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Forty-nine children who received peripheral blood stem cell autografts (PBSCT) were observed for the development of transfusion-related disease (TRD), including cytomegalovirus (CMV) disease, alloimmunization, and graft-versus-host disease (GVHD). Cytoreductive regimens did not include total body irradiation. A comprehensive prophylactic strategy including alkylated hyperimmune CMV globulin and filtered, nonirradiated, leukocyte-poor blood products was applied. Two patients showed temporary refractoriness to platelet transfusion, but none suffered from symptomatic CMV infection or GVHD. Thus, under these conditions the incidence of TRD appears to be low in children undergoing PBSCT.

A 5-year-old girl with stage IV neuroblastoma with diesseminated marrow involvement was treated with high-dose chemotherapy and autografts with peripheral blood stem/progenitor cells (PBSC), without local or total-body irradiation. After PBSC autograft, hematologic recovery was complete. Without further anticancer therapy, she remained tumor-free for 38 months until a local recurrence was identified. This case illustrates that late relapse can occur in advanced-stage neuroblastoma after long-term remission following high-dose chemotherapy without irradiation.

Purified peripheral blood hematopoietic progenitors from children in early remission from cancer respond to recombinant human interleukin-3 (IL-3), but not to granulocyte colony-stimulating factor (G-CSF). With these purified cells as a target, we studied the effect of recombinant human interferon-gamma (IFN-gamma) on progenitor growth, using both liquid-suspension limiting dilution assay (LDA) and regular methylcellulose culture of progenitors. We found that in LDA with IL-3, IFN-gamma directly stimulated the growth of blood progenitors in a dose- dependent manner with single-hit kinetics, whereas IFN-gamma suppressed the growth of G-CSF-supported progenitors obtained from bone marrow. The stimulatory effect was also observed in methylcellulose culture, but the addition of antibodies for G-CSF, granulocyte-macrophage CSF, IL-1 alpha, IL-1 beta, IL-6, or tumor necrosis factor did not result in a decrease of the colony number, supporting further the possible direct effect of IFN-gamma on progenitor growth. These results suggest that the inhibitory effect of IFN-gamma on hematopoietic progenitors is limited to those in an advanced stage of maturation. IFN-gamma may be one of the essential lymphokines upregulating the growth of human hematopoietic progenitor cells.

Background: We previously reported that interleukin (IL)-8 increased up to 207 pg/ml in vivo after administration of granulocyte colony-stimulating factor (G-CSF) at a dose 5μg/kg for five days to mobilize peripheral blood stem cells (PBSC) in hematopoietic stem cell transplantation in cancer patients or healthy donors. Since pre-apheresis serum IL-8 levels correlated with the yield of CD34-positive cells, IL-8 production may be critical to G-CSF induced stem cell mobilization. IL-8 is known to be surged from granules of endothelial cells (EC) by stimulation of GM-CSF, however, precise mechanisms of increased production of IL-8 by G-CSF in vivo is unclear. First, in this study we examined the relationship between G-CSF and IL-8 in vitro. Method: Peripheral blood was drawn from healthy donor with consent. Neutrophils were separated by density centrifugation method. Human umbilical vein endothelial cells (HUVECs) and G-CSF were purchased. HUVECs were cultured under serum-containing medium and stimulated by G-CSF or HUVECs were co-cultured with neutrophils. IL-8 in culture media was measured by ELISA. Results: G-CSF alone does not increase IL-8 production in the culture medium either of neutrophils or HUVECs alone, in either short time (6 hours) or long time (up to 4 days) stimulation. However, when HUVECs were co-cultured with neutrophils, IL-8 started to increase 10 hours after starting co-culture and gradually increased up to 3000pg/ml after 4 days culture. IL-8 in co-culture media increased five fold compared to that in the single culture of HUVECs. This increased production of IL-8 was dependent on the number of neutrophils. Further, when neutrophils were pre-incubated by G-CSF at a dose 50ng/ml, IL-8 production in co-culture media increased 70 percent compared to pre-incubation without G-CSF. This increased production of IL-8 was dependent on G-CSF dose. IL-8 production remarkably decreased to 15% when HUVECs and neutrophils were cultured separately by mesh to avoid direct contact. Conclusion: These data indicates that direct contact between HUVECs and neutrophils is necessary for production of IL-8. Some molecule(s) induced by G-CSF might play an important role in IL-8 production. The effect of stimulation by G-CSF seems less important than that of direct interaction between neutrophils and HUVECs. This correlates well to the fact that high WBC count after G-CSF administration in vivo correlates with the yield of CD34-positive cells in peripheral blood stem cells mobilization. Molecular mechanisms of interaction between HUVECs and neutrophils need to be clarified further.

