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Background Lymphoblastic lymphoma (LBL) and acute lymphoblastic leukemia (ALL) are categorized as the same entity under precursor lymphoid neoplasms in the World Health Organization classification. However, compared to B-cell ALL, the molecular genetic makeup of B-cell LBL remains to be understood, mainly due to its rarity. We performed whole exome sequencing (WES) on seven patients with TCF3-PBX1-positive B-cell LBL. Methods WES was performed using DNA extracted from tumor specimens and paired blood samples at remission for six patients, and tumor-only analysis was performed for one patient whose remission sample was not available. For one patient, a relapsed sample was also analyzed. Results KMT2D variants and 6q LOH were found as recurrent alterations. Somatic variants of KMT2D were identified in three of the seven patients. Of note, the two patients with heterozygous nonsense variant of KMT2D were at stage III, without bone marrow infiltration. 6q LOH was also identified in two others, out of the seven patients. The common 6q deleted region of the two patients ranged from 6q12 to 6q16.3. Both patients had bone marrow infiltration. Analysis of recurrent case also revealed that the relapsed clone might be derived from a minor clone of the bone marrow at diagnosis. Conclusion In this study, through WES for seven patients with TCF3-PBX1-positive B-LBL, we identified KMT2D mutations and 6q LOH as recurrent alterations. In order to elucidate the relationship between these recurrent alterations and disease specificity or outcomes, further studies comparing with TCF3-PBX1-positive B-ALL are required.

Background/aim Tacrolimus is an essential immunosuppressant for successful allogeneic haematopoietic stem cell transplantation (Allo-HSCT). This study aimed to examine the change in the blood concentration of tacrolimus during switching from intravenous to oral administration in allo-HSCT for paediatric cancer to predict the optimal dosage. Patients and methods We retrospectively examined the medical records of 63 patients who received allo-HSCT and were administered tacrolimus. To compare bioavailability among different dose ranges, the blood concentration was divided by the dose (C/D). Results Thirty-nine patients (age range=children 1-15 years, adults 17-67 years) were switched to oral administration of tacrolimus. The C/D after switching was significantly lower in children than in adults (p=0.039). There was a strong positive correlation between age and C/D in children, whereas no correlation was observed in adults. Conclusion In paediatric cancer patients, switching tacrolimus administration route may result in reduced blood concentrations. This tendency is more prominent in younger children.

Idiopathic pulmonary hemosiderosis (IPH) is a rare and life-threatening disorder. Early diagnosis and appropriate management are essential for their better prognosis and patients’ quality of life (QOL). It is considered that Down syndrome patients with IPH have a worse prognosis compared to other IPH cases. A 2-year-old girl with Down syndrome received the diagnosis of IPH after two episodes of massive pulmonary hemorrhage requiring assist ventilation, who suffered from recurrent IPH during tapering period of oral corticosteroid, started liposteroid therapy. We report here a case of successful control of recurrent IPH and improved QOL enormously with tapering dose of corticosteroid after starting liposteroid therapy.

Unrelated cord blood transplantation in aplastic anemia: is anti-thymocyte globulin indispensable for conditioning?

This study reviewed the clinical characteristics of 112 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients with TCF3-PBX1 fusion treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol (n = 82) and Children's Cancer and Leukemia Study Group (CCLSG) ALL 2004 protocol (n = 30). The 3-year event-free survival (EFS) and overall survival (OS) rates were 85.4 ± 3.9% and 89.0 ± 3.5% in JACLS cohort, and the 5-year EFS and OS were 82.8 ± 7.0% and 86.3 ± 6.4% in CCLSG cohort, respectively, which are comparable to those reported in western countries. Conventional prognostic factors such as age at onset, initial white blood cell count, and National Cancer Institute risk have also no impact on OS in both cohorts. Surprisingly, the pattern of relapse in JACLS cohort, 9 of 82 patients, was unique: eight of nine patients relapsed during the maintenance phase and one patient had primary induction failure. However, bone marrow status and assessment of minimal residual disease on days 15 and 33 did not identify those patients. Interestingly, the two patients with IKZF1 deletion eventually relapsed in JACLS cohort, as did one patient in CCLSG cohort. International collaborative study of larger cohort is warranted to clarify the impact of the IKZF1 deletion on the poor outcome of TCF3-PBX1 positive BCP-ALL.

Objective: Anti-TNF-α therapy is an effective therapy for Crohn’s disease (CD). Thus anti-TNF-α therapy may alter CD64 expression, one of the Fc receptors for IgG, in CD. We investigated CD64 expression on neutrophils before and after treatment with anti-TNF-α therapy in CD. Methods: A total of 11 patients with active CD treated with anti-TNFα antibodies were enrolled. The severity of CD was assessed with the CD activity index (CDAI). Peripheral venous blood was obtained before and 2 weeks after the initial administration of anti-TNF-α antibody. CD64 expression on neutrophils was measured by FACS analysis of whole blood samples. Results: CDAI, C-reactive protein value and CD64 expression decreased significantly after anti-TNF-α therapy. Both prior to and after anti-TNF treatment, there was a significant and positive correlation between CD64 expression and CDAI or CRP. Similarly, there was a significant and negative correlation between CD64 and albumin value. Conclusion: Anti-TNF-α therapy suppresses CD64 expression of neutrophils, which may account for the mechanism underlying the efficacy of the medication in CD.

t(17;19)-acute lymphoblastic leukemia (ALL) shows extremely poor prognosis. E2A-HLF derived from t(17;19) blocks apoptosis induced by the intrinsic mitochondrial pathway and has a central role in leukemogenesis and chemoresistance. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed on cytotoxic T cells and natural killer cells and binds with death receptors (DR4/DR5), inducing apoptosis by dual activation of intrinsic and extrinsic pathways, and TRAIL mediates the graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT). We found that cell lines and patients' samples of t(17;19)-ALL expressed death receptors for TRAIL, and recombinant soluble TRAIL immediately induced apoptosis into t(17;19)-ALL cell lines. E2A-HLF induced gene expression of DR4/DR5, which was dependent on the DNA-binding and transactivation activities of E2A-HLF through the 5' upstream region of the start site at least in the DR4 gene. Introduction of E2A-HLF into non-t(17;19)-ALL cell line upregulated DR4 and DR5 expression, and sensitized to proapoptotic activity of recombinant soluble TRAIL. Finally, a newly diagnosed t(17;19)-ALL patient underwent allo-SCT immediately after induction of first complete remission, and the patient has survived without relapse for over 3-1/2 years after allo-SCT. These findings suggest that E2A-HLF sensitizes t(17;19)-ALL to the GVL effect by upregulating death receptors for TRAIL.

