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Morphological changes in the testis induced by chemotherapy given according to the Tokyo Children's Cancer Study Group (TCCSG) regimens were studied in children with acute lymphoblastic leukemia (ALL). After informed consent, testicular biopsies were performed 14 times in 12 patients at the end of treatment. The testicular morphology in all cases had sustained a degree of damage. The tubular fertility index (TFI), calculated as the percentage of seminiferous tubules containing identifiable spermatogonia, was from 0 to 42.8% (mean 33.4%) below the normal value. Infiltration of leukemic cells was the most significant factor contributing to the decrease in TFI. There were no differences in the TFI among the TCCSG protocols. Formation of sperm was recognized in six cases, whose ages were 7, 8, 9, 10, 15 and 19 years. In two children, testicular biopsy was performed twice. In the second biopsy, TFI was elevated and sperm formation with the maturation of Leydig cells was observed. A number of other pathological changes were observed: modification of spermatogonia, Sertoli cells and inclusion bodies in spermatogonia, abnormal maturation of Leydig cells, evidence of interstitial fibrosis and thickening of the basement membrane. These results suggest that recent strong chemotherapy for the treatment of ALL might cause severe but not fatal damage to children's testicular tissue. As chemotherapy escalates, more investigation of testicular function will be necessary.

Encouraging results are reported with high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation in the treatment of advanced neuroblastoma. However, relapse remains a significant problem. We used high-dose chemotherapy, surgery, intraoperative radiation and an autologous bone marrow transplant treated in vitro to remove tumor cells followed by 13-cis-retinoic acid to treat 36 children with advanced neuroblastoma. This comprehensive treatment appears to improve the survival rate of patients with advanced neuroblastoma, including those with N-myc amplification and bony involvement. The disease-free survival rate was 66% (95% confidence interval, 49-84%) at 3 years. All patients who received 13-cis-retinoic acid developed cheilitis, but no bone marrow depression occurred in these patients. Five patients developed hemolytic uremic syndrome (HUS) post-transplant. This may have been related to the procedure used for total body irradiation. Patients who had their kidneys shielded during this procedure did not develop this syndrome. Patients who received local irradiation at the primary site showed no evidence of relapse in this region, indicating that such therapy may help to prevent a relapse. These data suggest a high rate of 3 year disease-free survival with this treatment strategy. The nonrandomized nature of the study and use of multiple modalities precludes analysis of the specific contribution of each.

Ex vivo expansion of hematopoietic progenitor cells in the umbilical cord blood mononuclear cells (CB-MNC) was investigated in liquid culture system with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO], and interferon [INF]-gamma). Non-lineage-committed hematopoietic progenitor cells and lineage committed hematopoietic progenitor cells were represented as CD34+CD38- and CD34+CD38+ subpopulations, respectively. Although absolute CD34+CD38- cell numbers decreased even in the presence of multicytokines, the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus IFN-gamma were significantly effective in maintaining CD34+CD38- cells than the other combinations (P < 0.05). After 4 weeks of culture. CD34+CD38- cells disappeared in all combinations of cytokines. Absolute CD34+CD38+ cell numbers increased in the presence of cytokines. Maximal expansion of CD34+CD38+ cells were observed in the combinations of SCF plus IL-3 plus IL-6 plus EPO (19.8 +/- 3.3-fold) and SCF plus IL-3 plus IL-6 plus G-CSF (18.3 +/- 2.6). The combination of SCF plus IL-3 plus IL-6 was also effective to expand CD34+CD38- cells (15.8 +/- 3.9). However, the expansion was transient and they decreased to zero within 3 weeks. In the combinations of SCF plus IL-6 and SCF plus IL-3 plus IL-6 plus INF-gamma, maximal expansion was inferior to the others but CD34+CD38+ cells were maintained more than 4 weeks. These results suggested that the indication of CBT can be expanded into older children by ex vivo augmentation of CB hematopoietic progenitor cells using multi-cytokines.

We compared the effects of various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) among the growth of human hematopoietic progenitor cells from cord blood (CB), bone marrow (BM), and peripheral blood mononuclear cells (MNC) mobilized by chemotherapy and G-CSF (PB) in a semi-solid medium. Macroscopic colonies, that were visible to the naked eye, were formed from PB-MNC within 1 week even without cytokines. They consisted of blasts containing macrophage-like cells with immature nuclei on Wright stain, and were strongly accelerated by IL-3. Macroscopic colonies were also formed from CB-MNC. However, they appeared after 1-3 weeks and synergistic effects of SCF with other cytokines, especially EPO, were prominent. Macroscopic colonies were not formed from BM-MNC. Granulocyte-colony stimulating factor was effective in increasing colony forming units of granulocyte macrophage from BM-MNC and they appeared between 1 and 2 weeks. These results suggested that the quality of hematopoietic progenitor cells was different among blood sources. This might lead to different bone marrow recovery patterns after transplantation of each blood source. The appropriate cytokines should be added to evaluate their exact potential.

