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MC710, a mixture of plasma-derived activated factor VII and factor X at a protein weight ratio of 1:10, is a novel bypassing agent for haemostasis in haemophilia patients with inhibitors. In a Phase II trial, we evaluated the haemostatic efficacy and safety of single doses of MC710, and investigated pharmacokinetic and pharmacodynamic parameters in nine joint bleeding episodes in six male haemophilia patients with inhibitors. This trial was a multi-centre, open-label, non-randomized study of two doses (60 and 120 μg kg(-1) as FVIIa dose), allowing the re-administration of different MC710 dosages to the same subjects. Haemostatic efficacy was assessed by evaluating reduction in pain and swelling, as well as increase in range of motion in a bleeding joint. The results of the study showed that in nine bleeding episodes, seven treatments were rated as 'excellent' or 'effective' according to investigator's rating system of efficacy at 8 h after administration. No serious or severe adverse events were observed after administration; furthermore, measurement of several diagnostic markers revealed no signs or symptoms of disseminated intravascular coagulation (DIC). The haemostatic potential of MC710 was confirmed at doses of 60 and 120 μg kg(-1) in this trial. MC710 is thus expected to be a safe and efficacious novel bypassing agent for controlling bleeding in haemophilia patients with inhibitors.

Minimal residual disease (MRD) is derived from tumor-initiating cells (TICs) and is responsible for tumor relapse. Neuroblastoma is characterized by extreme tumor heterogeneity, and more than half of high-risk patients experience tumor relapse. To overcome tumor heterogeneity and achieve more sensitive detection of MRD, several sets of real-time RT-PCR markers have been reported for MRD monitoring in neuroblastoma patients from different centers. However, these markers vary across centers and are still being validated. In the present study, we validated the ability of 14 commonly used real-time RT-PCR markers to detect MRD based on their expression in neuroblastoma TICs, and we developed a novel MRD detection protocol, which scored the samples as MRD-positive when the expression of one of the 11 real-time RT-PCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) exceeded the normal range. By using this protocol, we prospectively monitored MRD in 73 bone marrow (BM), 12 peripheral blood stem cell and 8 peripheral blood samples from 14 neuroblastoma patients treated at a single center. We scored 100, 56, 56 and 57% BM cytology-positive, elevated vanillylmandelic acid (VMA), elevated homovanillic acid (HVA) and elevated neuron-specific enolase (NSE) samples as MRD-positive, respectively. MRD was also positive in 48, 45, 46 and 43% of the BM cytology-negative and normal VMA, normal HVA and normal NSE samples, respectively. These results suggest that the present MRD detection protocol based on the expression of a set of 11 real-time RT-PCR markers in neuroblastoma TICs achieves sensitive MRD monitoring in neuroblastoma patients.

Purpose Perifosine is an alkylphospholipid analog that inhibits or modulates signaling through signal transduction pathways such as Akt, which is enhanced in neuroblastoma (NB) by activation of tyrosine kinase receptors. We conducted a phase I study of perifosine in Japanese patients with recurrent or refractory NB. Experimental Design All patients enrolled were over 2 years of age; all had refractory or relapsed NB and a performance status of greater than 50%. Perifosine was orally administered at a loading dose (100–300 mg) on day 1 and at a maintenance dose (50–150 mg) from day 2 onward. Dose-limiting toxicity (DLT) and pharmacokinetics were assessed in Step 1 and safety and efficacy in Step 2. Results Nineteen patients were recruited. No DLT was observed. Adverse reactions occurring in more than 30% of the patients were vomiting (63%), nausea (53%), and diarrhea (37%). The mean plasma concentration of perifosine was 27.5 ± 9.8 μM on day 15 and 27.3 ± 11.5 μM on day 29. The response rate (RR) in 18 patients evaluable according to modified International Neuroblastoma Response Criteria was 0%; the disease control rate (DCR) was 56%. Median progression-free survival (PFS) was 122 days. In 11 patients evaluable according to the Response Evaluation Criteria in Solid Tumors, the RR and DCR were 9% and 55%, respectively. The median PFS was not reached. Conclusions Perifosine monotherapy was well tolerated in Japanese patients with recurrent/refractory NB. Further investigations in combination with other anticancer or molecular targeted agents are warranted.

Central nervous system involvement in hemophagocytic lymphohistiocytosis (HLH) is associated with a poor outcome. For such patients, it is unknown whether more aggressive therapies, such as intrathecal methotrexate or hydrocortisone, are inevitably required. We present a very rare case of 3-year-old Japanese girl who developed mild encephalitis/encephalopathy with a reversible splenial lesion, accompanied by Epstein-Barr virus-associated HLH, and review previous similar reports. Our case and previous reports suggest that mild encephalitis/encephalopathy with a reversible splenial lesion accompanied by Epstein-Barr virus-associated HLH has a relatively good prognosis, even in the absence of intrathecal treatments.

BACKGROUND Optimal prevention of chemotherapy-induced vomiting (CIV) has not been established for patients receiving cisplatin in divided doses. The aim of this study was to describe the incidence and risk factors of CIV in children who received multiple-day cisplatin chemotherapy. PROCEDURE A total of 24 consecutive pediatric patients (age 0-19 years) who received multiple-day cisplatin chemotherapy in our hospital were enrolled. Patients with relapsed disease or primary intracranial tumor and those who received concurrent radiation therapy were excluded. The number of chemotherapy cycles reviewed was 107, with a median of five per patient. All patients received granisetron. Dexamethasone and NK-1 receptor antagonists (NK1RA) were used as additional antiemetics for prophylaxis of CIV. RESULTS CIV was observed in 22 of 24 (92%) patients, and 61 of 107 (57%) cycles. Patients who developed CIV had a higher incidence of other chemotherapy-related adverse events (87 vs. 41%, P < 0.001). The incidence of CIV was lower in patients administered with NK1RA than those without (32 vs. 68%, P < 0.001). Multivariate logistic regression identified age less than or equal to 2 years (odds ratio [OR] = 0.25, 95% confidence interval [CI] = 0.10-0.63) and administration of NK1RA (OR = 0.16, 95% CI = 0.06-0.43) as independent factors for CIV. CONCLUSIONS These results suggest that NK1RA is crucial to reduce CIV in children who receive multiple-day cisplatin chemotherapy.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the best therapeutic option for childhood high-risk acute leukemia. However, which donor source is optimal for children lacking an identical sibling remains unclear. To evaluate the clinical impact of donor source on allo-HSCT in childhood acute leukemia, we analyzed data from 577 children who underwent allo-HSCT after a myeloablative regimen during first or second complete remission from 2005 to 2012, using registry data of the Japan Society for Hematopoietic Cell Transplantation, and we compared outcomes of 7/8 to 8/8 HLA allelic–matched unrelated bone marrow transplantation (UR-BMT, n = 218) and 4/6 to 6/6 HLA allelic–matched unrelated cord blood transplantation (UR-CBT, n = 200) to those of HLA-identical related bone marrow transplantation (ID-BMT, n = 159). The median follow-up of survivors was 40.0 months. Three-year overall survival (OS) and leukemia-free survival (LFS) rates for ID-BMT, UR-BMT, and UR-CBT were 74.8% and 69.0%, 75.0% and 69.6%, and 71.8% and 63.8%, respectively. The multivariate analysis demonstrated that OS and LFS for the 3 groups are comparable, although UR-CBT carries a greater risk of nonrelapse mortality (hazard ratio, 2.20; P = .03, compared to ID-BMT) in the myeloablative setting for childhood high-risk acute leukemia.

We herein describe a 2-year-old boy with severe congenital neutropenia (SCN) who was successfully treated with reduced-intensity bone marrow transplantation (HSCT). He had suffered recurrent episodes of bacterial pneumonia from 12 months of age, and was found to have severe neutropenia with white blood cell counts below 100/μl. The patient harbored a heterozygous missense mutation in ELANE exon 4 (p.Gln134Pro, NM_001972.2: c.401AC). This was a novel mutation. Due to intractable pneumonia and severe persistent neutropenia, reduced-intensity HSCT was performed from an HLA-matched sibling donor. The preparative regimen consisted of melphalan, fludarabine, and 4 Gy of total body irradiation. Hematopoietic engraftment was rapidly obtained, i.e., by day +14, and complete donor chimerism was subsequently achieved. The lung complications observed pre-transplantation markedly improved after neutrophil recovery, i.e., by day +60. We concluded that HSCT is a useful treatment for SCN patients, especially for those at high risk of leukemic transformation. Fludarabine-based reduced-intensity HSCT may represent a safe and effective therapeutic option for patients with SCN who need HSCT even if they have intractable infectious complications.

It is extremely rare for metastasised medulloblastoma to form a large tumour in the suprasellar region. We present a case of medulloblastoma with large suprasellar metastasis at initial presentation. A 3-year and 5-month-old boy presented with a 1-month history of vomiting and loss of appetite, and body weight. Computed tomography and magnetic resonance imaging revealed a 20 mm × 20 mm mass in the suprasellar region and a 30 mm × 30 mm mass in the fourth cerebral ventricle. We performed endoscopic biopsy of the suprasellar tumour, and subsequently totally removed the vermian tumour through a suboccipital craniotomy. The histopathological findings revealed that both the suprasellar and vermian tumours were classic type and non SHH/WNT type medulloblastoma. The postoperative course was uneventful. The patient showed complete remission after chemotherapy. The tumour in the suprasellar region was most likely metastatic from the vermis. Endoscopic biopsy of the tumour in the suprasellar region and total removal of the tumour in the vermis in a one-stage operation followed by intensive chemotherapy with reduced dose radiotherapy may provide a satisfactory outcome.