Background: Inhibitor formation is a severe complication of hemophilia B associated with poor response to factor replacement and uncontrolled bleeding, although an inhibitor formation in hemophilia B is a rare event compared to hemophilia A. Currently, immune tolerance is the only proven method for inhibitor eradication. However, immune tolerance induction in patients with hemophilia B is a rare occurrence. We report a case of a hemophilia B patient who had a reduction of inhibitor titer during continual use of FEIBA. Case report: Patient is an 11-year-old boy with severe FIX deficiency and high titer inhibitor (historical peak: 65 BU/mL) since age 3 years. Since inhibitor formation, the patient had received FEIBA or FVIIa on demand. FVIIa became therapeutically not effective in 1999 October, and bleeding frequency increased as he developed a target joint. In 1999 November, 2002 February, and 2004 September, he received high dose of FIX concentrate for neutralization of inhibitor and then cyclophosphamide for 8 weeks. We achieved only a transient effect in reducing the inhibitor titer. The inhibiter titer fluctuated between 20 and 60 BU/mL. Since 2005 January, subsequently, he had received FEIBA 1,000–2,000 IU per dose (30–50 units/kg) on demand. He needed FEIBA twice or thrice a week, and continued to receive this dose till now. The inhibitor titer was gradually getting lower after a transient rise up to 39 BU/mL on 2005 February. On 2006 February, the inhibitor titer became below 1.0 BU/mL, and finally undetectable on 2006 March, despite the longevity of the inhibitor (8 years). Factor IX recovery was normalized, and bleeding frequency dramatically decreased. Elimination of the inhibitor by the continued administration of FEIBA was observed. Anaphylactic reaction and the development of nephrotic syndrome were not seen. Conclusion: The continuing use of FEIBA safely and effectively might decrease the inhibitor titer and the frequency of bleeding episodes in hemophilia B patients. This case may provide us the optimization of the FEIBA dose, and duration of treatment for inducing tolerance by using FEIBA.

Recently, we reported that genetic polymorphisms within the human IL18 gene were associated with disease susceptibility to adult-onset Still's disease (AOSD), which is characterized by extraordinarily high serum levels of IL-18. Because high serum IL-18 induction has also been observed in the systemic type of juvenile idiopathic arthritis (JIA), we investigated whether similar genetic skewing is present in this disease. Three haplotypes, S01, S02, and S03, composed of 13 genetic polymorphisms covering two distinct promoter regions, were determined for 33 JIA patients, including 17 with systemic JIA, 10 with polyarthritis, and 6 with oligoarthritis. Haplotypes were also analyzed for 28 AOSD patients, 164 rheumatoid arthritis (RA) patients, 102 patients with collagen diseases, and 173 healthy control subjects. The frequency of individuals carrying a diplotype configuration (a combination of two haplotypes) of S01/S01 was significantly higher in the JIA patients, including all subgroups, than in the healthy controls (P = 0.0045, Fischer exact probability test; odds ratio (OR) = 3.55, 95% confidence interval (CI) = 1.55–8.14). In patients with systemic JIA, its frequency did not differ statistically from that of normal controls. Nevertheless, it is possible that haplotype S01 is associated with the phenotype of high IL-18 production in systemic JIA because the patients carrying S01/S01 showed significantly higher serum IL-18 levels compared with patients with other diplotype configurations (P = 0.017, Mann-Whitney U test). We confirmed that the frequency of the diplotype configuration of S01/S01 was significantly higher in AOSD patients than in healthy control subjects (P = 0.011, OR = 3.45, 95% CI = 1.42–8.36). Furthermore, the RA patients were also more predisposed to have S01/S01 (P = 0.018, OR = 2.00, 95% CI = 1.14–3.50) than the healthy control subjects, whereas the patients with collagen diseases did not. In summary, the diplotype configuration of S01/S01 was associated with susceptibility to JIA as well as AOSD and RA, and linked to significantly higher IL-18 production in systemic JIA. Possession of the diplotype configuration of S01/S01 would be one of the genetic risk factors for susceptibility to arthritis in the Japanese population.