Since January 1987, chemoprophylaxis and empiric therapy with antifungal drugs have been used routinely in neutropenic children with neoplasms. The incidence of terminal invasive fungal infection (IFI) diagnosed by autopsy findings is compared in two groups: group A, consisting of 25 patients autopsied between January 1982 and December 1986, and group B, consisting of 14 patients autopsied between January 1987 and December 1991. There was no difference in the incidence of IFI between the two groups, but the characteristics of IFI appeared to differ. With the recent intensification of chemotherapy, the patients in group B had more risk factors for IFI. It might therefore have been anticipated that group B patients may have shown an increase in IFI. No such increase was observed, suggesting that the prophylaxis was effective. Autopsy findings also showed that the target organs of IFI were localized and that systemic candidiasis was decreased in group B. It is thus concluded that the present antifungal countermeasures are effective, although some problems still remain.

A total of 91 peripheral blood stem cell collections were performed in 26 children with various malignant tumors and peripheral blood stem cell transplantations (PBSCT) were performed in 15 of the children. There was a positive correlation between logarithm of total CD34+ cells/kg and logarithm of colony-forming unit-granulocyte macrophage (CFU-GM)/kg (r = 0.86). The time elapsed until the white blood cells (WBC) exceeded 1000/microL was related to both CFU-GM (r = 0.67) and CD34+ cell count (r = 0.60). The number of days elapsed until platelet count exceeded 5 x 10(4)/microL was not related to the logarithm of CFU-GM count/10(5) per kg transfused (r = 0.47), but was related to the logarithm of CD34+ cell counts/10(6) per kg transfused (r = 0.73). The number of days elapsed until the reticulocytes exceeded 10% was not related to the logarithm of CFU-GM count/10(5) per kg (r = 0.52), but was related to the logarithm of CD34+ cell counts/10(6) per kg transfused (r = 0.91). Although CD34+ cell counts correlated with the number of CFU-GM, bone marrow regeneration rates in three lineages were predicted more accurately by the number of CD34+ cells transfused than by the number of CFU-GM. These results suggest that measurement of the CD34+ cell count may be useful in predicting bone marrow regeneration rate after PBSCT.

PATIENTS AND METHODS We report patients who were treated for non-Hodgkin's lymphoma (NHL) or Ki-1 antigen-positive (Ki-1) lymphoma with a T-8801 protocol that included etoposide (VP-16) and behenoylcytosine arabinoside. RESULTS Secondary acute myeloid leukemia (AML) developed in 5 of 38 NHL and Ki-1 lymphoma patients, and the cumulative risk at 4 years was 18.4%. The median time from the initiation of the chemotherapy to the development of AML was 21 months (range, 13-30). Four patients had a FAB M5 morphology, and one had FAB M2. In four of five examined cases, chromosomal alterations involving the long arm of chromosome 11 were demonstrated at the time of development of AML. None of the 46 NHL patients who we treated with another protocol (B-8801), using significantly higher cumulative doses of VP-16 than in the case of the patients with T-8801 and a different schedule of VP-16 administration, developed secondary AML. CONCLUSIONS The risk of secondary AML possibly related to the use of VP-16 given twice weekly.

A 2-year-old boy with B-cell lymphoma (stage IV) in the first complete remission received myeloablative treatment followed by peripheral blood stem cell autotransplantation and is surviving event-free for 18 months. Reconstitution of all cell lines occurred very rapidly, ie, a level of 1000 leukocytes per microliter was reached after 9 days, that of 500 granulocytes per microliter after 10 days, and platelets were substituted only once. This result suggests that PBSCT can be safely employed during consolidation therapy for malignant disorders because of its short duration of bone marrow suppression.

