An emergence of drug resistance is one of the major obstacles in treating children with various cancers to cuve. The understanding of its mechanism will be the first step to overcome this obstacle. Since Dr. V. Ling discovered that the membrane bound P-glycoprotein was associated with pleiotropic drug resistance and its expression was encoded by multi drug resistance gene (MDR gene), light had been focused on the molecular mechanisms of the multi drug resistance, and numerous information could be collected. It is hence time to review the mechanisms of drug resistance including classical and newer entities. The over expression of MDR gene always results in the phenomenon of multi drug resistance, but the P-glycoprotein can also be found in normal and preneoplastic tissues. This glycoprotein is known to decrease in the intra-cellular drug accumulation by ATP dependent efflux mechanism with broad substrate specificity. P-glycoprotein can be detected using monoclonal antibodies with cell lines and clinical materials. The mechanisms of classical drug resistance are, on the other hand, widespread and multifactorial, and its clinical implication is more important and practical to design chemotherapy. In this review, the mechanisms of drug resistance were elucidated with methotrexate and cytosine arabinoside.

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Peripheral blood lymphocytes were investigated by two-color analysis in acute lymphoblastic leukemia (ALL) in order to elucidate the effects of anticancer chemotherapy on immune systems. The proportions of CD8⁺ cells, CD4⁺HLA-DR⁺ cells, CD8⁺HLA-DR⁺ cells and Leu7⁺ cells during maintenance chemotherapy were significantly higher than in age-matched normal controls, and decreased to nearly normal levels after cessation of chemotherapy while CD4⁺HLA-DR⁺ cells and CD8⁺HLA-DR⁺ cells in the patients were still higher than in normal controls. The proportions of CD4⁺ cells, CD 16⁺ cells and Leu7--CD16⁺ cells during maintenance chemotherapy were as high as in the off-therapy group, and CD4⁺cells in both groups were significantly lower than in normal controls. CD4/CD8 ratio during maintenance chemotherapy was significantly lower than in normal controls and elevated to nearly normal levels in the off-therapy group, though they were still significantly lower. These changes, observed during maintenance chemotherapy, might be reactive changes which reflect bone marrow and lymphoid regeneration.

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Five children with refractory chronic idiopathic thrombocytopenic purpura (ITP) were treated with danazol and the clinical effectiveness of the drug was evaluated. Patients comprised 2 boys and 3 girls, whose age ranged from 6 to 14 years. Their platelet counts continued to be less than 50, 000/μl over 6 months after onset of the disease, and no improvement was observed in spite of various therapies. Danazol was given at the doses of 200-600 mg daily for 2 to 6 months. Four of the five patients exhibited increases in platelet counts to more than 50, 000/μl within 2 weeks after administration of the drug : three of these patients (2 boys, 1 girl) showed sustained levels of more than 100, 000/μl over 2 months, suggesting that the effective rate of danazol therapy is 60%. Two patients showed side effects : one with slight acne on the face, and the other with transient mild menorrhagia, but no abnormal liver function was noted. The effect of danazol on the recovery of platelet counts in ITP patients seems to be attributed to inhibition of platelet antibody production ; this was deduced from the fact that the high level of platelet-associated IgG on pretreatment stage in 3 effectively treated patients declined to the normal range during or on completion of treatment. In conclusion, danazol therapy is thought to be an effective and safe treatment for children with refractory chronic ITP.

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Prophylactic effect of aztreonam (AZT) was compared with a control group by injecting AZT intravenously to the pediatric patients with malignant tumor in the neutropenic conditions after aggressive anticancer chemotherapy. Whether AZT should be administered or not was randomly determined by the envelope method. AZT 80-100 mg/kg/day was injected to the nonfebrile patients with neutrophil count less than 250/μl. Amphotericin B, polymyxin B and a combination of sulfamethoxazole/trimethoprim were given to the both groups for intestinal decontamination. Total number of cases evaluated was 44 each in the AZT group and the control group. Underlying diseases were hematological malignancy 27 and solid tumor 17 cases in the AZT group, and 22 cases of each tumor in the control group. Average minimum neutrophil count and the duration of neutrophil count less than 250/μl were 27.2/μl±45.7 and 11.5 days ±6.3 in the AZT group and 26.1/μl±34.4 and 12.3 days ±6.7 in the control group, respectively. Overall prophylactic efficacy-rate of the AZT group was 72.7% and that of the control group was 50.0%, indicating significantly superior prophylactic effect of the AZT group (p<0.05). Prophylactic efficacy-rate by minimum neutrophil count was 66.7% in the AZT group and 36.4% in the control group with neutrophil count less than 10/μl; 66.7% and 50.0% with the count less than 10-50/Al; and 100% and 80% with the count 50-250/μl, respectively. Side effect was not observed. The above results suggested the usefulness of prophylactic intravenous injection of aztreonam in the cases with neutrophil count less than 50/μl.