The purpose of this study was to examine the yield of PBSC, mobilized with G-CSF alone, and engraftment kinetics after autologous transplantation in pediatric cancer patients. In 55 patients (median age; 7 years, range 0–21) with various pediatric and adolescent solid tumors, PBSC were mobilized with G-CSF alone, and the yields of PBSC and engraftment following autologous PBSCT were evaluated retrospectively. Patients were categorized according to prior treatment; patients who had received less than 4 or 4 cycles of chemotherapy with/without local irradiation (Group 1: N= 21), patients who received more than 4 cycles of chemotherapy or 3 or more cycles of chemotherapy with extended irradiation (Group 2: N= 23), and patients who received high-dose chemotherapy with stem cell support (Group 3: N= 11). Ten microgram per kg of G-CSF was injected subcutaneously for mobilization when patients showed no influence of previous chemotherapy, and administration was continued for five days. The peaks of CD34+ cells and CFU-GM were observed on day 5 of G-CSF administration essentially in all patients. Aphereses were performed on days 5 and 6 of G-CSF treatment. Mobilization failure was observed in four patients in all groups. Compared with the results in patients mobilized by chemotherapy plus G-CSF (N=18), the progenitor cell yields were lower in those mobilized with G-CSF alone. However, there were no significant differences in WBC engraftment speed compared to the chemotherapy plus G-CSF mobilization group, although platelet recovery was delayed in patients with G-CSF alone, especially in patients in Group 3. The median time taken for ANC and platelet counts to reach 500 and 20K was 12 days (range 8–28) and 15 days (8–55), respectively, in all patients who were mobilized by G-CSF alone except for patients with progressive disease. In summary, mobilization with G-CSF alone can mobilize a sufficient number of CD34+ cells for successful autografting and sustained hematological reconstitution in pediatric patients with cancer, even in heavily pre-treated patients. Mobilization with G-CSF alone might offer some advantages, such as ease of determining a collection schedule without a daily determination of CD34+ cells in the blood, and the avoidance of neutropenic fever and additional transfusion.

Objective: We conducted a randomized study to compare the effectiveness of two maintenance regimens, namely the 6-MP/MTX-based regimen (A-arm) and the LSA2L2 type regimen (B-arm). Patients and Methods: A total of 218 newly diagnosed patients between 1 and 15 years of age were enrolled from August 1996 to August 2002. The patients characteristics was as follows: median age, 5; T-cell/B-cell precursor phenotype, 22/196; median WBC, 8,100/μl (range, 600–809, 200). The high-risk group (HR) was defined as having a WBC of over 50,000/μl, a T-cell precursor phenotype, CNS disease, hepatosplenomegaly of more than 5 cm under the costal margin while the standard-risk group (SR) was defined as having no HR features. Any cases with t(9;22) and t(4;11) were excluded from the present protocol. After successful induction treatment, the patients received early intensification, consolidation and late intensification treatment. Before late intensification, the patients were randomly assigned to receive one of two maintenance regimens: the A-arm consisted of daily 6-MP with weekly MTX pulsed with DEX and VCR for 2 weeks irrespective of the risk group. The B-arm consisted of 4 blocks of 6-MP with CPM, 6-MP with CA, 6-MP with VCR and PSL, and intermediate-dose (ID)-MTX in the SR group, while in the HR group, high-dose CA was substituted for ID-MTX. The total treatment duration was 3 years. Eleven cases with t(9:22) and t(4;11), 5 with a disagreement to randomization, while the other 6 did not meet the eligibility criteria and thus were excluded from the protocol. As a result, 196 cases were evaluable (98 cases for each risk-group). Of these, 95 and 94 cases, excluding 7 who developed relapses before randomization, were assigned to the A and B-arms, respectively. Results: CR was successfully achieved in 195 of 196 patients (99%). Relapse occurred in 36 cases (isolated BM, 30; others, 6). No toxic death was observed during the entire treatment period. The event-free survival (EFS) rates in all 196 eligible cases, SR and HR cases were 75.5±5.1 (SE) %, 88.1±3.6% and 62.5±7.5% (p=.0004), respectively. The EFS rates for SR were 86.8±5.6% in the A-arm and 90.6±4.6% in the B-arm (p=ns). In the HR cases, the EFS rate in the A-arm was tended to be superior to that in the B-arm (81.2±5.6% and 52.3±13.1%, p=.13). Conclusion: No significant difference was observed in the EFS rates between the two maintenance regimens. However, the late adverse effects of each regimen should be carefully evaluated by a long-term follow-up of the patients.