Introduction Hematopoietic stem cell transplantation (HSCT) is one of the promising treatment strategies for children with refractory Langerhans cell histiocytosis (LCH), because of its immunomodulatory effects.Efficacy and indication of HSCT has been still undetermined. We analyzed the outcomes of HSCT in children with refractory LCH registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation. Patients and methods Between 1996 and 2014, 30 patients Results Patients' characteristics The male/female ratio was 18/12. Ages of onset of LCH and at BMT were median 9 months (range 3-23 months) and median 1 years (range 0-11 years), respectively. At diagnosis of LCH, 19 patients were positive for risk organ involvement, 6 were negative. Eleven patients underwent HSCT using myeloablative conditioning (MAC) regimen, whereas 19 patients reduced intensity conditioning (RIC) regimen. Eight patients received total body irradiation (TBI) > 8Gy (10-12Gy) regimen, 3 received full dose busulfan (BU: 16mg/kg po or 17.6mg/kg iv) regimen. Seven received fludarabine (FLU: 125-180mg/m2) + melphalan (MEL: 70-180mg/m2) without TBI and 9 received FLU(120-180mg/m2)+MEL (140-180mg/m2) with low dose irradiation (2-5.1Gy) and the remaining 3 received other RIC regimens. Cyclosporine was used in 13 patients and tacrolimus was used in 16 patients for graft versus host disease (GVHD) prophylaxis. Donor sources were related donor in 9 patients and unrelated in 21 patients (cord blood 19 and bone marrow 2). Four related donors were father or mother and 3 of them were haplo-identical. In these patients, induction therapy at onset achieved a good (5/25), partial (8/25), no (4/25) and progressive (8/25) diseases. In regard to disease status at HSCT, recipients with no active diseae (NAD) were 4/25, active disease-regression (AD-r) 2/25, active disease-stable (AD-s) 4/25 and progressive (AD-p) 15/25. Eight patients received 2-chlorodeoxyadenosine (2-CdA), including 4 patients who received the combination of 2-CdA and high dose Ara-C before HSCT as salvage therapy. At HSCT, 15/23 (65%) patients were in primary induction failure and 8/23 (35%) experienced first or additional relapse, respectively. Transplantation outcomes Neutrophil recovery was observed in 24 patients and the median time to engraftment was 21 days. Platelets engraftment was observed in 17 patients and the median time to engraftment was 52 days. Acute GVHD of grade II - IV, chronic GVHD (cGVHD) were observed in 6 and 4 patients, respectively. Extensive cGVHD was observed in 3 patients who received MAC regimens (p= 0.079). Relapse after HSCT were observed 1 patient in RIC regimens and 2 patients in MAC (P=0.613). With follow-up of median 433 days (range 9-5307days) after HSCT, 17/30 (57%) patients are alive and 13 died. Death occurred within 3 months after HSCT in 8/13. The overall survival (OS) was not different between RIC and MAC (56.8% vs 63.6%, p=0.843). In regard to the correlation of disease status at HSCT and outcome, 6 patients with NAD/ AD-r had better outcome than 19 with AD-s/ AD-p (5-year OS 100% vs 52.1%, p=0.035). The 5-year FFS of the 16 patients who received FLU+ MEL based regimen were 68.8%, and were marginally better than those of the 14 patients who were conditioned with other regimens (68.8% (95%CI: 46.0-91.5) vs 42.9% (95%CI: 16.9-68.8), P=0.209). Discussion In conclusions, of 30 HSCT-recipients for refractory LCH, 17/30 (57%) are alive while post-transplant death occurred in 13/30 (43%). Our study showed that in regard to the correlation of disease status at HSCT and outcome, 6 patients with NAD/ AD-r had better outcome than 19 with AD-s/ AD-p (5-year OS 100% vs 52.1%, p=0.035). Novel bridging measures, such as targeted inhibition of the MAPK pathway, are required to stabilize the disease activity before HSCT. Disclosures No relevant conflicts of interest to declare.

免疫抑制療法に反応しない重症血球貪食性リンパ球組織球症の小児2例に持続血液透析濾過を施行した。 症例1は6歳の男児。若年性特発性関節炎の経過中に本疾患が再燃した。ステロイドパルス療法で解熱せず，低血圧，腎機能障害をきたした。 症例2は14歳の女子。EBウイルス感染による本疾患が再燃し，ステロイドパルス療法，シクロスポリン，エトポシドなどの投与でも全身状態は改善せず，低血圧，腎機能障害，播種性血管内凝固を合併した。 両症例とも持続血液透析濾過を開始後に症状と検査値が改善した。持続血液透析濾過は，低血圧，腎機能障害，播種性血管内凝固などを合併した重症血球貪食性リンパ球組織球症に対して有効であった。

Transient leukemia (TL) has been observed in approximately 10% of newborn infants with Down syndrome (DS). Although treatment with cytarabine is effective in high-risk TL cases, approximately 20% of severe patients still suffer early death. In this study, we demonstrate abundant KIT expression in all 13 patients with GATA1 mutations, although no significant difference in expression levels was observed between TL and acute myeloid leukemia. Stem cell factor (SCF) stimulated the proliferation of the TL cells from five patients and treatment with the tyrosine kinase inhibitor imatinib suppressed the proliferation effectively in vitro. To investigate the signal cascade, we established the first SCF-dependent, DS-related acute megakaryoblastic leukemia cell line, KPAM1. Withdrawal of SCF or treatment with imatinib induced apoptosis of KPAM1 cells. SCF activated the RAS/MAPK and PI3K/AKT pathways, followed by downregulation of the pro-apoptotic factor BIM and upregulation of the anti-apoptotic factor MCL1. Although we found novel missense mutations of KIT in 2 of 14 TL patients, neither mutation led to KIT activation and neither reduced the cytotoxic effects of imatinib. These results suggest the essential role of SCF/KIT signaling in the proliferation of DS-related leukemia and the possibility of therapeutic benefits of imatinib for TL patients.