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There are many reports about what kinds of growth factors act on stem cell in hematopoiesis, but only a few of them refer to metabolic dynamics in cell and environment after the action of growth factors. The purpose of this study was investigation of dynamics of fat metabolism on erythropoiesis. Rats were used for this studyin vivoandin vitro. They were established as a hemolytic anemia model in our department. (1) Variation of fatsin vivo: Most of fats in serum, supernatant of bone marrow and bone marrow cell decreased in the early stage and increased in the intermediate stage on erythropoiesis. But only phospholipid in supernatant of bone marrow and PI + PS in bone marrow cell increased from the early stage. And the recovery of unsaturated fatty acids in bone marrow cell was slower than that of saturated fatty acids. (2) Variation of fatsin vitro: Erythropoietin produced the decrease of archidonic acid in bone marrow cell. The concentration of IP₃in bone marrow cell at the 4th day was higher than at the first day. But Epo did not produce the increase of IP₃ in bone marrow cell. These data suggest the possibility that archidonic acid and phospholipids play a role in hematopoiesis. IP₃ might not be concerned with the intracellular response for the maturation of erythroid progenitor cell by erythropoietin.

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The production of tumor necrosis factor-a (TNFα) was examined in children with acute lymphoblastic leukemia (ALL) in order to assess the peripheral blood monocyte function. Peripheral blood mononuclear cells (PB-MNC) obtained from ALL children were stimulated with OK432 or autologous ALL cells. TNF α in culture supernatant was quantitated by enzyme-linked immunosorbent assay. Sequential study showed that TNFα production following OK432 stimulation reached the peak within 24 hours and declined thereafter. Major producing cells of TNFα in peripheral blood were plastic-adherent cells which were shown to be monocytes morphologically and cytochemically. Most of TNFα was released into culture supernatant, whereas solubilized fraction of OK432-stimulated PB-MNC contained little TNFα. The production of TNFα was well maintained in both ALL cases on chemotherapy and off chemotherapy. There was no significant difference in terms of TNFα production between ALL cases and normal subjects. Instead, inter-patient variation was remarkable in the group of patients on chemotherapy. In mixed cultures with autologous ALL cells, significant TNF-α production was observed in one out of 14 cases, suggesting that the specific anti-tumor immunity might be established.

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Seventeen patients with Hodgkin's disease were observed at our department between 1958 to 1988. Their ages were from 2 years and 2 months to 15 years and 8 months (median of 6 years and 3 months). Eleven were male and 6 were female. Mixed cellularity was the most frequent histopathological subtype and comprised 51 %. Nodular sclerosis comprised only 21 %. The time from the onset to diagnosis was not different between 10 cases of early stage (IA and IIA) and 7 cases of advanced stage (IIIA, IVA and B stage). The overall survival curve reached a plateau at 55.2% 2 years from diagnosis. A majority of the cases of early stage diagnosed before 1977, when the treatment was inadequate, were alive, although many of them had multiple relapses and the duration of treatment was around 6 years. All but 1 of the cases diagnosed after 1977 including 1 with B stage were alive without relapses after treatment of 2 years by combination chemotherapy with and without low-dose radiotherapy. The necessity of pathological staging procedure may be eliminated by a combination of combination chemotherapy and low-dose radiotherapy.

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To evaluate the efficacy of prophylactic systemic antibiotics with fosfomycin for prevention of post-transplant infections, 45 pediatric patients treated by bone marrow transplantation were allocated to prophylactic systemic antibiotics (PSA) group (22 patients) or control group (23 patients). The incidence of febrile episodes was lower in the PSA group (54.5%) than in the control group (78.3%), but it was not statistical significant. The afebrile period from the beginning of granulocytopenia (<500/μl) was significantly longer in the PSA group (11.4 ± 8.6 days) than in the control group (7.6±5.4 days). The febrile period during granulocytopenia was shorter in the PSA group (3.1 ±3.5 days) than in the control group (6.0 ±5.7 days). In bacterial examinations from throat swab, stool and urine cultures, the incidence of gram-negative bacteria isolation decreased but the incidence of fungus isolation increased in PSA group. Although prophylactic systemic antibiotics for prevention of bacterial infections in bone marrow transplantation is not common today, this result suggests its effectiveness and further study is needed.