A point mutation of the N-ras gene is one of the known genetic alterations identified in patients with acute lymphoblastic leukemia (ALL), but its clinical importance is still controversial. Using polymerase chain reactions, we examined codons 12, 13 and 61 of this gene in 125 Japanese childhood ALL patients (64 common-ALL, 22 pre-B-ALL, 33 T-ALL, 2 B-ALL, 3 undifferentiated ALL, and 1 unclassified ALL) including 9 relapsed patients. An N-ras point mutation was observed in 14 (11%) patients (9 common-ALL, 3 T-ALL, and 2 undifferentiated ALL; 13 patients at diagnosis and 1 at relapse). The patients with undifferentiated ALL harbored an N-ras mutation at a significantly higher rate. However, no correlation was found between the presence of an N-ras mutation and sex, age, or white blood count. There was no significant difference in the event-free survival rate between 13 fresh patients with an N-ras mutation and 103 patients with a wild-type configuration. The N-ras mutation was present in about 10% of childhood ALL cases but it did not have a prognostic impact. The sequence analyses revealed that the majority of the patients (13/14) had an N-ras mutation of a G to A transition. This finding was consistent with previous reports on N-ras mutations in acute leukemias in which the incidence of a G to A mutation was significantly higher in ALL than in myeloid malignancies.

Autografts with G-CSF mobilized blood stem cell (PBSC) has been widely applied for cancer therapy. However, its dose response effect has not been fully tested. To address this, we gave various doses of G-CSF to 31 children with solid tumors (age, 1 to 15 y) including 17 neuroblastoma, with concomitant measurement of circulating CD34 + cells. Eleven of 31 patients has > 6 mo history of chemotherapy. They received a regimen incorporating CDDP and G-CSF was started from the nadir of WBC. Blood was drawn 3 times a week for CD34 + cell assay and CBC. There was no dose response effect for shortening the duration of neutropenia with 2 to 12.5 μ g/kg of G-CSF. Increase of circulating CD34 + cells (> 100/ μ l) was observed in patients whose platelet recovery was fast, reaching to a level of 10 × 10 9 /1 within 2 weeks (n = 14), but no apparent dose response effect was observed. While in the rest of slowly recovering patients, its effect was marginal. Comparing our historical data for children with ALL/NHL, these may suggest that G-CSF has only a limited effect for mobilizing PBSC in children with a solid tumor and/or those who were treated with a regimen specific for solid tumors.

We have used limiting-dilution clonal analysis (LDA) in microwells to study the inhibitory effects of T lymphocytes (T-cells) or natural killer (NK) cells on human marrow progenitor cell growth. In four subjects with normal hematopoiesis, the growth of progenitors showed single-hit kinetics both before and after T-cell removal, indicating that, in the presence of colony-stimulating activity, T-cell have no effect on progenitor growth. In a patient with marrow hypoplasia associated with thymoma, hypogammaglobulinemia, and an increased number of suppressor T-cells (Good's syndrome), the progenitor growth deviated from linearity, demonstrating the presence of cells with suppressor activity. After T-cells were removed from this sample, the progenitor growth showed single-hit kinetics. The suppressive action of E-rosette-positive cells with NK or cytotoxic activities was also suggested in a patient with severe combined immune deficiency and in a patient with T gamma lymphocytosis. Poor progenitor-cell growth in three other patients with aplastic anemia was not significantly altered by T-cell removal. Thus, LDA of human hematopoietic progenitors is useful for evaluating cell-mediated interactions affecting hematopoiesis. This method may facilitate elucidation of mechanisms of myelosuppression in clinical settings.