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS-AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient-derived JAK3 mutation (JAK3(I87T)), as well as two novel JAK3 mutations (JAK3(Q501H) and JAK3(R657Q)) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof-of-principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS-AMKL patients carrying activating JAK3 mutations.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (20 x 10(9)/l), 15 showed hemoglobinor =8 g/dl and 14 showed platelet countor =100 x 10(9)/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

Several approaches for the detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) have shown the importance of determining the level of MRD precisely. In the present study, we tested a new real-time quantitative polymerase chain reaction (RQ-PCR) strategy with minor groove binder (MGB) technology for immunoglobulin heavy chain gene rearrangements by positioning a MGB probe at the germline JH segments and one of the primers at the downstream introns in combination with an allele-specific oligonucleotide (ASO) primer complementary to the VH-DH or DH-JH junctional region. A MGB probe forms extremely stable duplexes with single-stranded DNA targets, allowing the use of shorter probes for hybridization-based assays. Therefore, it shows positional flexibility. We have designed two novel consensus MGB JH germline probes for analyzing all of the germline rearrangements registered in the V BASE database, and demonstrated that the MRD was detectable with the probes in 17 cases of childhood ALL. The actual copy number for the targets and dynamic changes before and after treatment were almost identical between the JH MGB probe and conventional non-MGB probes in each patient. MGB technology will undoubtedly contribute to MRD-PCR studies of childhood ALL.

Incidence rates of Wilms' tumor (WT) markedly differ in East Asian and Caucasian children. In the present study, we examined WT1 deletions/mutations and loss of heterozygosity (LOH) on 11p and 11q in a large number of WTs and compared our findings with those from 4 series of Caucasian WTs. Incidence rates of the subtle WT1 mutation in 3 of the 5 series of sporadic and unilateral WTs including ours were 4.3–6.2% and similar. However, gross homozygous WT1 deletion was more frequent in our series than in some others. In addition, our series tended to show a higher incidence of LOH limited to 11p13 and a lower incidence of LOH including 11p15 than the Caucasian one. These findings indicate some genetic differences in WT between the 2 regions. One of the 4 Caucasian series reported a correlation of germinal WT1 mutation with the predominantly stromal histology. The present study not only confirms the correlation of germinal WT1 deletion/mutation with predominant stromal histology but also establishes a correlation with somatic WT1 deletion/mutations with predominant stromal histology. While WTs with WT1 abnormalities usually showed pseudodiploidy and predominant stromal histology, those without WT1 abnormalities showed various chromosome numbers and histologic subtypes. © 2001 Wiley-Liss, Inc.

The chromosomal inversion (16)(p13q22), which is associated with the M4-eosinophilia subtype of human acute myeloid leukemia, causes the fusion of two distinct genes. The polypeptide encoded by the chimeric gene, PEBP2p/CBFp-SMMHC, retains the ability to interact with, and dominantly interfere with the function of proteins possessing the Runt homology domain. The Runt protein homologs constitute the DNA binding subunit of the PEBP2/CBF transcription factor. We examined the subcellular localization of PEBP2beta/CBFbeta-SMMHC, as well as that of Runt protein homologs in leukemic cells carrying inversion 16 by immunoblot analysis. A significant amount of the PEBPbeta/CBFbeta-SMMHC protein was recovered from the nuclear fraction along with the Runt protein homologs. Furthermore, some of both polypeptides was retained in the DNA pellet that represents the material remaining after extraction of nuclear fraction with high salt. These observations suggest that the so-called dominant interfering effect of PEBPbeta/CBFbeta-SMMHC on PEBP2/CBF occurs inside the nucleus. In addition, we could detect PEBP2beta/CBFbeta-SMMHC in the cytoplasmic membrane fraction as well. The function of this membrane-located PEBP2beta/CBFbeta-SMMHC, if any, appears to be unrelated to that of Runt protein homologs.

Background The primary object of this study was to identify treatment-related variables that may predict relapse of acute myelogenous leukemia (AML) after autologous peripheral blood stem cell transplantation (PBSCT), which will be critical for the development of a suitable protocol for wider application. Methods A total of 28 children (age 0-18 years) with AML underwent PBSCT and have had a minimum follow-up of 25 months; including 24 patients in their first complete remission (CR) and four in their second CR. The patients were divided into two cohorts according to the study phase: 16 patients were treated in an early phase pilot study and 12 patients in their first CR were treated in a prospective trial. Fifteen of the first-CR patients had any of the cited high-risk features of high WBC count (>100x10(9)/l; n = 5), FAB M0/M4/M5/M7 subtypes (n = 11) or delayed achievement of CR (n = 9). Except in one patient, cytoreductive regimens did not include total body irradiation (TBI). Results After PBSCT, one patient died of veno-occulsive disease (VOD) and another patient relapsed early on day 43, but the remaining patients showed engraftment. Leukemic relapse was observed 1-29 months after PBSCT (median, 8 months); in all of the 4 children treated in their second CR and in 11 of the 24 patients (46%) treated in their first CR. The remaining patients have been disease-free for 24 to 97 months (median, 53 months). Using a multivariate analysis, the timing of apheresis was the most significant prognostic factor for those treated in their first CR (p = 0.03); 12 of the 16 patients whose PBSC were collected beyond 2.5 months of CR continue to remain in CR, while seven of the eight patients whose PBSC were harvested within 2.5 months of CR relapsed. Conclusion Although the small number of patients studied does not allow firm conclusions to be drawn regarding the relative effectiveness of this therapy, the results do suggest the feasibility of further studies of PBSCT for the treatment of childhood AML with high-risk features including the assessment of minimum residual disease.