Late effects of childhood acute leukemia and its treatment were studied in 776 patients (684 ALL, 73 ANLL, and 9 others) in Japan who had remained in remission for more than 1 year after their first complete remission. Delayed adverse sequelae involve a wide variety of organs and their functions. Short stature was present in 2.61%, obesity in 3.79%, abnormalities of growth hormone secretion in 1.5%, delayed secondary sex characteristics in 1.5% of males and 0.6% of females, motor disturbances in 1.17%, sensory disturbances in 0.91%, intellectual and learning disabilities in 2.48%, abnormal findings in routine neurologic examinations in 1.31%, EEG abnormalities in 4.30%, brain CT abnormalities in 5.09% and cardiac dysfunction in 1.07%. Various other disorders were seen in 20 patients. Many of these delayed adverse sequelae are caused by or related to central nervous system prophylaxis and systemic combination chemotherapy. The results suggest that it is needed to improve therapeutic methods through the stratification of patients by risk factors and detailed analysis of prognostic factors. Moreover it is important to render medical and psychosocial support to long-term survivors of childhood leukemia through interactions between the patient, parents and medical staff.

Since children with acute leukemia have prolonged periods of severe neutropenia due to the disease itself or its intensive chemotherapy, they often develop life-threatening infectious complications. A protected environment and prophylactic antibiotics for patients with severe neutropenia have been shown to decrease the incidence of infectious morbidity. In spite of the high mortality rate of infections, on the other hand, their diagnosis is not well established because the percentage of microbiological detectability by blood culture is low. The differentiation of infections may be possible by using some supplementary measures and particular laboratory parameters. Febrile neutropenic patients with leukemia should promptly receive empiric treatment with combinations of two or three broad-spectrum antibiotics that are bactericidal and synergistic toward the presumed pathogens, or with antifungal drugs if patients are resistant to antibiotics, as well as supportive measures such as granulocyte transfusion, intravenous gamma-globulin and granulocyte colony-stimulating factor.

The Tokyo Children's Cancer Study Group (TCCSG) has performed prospective randomized controlled studies since 1984 for childhood acute lymphoblastic leukemia (ALL). Four hundred and ninety-eight newly diagnosed patients were treated with 5 different regimens designated as S1, S2 for a standard risk group (SRG), H1 and H2 for a high risk group (HRG) and HEX for an extremely high risk group of patients. We added intermediate-dose methotrexate as early intensification therapy in the S2 and H2 groups to reduce extramedullary and medullary relapses. Event-free survival of all patients at 4 years 6 months from the start of this regimen (median follow-up period 32 months) was 67.5%. The CNS relapse rate was significantly decreased to 2.2% compared to previously reported studies and our own experience. There were some unexpected complications of the CNS such as myelopathy and/or leukoencephalopathy. For the SRG early ID-MTX seemed to be useful to improve the prognosis, but triple intrathecal injections (TIT) should be limited in number. The 24Gy cranial irradiation (CRX) was effective but possibly excessive for the HRG. As evidenced by the success of the HEX group regimen, more intensive multi-drug consolidation in the early post-remission phase might be necessary to improve further the prognosis and to make it possible to shorten the treatment periods.

We conducted a survey by questionnaire to clarify the actual conditions of neonates born to mothers with autoimmune thrombocytopenic purpura (ATP) in Japan. We investigated 93 pregnancies (1 resulting in twins) in 31 hospitals between 1985 and 1994. Forty-nine of the neonates (52%) had thrombocytopenia (below 150 x 10(9)/L). Nineteen neonates (20%) showed a bleeding tendency, but this was generally mild. In only one neonate (1%) (a case of asymptomatic intracranial hemorrhage, ICH), deep bleeding occurred due to thrombocytopenia. The lowest platelet count of neonates after birth occurred on day 4, not on day 0. There was no correlation between maternal and neonatal platelet counts. However, there was an apparent correlation between the neonatal platelet count on day 0 and the lowest platelet count after birth. Treatment of the mothers with intravenous high-dose gamma-globulin and prednisolone did not prevent risk of neonatal thrombocytopenia significantly.

We evaluated platelet associated immunoglobulin (PIag) G, PaIgM, platelet associated autoantibodies to platelet glycoprotein IIb/IIIa (Pa-GPIIb/IIIa), the percentage of CD5+ B cells and the amount of platelet-bound anti-GPIIb/IIIa monoclonal antibody (mAb) in the peripheral blood of 29 patients with childhood onset chronic immune thrombocytopenic purpura (c-ITP). The percentage of CD5+ B cells ranged from 2 to 8% (4.7 +/- 2.0) in control patients and 1 to 18% (6.2 +/- 4.2) in the ITP patients. There was no overall significant difference between the two groups, but the percentage of CD5+B cells in six of the ITP patients was higher than the mean + 2 s.d. of the controls. There was a significant correlation between the percentage of CD5+ B cells and PaIgM (y = 1.73x + 13.4, r=0.40, P < 0.05). This finding is the basis for the speculation that CD5+ B cells may play an important role in the production of PaIgM in vivo. There was no correlation between the amounts of PaIgG and Pa-GPIIb/IIIa). This suggests that the amount of PaIgG does not accurately reflect of the amount of Pa-GPIIb/IIIa. Furthermore, we have demonstrated that autoantibodies to GPIIb/IIIa are directed to more that one epitope.