A growing number of epidemiological studies have demonstrated that the consumption of green tea inhibits the growth of a variety of cancers. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has been shown to have an anti-cancer effect against many cancers. Most cancers are believed to be initiated from and maintained by a small population of tumor-initiating cells (TICs) that are responsible for chemotherapeutic resistance and tumor relapse. In neuroblastoma, an aggressive pediatric tumor that often relapses and has a poor prognosis, TICs were recently identified as spheres grown in a serum-free non-adherent culture used for neural crest stem cell growth. Although EGCG has been reported to induce growth arrest and apoptosis in neuroblastoma cells, its effect on neuroblastoma TICs remains to be defined.Gene expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The effects of EGCG on cell proliferation, apoptosis, and sphere formation were determined by cell counting, propidium iodide staining, and sphere (100 μm in diameter) counting, respectively.Neuroblastoma BE(2)-C cells showed increased expression of stem cell markers (nanog homeobox [NANOG] and octamer-binding transcription factor 4 [OCT4]), as well as decreased expression of neuronal differentiation markers (Cu(2+)-transporting ATPase alpha polypeptide [ATP7A] and dickkopf homolog 2 [DKK2]) in spheres grown in serum-free non-adherent culture, compared to parental cells grown in conventional culture. Although EGCG induced growth arrest and apoptosis in the parental cells in a dose-dependent manner, it was not effective against spheres. However, EGCG potently inhibited sphere formation in the BE(2)-C cells.The present results suggest that EGCG may inhibit the development of TICs in BE(2)-C cells.

Background Although the therapeutic outcome of acquired aplastic anemia has improved markedly with the introduction of immunosuppressive therapy using antithymocyte globulin and cyclosporine, a significant proportion of patients subsequently relapse and require second-line therapy. However, detailed analyses of relapses in aplastic anemia children are limited. Design and Methods We previously conducted two prospective multicenter trials of immunosuppressive therapy for children with aplastic anemia: AA-92 and AA-97, which began in 1992 and 1997, respectively. In this study, we assessed the relapse rate, risk factors for relapse, and the response to second-line treatment in children with aplastic anemia treated with antithymocyte globulin and cyclosporine. Results From 1992 to 2007, we treated 441 children with aplastic anemia with standard immunosuppressive therapy. Among the 264 patients who responded to immunosuppressive therapy, 42 (15.9%) relapsed. The cumulative incidence of relapse was 11.9% at 10 years. Multivariate analysis revealed that relapse risk was significantly associated with an immunosuppressive therapy regimen using danazol (relative risk, 3.15; P =0.001) and non-severe aplastic anemia (relative risk, 2.51; P =0.02). Seventeen relapsed patients received additional immunosuppressive therapy with antithymocyte globulin and cyclosporine. Eight patients responded within 6 months. Seven of nine non-responders to second immunosuppressive therapy received hematopoietic stem cell transplantation and five are alive. Eleven patients underwent hematopoietic stem cell transplantation directly and seven are alive. Conclusions In the present study, the cumulative incidence of relapse at 10 years was relatively low compared to that in other studies mainly involving adult patients. A multicenter prospective study is warranted to establish optimal therapy for children with aplastic anemia.

In aplastic anemia, predictive markers of response to immunosuppressive therapy have not been well defined. We retrospectively evaluated whether clinical and laboratory findings before treatment could predict response in a pediatric cohort from the multicenter AA-97 study in Japan. Between 1997 and 2006, 312 newly diagnosed children were enrolled and treated with a combination of antithymocyte globulin and cyclosporine. In multivariate analyses, lower white blood cell count was the most significant predictive marker of better response; patients with white blood cell count less than 2.0×10(9)/L showed a higher response rate than those with white blood cell count of 2.0×10(9)/L or more (P=0.0003), followed by shorter interval between diagnosis and therapy (P=0.01), and male sex (P=0.03). In conclusion, pre-treatment clinical and laboratory findings influence response to therapy. The finding that response rate worsens with increasing interval between diagnosis and treatment highlights the importance of prompt immunosuppressive therapy for patients with aplastic anemia.

A total of 27/28 (96%) immunocompromised Japanese children received ≥ 4 doses of palivizumab. No respiratory syncytial virus-associated hospitalizations occurred. Mean palivizumab trough concentrations were 59.0 and 91.8 μg/mL 30 days after the 1st and 4th doses, respectively. Of 28 subjects, 27 (96%) experienced ≥ 1 adverse event and 7 (25%) experienced ≥ 1 serious adverse event, none of which was considered related to palivizumab.

Introduction TP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors. Methods Mutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement. Result In total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing < 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P Conclusion TP53 mutations at diagnosis are common in Hypodiploid ALL and also found in a substantial fraction of MLL rearrangement and IGH-DUX4 ALL, where the mutations predict a poor prognosis. TP53 mutation is also found in NCI-SR cases but may not be associated with poor prognosis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Introduction The programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway is an inhibitory immune checkpoint that can suppress T-cell-mediated tumor cytotoxicity. Anti-PD-1 monoclonal antibodies have recently been recognized as promising therapy for adult patients with lymphoma, particularly in classical Hodgkin's lymphoma. However, little information is available regarding the expression patterns of PD-1 and PD-L1 in pediatric lymphoma. Therefore, this study aimed to investigate the expression patterns of PD-1 and PD-L1 in pediatric lymphoma. Methods Immunohistochemical analysis was performed on paraffin-embedded pretherapeutic tumor biopsies from 36 newly diagnosed pediatric patients (aged 0-15 years) with lymphoma or lymphoproliferative disorders treated at Kobe Children's Hospital (Kobe, Japan) from 2003 to 2018. Results Thirty-six samples comprising 11 of Burkitt lymphoma (BL), 7 of anaplastic large-cell lymphoma (ALCL), 6 of T-lymphoblastic lymphoma (T-LBL), 5 of diffuse large B-cell lymphoma (DLBCL), 3 of Hodgkin's lymphoma (HL), 2 of chronic active EBV-associated lymphoproliferative disorders (CAEBV-LPD), 1 of T cell/histiocyte rich B-cell lymphoma (T/HRBCL), and 1 of subcutaneous panniculitis-like T-cell lymphoma (SPTCL) were evaluated. PD-L1 and PD-1 staining results in each lymphoma type are explained below and in Table. Burkitt lymphoma None of the 11 samples stained for PD-L1 or PD-1 in BL cells. PD-1 was expressed in a small proportion of tumor-infiltrating lymphocytes (TIL) in 3 of the 11 samples. Anaplastic large-cell lymphoma PD-L1 was robustly expressed in ALCL cells in 5 of the 7 samples. However, PD-1 was not expressed in any ALCL cell samples but expressed in a small proportion of TIL in only 1 sample. T-lymphoblastic lymphoma None of the 6 samples stained for PD-L1 in T-LBL cells. PD-1 was stained in T-LBL cells in only 1 sample. Moreover, PD-1 was not stained in TIL in any samples. Diffuse large B-cell lymphoma None of the 3 samples with DLBCL-not otherwise specified (DLBCL-NOS) or 1 sample with DLBCL with IRF4 rearrangement expressed PD-L1 on tumor cells. Conversely, PD-L1 was overexpressed in tumor cells in 1 sample with DLBCL with an interfollicular pattern of proliferation (DLBCL-IF). However, PD-1 was not expressed in any DLBCL cell samples. PD-1 was expressed in a small proportion of TIL in 1 sample with DLBCL with IRF4 rearrangement. Hodgkin's lymphoma PD-L1 was overexpressed in HL cells in both nodular sclerosis classic HL (NScHL), whereas PD-L1 was not expressed in nodular lymphocyte predominant HL (NLPHL) cells. PD-1 was not expressed in HScHL or NLPHL cells but was expressed in a small proportion of TIL in 1 sample with NLPHL. Chronic active EBV-associated lymphoproliferative disorders PD-L1 was overexpressed in tumor cells in both samples with CAEBV-LPD. PD-1 was not expressed in tumor cells but was expressed in a small proportion of TIL in 1 sample. T cell/histiocyte-rich B-cell lymphoma PD-L1 was overexpressed on tumor cells in T/HRBCL. PD-1 was weakly expressed in a part of T/HRBCL cells but strongly expressed in TIL. Subcutaneous panniculitis-like T cell lymphoma PD-L1 was overexpressed in tumor cells in SPTCL. However, PD-1 was not expressed in SPTCL cells or TIL. Discussion In this pediatric cohort, PD-L1 was overexpressed in tumor cells in ALCL (5/7), DLBCL-IF (1/1), NScHL (2/2), CAEBV-LPD (2/2), T/HRBCL (1/1), and SPTCL (1/1), but not in BL, T-LBL, DLBCL-NOS, or NLPHL. While the PD-L1 expression in EBV-positive lymphoma cells has been reported before, this study demonstrated the PD-L1 overexpression in CAEBV-LPD. In addition, we demonstrated the PD-L1 overexpression on SPTCL and DLBCL-IF cells, whereas the PD-L1 overexpression in T/HRBCL cells was consistent with previous reports. This study demonstrated that the PD-1 expression in tumor cells was rare in pediatric lymphoma. In addition, PD-1 expressions in TIL tended to be low in pediatric lymphoma, except for NLPHL and T/HRBCL. Besides classic HL, PD-1 blockade might be a promising treatment strategy for ALCL, DLBCL-IF, CAEBV-LPD, T/HRBCL, and SPTCL in children. Indeed, anectodal reports showed promising efficacy in ALCL. Therefore, further investigations are required to assess the role of the PD-1-PD-L1 pathway in pediatric lymphoma. Disclosures No relevant conflicts of interest to declare.