There exists limited information about the usefulness of hemopoietic stem cell transplantation (HSCT) for the treatment of patients with refractory Langerhans cell histiocytosis (LCH). We report here four Japanese pediatric patients with multisystem LCH disease who underwent HSCT between 1994 and 1997. Two of the four patients are doing well without any relapse. However, neither of them shows improved sequelae 3 to 4 years after allogeneic HSCT, although the graft was rejected in one of the cases. The remaining two patients died of septic shock. A review of the literature of 11 patients revealed four fatalities after the use of HSCT in the treatment of LCH. Three of these were due to active LCH and three deaths occurred within 2 months after HSCT. To establish the usefulness of HSCT for refractory LCH, further studies are required.

This study reviewed the clinical characteristics of 112 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients with TCF3-PBX1 fusion treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol (n = 82) and Children's Cancer and Leukemia Study Group (CCLSG) ALL 2004 protocol (n = 30). The 3-year event-free survival (EFS) and overall survival (OS) rates were 85.4 ± 3.9% and 89.0 ± 3.5% in JACLS cohort, and the 5-year EFS and OS were 82.8 ± 7.0% and 86.3 ± 6.4% in CCLSG cohort, respectively, which are comparable to those reported in western countries. Conventional prognostic factors such as age at onset, initial white blood cell count, and National Cancer Institute risk have also no impact on OS in both cohorts. Surprisingly, the pattern of relapse in JACLS cohort, 9 of 82 patients, was unique: eight of nine patients relapsed during the maintenance phase and one patient had primary induction failure. However, bone marrow status and assessment of minimal residual disease on days 15 and 33 did not identify those patients. Interestingly, the two patients with IKZF1 deletion eventually relapsed in JACLS cohort, as did one patient in CCLSG cohort. International collaborative study of larger cohort is warranted to clarify the impact of the IKZF1 deletion on the poor outcome of TCF3-PBX1 positive BCP-ALL.

Children with Down syndrome have an increased incidence of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia. The majority of these cases harbor somatic mutations in the GATA1 gene, which results in the loss of full-length GATA1. Only a truncated isoform of GATA1 that lacks the N-terminal 83 amino acids (GATA1-S) remains. We found through genetic studies of 106 patients with TAM that internally deleted GATA1 proteins (GATA1-IDs) lacking amino acid residues 77-119 or 74-88 (created by splicing mutations) contributed to the genesis of TAM in 6 patients. Analyses of GATA1-deficient embryonic megakaryocytic progenitors revealed that the GATA1 function in growth restriction was disrupted in GATA1-IDs. In contrast, GATA1-S promoted megakaryocyte proliferation more profoundly than that induced by GATA1 deficiency. These results indicate that the internally deleted regions play important roles in megakaryocyte proliferation and that perturbation of this mechanism is involved in the pathogenesis of TAM.

Introduction Hematopoietic stem cell transplantation (HSCT) from an HLA-matched related donor is a first-line treatment for children with severe aplastic anemia (SAA). HSCT from an HLA-matched unrelated donor (MUD) is indicated as a salvage treatment for non-responders to immunosuppressive therapy (IST).Because unrelated cord blood transplantation (UCBT) has been considered as an experimental therapy, information of UCBT in patients with SAA is scarce, especially in pediatric patients. We analyzed the outcomes of UCBT in children with AA registered in the Transplant Registry Unified Management Program (TRUMP) conducted by the Japanese Society for Hematopoietic Cell Transplantation. Patients and Methods Between 1998 and 2013, 27 patients Results Patients' characteristics Among the 27 patients, 10 patients underwent UCBT during 1998 - 2005 and 17 patients underwent UCBT during 2006 - 2013. The median age at the time of UCBT was 6 years (range, 0 - 14). The male/female ratio was 12/15. The median interval between the diagnosis of AA and CBT was 212 days (range, 39 - 2,982). Sixteen patients received antithymocyte globulin (ATG) as immunosuppressive therapy (IST) before UCBT. The median total nucleated cell number and CD34 positive cell number at cryopreservation were 3.99 x 107/kg (range, 0.09 - 16.11 x 107/kg) and 1.31 x 105/kg (range, 0.39 - 6.17 x 105/kg), respectively. Ten patients received fludarabine (FLU) + cyclophosphamide (CY) + irradiation ± ATG. Six received FLU + CY + ATG. Four received FLU + melphalan (MEL) + irradiation and the remaining 7 received other various conditioning regimens. Cyclosporine was used in 9 patients and tacrolimus was used in 17 patients for graft-versus-host disease (GVHD) prophylaxis. Transplantation outcomes Neutrophil recovery was observed in 19 patients and the median time to engraftment was 20 days. Platelets engraftment was observed in 17 patients and the median time to engraftment was 50 days. One patient experienced secondary GF. Sustained engraftment was observed in 18 patients. Grade II - IV acute GVHD, limited chronic GVHD, and extensive chronic GVHD were observed in 6 (26%), 2 (10%), and 2 patients (10%), respectively. With median follow-up times of 18 months after CBT, 19 of 27 patients were alive. Causes of death included bacterial/fungus infections in 3, veno-occulusive disease (VOD) in 2, EBV-LPD, hematologic malignancy, and secondary GF in each 1 patient. The 5-year overall and failure-free survival (OS, FFS) of all patients were 69.5±9.0% and 59.3±9.5%, respectively. The 5-year OS and FFS of 17 patients who underwent UCBT after 2006 was 93.8±6.1% and 76.0±10.5%, respectively. Primary GF was observed in 8 patients, including 3 who underwent UCBT during 2006 - 2013. Two patients, who underwent UCBT after 2006, received FLU + CY + ATG without irradiation and remaining one patient received CY + ATG + total body irradiation (TBI). We screened anti-HLA antibodies in 8 patients but none of them were positive. The conditioning regimens including ATG exhibited a significantly worse FFS than those without ATG (33.3±12.2 % vs 91.7±8.0 %, P=0.004). On the other hand, the conditioning regimens including irradiation exhibited a better FFS than those without irradiation (75.0±9.7 % vs 14.3±13.2 % , P=0.01). The 5 year FFS of FLU + MEL + irradiation regimen (n=4) and FLU + CY + irradiation regimen (n=10) was 100% and 90%±9.5%, respectively. Univariate analysis resulted in no statistical significant differences in OS and FFS in HLA disparities, total nucleated cell number and CD34 positive cell number at cryopreservation, and methods of GVHD prophylaxis. One patient developed secondary malignancy. Discussion Our study showed that a reduced conditioning regimen with Flu, MEL/CY and low-dose irradiation may be an optimal regimen for SAA children receiving UCBT. An important finding is that ATG has no benefit in this setting. A prospective study with larger number of patients is warranted to confirm our promising results. Disclosures Kojima: SANOFI: Honoraria, Research Funding.