To investigate immaturity of hematopoietic progenitor cells in umbilical cord blood mononuclear cells (CB-MNC), the formation of macroscopic colonies and mixed-cell colonies was assayed by methylcellulose culture with various combinations of cytokines (stem cell factor [SCF], interleukin [IL]-3, IL-6, granulocyte-colony stimulating factor [G-CSF], erythropoietin [EPO]) and compared with bone marrow (BM)-MNC. Moreover, distribution of the subpopulations divided by CD34, CD38, HLA-DR and CD33 was compared by flow-cytometry. Colonies derived from CB-MNC were so large that they could be observed with the naked eye and consisted of a variety of types of hematopoietic cells. Mixed-cell colonies were formed to a much greater extent in CB-MNC than in BM-MNC. Addition of EPO, IL-3, and SCF had rapid effects on the growth of mixed-cell colonies. The subpopulations of immature hematopoietic progenitor cells (CD34+, CD38-, HLA-DR-), which are supposed to be able to differentiate into hematopoietic precursors and stromal cells, were significantly higher in CB-MNC (8.7 +/- 6.6%) than in BM-MNC (0.0 +/- 0.1%; P < 0.001). These results suggest that CB is a rich source of immature hematopoietic progenitor cells compared to BM.

This paper reports on a patient with lymphoma syndrome leukemia (LSL) who showed interesting findings on brain computed tomography (CT) and ultrasound scans of the abdomen at the initial presentation. The patient was a 5 year old girl. When she was admitted to our hospital, there were many lymph nodes palpable. The abdomen was distended and the liver and spleen were palpable below the umbilicus. Hematologic examinations revealed a leukocyte count of 275,800/microL with 98% lymphoblasts. Chest X-ray film revealed a mediastinal mass. The diagnosis of LSL was made. Her brain CT scan showed a low density area in the right thalamic region without contrast enhancement; infarction was suspected. Furthermore, her abdominal ultrasound scan showed hepatosplenomegaly, kidney swelling with increasing echogenicity and hydronephrosis and stones in the renal pelvis and bladder. These findings are unprecedentedly rare in cases of childhood acute lymphoblastic leukemia (ALL), much less in LSL.

This retrospective study attempts to clarify the optimal timing for peripheral blood stem cell (PBSC) collection after conventional chemotherapy followed by granulocyte-colony stimulating factor (G-CSF) administration. Leukapheresis was performed 32 times in nine children with various cancers during bone marrow recovery phase following transient pancytopenia after chemotherapy. (On two occasions, leukapheresis was excluded because many leukemic blasts were included). When the number of white blood cells (WBC) exceeded 1.8 x 10(10)/L after administration of G-CSF (200 micrograms/m2, continuous infusion), many more CD34+ cells were contained in the collected peripheral mononuclear cells (P > 0.02) and a sufficient number of PBSC for transplantation (> or = 10 x 10(8) CD34+ cells/kg) was obtained after one run in 15 of 17 leukapheresis sessions. In contrast, sufficient PBSC were obtained only in one of 13 runs of leukapheresis when the number of WBC was < 1.8 x 10(10)/L. The number of WBC on the day when PBSC were collected correlated with collected nuclear cell number (r = 0.60), but not with the CD34+ cell ratio. The ratio was higher only when both platelets and reticulocytes increased in parallel with WBC. We conclude that sufficient PBSC collection is possible after conventional chemotherapy using G-CSF, when hematopoietic recovery is parallel, without the use of high-dose chemotherapy.

Determining the site and severity of blood loss is important in the management of children with gastrointestinal (GI) bleeding. Blood urea nitrogen (BUN) and serum creatinine (Cr) were measured on the day of hospitalization and the ratio of BUN/Cr was calculated in 11 children with 16 episodes of upper GI bleeding and 49 with lower GI bleeding. There was a significant difference between the two GI bleeding groups with regard to BUN/Cr ratio (p less than 0.001). When the ratio was 30 or above, the specificity of upper GI bleeding was 98% with a sensitivity of 68.8%. A linear relationship was found between the BUN/Cr ratio and delta Hb (delta Hb = 0.08 x BUN/Cr +/- 0.8 g/dl) for bleeding originating from the upper GI tract. This study confirms that measurement of the BUN/Cr ratio is useful for localizing the source of bleeding to the upper GI tract and also demonstrates its usefulness as an estimation of the severity of blood loss from the upper GI tract.