Introduction: L-asparaginase (L-Asp) is one of the risk factor of thromboembolism during ALL chemotherapy. Although the pathogenesis has been still unclarified, L-Asp may have profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In addition, recent studies demonstrated that the age of over 10-years might be a risk factor for this L-Asp associated coagulopathy. In this study, we hypothesized that change of balance between coagulation and fibrinolysis contributes to hyper-coagulation condition during chemotherapy including L-Asp. In order to clarify our hypothesis, we investigated the global coagulation and fibrinolytic function during the ALL induction therapy with simultaneous thrombin and plasmin generation assay (T/P-GA). Patients: Seventy-two pediatric patients aged 1.0 to 15.2 years (43 males and 29 females) with newly diagnosed ALL were enrolled from Aug. 2014 to Mar. 2018 at four hospitals in Japan. All patients and their families had no thrombotic predisposition. Fifty-five cases (76.4%) (BCP-ALL; n=47, T-ALL; n=7, MPAL; n=1) received BFM-95 oriented protocol consisting of a total of 8 doses of E.coli L-Asp 5,000 U/m2, whilst the others (BCP-ALL; n=13, T-ALL; n=2, MPAL; n=1, Ph-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS)-ALL02-protcol consisting of a total of 6 doses of E.coli L-Asp 6,000 U/m2. Fifty-two cases (72.2%) were categorized to younger group (1-10 years; median, 4.3 years) and the other cases to older group (>10 years; median, 12.6 years). The percentage of patients of each category were similar in the two treatment protocols (p=0.764). Methods: The global functions of coagulation and fibrinolysis were evaluated using T/P-GA [Matsumoto et al. TH 2013]. This clotting was initiated by the mixture of recombinant human tissue factor (Innovin®, f.c. 1 pM), phospholipid vesicles (f.c. 4 μM) and tissue-type plasminogen activator (f.c. 3.2 nM). Thrombin and plasmin generation were monitored simultaneously using thrombin- and plasmin-specific fluorogenic substrate (Z-Gly-Gly-Arg-AMC and BOC-Glu-Lys-Lys-MAC, respectively) in separate microtiter wells. The first derivatives (velocity) of thrombin and plasmin generation were utilized to derive the parameters, lag time (LT), endogenous potential (EP), peak levels (peak), and time to peak (ttPeak). EP of thrombin generation (T-EP) and plasmin-peak (P-peak) were selected as parameters for evaluation in this study. A ratio of T-EP and P-peak of patients' plasmas to those of control normal plasma were calculated. The conventional laboratory markers of coagulation and fibrinolysis were also monitored by fibrinogen (Fbg), fibrin-Fbg degradation products (FDP), antithrombin (AT), thrombin-AT complex and plasmin-α2 plasmin inhibitor complex. Plasmas were collected at T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post induction phase. Results: None of the cases developed L-Asp associated coagulopathy, and two cases (2.8%) developed induction failure. Thirteen cases (18.1%) received fresh frozen plasma transfusion for low Fbg level, whilst 52 (72.2%) cases received AT supplement for low AT level. Fbg showed a median of 165, 99.0, 97.0 and 316 mg/dl at T0, T1, T2 and T3, respectively, and AT showed a median of 143, 80.9, 80.0 and 105%, respectively, whilst the value of other conventional markers remained within reference value. T-EP revealed a median of 1,134, 1,135, 1,221, 1,277 and 1,102 nM, whilst P-peak showed a median of 5.33, 3.75, 3.63, 4.91 and 5.51 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-peak ratios (Fig. 1). T-EP ratios showed significantly higher at T2 and T3 than at T0 and T1 (p Disclosures Nogami: Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.

Introduction: ETV6-ABL1 fusion represents a rare subgroup of pediatric acute lymphoblastic leukemia (ALL) with unfavorable outcomes. ETV6-ABL1-positive ALL is recently identified in Philadelphia chromosome (Ph)-like ALL and exhibits a gene expression profile similar to BCR-ABL1-positive ALL. Analogous to BCR-ABL1 fusion, ETV6-ABL1 fusion results in the formation of constitutively active non-receptor tyrosine kinases that can also be targeted by selective ATP-competitive tyrosine kinase inhibitors (TKIs). Since TKIs are currently incorporated into the standard treatment of BCR-ABL1-positive ALL, they will be a promising option also for the treatment ETV6-ABL1-positive ALL. However, TKI resistance becomes a common problem in TKI-treated patients, where it is frequently caused by BCR-ABL1-dependent alterations including mutations, genomic amplification, and enhanced expression of BCR-ABL1-fusion kinase. In addition, BCR-ABL1-independent alterations have also been reported to cause TKI resistance. It includes a variety of activating and/or inactivating alterations in RAS, NF-kB, PI3K-AKT, and JAK-STAT signaling pathways that mediate the oncogenic activity of BCR-ABL1 fusion kinase. In contrast, the molecular mechanisms of TKI resistance have been poorly described in ETV6-ABL1-positive ALL, except for T315I mutation of ETV6-ABL1 fusion gene in a single patient and K89M mutation of GNB1 gene in a cell line model. Patient and Results: A previously healthy 14-year-old girl was admitted to our hospital because of persistent fever. Laboratory data showed white blood cell count of 417,800 /µL and increased LDH level and uric acid level. Bone marrow examination showed nuclear cell count of 855,000 /µL with 90.0% blastic cells of lymphoid morphology. Bone marrow blasts at initial diagnosis were positive for CD10, CD19, CD20, CD34, cyCD79a, cyTdT, HLA-DR, and CD66c; had a karyotype of 45, XX, -7; and were sensitive to TKIs (imatinib and dasatinib) in vitro. A split signal analyzed by ABL1 FISH probe was positive (92.7%), while major and minor BCR-ABL1 fusion transcripts were not detected by RT-qPCR. She was treated with the high-risk protocol based on BFM 95 protocol because of prednisolone poor response. After induction chemotherapy, she achieved complete remission (CR) without ABL1 split signal and IgH gene rearrangement. However, she relapsed 19 months after initial diagnosis, and failed to achieve second CR by alternating administration of dasatinib and antileukemic drugs. Bone marrow blasts at initial diagnosis and after relapse were subjected to whole-transcriptome sequencing. ETV6-ABL1 fusion transcripts were identified in both initial diagnostic and relapsed samples, and their level of expression was not significantly changed. No known alteration in ETV6-ABL1 fusion and GNB1 genes was detected. These results suggested a novel mechanism of TKI resistance in ETV6-ABL1-positive ALL. Conclusion: To our knowledge, this is a first case of ETV6-ABL1-positive ALL who acquired ETV6-ABL1-independent TKI resistance. It will provide a foundation for the treatment of TKI-resistant ETV6-ABL1-positive ALL. Citation Format: Suguru Uemura, Daiichiro Hasegawa, Akemi Shono, Khin Kyae Mon Thwin, Nanako Nino, Satoru Takafuji, Takeshi Mori, Akihiro Tamura, Nobuyuki Yamamoto, Atsuro Saito, Kenji Kishimoto, Toshiaki Ishida, Yoshiyuki Kosaka, Kazumoto Iijima, Noriyuki Nishimura. A pediatric ETV6-ABL1-positive acute lymphoblastic leukemia case with ETV6-ABL1-independent resistance to tyrosine kinase inhibitor [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A11.

Most chromosomal rearrangements including the mixed lineage leukemia (MLL) gene are manifested as leukemia and predict a poor prognosis. Although more than 50 MLL-rearrangement partners are characterized, MLL-related leukemogenesis remains to be understood. Here we report a case of a 3-year old boy bearing a novel MLL-rearrangement with the suppressor of actin mutations 1-like (SACM1L) gene in the absence of leukemia. Bone marrow cells harboring the MLL-SACM1L rearrangement appeared during chemotherapy for acute lymphoblastic leukemia with hyperdiploidy and were continuously detected over 7 years without clonal expansion.