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.

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

Specific induction of CD33 expression by E2A–HLF: the first evidence for aberrant myeloid antigen expression in ALL by a fusion transcription factor

In pro-B cell acute lymphoblastic leukemia (ALL), expression of the E2A-HLF fusion gene as a result of t(17;19)(q22;p13) is associated with poor prognosis, hypercalcemia, and hemorrhagic complications. We previously reported that the E2A-HLF fusion protein protects interleukin-3 (IL-3)–dependent lymphoid cells from apoptosis caused by cytokine starvation. Here, we report that annexin II, a surface phospholipid-binding protein and one of the proposed causes of the hemorrhagic complications of acute promyelocytic leukemia (APL), is also implicated in t(17;19)+ ALL. Annexin II was expressed at high levels in APL cells and in each of 4 t(17;19)+ leukemia cell lines, and annexin II expression was induced by enforced expression of E2A-HLF in leukemia cells. In IL-3–dependent cells, we found that annexin II expression was regulated by IL-3 mainly by Ras pathways, including Ras/phosphatidylinositol 3-kinase pathways. Moreover, E2A-HLF increased annexin II expression in IL-3–dependent cells in the absence of the cytokine. These findings indicate that E2A-HLF induces annexin II by substituting for cytokines that activate downstream pathways of Ras. (Blood. 2004;103:3185-3191)

Regulatory T (Treg) cells are a subset of CD4+ T cells that play key roles in the maintenance of immunologic self-tolerance and negative control of immune responses. Treg cells are defined based on expression of CD4, CD25, and transcription factor forkhead box P3 (FoxP3). FoxP3 controls the development and function of Treg cells and can be used as a reliable marker of Treg cells, however, its intracellular localization prohibits its use for the isolation of viable Treg cells. Most FoxP3+ Treg cells express low levels of CD127 in adult peripheral blood, however, the expression of CD127 in cord blood (CB) Treg cells of preterm and term infants at each gestational age remains unknown. Being that CD127 is a cell surface marker, it enables the isolation of viable Treg cells by flow cytometric sorting for further analysis. We analyzed CD4+CD25+FoxP3+ and CD4+CD25+CD127− T cells in preterm and term CB at various gestational ages. CB samples were obtained at Iwate Medical University hospital from preterm and term neonates who had no hereditary disorders, hematologic abnormalities, or immunodeficiency diseases. Of 103 total samples, 36 were obtained from preterm (24–36 gestational weeks) neonates and 67 were obtained from term (37–41 gestational weeks) neonates. Out of 36 preterm infants, 9 were diagnosed with chorioamnionitis histologically. None of the mothers involved in the study had any specific underlying diseases. CB mononuclear cells were isolated and analyzed by FACSCalibur flow cytometer and CellQuestPro software (BD Biosciences). No differences were observed in the proportions of CD25+FoxP3+ (median 4.5%) and CD25+CD127− (median 5.0%) cells in the CD4+ T-cell population. A strong correlation was found between the proportions of CD25+FoxP3+ and CD25+CD127− cells in the CD4+ T-cell population (r=0.96). The proportion of CD25+FoxP3+ cells in the CD4+ T-cell population in the CB of neonates at 24-26, 27-29, and 30-32 gestational weeks was significantly higher than in the CB of neonates at 37-38 and 39-41 gestational weeks. The proportion of CD25+CD127− cells in the CD4+ T-cell population in the CB of neonates at 24-26, 27-29, and 30-32 gestational weeks was also higher than in the CB of neonates at 37-38 and 39-41 gestational weeks. The absolute number of both CD4+CD25+FoxP3+ and CD4+CD25+CD127− T cells did not differ between the CB of preterm and term neonates. Absolute lymphocyte counts in preterm CB were significantly lower than term infants (P We showed that CD127 is preferable to FoxP3 as a marker for identifying Treg cells in CB of various gestational ages and would be useful for separating Treg cells by flow cytometry for therapeutic applications. The proportions of Treg cells in the CB of preterm infants were significantly higher than in the CB of term infants. The high Treg proportion during early gestation suggests that fetal naive T cells have a high propensity to differentiate into Treg cells in response to antigenic stimulation, such as by maternal alloantigens. The functional implications of the high proportion of Treg cells in preterm CB are unknown but may be important for the suppression of undesirable alloimmune reactions to maternal derivatives in utero and the maintenance of tolerance during pregnancy. Disclosures No relevant conflicts of interest to declare.