Infection is the major complication occurring in children with cancer and is often fatal. Since the clinical manifestations of infection are slight, except for fever, a definitive diagnosis is difficult in the early phase of infection, but using laboratory parameters, the differentiation of bacterial infections may be possible to some extent. The infection cannot be treated only by antimicrobial chemotherapy, but supportive measures such as neutrophil transfusion, and other biological medications should be given in consideration of the deficiency of the immune defense mechanisms of the patient. Anticancer treatment is also needed, along with antimicrobial treatment. The prophylactic use of antimicrobial drugs is still controversial but it is effective, depending on the type of infection for which it is used, and on the status of the defective defense mechanism in the patient who is on cancer chemotherapy.

Platelet-associated IgG (PAIgG) levels were measured in 60 children with ITP (46-chronic, 14-acute) using Fab-anti Fab radioimmunoassay method described by McMillan et al. In some patients platelet binding IgG in serum (PBIgG) was also determined at the same time. Patients with ITP had significantly greater PAIgG levels than 30 normal subjects and 13 non-immune thrombocytopenic controls. Elevated PAIgG values did not correlate with parameters of platelet size (mean platelet volume; MPV and percentage of large platelet; PLP) and so these data indicated that high levels of PAIgG in ITP were not due to nonspecific adhesion of serum IgG to megathrombocytes usually increased in this disorder, but due to specific immunological reaction. PBIgG IgG values were also elevated in patients with pretreated chronic ITP, but high levels remained even after successful splenectomy. Furthermore, serial determination of PAIgG values were obtained in some patients with chronic ITP who underwent splenectomy and with acute ITP who achieved spontaneous remission. PAIgG returned to normal levels when thrombocytopenia disappeared. PAIgG seems to be the most reproducible indicator which reflects transition of the clinical picture in this disorder.

This work was supported in part by a grant-in-aid from the Japan Medical Research Foundation and by Scientific Research Grants (project No. 57770632) from the Ministry of Education, Science and Culture of Japan.

Platelet volume was measured in 20 healthy children, 18 patients with acute idiopathic thrombocytopenic purpura (ITP), 24 patients with chronic ITP, 22 patients with aplastic anemia, and 17 patients with acute leukemia in childhood. The authors compared the platelet volume of these patients by use of peak platelet volume (PPV or mode of the platelet volume), mean platelet volume (MPV) and the percent of large platelets (PLP) as parameters of the platelet volume, and then studied the relationship between the platelet volume and the specific gravity of platelets from these patients. The following was the platelet volume of normal children: PPV was 6.7 +/- 1.1 micrometer 3, MPV was 9.9 +/- 1.6 micrometer 3, PLP was 26.0 +/- 7.7%. All parameters of the platelet volume were remarkably large in chronic ITP, but only PPV was small in acute ITP. On the other hand, all parameters were small in both aplastic anemia and acute leukemia. In acute ITP and chronic ITP, the platelet volume returned to normal value after these thrombocytopenias disappeared. The platelet volume, however, still remained small even after remission in acute leukemia. There was better correlation between MPV and PLP than PPV and PLP. The density analysis of the platelets obtained from platelet-rich plasma (PRP) revealed that PLP in each medium (1.010 approximately 1.052) of normal PRP increased slowly in proportion to the increase of specific density of the media. On the contrary, most of large platelets obtained from the patients with acute ITP and chronic ITP were especially contained in the medium of high specific density (1.052). Large platelets of aplastic anemia and acute leukemia, however, were found in both low and high density media. The authors believe that analysis of the platelet volume using above mentioned three parameters would provide good weapon in early differential diagnosos of acute ITP, chronic ITP, and aplastic anemia. And these findings also may suggest the difference of thrombopoiesis between acute ITP and chronic ITP.

The pathogenesis of the anemia of chronic renal failure is multifactorial. Anabolic steroid has been reported to be of therapeutic value, but it is difficult to use because of its side effect especially in children. We used an analog of activated vitamin D (1α-OH-D3) in 6 haemodialysis patients with anemia. After lα-OH-D3 treatment, hemoglobin and hematocrit values didn't change in these patients, but frequency of blood transfusion has been decreased. We examined serum erythropoietin by in vitro bioassay and haemoaggulutination inhibition assay. And we also examined the effect of various vitamin D metabolites on in vitro growth of granulopoietic and erythropoietic colonies. lα-OH-D3 is suspected to have anabolic steroid like effect.