Background Diamond-Blackfan anemia is a rare, clinically heterogeneous, congenital red cell aplasia: 40% of patients have congenital abnormalities. Recent studies have shown that in western countries, the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes in about 50% of patients. There have been no studies to determine the incidence of these mutations in Asian patients with Diamond-Blackfan anemia. Design and Methods We screened 49 Japanese patients with Diamond-Blackfan anemia (45 probands) for mutations in the six known genes associated with Diamond-Blackfan anemia: RPS19 , RPS24 , RPS17 , RPL5 , RPL11 , and RPL35A. RPS14 was also examined due to its implied involvement in 5q- syndrome. Results Mutations in RPS19 , RPL5 , RPL11 and RPS17 were identified in five, four, two and one of the probands, respectively. In total, 12 (27%) of the Japanese Diamond-Blackfan anemia patients had mutations in ribosomal protein genes. No mutations were detected in RPS14 , RPS24 or RPL35A . All patients with RPS19 and RPL5 mutations had physical abnormalities. Remarkably, cleft palate was seen in two patients with RPL5 mutations, and thumb anomalies were seen in six patients with an RPS19 or RPL5 mutation. In contrast, a small-for-date phenotype was seen in five patients without an RPL5 mutation. Conclusions We observed a slightly lower frequency of mutations in the ribosomal protein genes in patients with Diamond-Blackfan anemia compared to the frequency reported in western countries. Genotype-phenotype data suggest an association between anomalies and RPS19 mutations, and a negative association between small-for-date phenotype and RPL5 mutations.

A new thermally expandable microcapsule was developed for use with foaming polypropylene (PP) by injection molding and extrusion processes at operating temperatures above 200°C. The microcapsule consists of a blowing agent as the core and a shell polymer. The rheological properties of the shell polymer were controlled by a crosslinking agent to design the expandability and shrinkage. The effects of rheological properties on the expandability and the surface appearance of foam products were thoroughly investigated. It was found that storage modulus G′ and tan δ significantly affected the expandability and shrinkage and were controllable through crosslinking polymerization. Visual observation of batch foaming, rheological measurement, and experiments of foam injection molding and extrusion elucidated the existence of the optimal degree of crosslinking that could realize more than 30% density reduction while maintaining a smoothsurface at PP foam injection molding and extrusion. POLYM. ENG. SCI., 2010. © 2009 Society of Plastics Engineers

There exist three kinds of methods to determine the scattering coefficients of architectural surfaces: I) the reverberation room method, using coherent averaging of room impulse responses during sample rotation, 2) the free-field method, using coherent averaging of reflected responses during sample rotation, 3) the free-field method, using directivity correlation between reflected fields with a sample and with a flat reference surface. This paper discusses the relationship among the scattering coefficients determined by the three methods. Numerical simulation demonstrates that the two free-field methods give little different values at mid and high frequencies for a 1D sinusoidal surface, but not to the same extent for a 2D sinusoidal surface. Subsequently, this disagreement is examined in comparison with experimental results obtained by the reverberation room method and with theoretical ones. Theoretical consideration reveals that the dominant reason for the disagreement is due to different averaging processes in the three methods, which take the arithmetic mean or the quadratic mean of specular reflection factors with respect to azimuthal incidence angle, respectively.

We evaluated the efficacy of a treatment strategy in which infants with acute lymphoblastic leukemia (ALL) were stratified by their MLL gene status and then assigned to different risk-based therapies. A total of 102 patients were registered on two consecutive multicenter trials, designated MLL96 and MLL98, between 1995 and 2001. Those with a rearranged MLL gene (MLL-R, n=80) were assigned to receive intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), while those with germline MLL (MLL-G, n=22) were treated with chemotherapy alone. The 5-year event-free survival (EFS) rate for all 102 infants was 50.9% (95% confidence interval, 41.0-60.8%). The most prominent late effect was growth impairment, observed in 58.9% of all evaluable patients in the MLL-R group. This plan of risk-based therapy appears to have improved the overall prognosis for infants with ALL, compared with previously reported results. However, over half the events in patients with MLL rearrangement occurred before the instigation of HSCT, and that HSCT-related toxic events comprised 36.3% (8/22) of post-transplantation events, suggesting that further stratification within the MLL-R group and the development of more effective early-phase intensification chemotherapy will be needed before the full potential of this strategy is realized.

We initially conducted a multicenter, randomized trial (n=43), and subsequently a questionnaire study (n=209) of participating hospitals, to evaluate whether infused fresh frozen plasma (FFP) could prevent the occurrence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT). Forty-three patients were divided into two groups: 23 receiving FFP infusions and 20 not receiving it. VOD developed in three patients not receiving FFP. Plasma von Willebrand factor (VWF) antigen levels were lower at days 0, 7 and 28 after SCT in patients receiving FFP than in those not receiving it, whereas plasma ADAMTS13 activity (ADAMTS13:AC) did not differ between them. Plasma VWF multimer (VWFM) was demonstrated to be defective in the high approximately intermediate VWFM during the early post-SCT phase, but there was a significant increase in high VWFM just before VOD onset. This suggests that a relative enzyme-to-substrate (ADAMTS13/high-VWFM) imbalance is involved in the pathogenesis of VOD. To strengthen this hypothesis, the incidence of VOD was apparently lower in patients receiving FFP infusions than in those not receiving it (0/23 vs 3/20) in the randomized trial. Further, the results combined with the subsequent questionnaire study (0/36 vs 11/173) clearly showed the incidence to be statistically significant (0/59 vs 14/193, P=0.033).

We evaluated the efficacy of treatment using reduced cumulative doses of anthracyclines in children with acute promyelocytic leukaemia (APL) in the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-P05 study. All patients received two and three subsequent courses of induction and consolidation chemotherapy respectively, consisting of all-trans retinoic acid (ATRA), cytarabine and anthracyclines, followed by maintenance therapy with ATRA. Notably, a single administration of anthracyclines was introduced in the second induction and all consolidation therapies to minimize total doses of anthracycline. The 3-year event-free (EFS) and overall survival rates for 43 eligible children were 83·6% [95% confidence interval (CI): 68·6-91·8%] and 90·7% (95% CI: 77·1-96·4%), respectively. Although two patients died of intracranial haemorrhage or infection during induction phases, no cardiac adverse events or treatment-related deaths were observed during subsequent phases. Patients not displaying M1 marrow after the first induction therapy, or those under 5 years of age at diagnosis, showed inferior outcomes (3-year EFS rate; 33·3% (95% CI: 19·3-67·6%) and 54·6% (95% CI: 22·9-78·0%), respectively). In conclusion, a single administration of anthracycline during each consolidation phase was sufficient for treating childhood APL. In younger children, however, conventional ATRA and chemotherapy may be insufficient so that alternative therapies should be considered.

Neuroblastoma is an aggressive solid tumor that leads to tumor relapse in more than half of high-risk patients. Minimal residual disease (MRD) is primarily responsible for tumor relapses and may be detected in peripheral blood (PB) and bone marrow (BM) samples. To evaluate the disease status and treatment response, a number of MRD detection protocols based on either common or distinct markers for PB and BM samples have been reported. However, the correlation between the expression of MRD markers in PB and BM samples remains elusive in the clinical samples. In the present study, the expression of 11 previously validated MRD markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) was determined in 23 pairs of PB and BM samples collected from seven high-risk neuroblastoma patients at the same time point, and the sample was scored as MRD-positive if one of the MRD markers exceeded the normal range. Although the number of MRD-positive samples was not significantly different between PB and BM samples, the two most sensitive markers for PB samples (CRMP1 and KIF1A) were different from those for BM samples (PHOX2B and DBH). There was no statistically significant correlation between the expression of MRD markers in the PB and BM samples. These results suggest that MRD markers were differentially expressed in PB and BM samples from high-risk neuroblastoma patients.

Currently, the standard management of moderate aplastic anemia (MAA) has not been well described, although the superiority of the combination of antithymocyte globulin (ATG) and cyclosporine (CyA) over CyA alone has been demonstrated in terms of hematological responses and failure-free survival (FFS).We adopted this therapeutic strategy and treated 95 children with MAA who were enrolled in two consecutive prospective studies between October 1992 and August 2009.For these patients, the 6-month response rate was 54.7% (complete response, 13.7%; partial response, 41.1%). There were no statistically significant differences in the overall response rates between the transfusion-dependent (48.8%, n = 41) and transfusion-independent groups (59.3%, n = 54; P = 0.4). Treatment failure was defined as the requirement of salvage treatment, and was observed in 52 patients. The 10-year FFS was 44.0% (95% confidence interval [CI], 32.9%-54.6%). Of the 22 patients who underwent a second immunosuppressive therapy (IST), 12 responded. Forty patients underwent hematopoietic stem cell transplantation as second- or third-line therapy and three died of complications. Consequently, the 10-year overall survival rate was 96.0% (95% CI, 88.0%-98.7%) with a median follow-up period of 103 months (range, 29-221 months).Although current guidelines recommend only observation for patients with transfusion-independent MAA, the results of our study justify early intervention with ATG and CyA in those patients. A prospective randomized trial is warranted to clarify the risks and benefits of early intervention with IST and observation alone until progression to severe AA in patients with MAA.