Background: In pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), aberrant JAK-STAT signaling is one of the key leukemogenic mechanisms. Although activating mutations of JAK2 are found in high-risk BCP-ALL patients in western countries, such mutations are rare in Japanese cohorts, led to speculation of other factors contributing abnormal activation of JAK-STAT pathway. The adaptor protein LNK (SH2B3) is one of the negative regulator of JAK-STAT signaling, and its loss of function mutations have been identified in myeloproliferative neoplasms and high-risk BCP-ALL. In addition, the loss of function of LNK has been demonstrated to increase proliferation of B cells in vivo. Based on these findings, we conducted genetic analysis to determine the prognostic impact of LNKin BCP-ALL, and functional analysis to investigate its possible mechanisms. Methods: For genetic analysis, we evaluated diagnostic bone marrow or peripheral blood samples of 164 pediatric BCP-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol along with peripheral blood samples from 9 healthy volunteers. The LNK expression level was determined by qRT-PCR. Deletion of IKZF1 was determined by MLPA, and direct sequencing was employed to detect LNK mutations in patients with IKZF1 deletion. For functional analysis, thrombopoietin (TPO)-dependent BaF3-MPL cells expressing LNK (BaF3-MPL-LNK) was established by retroviral transduction to investigate if LNKexpression levels impact on drug sensitivity for prednisolone (PSL), doxorubicin (DOX) or vincristine (VCR). For growth assay, BaF3-MPL cells with or without LNK were cultured for four days in the presence of TPO and the viable cell number was counted. For drug sensitivity test, BaF3-MPL cells with or without LNK were treated with each drug for 48 hours in the presence of TPO, then the IC50 was calculated. Phospho-specific flow cytometory (Phos-Flow) was performed to measure JAK-STAT activation. Results: The LNK expression levels in pediatric BCP-ALL patients’ samples were significantly lower than in samples from healthy donors (P < 0.01). When analyzing all 164 cases, the expression level of LNK was decreased in relapsed patients but there was no statistical significance (P = 0.067). IKZF1 deletion was found in 25 (15% of all) patients, and in these patients, LNK expression level dose not relate to relapse (P = 0.39). Intriguingly, when patients with known high-risk factor (i.e., IKZF1 deletion or poor response to PSL) were excluded, the expression level of LNK was significantly higher in non-relapsed patients (P < 0.05). In functional assay, we observed inhibition of TPO dependent growth of BaF3-MPL cells by expression of LNK (P < 0.01), consistent with previous reports. Phos-Flow analysis revealed that LNK expression suppressed TPO–induced phosphorylation of STAT5 in BaF3-MPL cells. In drug sensitivity test, we found that IC50 of PSL and DOX were substantially lower in BaF3-MPL-LNK cells from in BaF3-MPL-mock cells (0.70 vs 3.93 nM, P < 0.01 and 0.61 vs 1.14 nM, P < 0.05, respectively). Decline in IC50 of VCR by LNK expression was not statistically significant (1.38 vs 2.45 nM, P = 0.056). We next compared the impact of LNK with Ruxolitinib (RUX), a potent synthesized JAK2 inhibitor. The diminution in IC50 of PSL in BaF3-MPL-mock cells treated with RUX (50 nM) was comparable of that in BaF3-MPL-LNK cells (0.31 nM, combination index (CI) = 0.39), consistent with our hypothesis that LNK is working as a JAK2 inhibitor. Since we identified two amino-acid substitutions in N-terminal proline-rich dimerization domain (R139H) and PH domain (P242S), we also examined their function. Transductions of these genes in BaF3-MPL cells, however, did not alter cell growth, suggesting they are single nucleotide variants. Discussions: Our findings that high LNK expression is associated with low relapse rate in intermediate risk (IKZF1 intact, good PSL response) patients indicate potential of LNK to restrain relapse in such patients, presumably by suppressing JAK-STAT signaling. Since we proved the impact of LNK expression to improve sensitivity of PSL in vitro which was comparable to RUX, RUX could compensate lack of internal LNK expression to induce cell death of BCP-ALL cells. Collectively, targeting JAK-STAT pathway will be promising therapeutic option for intermediate risk BCP-ALL patients with low expression level of LNK. Disclosures No relevant conflicts of interest to declare.

We studied retrospectively the clinical feasibility of minimal residual disease (MRD) monitoring by reverse transcription-polymerase chain reaction (RT-PCR) detecting the PML/retinoic acid receptor alpha (RARalpha) chimeric gene in children with acute promyelocytic leukemia (APL). MRD monitoring of APL was performed with standard and nested RT-PCR for PML/RARalpha gene, the sensitivity of which was 1 leukemic cell in 10(3)-10(4) and 1 in 10(4)-10(5) cells, respectively. Patients were nine children with APL (average age: 8.3 year; average period of follow-up: 69.2 months) who, after achieving remission with all-trans retinoic acid (ATRA), received treatment either with multidrug chemotherapy or with a combination of chemotherapy and ATRA. Out of six patients treated with multidrug-combined chemotherapy, two patients exhibited PCR positivity after six months of post- remission therapy, which shifted from the detectable range of the nested PCR to that of the standard PCR. These two patients subsequently relapsed and, together with two of the other patients receiving multidrug-combined chemotherapy, underwent allogeneic bone marrow transplantation. No MRD was detected in these patients after transplantation. In the remaining three patients who underwent cyclic treatment with alternative chemotherapy and ATRA, two showed positive RT-PCR at the nested or standard level, respectively, after six months of combined therapy, and one of them relapsed. Overall, three of four patients with MRD detected in post-remission period ultimately relapsed, while all of five patients without detectable MRD had a good prognosis. These findings suggest that impending relapse may be predicted by the detection of preceding PCR positivity with an increasing quantity of the PML/RARalpha mRNA that appears beyond six months of post-remission chemotherapy, with or without combined ATRA therapy.

