevated liver enzymes and coagulopathy. He was treated with two courses of etoposide and prednisolone and one course of aclarubicin, etoposide and cytarabine, but each time his disease recurred. Thus we tried high dose cytarabine combined with mitoxantrone and etoposide, which resulted in long-term remission without any consolidation therapy. It is suggested that a short-term intensive chemotherapy can improve the prognosis of severe recurrent HPS. The etiology of HPS is also discussed.

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Administration of high-dose cytosine arabinoside combined with mitoxantrone (HAM therapy) was used as remission reinduction therapy for five children with relapsed acute non-lymphoblastic leukemia (ANLL) including two patients after bone marrow transplantation. Complete remission (CR) was achieved in four patients. Severe bone marrow supression, bacterial or fungal infection and gastrointestinal complications were observed in all cases, but they were improved by appropriate supportive therapy. Fatal cardiac toxicity was observed only in one patient who failed to achieve CR, in the following therapy with daunomycin. 'Three of four patients who were successfully treated by HAM therapy died due to relapse or to treatment-regimen related toxicity of subsequent chemotherapies. Even though HAM therapy is safe and effective as a reinduction regimen for relapsed ANLL, it does not seem to eradicate the residual leukemic cells enough to maintain CR for a long period. Thus, further intensive consolidation chemotherapy in combination with stem cell transplantation is required to improve the prognosis.

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