Recent studies have reported that the absolute lymphocyte count (ALC) during induction therapy is predictive of treatment outcome in de novo acute lymphoblastic leukemia (ALL); however, the significance of ALC on outcomes remains controversial. In the present study, we assessed the significance of ALC at day 29 (ALC-29), the end of induction therapy, on outcomes in our Japanese cohort. The outcomes of 141 patients aged ≤18 years with newly diagnosed ALL who were enrolled on the JACLS ALL-02 at our hospitals were analyzed in terms of ALC-29. Patients with ALC-29 ≥750/μL (n = 81) had a superior 5-year EFS (95.2 ± 2.7 vs 84.3 ± 4.8 %, P = 0.016) and OS (100 vs 87.0 ± 4.7 %, P = 0.0062). A multivariate analysis identified ALC-29 ≥750/μL as a significant predictor of improved EFS and OS after controlling for confounding factors. A multiple linear regression model revealed a significant inverse relationship between the percentage of blasts in bone marrow on day 15 and ALC-29 (P = 0.005). These results indicate that ALC is a simple prognostic factor in childhood ALL, and, thus, has the potential to refine current risk algorithms.

Background Familial hemophagocytic lymphohistiocytosis (FLH) is an autosomal recessive disorder of immune regulation that leads to a hyperinflammatory syndrome. Fetal onset FHL is extremely rare and is considered to be the most severe form of FHL. Case We report a preterm case of FHL that presented as hydrops fetalis. The infant was treated with a chemotherapy regimen based on the HLH-2004 protocol from the third day of life. However, he had persistent cytopenia and died on the 18th day of life due to bacteremia. The detection of defective perforin expression in the patient's natural killer cells and mutations in the PRF1 gene resulted in a molecular diagnosis of FHL. Conclusion We suggest that early diagnosis and the development of an appropriate immunosuppressive strategy that can induce and maintain remission until hematopoietic stem cell transplantation can be performed are required to improve the outcomes of fetal onset FHL.

8施設の血友病患者の入院医療コストを集計し, その特徴と診断群分類(DPC)点数表の導入に対する問題点を考察した. 2002年4月から2004年3月までの8施設における患者の各入院に対し, 血友病の種類, 体重, インヒビターの有無, 手術の有無, 入院日数, 請求点数などを集計した. さらにそれらのデータからそれぞれの因子により請求点数を解析し, どの因子が請求点数に関与しているか統計学的検定を行った. 入院日数, 請求点数は施設間にばらつきがみられ, 施設別1日あたりの平均医療コストは最少が124,110円, 最大が222,080円であった. 体重別では25kg未満127,280円, 25－50kg 210,720円, 50-75kg 280,861円, 75kg以上526,958円で有意差がみられた. インヒビターの有無(524,416円, 147,534円)でも有意差がみられた. 2004年度の血友病類縁疾患のDPC導入案と比較すると, 全施設で病院側の赤字になることが分かった. 以上より, 血友病患者の入院医療コストは患者の体重や状態により多種多様で, 安易にDPCを導入することは問題と思われた.

We report a 4-year-old boy with severe congenital neutropenia (SCN), who was successfully treated with hematopoietic stem cell transplantation (HSCT). The patient had frequently developed bacterial infections since 6 months of age, and showed severe neutropenia below 100/μl at 1 year and 4 months of age. The patient harbored a heterozygous missense mutation in ELANE exon 3 (p.Q73P, g.2253 AC). This was a novel de novo mutation, and he was thus diagnosed as having SCN. Because of failure to respond to granulocyte colony-stimulating factor treatment and repeated admissions due to bacterial infections, allogeneic HSCT was performed from a serologically matched unrelated donor following the conditioning regimen: fludarabine/melphalan/anti-thymocyte globulin and a low dose of total body irradiation. Tacrolimus and a short course of methotrexate were used for graft-versus-host disease prophylaxis. Engraftment was achieved at day 12, and the patient maintained normal hematopoiesis for over 15 months after HSCT. We concluded that HSCT is a useful treatment for SCN patients, especially those who are at high risk for leukemic transformation. However, a larger number of SCN patients and longer follow-up are necessary to identify appropriate conditioning regimens and long-term prognosis.

Neuroblastoma is an aggressive pediatric tumor accounting for ~15% of cancer-associated mortalities in children. Despite the current intensive therapy, >50% of high-risk patients experience tumor relapse or regrowth caused by the activation of minimal residual disease (MRD). Although several MRD detection protocols using various reverse transcription-quantitative polymerase chain reaction (RT-qPCR) markers have been reported to evaluate the therapeutic response and disease status of neuroblastoma patients, their clinical significance remains elusive. The present study reports two high-risk neuroblastoma patients, whose MRD was consecutively monitored using 11 RT-qPCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) during their course of treatment. The two patients initially responded to the induction therapy and reached MRD-negative status. The patients' MRD subsequently became positive with no elevation of their urinary homovanillic acid, urinary vanillylmandelic acid and serum neuron-specific enolase levels at 13 or 19 weeks prior to the clinical diagnosis of tumor relapse or regrowth. The present cases highlight the possibility of consecutive MRD monitoring using 11 markers to enable an early detection of tumor relapse or regrowth in high-risk neuroblastoma patients.

In order to evaluate the scattering coefficients of architectural surfaces, a numerical technique based on Mommertz’s definition is developed with employing a 3-dimensional boundary element method. Numerical examination on the setting of parameters in computation and the conditions of samples is performed so as to ensure accurate calculation with this technique. As a result, criteria for the numerical parameters and for the test arrangement are clarified, and additionally, illustrating the behavior of directional and random-incidence scattering coefficients. Furthermore, in comparison between the values with the present method and those for an infinite periodic surface, general correspondence is confirmed although some differences appear due to the edge diffraction by the finite sample.

Hepatoblastoma is a common hepatic tumor in children. Although evidence regarding cytogenetic and molecular genetic alterations in hepatoblastomas has been reported, the molecular events affecting the biologic characteristics of this tumor, including alterations of the gene expression profile, are largely unknown. To identify genes differentially expressed between nondiseased liver (NDL) and hepatoblastoma tumor (HBT), we analyzed the gene expression profile in 14 NDL and 16 HBT samples using a high-density oligonucleotide DNA array. Using Mann-Whitney U test followed by the k-nearest neighbor algorithm, we identified 26 genes (predictor genes) that were able to assign unknown samples derived from NDL and HBT to either the NDL group or HBT group with 100% accuracy. Using a cross-validation approach, we confirmed that the k-nearest neighbor algorithm assigned the particular samples derived from NDL and HBT to either the NDL or HBT group with 93.3% (28/30 samples) accuracy. In the 26 predictor genes, we found alteration of the expression of genes regulating cell division (NAP1L1, STMN1, CCNG2, and CDC7L1) and tumor cell growth (IGF2 and IGFBP4) in HBT. Four predictor genes (ETV3, TPR, CD34, and NR1I3) were also found to be mapped to the chromosomal region 1q21 approximately q32, which has been reported to be frequently involved in the development of hepatoblastoma. The findings obtained in this study suggest that alteration of the expression of some genes regulating cell division and tumor cell growth may be characteristics of the gene expression profile in HBT, and that alteration of the expression of the four predictor genes mapped to chromosomal region 1q21 approximately q32 may also contribute to the differences in gene expression profile between NDL and HBT.

We studied the effectiveness of risk-directed therapy for infants younger than 13 months of age with acute lymphoblastic leukaemia (ALL). Fifty-five infants were assigned to different treatment programs (from December 1995 to December 1998) on the basis of their MLL gene status at diagnosis. Forty-two cases (76.3%) had a rearranged MLL gene (MLL+) and were treated with remission induction therapy followed by sequential intensive chemotherapy, including multiple genotoxic agents (MLL9601 protocol). Haematopoietic stem cell transplantation (HSCT) was attempted if suitable donors were available. Thirteen infants (23.7%) were classified as MLL- and treated for 2.5 years with intensive chemotherapy for high-risk B-ALL (MLL9602 protocol). Complete remission was induced in 38 of the 42 infants (90.5%) with MLL+ ALL and in all 13 patients (100%) with MLL- disease. In the MLL+ subgroup, the estimated event-free survival (EFS) rate at 3 years post diagnosis was 34.0% +/- 7.5%, compared with 92.3% +/- 7.4% in the MLL- subgroup (overall comparison, P = 0.001 by log-rank analysis). Both age less than 6 months (hazard ratio = 6.87, 95% CI = 0.91-52.3; P = 0.013) and central nervous system (CNS) involvement at diagnosis (hazard ratio = 2.92 95% CI = 1.29-6.63; P = 0.015) were significant independent predictors of an inferior outcome. These findings indicate a strategic advantage in classifying infant ALL as either MLL+ or MLL- early in the clinical course and selecting therapy accordingly. Standard chemotherapy for high-risk B-lineage ALL appeared adequate for MLL- cases. Novel therapeutic initiatives are warranted for infants with MLL+ disease, particularly those with initial CNS leukaemic involvement or age less than 6 months, or both.