Abstract 4685 The high affinity Fc gamma receptor I (Fc gamma RI, cluster of differentiation 64 [CD64]) is normally expressed to a very low extent by neutrophils; during bacterial infections, the expression has been shown to increase in adults and newborn infants. However, the normal range in steady status remains unknown. To ultimately develop the novel measurement technique and clinical application of CD64 expression as an early diagnostic marker for bacterial infection, normal CD64 expression levels were determined in normal neonates, infants, young children, and adults who had no symptoms of infection, stable temperature, normal leukocyte counts and normal C-reactive protein levels, and who were not using steroids, immunosuppressive drugs, aspirin, or other non-steroid anti-inflammatory drugs in this study. To quantify the expression levels of neutrophil CD64, mean fluorescence intensity (MFI) on neutrophils was measured by flow cytometry in 420 samples. Subjects were assigned to 7 groups by age: neonates (group A, n = 76); 1 – 11 months (group B, n = 51); 1 – 5 years (group C, n = 87); 6 – 10 years (group D, n = 76); 11 – 15 years (group E, n = 77); 16 – 20 years (group F, n = 33); and over 21 years (group G, n = 20). Each expression level in groups A, B, and C was significantly higher than that in group G (P < 0.01); no differences were found among groups D, E, F, and G. These results suggest that normal cutoff levels of CD64 should be determined according to age to apply to the clinical setting because CD64 expression has a tendency to increase according to age, especially over 21 years. Disclosures: No relevant conflicts of interest to declare.

Abstract 1491 Five to ten percent of neonates with Down syndrome (DS) develop transient abnormal myelopoiesis (TAM), which is characterized by a rapid growth of abnormal blast cells with the erythroid/megakaryocytic phenotype. In most cases, it resolves spontaneously within three months. However, 20 to 30% of patients develop acute megakaryocytic leukemia referred to as myeloid leukemia of DS (ML-DS) within four years. Blast cells in most of TAM and ML-DS patients have mutations in exon 2 of the gene encoding transcription factor GATA1, which is essential for the normal development of erythroid and megakaryocytic cells. These mutations lead to expression of a truncated GATA1 protein lacking the N-terminal 83 amino acids (GATA1s). However, the molecular mechanism whereby GATA1s contributes to the genesis of TAM and ML-DS remains elusive. From 2003 to 2010, we screened GATA1 mutations in clinical samples obtained from 106 patients with TAM upon request from referring hospitals. We performed direct sequencing analysis using cDNA prepared form total RNA extracted from white blood cells in peripheral blood and genomic DNA. Acquired GATA1 mutations were detected in a total of 99 (93.4%) patients among them. Most of these mutations were coding GATA1s. However, the mutations coding GATA1 mutants with internal deletion of 43 and 15 amino acids were detected in five and one patients, and we referred to them as GATA-ID type-1 and GATA1-ID type-2, respectively. Sequences of exon 3 were discrepant with that expected from the corresponding cDNA sequence. Instead, a 2-nucleotide insertion, a 2-nucleotide deletion and a 7-nucleotide deletion with a single-nucleotide substitution were detected in exon 3 of the GATA1 gene in the GATA1-ID type-1 patients, and a 21-nucleotide deletion encompassing the boundary of intron 2 and exon 3 was found in the GATA-ID type-2 patient. These findings suggest that the mutations in exon 3 lead to expression of alternative splicing forms of mRNA encoding GATA1-ID. To investigate the role of GATA1-ID on the pathogenesis of TAM and ML-DS, we next expressed GATA1-ID by retrovirus in a ML-DS cell line, KPAM1, or primary GATA1-deficient fetal megakaryocytic progenitors. Expectedly, the cell growth was markedly reduced upon transduction with full-length GATA1-expression retrovirus. However, ectopic expression of GATA1-ID type-1 and type-2 failed to restrict the proliferation of cells as similar as expression of GATAs. Interestingly, all these patients had high white blood cell counts in the peripheral blood at diagnosis. Furthermore, three patients developed liver fibrosis and two had effusion. All these factors are significantly associated with early death. Three patients were treated with low dose of Ara-C. However, early death occurred in three patients. In this study we found that GATA1-ID proteins are created by somatic mutations in DS patients and contribute to the generation of TAM phenotypes. Our newly identified GATA1-ID mutants have highlighted a much narrower set of sequences responsible for the pathogenesis of than was previously suggested. The missing region identified by the GATA1-ID proteins contains a consensus motif (LxCxE, amino acids 81–85) essential for the interaction with pRB protein and this motif is also lost in GATA1s. Our results suggested the lack of Rb-GATA1 interaction as the most likely pathogenesis for onset of TAM. Disclosures: No relevant conflicts of interest to declare.

Abstract 4930 Objectives: To develop the use of CD64 expression as an early diagnostic of adult collagen disease, we elucidated normal levels in neonates, infants and children. Methods: Blood samples were collected from a total of 62 symptom- and infection-free subjects recruited at our outpatient department: neonates (n=80), infants (n=41), 1- to 5-year-old children (n=77), 6- to 10-year-old children (n=58), children 11 years old or older and adults (n=95). CD64 expression per neutrophil was quantitatively determined by flow cytometry using a BD FACS Calibur HG flow cytometer (Becton Dicknson, BD) and BD CellQuest Pro analysis software (BD). Fluorescence was measured and converted to number of CD64 molecules per cell using a phycoerythrin (PE) fluorescence kit (Quantibrite PE; BD Biosciences) according to the manufacturer's instructions. This study was approved by the ethics committee of Iwate Medical University. Results: CD64 expression level in neonates (median, 1981 molecules per neutrophil; range, 908–9844), infants (1666.5, 572–12941), and 1- to 5-year-old children (1778, 545–11283) was significantly higher than that in children 11 years old or older and adults (1280, 533–11205) (p Conclusion: Our results suggest that the normal value for adults can not be applied for neonates, infants, and 1- to 5-year-old children. To use CD64 expression level on neutrophils as an infection marker in neonates, infants and 1- to 5-year-old children cut-off values higher than that in adults must be established. Disclosures: No relevant conflicts of interest to declare.

In this study we examined the proper pressure of air boots for a person in a sitting position. Air boots can improve calf congestion by pulsatile calf compression. Higher pressure may improve calf congestion more, though it might decrease arterial blood flow. A photo-electric plethysmometer was put on the second toe of a foot to measure the amplitude of the pulse which represent arterial blood flow. A sensor of a near infrared oxygen monitor was put on the calf just above an air boot. A near infrared oxygen monitor can detect tissue reduced hemoglobin which exists almost only in venous blood. So, the concentration of tissue reduced hemoglobin could represent tissue venous blood concentration.When the air boots pressure was more than 140mmHg, the amplitude of the arterial pulse decreased to half of that without massage. Pressure less than 70mmHg didn't affect arterial blood flow. Meanwhile pressures of more than 70mmHg didn't show any difference in the improvement of calf congestion. So, we concluded that the proper pressure for air boots massage for calf congestion in a sitting position is less than 70mmHg.

Graft versus leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT) is mediated by the cytotoxic factors on cytotoxic T-cells and NK cells. Recent analysis demonstrates that TNF-related apoptosis inducing ligand, TRAIL, plays an important role in the GVL effect. TRAIL induces apoptosis of leukemia cells by the binding to the death receptors, DR4 and DR5. Thus, although the precise mechanism remains unclarified, the regulation of DR4/DR5 expression could be one of critical factors for susceptibility of leukemia cells to the GVL effect. t(17;19)-positive acute lymphoblastic leukemia (ALL) has an extremely poor prognosis, but long-term disease-free survival was exceptionally observed in the case who underwent allo-SCT early in the first complete remission, suggesting that t(17;19)-ALL cells might be susceptible to TRAIL and eventually vulnerable to GVL effect. Accordingly, we examined the sensitivity of t(17;19)-ALL to recombinant human soluble (rhs) TRAIL using 4 cell lines. All 4 cell lines immediately underwent apoptosis by the treatment with rhsTRAIL, and the activation of Caspases 3 and 8, Bid, and PARP was confirmed with Western blotting. Of note, 4 t(17;19)-ALL cell lines showed a significantly higher TRAIL-sensitivity than did other 25 B-precursor ALL cell lines including Ph1, t(1;19), and MLL-rearrangement-positive ALLs. Cell surface expression of DR4/DR5, but not decoy receptors, was confirmed on all 4 cell lines by flow cytometry. Of importance, the levels of cell surface DR4/DR5 expression in 4 t(17;19)-ALL cell lines were significantly higher than those in other 25 B-precursor ALL cell lines. Real time RT-PCR analysis also demonstrated a significantly higher DR4/DR5 gene expression in t(17;19)-ALL cell lines. Since E2A-HLF chimeric transcription factor derived from t(17;19) plays an essential role in the leukemogenesis, we introduced E2A-HLF into the B-precursor ALL cell line 697, whose DR4/DR5 expression level is low, using Zn-inducible vector. When E2A-HLF expression was induced by Zn, the levels of DR4 and DR5 transcripts were immediately upregulated by 10-fold and 40-fold of baseline, respectively. The induction of DR4/DR5 expression was dependent on the DNA-binding and transactivation activity of E2A-HLF, since the mutants lacking either the DNA-binding or transactivation domains failed to induce DR4/DR5 expression. Further, binding of E2A-HLF to the consensus sites separated from the promoters of DR4/DR5 genes was demonstrated in EMSA. These observations indicate that E2A-HLF directly induces expression of DR4/DR5 and sensitizes leukemia cells to TRAIL and eventually GVL effect. This is the first presentation that oncogenic fusion product of translocation regulates the expression of death receptors.

CD33 (Siglec-3), one of myeloid-specific antigens, belongs to a family of sialic acid-binding, immunoglobulin-like lectins (Siglecs), which is adhesion receptor for sialic-acid bearing ligands and regulates a signal transduction through its cytoplasmic tyrosine residues designated immunoreceptor tyrosine-based inhibitory motif (ITIM) as representatively well-characterized in CD22 (Siglec-2). CD33 is ectopically expressed in 5–20% of B-precursor ALL, and has been reported to be potentially associated with poor prognosis. This myeloid-specific antigen expression in B-precursor ALL is considered as lineage promiscuity or lineage infidelity, but underlying mechanism remains totally unknown. We have recently found that B-precursor childhood ALL with t(17;19), which has extremely poor prognosis, is principally CD33 positive, suggesting that CD33 expression might be regulated by E2A-HLF fusion transcription factor generated by t(17;19). To test this hypothesis, using zinc inducible expression vector, we have introduced E2A-HLF into Reh cells, which is a CD33-negative B-precursor ALL cell line. In flow cytometric analysis, by the addition of zinc, CD33 expression was gradually induced in Reh cells transfected with E2A-HLF but not in control cells, and returned to baseline after deprivation of zinc. Of note, mutant of E2A-HLF defective in its DNA-binding domain did not induce CD33 expression. Induction of CD33 expression was not due to “myeloid differentiation”, since upregulation of other myeloid markers including CD11b, CD13, CD14, CD15 and cytoplasmic myeloperoxidase was not observed. In real-time PCR analysis, despite the immediate expression of E2A-HLF protein after addition of zinc, the CD33 mRNA was slowly induced and progressively upregulated to the level approximately 20 times higher than baseline at 5 days after addition of zinc. In promoter region of the CD33 gene, there was no authentic HLF-binding consensus sequence. These observations suggest an indirect induction of CD33 transcription by E2A-HLF. As reported in myeloid cells, when CD33-positive t(17;19)-ALL cell lines were treated with a phosphatase inhibitor pervanadate and analyzed with immunoprecipitaions and Western blotting assay using anti-CD33 antibody, CD33 was confirmed to be tyrosine-phospholyrated and recruit both the protein-tyrosine phosphatases, SHP-1 and SHP-2. This is a first report indicating that an oncogenic fusion transcription factor is evidenced to be associated with ectopic expression of CD33 in B-precursor ALL and that CD33 might play some roles in signal transduction in B-precursor ALL cells.