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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.

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.

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.

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.

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.