Marked and persistent cytopenia was observed in two 3-year-old children with cancer, after lymphopheresis to collect peripheral blood stem cells (PBSC) for autotransplantation after high-dose chemotherapy. This finding suggests that PBSC are a major component of the hematopoietic cells in small children and, hence, that special care is necessary in the construction of a protocol for collection of stem cells from young children.

Kawasaki disease associated with agranulocytosis was observed in a one-year-old girl. The agranulocytic state continued for a month, and granulocyte transfusions were required. We discuss the relation between agranulocytosis and drugs, viral infections, Kawasaki disease, etc. The systemic vasculitis of Kawasaki disease seems to be the cause of the bone marrow disturbance.

Peripheral blood stem cells (PBSC) were collected for autotransplantation by a total of 46 continuous-flow leukaphereses in 17 children with various types of cancer in whom the stem-cell pool had been expanded by chemotherapy. As the cells collected by leukapheresis were contaminated with many visible cell clumps, platelets, and erythrocytes, they were separated from the platelet-rich plasma (PRP) by slow-speed centrifugation and fractionated on a discontinuous gradient of Percoll. All the hematopoietic progenitors (CFU-GM, CFU- GEMM) in the starting samples were recovered at the interface of 40% and 60% Percoll solutions largely free of other cellular components and with a substantial reduction in volume. The separation and freezing procedures could be completed within three hours after obtaining cells by leukapheresis. After their fractionation and storage, these PBSC were shown to be able to reconstitute normal hematopoiesis in ten children with poor prognosis leukemia or neuroblastoma for whom no HLA- compatible marrow donors were available and who had been subjected to marrow-ablative therapy. This separation procedure is simple, efficient, and readily available and can be used for children as a routine procedure for PBSC autotransplantation.

Summary Antilymphocyte globulin (ALG) and antithymocyte globulin (ATG) have an established role in the treatment of severe aplastic anaemia. The response rate ranges from 40% to 80%. Its mode of action is believed to be complement dependent lysis of immunocompetent cells which inhibit haemopoietic maturation. This might not be the sole mechanism. We have tested four different preparations of ALG/ATG for their mitogenic effect on normal peripheral blood cells and on enriched T-cells in vitro by 3H-thymidine incorporation. We found marked differences between the four preparations. One was strongly mitogenic and able to induce profound release of haemopoietic growth factors. This mitogenic effect could be detected in the serum of patients during ALG treatment. Clinical response rates of this preparation are about 80%. Three other preparations were of lower or no stimulatory effect. Clinical response rates with these preparations vary between 40% and 60%. From our results, we postulate that the beneficial effect of ALG could be partially due to its ability to stimulate release of haemopoietic growth factors. The mitogenicity of different ALG/ATG preparations should be tested as an in vitro parameter of clinical efficacy.

The in vivo dose-response potential of recombinant human granulocyte colony-stimulating factor (rG-CSF) in facilitating reconstitution of granulopoiesis was evaluated in an 11-year-old girl who received autotransplantation of peripheral blood stem cells (PBSC) after marrow-ablative therapy for relapsing acute lymphoblastic leukemia. The recovery of hematopoiesis was slow when a small number of progenitors were collected and reinfused into the patient, but administration of rG-CSF led to significant dose-dependent increase in peripheral blood granulocytes.

To evaluate the toxicity and efficacy of an i.v. preparation of BU (12.8 mg/kg), combined with CY (120 mg/kg), a prospective study was performed on 30 Japanese patients (median age, 30 years) with hematologic malignancies undergoing hematopoietic SCT (28 allogeneic transplants from an HLA-matched donor and 2 autologous transplants). There were no significant toxicities, and all but one patient showed evidence of granulocyte engraftment at a median of 14 days for allogeneic and 11 days for autologous transplantation. Grades II-IV acute and chronic GVHD occurred in 9 (9/27, 33%) and 16 patients (16/27, 59%), respectively. Non-relapse mortality at days 100 and 365 was 3 and 17%, respectively. The pharmacokinetics of i.v. BU showed close inter- and intrapatient consistency; the area under the plasma concentration-time curve of the first administration remained at less than 1500 micromol min/l in 27 of the 29 patients (93%), and between 900 and 1350 micromol min/l in 22 patients (73%). As all of the profiles overlap with data from non-Japanese patients, we conclude that racial factors may not seriously influence the bioactivity of i.v. BU.