The production of factor VIII (FVIII) inhibitory antibodies is a serious problem in patients with hemophilia A. Immune tolerance induction (ITI) is the only strategy proven to eradicate persistent inhibitors and has been shown to be successful in 70 % of patients with hemophilia A. However, a minority of hemophilia patients present life-long inhibitors. To eliminate such inhibitors, we designed an intravenous immunoglobulin (IVIG) strategy in combination with high dose recombinant FVIII for ITI in hemophilia A children with inhibitors. Four previously untreated patients produced inhibitors within 16 exposures to FVIII. The peak inhibitor titers in these patients ranged from 3 to 14 BU/mL. The patients received ITI combined with IVIG within 1.5 months after the inhibitors were detected. All patients showed a negative titer for inhibitors by 28 days, with no anamnestic responses. The recovery of FVIII in the plasma concentration was normalized within three months after initiation of ITI. An additional course of IVIG administration led to induction of complete tolerance by 20 months after initiation of ITI therapy in all patients. ITI treatment with high-dose FVIII combined with IVIG may be effective for the early elimination of inhibitors.

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Pediatric blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy that has an extremely poor prognosis despite the use of intensive chemotherapy. Recently, treatment of BPDCN with bone marrow transplantation (BMT) using myeloablative conditioning has been reported to increase survival in adults. We report a 9-year-old girl with cutaneous BPDCN who was successfully treated with combination chemotherapy followed by BMT using reduced intensity conditioning (RIC), without any adverse complications. The success of this treatment regimen suggests that BMT with RIC may be a feasible option for treating children with cutaneous BPDCN.

The ELANE is known as the responsible gene for both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). However, relations between mutations in the ELANE gene and abnormal myelopoiesis in the different phenotype of these diseases still remain unclear. It has been reported that induced pluripotent stem cell (iPSC) from an individual patient with SCN (SCN-iPSC) demonstrated maturation arrest of myeloid progenitor cells and poor response to granulocyte-colony stimulating factor as similarly observed in patient's bone marrow cells. Thus, the study on myelopoiesis using disease specific iPSC seems to provide disease pathogenesis as a novel in vitro experimental model. In this study, we established iPSC line from an individual patient with CyN (CyN-iPSC) with heterozygous mutation in ELANE gene (Exon5, R191Q point mutation). Then we compared myelopoiesis among healthy Control-iPS (253G1), SCN-iPS (Exon5, C194X point mutation) , and CyN-iPSC. Undifferentiated colonies derived from CyN-iPSC were staind with pluripotency markers (OCT3/4 and NANOG). CyN-iPSC retained a normal karyotype and ELANE locus mutation of the original samples. In vitro myelopoiesis was examined by using a serum- and feeder-free monolayer hematopoietic culture system. iPSC colonies were cultured on growth factor-reduced Matrigel-coated cell culture dishes in modified Tenneille Serum Replacer 1 (mTeSR™1) medium (StemCell Technologies, Inc.), containing BSA, rh bFGF, rh TGFβ, Lithium Chloride, Pipecolic acid, GABA. Medium was replaced every four days. Then medium was changed to StemPro®-34 SMF Complete Medium plus nutrient supplement (Life technologies Corp.). The iPSC were cultured with BMP4 (80 ng/mL) for four days, and then replaced with VEGF165 (80 ng/mL), bFGF (25.7 ng/mL), and SCF (100 ng/mL) on Day 4. On Day 6, cytokines were replaced with a combination of SCF (50 ng/mL), IL-3 (50 ng/mL), and G-CSF (50 ng/mL). Medium was replaced every 3 - 4 days. No significant difference in the ratio of proliferating CD33+ cells were noted between CyN-iPSCs and Control-iPSCs. CyN-iPSCs showed less capability in the proliferation and maturation for CD15+ cells on days 20 to 40 than Control-iPSCs. The decreased number of CD15+ cells derived from CyN-iPSc implies the defect in mature neutrophil survival. In contrast, CD15+ / CD33+ cells derived from SCN-iPSCs were hardly observed in this culture condition, suggesting the defects of proliferation and maturation in SCN-iPSCs. We next examined the colony formation of CD34+ cells derived from CyN-iPSCs, Control-iPSCs, and SCN-iPSCs. CD34+ cells were obtainded at the day 12 of primary culture of iPSCs and purified by cell sorting using FACS-Aria®. No significant differences in the number of G-colony and GM-colony between CD34+ cells from CyN-iPSCs and Control-iPSCs. In contrast, CD34+ cells from SCN-iPSCs gave rise to the significantly decreased number of G-colony and GM-colony. The observations of myeloid proliferation/maturation and colony formation of CD34+ cells were almost compatible with those obtained from bone marrow cells in patients with SCN and CyN. Furthermore, neutrophils differentiated from CyN-iPSCs showed the excessive cell death, whereas SCN-iPSCs presented the defective myelopoiesis. These results suggest that the analyses using CyN-iPSCs and SCN-iPSCs may be useful tool for investigating the relation of gene mutation and pathophysiology in both diseases. Disclosures No relevant conflicts of interest to declare.

Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by severe chronic neutropenia, with absolute neutrophil counts below 0.5×109/L, and by recurrent bacterial infections from early infancy. Granulocyte colony-stimulating factor (G-CSF) is widely used for the treatment of neutropenia in patients with SCN. However, the long-term G-CSF therapy has a relative risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The only curative treatment available for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning (RIC) regimens have been applied to the treatment of SCN patients without malignant transformation who have become G-CSF refractory. However, the optimal conditions of HSCT for SCN patients have not been established. In this study, we conducted bone marrow cell transplantations (BMT) in ten patients with SCN using an immunosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. Ten patients with a total of 11 HSCT procedures in our institution (performed from 2007 to 2015) were enrolled in this study. Four of the ten patients had experienced engraftment failure of the initial HSCT and three of them were referred to our hospital for re-transplantation. Heterozygous mutation inthe ELANE gene was identified in nine of ten patients. These nine patients received BMT less than 10 years of age. All ten patients had recurrently experienced moderate to severe bacterial or fungal infection before HSCT and received temporal or regular administration of G-CSF. Bone marrow cells (BM) were obtained from five HLA-matched related (MRD), three HLA-matched unrelated (MUD), and three HLA-mismatched unrelated (7/8) donors (MMUD), respectively. The conditioning regimen basically consisted of fludarabine (100 to 125 mg/m2), cyclophosphamide (100 to 150 mg/kg), melphalan (70 to 90 mg/m2), total body irradiation (3 to 3.6 Gy), and/or anti-thymocyte globulin (10 to 12 mg/kg). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was successfully observed within 24 days of post-transplantation in all patients. All patients achieved complete chimerism at the time of engraftment. Two patients who underwent BMT from MRD and one patient who underwent BMT from MUD showed the gradual decrease of donor-derived cells. Donor lymphocyte infusion treatment successfully achieved the complete chimerism or stable mixed chimerism in these 3 patients. Although 3 patients experienced the acute GVHD (Grade I-II), the addition of glucocorticoids to tacrolimus prevented the extension of acute GVHD. Only one patient developed mild chronic GVHD presenting limited type of skin involvement. All patients are alive for 9 months to 9 years after HSCT with no signs of severe infections or transplantation-related morbidity. Our results demonstrate that BMT together with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. Although our results through the small number of cohort is limited to conclude, the BMT with the optimal donors may lead to the increased opportunity for lower risk of SCN patients especially at younger age as a curative treatment. The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.