BACKGROUND: Recurrent somatic Dicer1 mutations were observed in patients with pulmonary blastoma, Sertoli-Leydig cell tumor and less commonly in several rare cancers including pineoblastoma and pituitary blastoma. Some of the patients have germline Dicer1 mutations, known as a Dicer1 syndrome. Here, we report the first case in the literature of immature teratoma harboring Dicer1 mutation. CASE REPORT: A week-old boy was admitted for paraplegia. His prenatal period and family history were unremarkable. MRI showed extradural tumor extended from L2 to sacral region. Levels of serum AFP and hCG were not elevated. The tumor was hemorrhagic and partially resected at the initial operation. Pathologically, the tumor consisted of immature mesenchymal tissue and primitive neuroepithelial elements. Primitive cartilage and skeletal muscle components were also observed. The tumor was diagnosed as immature teratoma after central pathology review. The patient received one course of chemotherapy with etoposide and carboplatin and then underwent total resection. Eight months later, however, the tumor recurred. The patient received partial resection and following local radiotherapy (45Gy) and chemotherapy with cisplatin, cyclophosphamide and etoposide. Although the patient transiently responded to the salvage therapy, he died of the disease with intracranial dissemination 25 months after initial diagnosis. MOLECULAR ANALYSIS: Analysis of frozen tumor sample by Ion Torrent sequencing revealed Dicer1 mutations (p.Arg745* in exon14 and p.Glu1813Lys in exon 25), confirmed by Sanger sequencing. Matched non-neoplastic material was not available. DISCUSSIONS AND CONCLUSIONS: The existence of Dicer1 mutation may reflect some embryonal components in the immature teratoma. Furthermore, the mutation in exon 14 may be germline mutation because previously reported somatic Dicer1 mutations are almost always restricted in RNaseⅢ domain (exon 24 and 25). Our case suggests that a subset of immature teratoma may be related to Dicer1 syndrome.

Despite the introduction of 13-cis-retinoic acid (13-cis-RA) into the current chemotherapy, more than half of high-risk neuroblastoma patients have experienced tumor relapses driven by chemoresistant cancer stem cells (CSCs) that can be isolated by their ability to grow as spheres. Although aldehyde dehydrogenase (ALDH) has been used to characterize CSCs in certain cancers, ALDH remains elusive in neuroblastoma. In the present study, we determined ALDH activity and expression of its 19 isoforms in spheres and parental cells of neuroblastoma. ALDH activity and several ALDH isoforms were consistently induced in spheres of different neuroblastoma cells. While ALDH1A2, ALDH1L1 and ALDH3B2 expression was consistently induced in spheres and associated with the sphere and colony formation, only ALDH1A2 expression was significantly correlated with the poor prognosis of neuroblastoma patients. ALDH1A2 expression was further associated with the growth and undifferentiation of neuroblastoma xenografts and the resistance of neuroblastoma cells to 13-cis-RA. These results suggest that ALDH1A2 is involved in the regulation of CSC properties in neuroblastoma.

For an overall characterization of pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) with CRLF2 overexpression (OE), we conducted genetic analysis of CRLF2 in 167 pediatric BCPALL patients. CRLF2 OE was detected in 30 (18%) of 167 patients, the P2RY8-CRLF2 fusion was identified in only 3 (1.8%) of 167 patients, all of which demonstrated CRLF2 OE. Moreover, CRLF2 gain was identified in 18 (11%) of 167 patients. Messenger RNA sequencing revealed a novel fusion transcript, CSF2RA-CRLF2, in a case with CRLF2 OE, suggesting that this fusion is associated with CRLF2 OE. In survival analysis, no significant differences in 5-year event-free survival (EFS) and overall survival were observed between patients with and without CRLF2 OE (70.7 vs. 75.4%, log rank P = 0.68 and 96.4 vs. 82.1%, log rank P = 0.11, respectively). However, a significant difference in 5-year EFS between CRLF2 OE patients with and without IKZF1 deletion was observed (44.4 vs. 83.1%, log rank P = 0.02). In multivariate analysis, only IKZF1 deletion was a significant predictor of inferior OS (hazard ratio: 2.427, P = 0.04).These findings suggest that CRLF2 OE is not an independent prognostic factor in pediatric BCPALL.

In Western countries, gene alterations involving the CRLF2-JAK signaling pathway are identified in approximately 50-60% of patients with Down syndrome-associated acute lymphoblastic leukemia (DS-ALL), and this pathway is considered a potential therapeutic target. The frequency of BTG1 deletions in DS-ALL is controversial. IKZF1 deletions, found in 20-30% of DS-ALL patients, are associated with a poor outcome and EBF1 deletions are very rare (∼2%). We analyzed 38 patients to determine the frequencies and clinical implications of CRLF2-JAK pathway genetic alterations and recurrent gene deletions in Japanese DS-ALL patients. We confirmed a high incidence of P2RY8-CRLF2 (29%) and JAK2 mutations (16%), though the frequency of P2RY8-CRLF2 was slightly lower than that in Western countries (∼50%). BTG1 deletions were common in our cohort (25%). IKZF1 deletions were detected in 25% of patients and associated with shorter overall survival (OS). EBF1 deletions were found at an unexpectedly high frequency (16%), and at a significantly higher level in P2RY8-CRLF2-positive patients than in P2RY8-CRLF2-negative patients (44% vs. 4%, P=0.015). Deletions of CDKN2A/B and PAX5 were common in P2RY8-CRLF2-negative patients (48 and 39%, respectively) but not in P2RY8-CRLF2-positive patients (11% each). Associations between these genetic alterations and clinical characteristics were not observed except for inferior OS in patients with IKZF1 deletions. These results suggest that differences exist between the genetic profiles of DS-ALL patients in Japan and in Western countries, and that P2RY8-CRLF2 and EBF1 deletions may cooperate in leukemogenesis in a subset of Japanese DS-ALL patients.

This study analyzed data on 35 infants with acute myeloid leukemia (AML) who were treated with intensive chemotherapy between 1995 and 1998 in Japan. The incidence of boys, younger age (< 6 months old), and hyperleukocytosis at onset was high in patients with the M4/M5 subtype (n = 23) in the French-American-British classification, compared with the non-M4/M5 subtype (n = 12). Thirteen (56%) and 16 (70%) patients with the M4/M5 subtype also showed 11q23 translocations and MLL gene rearrangements, respectively, whereas only one patient with the non-M4/M5 subtype had this rearrangement. All 35 patients were treated with the ANLL91 protocol consisting of etoposide, high-dose cytarabine, and anthracyclines. Overall survival and the event-free survival (EFS) rates at 3 years of all patients were 76% (95% confidence interval [CI], 61.3%-90.7%) and 72% (95% CI, 56.4%-87.9%), respectively. EFS showed no significant difference between 2 subgroups divided by age, gender, presence of the MLL gene rearrangements, and white blood cell count at onset; EFS in patients with the M4/M5 subtype tended to be better than those with the non-M4/M5 subtype. Although all 6 patients who underwent allogeneic stem cell transplantation (SCT) have been in complete remission, no benefit of SCT was confirmed. These findings suggest that the intensive chemotherapy with the ANLL91 protocol might have been responsible for the observed good outcome of infant AML, even without SCT. The presence of the MLL gene rearrangements or the age at onset had no impact on the outcome of infant AML.

Between April 1994 and March 1997, 143 children (age range, 1-15 years) with newly diagnosed acute lymphoblastic leukemia (ALL), except for those patients with t(9;22), were treated according to protocol-94 of the Osaka Childhood Leukemia Study Group. In this trial, the intensity of chemotherapy was enforced in the consolidation and reinduction phases by introducing AML-type block therapies consisting of concentrated administration of 4 to 6 drugs during 5 or 6 days. For patients in the higher risk groups, rotational combination chemotherapy was introduced following the early phase. A total of 124 children with B-cell precursor ALL (B-pre ALL) were classified into 3 groups, the ultrahigh-risk group (UHRG) (15 patients), the high-risk group (HRG) (61 patients), or the standard-risk group (SRG) (48 patients), based on age. leukocyte count, immunophenotype, central nervous system leukemia, response to treatment, and selected chromosomal abnormalities. The complete remission rate was 93%, and the 6-year event-free survival (EFS) rate was 79%+/-4%. EFS rates for the UHRG, HRG, and SRG groups were 67%+/-12%, 80%+/-6%, and 81%+/-6%, respectively. Nineteen patients with T-cell ALL were treated with the protocol for the UHRG. Thirteen patients (68%) attained complete remission, and the 6-year EFS rate was 55%+/-12%. Thus, intensification of chemotherapy improved the EFS rate and AML-type block therapies appeared to be effective as the consolidation and reinduction therapies for B-pre ALL.

The extent of red blood cell fragmentation in peripheral blood is useful for diagnosis and follow-up in many diseases, e.g. haemolytic uremic syndrome, transplantation-associated thrombotic microangiopathy (BMT-TMA). However, this quantification still relies on manual counting of fragmented red cells on blood smears. We have developed a quantification system by gating a fixed area of fragmented red blood cells (Gate 1) on an automated haematology analyser (XE-2100, Sysmex Co., Kobe, Japan). The fragmented red cell percentage (FRC%) calculated with this system, from 100 samples, was highly correlated with the manual count (r=0.902, P < 0.0001). Because microcytic anaemia specimens usually occupy a lower position on the XE-2100 scattergram, with microcytic cells overlapping Gate 1 and causing a spuriously high FRC% calculation, a supplementary gate (Gate 2) was added. Using the particle number in this gate as well as in Gate 1, a revised method for such samples was developed and its validity confirmed (revised FRC% correlated with a manual count for 10 subjects (P < 0.001). Because this gating system can be programmed on any XE-2100, it is likely to prove useful for accurate quantification of red blood cell fragmentation and for the monitoring of the development of BMT-TMA.