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We studied the different hematopoietic recovery after allogeneic and autologous bone marrow transplantation. Marrow recovery of myeloid (CFU-C) and erythroid (BFU-E) progenitor cells showed different repopulation kinetics between allogeneic (allo-BMT) and autologous (auto-BMT) bone marrow transplantation. Following allo-BMT almost normal numbers of CFU-C and BFU-E were detected at 2 weeks and 4 weeks, respectively.On the contrary, in auto-BMT, numbers of CFUC and BFU-E increased slowly and still reduced at 2-3 months after transplantation. Statistically, numbers of CFU-C at 2 weeks and BFU-E at 4 weeks were different significantly between the two groups. We suppose that the delayed recovery of hematopoiesis is due to suboptimal cryopreservation technique or hematopoietic impairment as a result of the chemotherapy or purging treatment, since transfused numbers of CFU-C and BFU-E were not different between allo-BMT and auto-BMT.

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One characteristic of Philadelphia chromosome (Ph1) -positive acute leukemia is the occasional presence of both lymphoid and myeloid features in the same leukemia. This phenomenon has provided evidence supporting the theory that this subtype of acute leukemia arises from lymphoidmyeloid stem cell/pluripotent progenitors. Very few reports, however, have described immunophenotype, especially CD34 antigen, of Ph1+ acute lymphoblastic leukemia (ALL). It has been reported that CD34, human progenitor cell antigen (HPCA-1), is found on a multipotent progenitor cell, whereas CD34 is not found on normal peripheral blood cells. Here, we report that high frequency of CD34 expression was found in children with Ph1+ ALL : CD34 was positive for six out of six patients tested. These findings suggest the involvement of a pluripotent stem cell in Ph 1-positive ALL.

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Effects of chemoimmunotherapy, including cranial irradiation for central nervous system (CNS) -directed therapy, on children with acute lymphoblastic leukemia (ALL) were investigated. Fifty-six children with ALL in continuous complete remission (> 5 yr) and without evidence of current or past CNS diseases were evaluated in this retrospective study. Using standard measures of intelligence quotient (IQ), we repeatedly (1-4 times/person) evaluated IQ in the cohort of patients for the mean follow-up time of 10.1 yr, ranging from 5.1 to 15.5 yr. The 56 patients received a total number of 121 IQ testings and 42 patients received them more than twice. They were examined periodically at intervals of 1.4 to 10.0 yr (mean 4.5 yr) following diagnosis. This report confirms and extends the previous findings : decreased IQ was related to the younger age at the time of diagnosis and irradiation (<5 yr of age at diagnosis), the longer irradiation-examination interval, and female sex. Further longterm follow-up study is needed in these groups, since their IQ is still on the decline even after 10 yr of diagnosis.

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The production of interleu kin-1 (IL-1) was examined in children with acute lymphoblastic leukemia (ALL) in order to assess the peripheral blood monocyte function. Peripheral blood mononuclear cells (PB-MNC) obtained from ALL children were stimulated with OK432 or autologous ALL cells. IL-1 in culture supernatant was quantitated with bioassay using peanut agglutinin-negative mouse thymocytes. Sequential study showed that IL-1 production following OK432 stimulation reached the peak within 24 hours. Major producing cells in peripheral blood were plastic-adherent cells which were shown to be monocytes morphologically and cytochemically. Most of IL-1 was released into culture supernatant, whereas solubilized fraction of OK432-stimulated PB-MNC contained little IL-1 activity. The production of IL-1 was well maintained in ALL cases both on chemotherapy and off chemotherapy. There was no significant difference in terms of IL-1 production between ALL patients and healthy subjects. Instead, inter-patient variation was remarkable in the group of patients on chemotherapy. In mixed cultures with autologous ALL cells, significant IL-1 production was observed in one out of 10 cases, suggesting that the specific anti-tumor immunity might be established.

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The Bone Marrow Transplantation Committee of the Society has conducted an annual registry of bone marrow transplantation in children in Japan since 1983. As of June 30, 1990, 732 patients had received stem-cell transplantation and were registered from 57 institutions. Among them, autologous bone marrow transplantation and autologous peripheral blood stem-cell transplantation were mainly performed in hematological malignancies or malignant solid tumors, and the former procedure was done in 192 children (88 alive) and the latter was done in 42 children (32 alive), respectively. Allogeneic or syngeneic bone marrow transplantation including fetal liver cell transplantation were given to 498 children, i.e. 133 cases of acute lymphocytic leukemia (65 alive), 129 cases of acute non-lymphocytic leukemia (85 alive), 39 cases of adult-type chronic myelocytic leukemia (29 alive), 6 cases of juvenile-type chronic myelocytic leukemia (4 alive), 19 cases of nonHodgkin lymphoma (11 alive), 11 cases of malignant solid tumors (4 alive), 99 cases of aplastic anemia (88 alive), 29 cases of severe combined immune deficiency (12 alive), and 33 other cases (28 alive). The total number of transplanted cases has increased from 540 to 732 during the past one year. The details are reported in this paper.

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We present 4 cases of myelodysplastic syndrome (MDS) in children : 1 case of refractory anemia (RA), 1 case of RA with excess of blasts (RAEB) and 2 cases of RAEB in transformation (RAEB-T) at the time of diagnosis. RAEB-T eventually transformed to acute myeloblastic leukemia (AML). One case of the RAEB-T had hypoplastic marrow. Chromosomal analysis of 3 cases showed abnormalities of the 7th and/or 8th chromosomes. Bone marrow progenitor cell assay showed decreased colony-forming units in all cases, but many clusters were observed in 2 cases. The cases of RAEB and RAEB-T were treated with anti-leukemic drugs and complete remission was induced. They relapsed 7-8 months later and died in 14-62 months from the initial diagnosis. One case of RA showed transient improvement of anemia with Ubenimex. In conclusion, further propspective group study of chemotherapy according to the types of MDS should be performed in order to improve the prognosis.

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A 13-year-old girl with chronic myelogenous leukemia developed hyponatremia and psychotic reaction by SIADH during preconditioning regimen of allogeneic marrow transplantation. It was suggested that the most likely cause of this disease was the high-dose cyclophosphamide therapy and emotional stress in a laminar air flow unit. Whenever the high-dose cyclophosphamide therapy is used as preconditioning therapy in an older child, we should constantly pay attention to the occurrence of SIADH and try to relieve the patient's emotional stress. In our case, allowing her mother to stay in the clean unit effectively relieved the patient's emotional stress.

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A 12-year-old girl with refractory anemia with excess of blasts in transformation (RAEB in T) received allogeneic bone marrow transplantation. Conditioning regimen consisted of busulfan 16 mg/kg and cyclophosphamide 120 mg/kg. Engraftment was documented. In spite of prophylactic treatment with cyclosporin A and methotrexate, she developed acute and chronic graft-versus-host disease (GVHD). Her cutaneous symptom was severe (stage IV), but no liver disease nor gastrointestinal disease have been observed in her clinical course. This makes it difficult to diagnose her GVHD grade, when one uses clinical grading system of aute GVHD by the Seattle group. Her serum IgE level correlated well with skin lesion of acute and chronic GVHD. This suggests that serum IgE level could be useful for early diagnosis of skin lesion of GVHD as well as that of liver disease. Her skin symptom is improved by treatment with prednisolone and azathiopurine.

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Bone marrow transplantation (BMT) was done three times for a 6-year-old girl with myelodysplastic syndrome (MDS). She showed progressive bicytopenia and constitutional abnormalities including short stature and skin pigmentation. We initially diagnosed the patient as having Fanconi's anemia, although the chromosome fragility was not demonstrated by the mitomycin C stress test. The patient received conditioning regimens which consisted of 3 Gy of total body irradiation followed by decreased dose of cyclophosphamide (CY, 10 mg/kg for 4 days) and anti-lymphocyte globulin (ALG, 30 mg/kg for 5 days) and marrow cells from HLA-identical 8-year-old brother were given. However, the graft was rejected. Reviewing the previous bone marrow smears disclosed the abnormalities in the three major cell lines : erythroblasts, granulocytes, megakaryocytes. Therefore, we considered her to have MDS. The second BMT was done with busulfan (4 mg/kg for 4 days) followed by CY (60 mg/kg for 2 days); however, no evidence of engraftment occurred until day 25. Therefore, she received CY (50 mg/kg for 4 days), ALG (40 mg/kg for 4 days) and the third BMT with successful engraftment which was confirmed by the chromosomal karyotype on day 19. She showed remarkable hematological recovery and bone marrow examination on day 54 showed complete disappearance of morphological abnormalities of all cell lineages seen prior to BMT. She is well, with full performance state.

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A 12-year-old boy was admitted because of petechia, hemorrhage in the oral cavity, swelling of the gingiva, and hematuria on Sep. 19, 1988. White blood cell count (WBC) was 98.2 × 103 cells/μl with 41 % blasts and aspirated bone marrow contained 34% blastic cells. He was diagnosed as acute nonlymphocytic leukemia [ANLL (FAB-M4)]. Lysozyme level was high and PAIgG 413 ng/107 cells. Immunological marker studies revealed that CD4, CD13, CD14, CD33 and HLA DR were positive. However, these findings changed during the clinical course, with leukemic cells at the terminal stage positive only for CD33 and HLA DR. Chromosomal studies showed that 100% of analyzed bone marrow metaphyses was 46XY, -12, + mar. His ABO Blood Group was Am type of A type-variant. Initial therapy consisted of the 12th ANLL TCCSG protocol, after which suppressive effects in peripheral blood persisted for about 6 months. After he acquired herpes zoster, leukemic cells proliferated. Complete remission could not be obtained with any therapy and he died on Oct. 27, 1989.

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A 14-year-old boy was admitted because of fever and paleness. Based upon hematological findings of bone marrow, acute lymphocytic leukemia was diagnosed. Patient was treated with vincristine, prednisolone, l-asparaginase and daunorubicin, and complete remission was achieved. Post remission chemotherapy with 6-mercaptopurine, cytosine arabinoside and cyclophosphamide was begun, and he developed spiky fever after chemotherapy. He was treated with intravenous administration of amphotericin B and recombinant human G-CSF, but developed jaundice, right upper quadrant pain, hepatomegaly and ascites. These signs and symptoms were similar to veno-occlusive disease of the liver. Ultrasonic tomography revealed no liver abscess. He died of hepatic failure in June 1989. Necropsy of the liver showed multiple occlusion of small portal veins by Aspergillus, with resulting veno-occulsion in liver.

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This case illustrates that treatment with rhG-CSF was effective for increasing the numbers of neutrophils and for infectious disease in a patient with infantile genetic agranulocytosis (Kostmann type). The patient is a 2 years and 3 months old boy who was diagnosed as infantile genetic agranulocytosis (Kostmann type) at the age of 4 months. He was admitted to our hospital for pneumonia at the age of 2 years and 3 months. Bone marrow aspiration on admission revealed a complete absence of neutrophils. At day 38, rhG-CSF (KRN8601) therapy was started at the dose of 400 μg/m² with 30 min continuous infusion. At day 13 after treatment, WBC reached 7, 600/μl with 13 percent band and 18 percent segmented neutrophils. At day 21, bone marrow showed increased granulocytes. Nuclear cell count was 11.6 × 10⁴/μl, granulocytes was 17.9 percent with 2.1 percent band and 2.2 percent segmented neutrophils, and erythrocytes was 15.8 percent. Within days of the start of rhG-CSF therapy, the inflammatory findings were improved markedly. We conclude that treatment with rhG-CSF leads to increase in the numbers of neutrophils temporarily in a patient with infantile genetic agranulocytosis (Kostmann type), and it is effective for the treatment of infectious disease in that patient.

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We report a 15-year-old girl with acute lymphoblastic leukemia who had undergone Kasai's operation for congenital biliary atresia. She had obstructive liver dysfunction and suffered from severe side effect on antileukemic drugs. So we examined pharmacokinetics of antileukemic drugs. We examined the plasma disappearance rate of ICG as the index of liver blood flow and pharmacokinetic study of caffeine and acetaminophen as the index of residual liver drug metabolic ability. Caffeine is mainly metabolized by cytochrome P450-mediated metabolism (phase 1 reaction), and the half-life of caffeine was prolonged. Acetaminophen is excreted mainly as glucuronide conjugation (phase 2 reaction), and the half-life of acetaminophen was prolonged. We twice examined pharmacokinetics of 6MP, but we could not calculate the half-life of 6MP because of absorption delay. The half-life of prednisolone was 1.7 times as acetaminophen. Methotrexate is excreted from kidney without hepatic biotransformation. So the half-life of MTX (β phase) was 1.5 hr and it was within normal range. The examination of pharmacokinetic study of caffeine and acetaminophen is the index of own liver drug metabolic ability and can avoid severe side effect. We can decide optimal dosage for the patient.

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