When natural killer (NK) cells work as effectors for cytolysis, they first recognize and bind to a target cell, and then lyse the bound target by releasing lytic materials such as NK soluble cyto-toxic factors (NKCF). After killing the target, NK cells detach from the dead target and repeat this lytic sequence by the recycling capacity. Thus, the impairment of NK cell activity results from (1) a decreased number of NK cells, (2) defective binding, (3) defective lysis, or (4) impaired recycling. Ac-tually, in the case of (1), numbers of total NK cells or active NK cells are decreased or absent in new-borns, Chediak-Higashi syndrome, severe combined immunodeficiency, and hemophagocytic lymphohistiocytosis ; in (2), NK cells have defective target binding capacity in newborns and LFA-1 (lymphocyte function-associated antigen-1) /Mac-1 /p 150, 95 deficiency; in (3), there is defective NK cell cytolysis in newborns, leukemia, myelodysplastic syndrome (MDS), erythematosus, Chediak-Higashi syndrome, and a familial NK cell deficiency with defective NKCF production; and in (4), NK cells are impaired in the recycling in newborns, leukemia, Chediak-Higashi syndrome, and congenital neutropenia. Some clinical and laboratory data of our representative cases are presented for better understanding of NK cell dysfunctions.

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The clinical usefulness of the Toxicolor test (T-test) and Endospecy test (E-test) was evaluated in 96 children with fever of unknown origin. T-test detects endotoxin from gram negative rod (GNR) and β-D-glucan from fungus, while the E-test detects only endotoxin. Fifteen patients were considered to have fungal infections by the difference which was larger than 10 pg/ml between the T-test and E-test (T-E) values. Three cases (T-E= 117.7-217.8 pg/ml) were proved to have fungal infections of internal organs. Four cases (T-E=23.8-31.7 pg/ml) were proved to have skin and mucous membrane fungal infections. Thirty-eight patients (E-test >3 pg/ml) were suggested to have GNR infections. In these patients, 6 (E-test =18.7-96.9 pg/ml) were proved to have GNR infections. Some cases showed false posibility of the T-test was related with the use of gammaglobulin, glycerol, transfer factor and interferon. The T-test and E-test were a useful methods for the possible diagnosis of fungal and GNR infections, but the effect of the above substances on the test results should be born in mind.

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We examined the somnolence syndrome, which is one of the side effects of cranial irradiation. Out of 53 patients in acute leukemia who had received cranial irradiation, nine patients (17%) developed the somnolence syndrome. Patients with the somnolence syndrome showed slow waves on EEG. Some patients had ventricular dilatation and widening of sulci before cranial irradiation on CT findings, but these findings improved after cranial irradiation. Out of nine cases with the somnolence syndrome, 6 patients survived and did not experience difficulties in school. But one patient showed calcification on CT brain scan. It is considered that the cause of the somnolence syndrome is a trasient inhibition of myelin synthesis and most patients improved without serious sequelae. It is necessary to follow up many cases of somnolence syndrome.

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Severe neurological complications during and after chemotherapy were surveyed in 426 patients with acute lymphocytic leukemia (ALL) who were registered in the Children's Cancer and Leukemia Study Group (CCLSG) during 1981 to 1987. Eleven of the 426 cases developed neurological dysfunction or encephalopathy. Seven patients with overt central nervous system (CNS) leukemia, treated with frequent intrathecal methotrexate (MTX) and/or skull irradiation, developed seizure, ataxia, and gradually an unconscious condition, and were diagnosed as having necrotizing leukoencephalopathy. However, four patients out of 11 cases who received early consolidation therapy with 3 courses of high-dose MTX (1.5-6.0 g/m² with 24-hour infusion) and intrathecal MTX (15 mg/m² for 6 doses) for CNS-prophylaxis, developed temporary neurological complications, such as abrupt episodes of convulsion and loss of consciousness. All neurological abnormalities, including transient, nonspecific abnormalities in CT scan and EEG findings, recovered to normal within 30 days without any sequelae. After the modification of dosage and number of intrathecal injections of MTX (reduced to 12 mg/m² for one dose), these neurological complications were not observed. These data suggested that transient subacute neurotoxicities of MTX had a mechanism that was not similar to necrotizing leukoencephalophathy.

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Conventionally, multidrug therapies have been applied extensively for the purpose of increasing efficacy of antitumor agents. To obtain favorable therapeutic results it is necessary to know the interaction among the drugs. Methotrexate (MTX) and 1-β-D-arabinofuranosyl cytosine (Ara-C) are representative drugs for leukemia therapy. Although some inconsistency is seen among the reports of various authors, in many papers these two drugs have been reported to act antagonistically when used at the same time. In the present study, we investigated interaction between MTX and Ara-C using CCRF-CEM cells already established. MTX and Ara-C were cytotoxic against CCRF-CEM cells in both a concentration-dependent and time dependent manner in respective use. When MTX and Ara-C were administered simultaneously or administration of Ara-C preceded that of MTX, colony count showed even an increasing tendency when compared with administration of Ara-C alone in methylcellulose culture. Under these conditions both drugs are considered to interact antagonistically.

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Ara-C-resistant cells have increased cytidine deaminase activity and deoxycytidine kinase activity compared with cells sensitive to Ara-C. From these results it was found that both enzyme activities are closely related to the mechanism for the development of resistance to Ara-C. A remarkable decrease in incorporation of Ara-C into these cells was also found. After incubation of Ara-Cresistant cells with MTX, their cytidine deaminase activity decreased and deoxycytidine kinase activity increased. These findings indicate that treatment with MTX is effective to restore sensitivity of cells to Ara-C. Moreover, the pretreatment with MTX increased incorporation of Ara-C into cells. The results may indicate that administration of MTX followed by that of Ara-C 1 or 2 hr later provides synergism of both drugs in cell toxicity and that this combination therapy is effective even against Ara-C-resistant cells. Then, a sequential ACMP regimen (S-ACMP) was designed and applied clinically. For 19 cases of intractable leukemia, S-ACMP regimen was applied. CR was obtained in 9 cases (47%) and PR in 6 cases (32%), with an efficacy rate of 79%.

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In this report, we studied the side effects of methotrexate (MTX) in patients with ALL who had maintained complete remission with treatment by the 10th or 11th TCLSG protocol. 1 : In vivo administration dose of MTX to ALL patients must be modified in respect to the appearance of liver dysfunction, leukopenia, and exanthema, the necessity of the modification increased according to the clinical course. 2 : MTX concentration in serum at 4 hours after per os administration was observed between 1.3 × 10^-7 mol/ml and 5.8 × 10^-7 mol/ml. 3 : Effects of MTX on TdR-incorporation were studied on 20 cases and different effects were observed. The effects could be tentatively divided into three groups : TdR-incorporation was increased in 5 cases, decreased in 2 cases, and was not influenced in 13 cases. 4 : Some side effects observed in ALL patients were thought to be induced by allergic reaction according to the clinical signs and the experimental results.

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We evaluated delayed neurotoxicities in treatment of childhood acute leukemia. Of 28 patients treated over 2 years who were examined on computed tomography of brain scans, 7 patients had abnormal findings. These abnormalities included two cases of leukoencephalopathy, three cases of intracranial calcifications, and two of ventricular dilatation. These patients were under 6 years old at the onset of disease, especially under 3 years old. Also, delayed neurotoxicities developed after relapse of leukemia, especially CNS relapse. It was considered that these were caused by cranial irradiation, intravenous methotrexate injection, intrathecal methotrexate, and sometimes high-dose Ara-C therapy, etc. Most of the cases of leukoencephalopathy were associated with treatment of intermediate-dose or high-dose methotrexate after relapse. These abnormalities must be carefully considered in the treatment of younger children with leukemia and patients with relapse.

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Seven pediatric patients with refractory acute leukemia (five with acute lymphoblastic leukemia, one with acute non-lymphoblastic leukemia, one with leukemic transformation of malignant lymphoma) received mitoxantrone as a single agent. The median age of the patients was 6 years (range, 2 years to 16 years). The median dose of the cumulative anthracyclines (i.e., adriamycin and/or daunorubicin and/or aclarubicin) was 300 mg/m² (range, 195 mg/m² to 1575 mg/m²). Mitoxantrone was administered intravenously at a dose of 5-10 mg/m² daily for five consecutive days. Of the six evaluable patients, one who achieved complete remission had received cumulative doses of aclarubicin at 1575 mg/m² before the mitoxantrone course. Five of the six evaluable patients, that had survived four weeks or longer, had suffered from severe myelosuppression. All five patients had received the antibiotics because of suspected infection; two had documented sepsis, one had fungal pneumonia resulting in death. Further study is needed to determine the value of mitoxantrone in the treatment of acute leukemia in children.

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BHAC is a newly synthesized lipophilic derivative of ara-C, and it has been widely used in the treatment of acute leukemia in Japan. To clarify its pharmacological mode of action, P388 murine leukemic cells were incubated with two different types of ¹⁴C-labeled BHAC- (cytosine-2-¹⁴C) BHAC and (acyl-1-¹⁴C) BHAC-and DNA was extracted with phenol. The phenol-extracted DNA was then hydrolyzed by nuclease P₁ and analyzed with high-performance liquid chromatography (HPLC). The radioactivity of DNA, from the cells incubated with (cytosine-2-¹⁴C) BHAC, was detected as ara-CMP. But the radioactivity of DNA, from the cells incubated with (acyl-1-¹⁴C) BHAC, was hardly detected. On the other hand, the main radioactivity of the acid-soluble fraction was determined as ara-CTP. On the basis of our results, BHAC is not phosphorylated directly to produce N⁴-behenoyl-ara-CTP, but is mainly converted to ara-C which subsequently produces ara-CTP, the active metabolite of the drug, and which is then incorporated into DNA.

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Serum level of IgG subclass was measured in 11 children with acute lymphocytic leukemia (ALL) and age-matched (5-12 years) normal children in order to analyze the effects of maintenance chemotherapy on immune status of ALL. The results were as follows : Serum IgG₁ and IgG₃ levels in the patients were 7.28 (4.41-12.02 : geometric mean±SD) mg/ml and 0.37 (0.16-0.90) mg/ml, and those in normal controls were 8.38 (5.94-11.80) mg/ml and 0.35 (0.20-0.61) mg/ml, respectively. The differences were not significant. Serum IgG₂ and IgG₄ levels in the patients were 0.64 (0.21-1.98) mg/ml and 0.018 (0.011-0.029) mg/ml, and those in normal controls were 1.99 (1.04-3.81) mg/ml and 0.10 (0.041-0.24) mg/ml, respectively. The differences were statistically significant (p<0.05 and p <0.005). IgG₂ levels of 4 patients were less than-2SD the level of normal controls, and IgG₄ was not detectable in 6 patients. Though no recurrent or severe infection occurred during maintenance chemotherapy, this IgG subclass deficiency may cause severe infection. In conclusion, gammaglobulin preparations should be appropriately used in infectious episodes in ALL during chemotherapy.

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We studied the efficacy of antilymphocyte globulin (ALG) and antithymocyte globulin (ATG) in the treatment of 40 cases of moderate to severe aplastic anemia in children. These included 32 cases of idiopathic type, one case of Fanconi's anemia, 3 cases of secondary type, and 4 cases of Blackfan-Diamond's anemia (PRCA). ALG was administered to 39 patients and ATG was administered to one patient. The ALG or ATG was infused to the patients at a rate of 20-40 mg/kg/day f or 5 days. Ten of them received a combination of ALG and high-dose methylprednisolone. Six out of 25 severe cases and one out of 10 moderate cases had a complete or partial response within 3 months, respectively. Statistically, the response rate and survival rate were not influenced by the combination of ALG and high-dose methylprednisolone. The transient improvement was observed in one case out of 4 cases of PRCA. The proportion of CD4, CD8, and CD4/CD8 ratio did not correlate with clinical response before and after ALG or ALT therapy. Complications were minimal and included moderate fever, urticaria, exanthema, and glucosuria. Combination therapy with ALT or ALG and high-dose methylprednisolone should be considered in childhood severe aplastic anemia when bone marrow transplantation is not possible.

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A two-year-old boy with severe aplastic anemia underwent successfully the second marrow grafting from his HLA-identical sister after the rejection of the first graft from the same HLA-identical donor. A skin rash and conjunctivitis developed as acute GVHD. Following the tapering off of oral prednisolone for chronic GVHD by day 538, he developed recurrent cough on exertion. Bronchodilators and antibiotics were administered without improvement. Pulmonary ventilation scans showed abnormal areas of decreased activity, corresponding to similar areas of decreased activity on perfusion scans. Dryness of the eyes and chronic infection of lacrimal sacs due to the obstruction of the nasolacrimal ducts were also noted. Oral prednisone following intravenous methylprednisolone was begun with clinical improvement. Although air-trapping remained on lung volume studies, with enlarged residual volume and reduced forced expiratory volume, quality of life of the patient markedly improved following the oral prednisone. It is suggested that prompt clinical diagnosis and administration of immunosuppressive drugs might be very important for the treatment of the pulmonary manifestation of chronic GVHD.

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We observed two infants with CNS leukemia in which CT brain scans show chronic subdural hematoma. Later, the one relapsed hematologically, and died due to severe infection six months after onset. The other obtained complete remission and received cranial radiation of 24 Gy at the age of one year. Three months after radiotherapy, she developed severe generalized convulsion. CT evaluated diffuse intracranial calcifications and co-existing subdural hematoma. We suspect that in the latter case, the causative factors of early progession of the adverse effects following cranial radiation are that the onset of her disease is in infancy and with CNS leukemic infiltration, and that the age at which radiation was received is low (one year). Moreover, we suspected that the existence of chronic subdural hematoma contributed to progression of the adverse effects.

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We describe a 4-year-old boy with biphenotypic leukemia. At the onset of the disease, he was diagnosed as FAB-L1 and non-T, non-B ALL (common ALL), on the basis of morphology of small lymphoblastoid cells negative for peroxidase staining (PDX) on the bone marrow smear, and marker profiles (Ia⁺, CD 10⁺, CD5^-, CD 13^-, and E rosette^-). Complete remission was achieved by TCLSG 10th, ALL standard risk protocol. However, relapses were observed at 17, 26, and 31 months after the first remission. At the third relapse, PDX⁺ large blasts appeared in bone marrow together with PDX-small blasts. The surface marker analysis revealed the blasts with Ia⁺, CD 10⁺, CD 19⁺, and CD 13⁺. By double staining with CD 19 and CD 13, about 30% of the blasts reacted to both antibodies. As indicated in this case, biphenotypic leukemias may exist among early relapsed ALL, and surface marker analysis should be repeated in detail for detecting such type of leukemias.

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A 4-year-old girl with common ALL exhibited marked enlargement of bilateral kidneys and swelling and pain of multiple joints as observed in juvenile rheumatoid arthritis. Her kidneys, which were smooth-surfaced, elastic, and hard, were palpated in the bilateral hypochondrial regions on admission. Erythema was observed on dorsa and palms of bilateral hands and bilateral foot joints, and swelling and pain were noted in the DIP and PIP joints of the first and second fingers of the left hand, left knee, and bilateral foot joints. The peripheral WBC count was 16, 400/mm³, and no leukemic cells were observed. Bone marrow blood showed 79.2% blasts, and their morphology was equivalent to L1 of the FAB classification. A large number of nuclear pockets were observed under electron microscopy. The patient was treated by the high risk regimen H-1 for ALL according to the 11th protocol of TCLSG, and the bilateral enlargement of kidneys and joint symptoms were resolved and complete remission was obtained.

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A 10-year-old girl with acute mixed lineage leukemia (AMLL) is presented. Bone marrow examination at diagnosis showed 66.5% small leukemic cells with lymphoblastoid feature and 30.0% large leukemic cells with monocytoid feature. Surface marker analyses revealed that large leukemic cells expressed CD2, CD 19, HLA-DR, CD33, and LeuM3, whereas small leukemic cells bore only CD2 antigen. Both of these leukemic cell populations were negative for periodic acid-Schiff, peroxidase, and alpha naphthyl butyrate esterase. Large leukemic cells phagocytized ox red blood cells coated with goat antimouse immunoglobulin antibody, but small leukemic cells did not. These findings were compatible with those of AMLL composed of two separate leukemic cell populations. Additional studies using X-chromosome-linked DNA polymorphism to determine the clonal origin of the leukemic cells supported the idea that these two leukemic cell populations originated from two independent clones in the bone marrow. Thus, recombinant technology is helpful for the study of clonal origin of leukemias with a mixed lineage phenotype.

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