Introduction B-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner. Methods We enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection. Results The median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]). Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation. Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified. Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q < 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL. Conclusions We revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Background: It is important to establish proper intensity of chemotherapeutic regimen for low risk group of childhood B-precursor ALL (BCP-ALL) to avoid late effects. For this purpose, in Japan Association of Childhood Leukemia study (JACLS) ALL-02 clinical trial, we defined standard risk (SR) group as the patients ranged from 1 to 10 years showing white blood cell (WBC) count of less than 10,000/μL at the diagnosis without predonisolone poor responder, t(4;11), t(1;19) and CNS3 to treat with less intensive chemotherapy (JACLS ALL02 SR protocol). Herein, we report the outcome of JACLS ALL02 SR protocol and analyzed prognostic factors to identify super low risk group which is curable by less intensive chemotherapy. Patientss and treatment: JACLS ALL02 SR protocol consisted of early phase therapies for total 4 months including an induction therapy using vincristine (VCR), steroids, pirarubicin (THP-ADR), and L-asparaginase (L-asp) for 4 weeks, a consolidation therapy using cyclophosphamide and a combined administration of cytarabine and dexamethasone (DEX) or 6-mercaptopurine (6-MP), a sanctuary therapy with only two cycles of high-dose methotrexate (MTX) of 3g/m2, one course of 4 drugs re-induction therapy and maintenance phase with 6-MP and oral MTX combined with VCR and PSL every 5 weeks by the end of the second year. In addition, total of 12 times of triple intrathecal therapy were administered by the end of the first year. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test. Multivariate analysis was performed using a Cox regression model. Results: 1252 newly diagnosed ALL patients were enrolled from April 2002 to May 2008 in JACLS ALL02 clinical trial, which include 388 patients (192 males and 196 females) in SR group (31.0%). The median age at diagnosis was 4.2 years (range, 1.0 to 9.9 years) and the median WBC count was 3,700/μL (range, 370 to 9,984 /μL). The median follow-up time was 6 year and 5 months. 4y / 8y EFS and OS (±SE) of the SR group are 91.5±1.4% / 89.4±1.7% and 97.9±0.7% / 95.2±1.2%, respectively. 4y / 8y EFS of the patients of 1 to 5 years old (n=300) and 6 to 9 years old (n=88) are 93.5±1.4% / 93.0±1.5% and 84.9±3.9% / 75.8±5.8%, respectively (log rank p=0.0003). 4y / 8y EFS of the patients with day 15 M1 marrow (n=287 ) was 92.8±1.6% / 91.6±1.8%, while those of the patients with day 15 M2 marrow (n=101) was 88.0±3.3% / 83.7±3.9% (log rank p=0.0446). When 388 patients were further classified into 4 subgroups based on genetic abnormalities, such as ETV6-RUNX1 (n=89, 22.9%), high hyperdiploid (HHD) (n=97, 25%), normal karyotype (n=147, 37.9%), and others (n=55, 14.2%), 4y-EFS/OS of each group was 97.6±5.9 /100% (ETV6-RUNX1), 91.5±2.9 /100% (HHD), 89.6±2.5 /95.1±1.8% (normal karyotype), and 86.9±4.6 /98.2±1.8% (others), suggesting poor EFS of B-others group. When HHD subgroup was divided into two groups, such as triple trisomy (TT; n=35) (trisomy of chromosome 4,10,17) and non-TT (n=62), 4y-EFS of each sub-group was 100% and 84.5±4.5%, respectively (log rank p=0.0161). Interestingly, day 15 M2 marrow was associated with poor 4y/8y EFS only in the normal karyotype subgroup (day 15 M1 vs M2 92.0±2.6% / 90.2±3.1% vs, 81.3±6.9% / 77.2±7.7%, log rank p=0.0497). The rates for NCI-CTC grade 4 infection, allergy, increase of transaminase were 1.80%, 0.26%, and 22.9%, respectively. Multivariate analysis identifies the patients of 6 to 9 years old (HR=3.178, p= Discussion: Although JACLS ALL-02 SR protocol was less intensive chemotherapeutic regimen compared to the contemporary protocols used for NCI-SR patients, good outcome of the 4y EFS and OS (91.5±1.4 % and 97.9±0.7 %) was achieved with good feasibility. Because the specific genetic subgroups such as B-others and HHD without TT were associated with relatively poor outcome, those patients should be treated with more intensive chemotherapy, especially for the patients showing initial poor response (day 15 M2). On the other hand, ETV6-RNUX1 and TT subgroups showed excellent outcome. Thus, appropriate reduction of therapy should be explored for these subgroups. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Congenital pure erythroid leukemia (M6b) is exceedingly rare with only a few reported cases to date. Because of the extreme rarity, almost nothing is known about the pathogenesis, appropriate therapy and prognosis. Diagnosis of erythroid leukemia is usually based on the positivity for Glycophorin A, Glycophorin C or PAS staining. We report a first case of congenital pure erythroid leukemia expressing E-cadherin in the absence of Glycophorin A, Glycophorin C and PAS staining. We analyzed the cytogenetic abnormalities of this extremely rare disease. RESULTS: The patient was the first daughter of healthy and non-consanguineous Japanese parents, born at 40 weeks of gestation by emergency cesarean section in non-reassuring fetal state after uncomplicated pregnancy. Apgar score was 8/9. Characteristic facial appearance was not recognized. At birth, she presented with marked hepatomegaly, purpura and disseminated intravascular coagulation. White blood cell (WBC) count was 63.5x109/L with blastic cells with vacuoles. Although congenital leukemia was suspected, flow cytometric analyses using CD45 blast gating failed to demonstrate leukemic cells. Karyotype was 46, XX. Fluorescence in situ hybridization (FISH) for trisomy 21 and MLL split signal were negative. GATA1 mutation was not detected. WBC count has gradually decreased within 3-4 weeks with supportive care.However, liver failure, hemophagocytic lymphohistiocytosis and schistocytosis developed. Although treatment with dexamethasone and etoposide has started, multiple nodules appeared in the liver 11 weeks after birth. Liver biopsy demonstrated small round cell tumor with high N/C ratio and vacuoles infiltrating the liver. The tumor cells were immunohistochemically positive for CD43, CD71, E-cadherin, beta-catenin, Ki-67 and c-Myc and negative for CD45, CD20, CD10, PAX5, CD3, CD4, CD8, TdT, CD1a, CD34, CD56, cyMPO, c-kit, CD42b, CD61, Glycophorin A, Glycophorin C, tyrosine hydroxylase, PGP9.5, myogenin, glypican3, NKX2.2, CAM5.2 and Periodic Acid Schiff (PAS) staining etc. Flow cytometric analysis revealed CD43+ CD71+ CD36+ CD58+ cells within large CD45 negative cell population. These cells expressed almost no other hematopoietic cell markers used to screen for leukemia. These cells were indistinguishable from normal erythroblast based on surface markers only. However, flow cytometric cell sorting revealed these cells are blasts with vacuoles. Karyotype of tumor cells has changed to 50, XX, +7, +8, add(15)(q22), +19, add(19)(q13.1-13.3)×2, +21. Based on these results, she was diagnosed with pure erythroid leukemia. Low dose Cytosine Arabinoside improved her clinical symptoms. She is alive at 5 months of age. DISCUSSION: E-cadherin is a selective marker of immature erythroblast. In our case, E-cadherin was key in erythroid lineage assignment. To our knowledge, this is the first reported case of infantile pure erythroid leukemia expressing E-cadherin in the absence of Glycophorin A, Glycophorin C and PAS staining. These results suggest that the tumor cells originated from undifferentiated erythroblast. This disease entity should be recognized. Immunohistochemical staining of c-Myc showed strong positivity. The c-Myc gene is located on chromosome 8. FISH for c-Myc split signal was negative. G-banding and FISH revealed trisomy 8. Overexpression of c-Myc may be involved in the pathogenesis of this undifferentiated pure erythroid leukemia. At birth, karyotype was 46, XX and blasts in peripheral blood decreased with supportive care only. However, we observed changes in karyotype of blasts. We assume that second hit was added during clinical course. Whole exome sequencing analysis is in progress to reveal somatic and germline mutations underlying this unrecognized disease. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: The trial JACLS ALL-02 for treatment of childhood acute lymphoblastic leukemia (ALL) was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in unfavorable-risk groups by treatment intensification. These aims were pursued through a refined stratification strategy using white blood cell count, age, immunophenotype, unfavorable genetic aberrations, and treatment response providing an excellent discrimination of risk groups. PATIENTS AND METHODS: Between April 2002 and March 2008, 1252 children with newly diagnosed de novo ALL with 1-18 years of age were enrolled to JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Patients with BCP-ALL were stratified into 3 groups: standard-risk (SR), high-risk (HR), extremely high-risk (ER), based on initial prednisolone (PSL) response and the modified National Cancer Institute(NCI) workshop criteria. Prednisolone poor response (PPR) was determined after 7 days of monotherapy with prednisone and one intrathecal dose of methotrexate. PSL good responders (PGR; < 1,000 blasts/microL) were divided into SR and HR according to the modified NCI workshop criteria by WBC 10K and age 10, and received conventional therapy. BCP-ALL with PPR (≥ 1,000 blasts/microL) or t(4;11), and acute mixed lineage leukemia/ acute unclassified leukemia were assigned to ER and received intensified post-induction therapy . Patients with T-ALL were treated by a specific protocol that was different from the protocol used for BCP-ALL. Bone marrow response was also evaluated in aspiration smears on day 15 and 33 of induction treatment, and those who had slow early bone marrow response, defined as an M3 marrow on day 15 or M2/3 marrow on day33, shifted to the higher risk and received augmented post-induction therapy. Cranial irradiation was restricted to patients with initial central nerve system involvement or T-ALL with high WBC (≥10K) at diagnosis. Alternatively, protracted TIT was given during induction, intensification and maintenance depending on the risk group (12 doses in SR/T and 15 in HR/ER). Slow early responders who had an M3 marrow on day 15 in ER/T or M2/3 marrow on day33 in any risk, underwent to stem cell transplantation (SCT). PPRs without slow early bone marrow response underwent to SCT only if a matched sibling donor was available. The probability of event-free survival (EFS) and overall survival were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. All statistical analyses were done according to intent-to treat methods. RESULTS: Estimated 4-year EFS and OS for all 1252 patients was 83.7% (SE=1.1) and 90.3% (SE=0.89), slightly better in EFS than the former study (ALL-97; 79.3%, SE=1.7, p=0.054). The SR group (N=457, 37%) achieved excellent 4-year EFS of 90.4% (95%CI: 87.2, 92.7) and 4-year OS 97.3% (95.3, 98.5); in SR without slow early bone marrow response, patients with hyperdiploid and triple trisomy showed excellent 4-year EFS/OS of 100%. In the HR group (N=542, 43%), the estimated 4-year EFS and 4-year OS were 84.9% (81.5, 87.7) and 89.3% (86.3, 91.6), respectively; older age (≥ 6 years) was predictive for inferior OS (hazard ratio=1.14, p CONCLUSIONS: Refined stratification and risk-adjusted therapy in the JACLS ALL-02 trial can bring about the abolition of prophylactic CRT in non-T ALL and the optimized indication of SCT without compromising the treatment results. Disclosures No relevant conflicts of interest to declare.

Since an excellent outcome was observed in a previous study (OCLSG-94) utilizing continuous cytarabine (CA) infusion in the consolidation phase for the treatment of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), a randomization trial was performed to test the superiority of continuous CA infusion to conventional repeated CA injection as consolidation therapy in Japan Association of Childhood Leukemia Study (JACLS) ALL02 clinical trial. Newly diagnosed children with standard-risk (SR) or high-risk (HR) BCP-ALL excluding patients with Ph+ ALL and infants were included in this study. Patients showing prednisolone good response (PGR) were assigned to SR or HR based on the age at onset and initial white blood cell (WBC) counts (SR: age 1 to 9 and WBC less than 10,000 /µL, HR: remaining patients). Patients achieving complete remission (CR) at the end of induction (day 33) proceeded to the randomization. HR patients with M3 marrow on day 15 were excluded. They were randomly assigned to Arm A (cyclophosphamide 750 mg/m(2) 1hr div: day 1 and 8, CA 75 mg/m(2) 1hr div: day 1-6, 8-13 and 6MP 50 mg/m(2) orally, day 1-14) or Arm B (cyclophosphamide 500 mg/m(2) 1hr div: day 1, 3 and 5, CA 100 mg/m(2) 24hr div: day 1-5 and DEX 5 mg/m(2) 1hr div twice a day, day 1-5). Intrathecal chemotherapy (MTX, HDC and CA) was administered twice in this phase in both arms. In the HR group two doses of pirarubicin (25 mg/m (2)) were added and this phase was repeated after re-induction therapy. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test. Between April 2002 and May 2008, 1252 children with newly diagnosed ALL were enrolled to JACLS ALL-02 study. Among these, the number of patients with SR and HR were 388 (arm A=200, arm B=188) and 513 (arm A=251, arm B=257, not assigned=5), respectively. The characteristics of eligible patients between arm A and B were not different statistically (Table). Among SR patients, the 4y-EFS were 89.7 +/- 5.2% and 93.4 +/- 4.7% in Arm A and B, respectively (p=0.70). Among HR patients, the 4y-EFS were 89.7 +/- 5.2% and 90.5 +/- 4.4% in Arm A and B, respectively (p=0.97). Although hematological toxicity was not different, in the next phase (sanctuary phase) following Arm A consolidation therapy the frequencies of hematological grade 3 and 4 adverse effects were higher in both SR and HR (SR: p Consolidation therapy with continuous infusion of CA (Arm B) was not superior to conventional CA regimen. Disclosures No relevant conflicts of interest to declare.

Introduction: Thromboembolism is a serious complication associated with ALL. The use of central venous catheter and treatment protocols involving corticosteroids and L-Asp is assumed as important thrombogenic factors at the induction phase. In particular, L-Asp has profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In this study, we hypothesized that change of coagulation and fibrinolytic function contributes to hyper-coagulation condition during the induction phase with L-Asp. In order to clarify this, we evaluated the dynamic change in coagulation and fibrinolysis by simultaneous measurement of both thrombin and plasmin generation assay (T/P-GA). Patients: Twenty-seven pediatric patients with newly diagnosed ALL were enrolled from Aug. 2014 to Oct. 2015 at 3 hospitals in Japan. All cases had no thrombotic predisposition. Eighteen cases (66.7%) (BCP-ALL; n=17, T-ALL; n=1) received Berlin-Frankfürt-Münster (BFM)-95 oriented induction therapy included prednisolone (and dexamethasone for T-ALL), vincristine, daunorubicin, and E.coli L-Asp (a total of 8 doses of 5,000 U/m2). The others (BCP-ALL; n=8, T-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS) ALL02 oriented induction therapy included prednisolone, dexamethasone, vincristine, daunorubicin, cyclophosphamide and E.coli L-Asp (a total of 6 doses of 6,000 U/m2). Methods: The individual hemostatic parameters were monitored by fibrinogen (Fbg), FDP, AT, TAT and PIC. Additionally, the global functions of coagulation and fibrinolysis were evaluated using T/P-GA established by our group [Matsumoto et al. TH 2013]. This assay was initiated by the addition of a mixture of optimized concentrations of tissue factor and tissue-type plasminogen activator. Thrombin and plasmin generation were monitored simultaneously using individual fluorescent substrates in separate microtiter wells. Standard curves were set using purified alpha-thrombin and plasmin. Patients' plasmas were collected at the following points, T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post-induction phase. Endogenous potentials of thrombin generation (T-EP) for coagulant activity and plasmin peak levels (P-Peak) of plasmin generation for fibrinolytic activity were selected as parameters for evaluation in this study. A ratio of T-EP and P-Peak of patients' plasmas to those of control normal plasma were calculated. Results: All cases obtained first remission, and none of them developed coagulopathy. Six cases received FFP transfusion for low Fbg level, whilst 21 cases received AT supplement for low AT level. Fbg showed a median of 170, 99.0, 99.0 and 328 mg/dl at T0, T1, T2 and T3, respectively, whilst the other individual parameters showed relatively unchanged. T-EP revealed a median of 1,126, 1,059, 1,175, 1,343 and 1,132 nM, whilst P-Peak showed a median of 6.67, 4.54, 4.12, 5.50 and 5.77 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-Peak ratios (Fig. 1). The most significant difference in both ratios demonstrated a median of 1.5-fold (range, 1.0 to 2.6) at T2, consistent with the lowest Fbg levels. The FFP transfusion group showed significantly lower T-EP ratios than non-transfusion group at T1 (a median of 0.87 vs. 1.01, P=0.041) and T2 (a median of 0.96 vs. 1.07, P=0.009), whilst P-Peak ratios revealed no significant changes. The AT supplement group showed no significant changes of both ratios. Conclusion: The results from decreased Fbg and unchanged FDP might reveal the hepatic synthesis disorder of Fbg, whilst the results from T/P-GA showed that their hemostatic dynamics appear likely to be thrombotic tendency, since their coagulation state was hyper-coagulation and anti-fibrinolysis at post-phase of L-Asp. These results suggest that the impaired balance of coagulation and fibrinolysis due to L-Asp therapy might play an important role of a thrombotic complication at induction phase. On the other hand both conventional FFP transfusion and AT supplement therapy might not dramatically repair this unbalance state. A further research would be required to examine the role of coagulant and fibrinolytic function using T/P-GA in the pathogenesis of coagulopathy associated with L-Asp therapy in order to establish the optimal supportive therapy. Figure 1 The Changes of Both T-EP and P-Peak Ratios Figure 1. The Changes of Both T-EP and P-Peak Ratios Disclosures Nogami: F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding.