Search

Intestinal epithelial cells (IECs) are implicated in the propagation of T-cell–mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell–recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell–produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

Treatment refusal and death as a result of toxicity account for most treatment failures among children with acute myeloid leukemia (AML) in resource-constrained settings. We recently reported the results of treating children with AML with a combination of low-dose cytarabine and mitoxantrone or omacetaxine mepesuccinate with concurrent granulocyte colony-stimulating factor (G-CSF) (low-dose chemotherapy [LDC]) for remission induction followed by standard postremission strategies. We have now expanded the initial cohort and have provided long-term follow-up. Eighty-three patients with AML were treated with the LDC regimen. During the study period, another 100 children with AML received a standard-dose chemotherapy (SDC) regimen. Complete remission was attained in 88.8% and 86.4% of patients after induction in the LDC and SDC groups, respectively (P = .436). Twenty-two patients in the LDC group received SDC for the second induction course. Significantly more high-risk AML patients were treated with the SDC regimen (P = .035). There were no significant differences between the LDC and SDC groups in 5-year event-free survival (61.4% ± 8.7% vs 65.2% ± 7.4%, respectively; P = .462), overall survival (72.7% ± 6.9% vs 72.5% ± 6.2%, respectively; P = .933), and incidence of relapse (20.5% ± 4.5% vs 17.6% ± 3.9%, respectively; P = .484). Clearance of mutations based on the average variant allele frequency at complete remission in the LDC and SDC groups was 1.9% vs 0.6% (P < .001) after induction I and 0.17% vs 0.078% (P = .052) after induction II. In conclusion, our study corroborated the high remission rate reported for children with AML who received at least 1 course of LDC. The results, although preliminary, also suggest that long-term survival of these children is comparable to that of children who receive SDC regimens.

Introduction Patients with E2A-PBX1 fusion are expected to have an aggressive disease course. Because of the rarity of this genotype (nearly 5% of pediatric and 3% of adult B-cell acute lymphoblastic leukemia (B-ALL) cases), a consensus on the clinical and prognostic characteristics of adult E2A-PBX1-positive B-ALL patients, especially in adult patients, has not yet been reached. Patients and Methods We retrospectively summarized our clinical findings from 137 B-ALL patients diagnosed with E2A-PBX at our centers from 2009 to 2019, including 56 adolescents/adults (≥15 years old) and 81 children ( Results The proportions of E2A-PBX1-positive B-ALL in our centers were 5.3% (81/1526) in children, 4.6% (43/925) in AYA and 2.1% (15/713) in older adults. The complete remission rate among all E2A-PBX1-positive B-ALL patients in this study was 94.9% (129/136) after one course of induction chemotherapy. The 5-year overall survival (OS) and disease-free survival (DFS) rates of the whole cohort were 68.6% and 61.0%, respectively. Allo-HSCT at CR1 in adolescents/adults could dramatically improve the 5-year prognoses (OS: 80.8% vs. 25.7%, P A total of 12 patients received CD19-targeted CAR-T cell therapy for disease progression (Figure 1C), and 91.7% (11/12) of patients achieved remission. Two patients died of relapse, and 3 patients died of complications (One died of grade 4 CRS, one died of cerebral hemorrhage after transfusion, and the other one died of infection after 14 months). Three patients received CAR-T bridging to allo-HSCT, and all of them remained in remission within the follow-up period. Univariate and multivariate analysis showed that t(1;19)(q23;p13) only (OS: P=0.020, HR=0.387, 95% CI: 0.174-0.862; DFS: P=0.004, HR= 0.375, 95% CI: 0.193-0.729; CIR: P=0.009, HR= 0.400, 95% CI: 0.200-0.799), Age (DFS: P=0.037, HR=1.009, 95% CI: 1.001-1.018; CIR: P=0.005, HR=1.025, 95% CI: 1.008-1.044) and the level of minimal residual disease (MRD) after induction chemotherapy (OS: P=0.020, HR=2.971, 95% CI: 1.185-7.452; DFS: P=0.002, HR= 3.218, 95% CI: 1.510-6.861; CIR: P=0.006, HR= 3.190, 95% CI: 1.406-7.246) were independent risk factors in E2A-PBX1-positive B-ALL (Figure 1D). In the diagnosis samples, mutations in PBX1, PAX5, CTCF and SETD2, amplification of AKT3, and deletion of CDKN2A/B were common in the total cohort, while transcriptome differences were found in the cell cycle, NGF signaling pathway and transcriptional regulation by TP53 between adolescents/adults and children (Figure 2A,B). More DNA repair gene mutations were detected in the relapse samples (7.9% vs. 57.1%, P Conclusions Our study indicated that age, the level of MRD and DNA repair gene mutations were associated with E2A-PBX1-positive B-ALL outcomes. Allo-HSCT, especially haploidentical-HSCT, could improve the prognosis of adolescent/adult patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Cases of pediatric acute myeloid leukemia (AML) with complex karyotypes including chromosome 5 abnormalities are rare and have a dismal prognosis. Management of AML with monosomy 5/del(5q) has not been uniform. We treated three adolescents with this AML subtype with combined low-dose cytarabine and mitoxantrone, concurrent with decitabine and G-CSF, for remission induction. Decitabine was also included in the conditioning regimen before hematopoietic cell transplantation (HCT). All three patients attained complete remission after treatment with this combination. The treatment was well tolerated, and the patients are alive and free of disease at 3.6, 3.2, and 3.0 years after HCT, respectively. Our experience suggests that HCT is required for the eradication of pediatric AML, and possibly MDS, with complex karyotypes including del(5q). Decitabine combined with regimens of low myelotoxicity for remission induction represents an alternative approach to decrease the risk of complications associated with post-remission chemotherapy before HCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Our understanding of acute lymphoblastic leukemia (ALL) has expanded tremendously in the past few years because of large-scale genomic studies.[1][2],[2][3] ALL is characterized by a relatively

Modulating T-cell alloreactivity has been a main strategy to reduce graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem-cell transplantation (HSCT). Genetic deletion of T-cell Ezh2, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), inhibits GVHD. Therefore, reducing Ezh2-mediated H3K27me3 is thought to be essential for inhibiting GVHD. We tested this hypothesis in mouse GVHD models. Unexpectedly, administration of the Ezh2 inhibitor GSK126, which specifically decreases H3K27me3 without affecting Ezh2 protein, failed to prevent the disease. In contrast, destabilizing T-cell Ezh2 protein by inhibiting Hsp90 using its specific inhibitor AUY922 reduced GVHD in mice undergoing allogeneic HSCT. In vivo administration of AUY922 selectively induced apoptosis of activated T cells and decreased the production of effector cells producing interferon γ and tumor necrosis factor α, similar to genetic deletion of Ezh2. Introduction of Ezh2 into alloreactive T cells restored their expansion and production of effector cytokines upon AUY922 treatment, suggesting that impaired T-cell alloreactivity by inhibiting Hsp90 is achieved mainly through depleting Ezh2. Mechanistic analysis revealed that the enzymatic SET domain of Ezh2 directly interacted with Hsp90 to prevent Ezh2 from rapid degradation in activated T cells. Importantly, pharmacological inhibition of Hsp90 preserved antileukemia activity of donor T cells, leading to improved overall survival of recipient mice after allogeneic HSCT. Our findings identify the Ezh2-Hsp90 interaction as a previously unrecognized mechanism essential for T-cell responses and an effective target for controlling GVHD.

Although majority of pediatric acute myeloid leukemia (pAML) undergo complete remission (CR) after chemotherapy, more than 30% of which eventually relapse, leading to a dismal outcome. Chemo-resistant cells at CR, measured as minimal residual disease (MRD), are thought to be origin for relapse. Although more sensitive detection of MRD has been achieved through genetic mutations, the pathological and clinical significance for most of the detected mutations remain unknown, largely due to a lack of understanding for the biological context where the mutation resides. The idea of retention of leukemia stem cells (LSCs) as a mechanism of chemo-resistance has not been demonstrated post-therapy in patients. Here we applied single cell RNA sequencing (scRNA-seq) on 14 pAML who met the criteria for clinical CR (n=11) and partial remission (PR, n=3) to determine the cellular heterogeneity and cancerous feature of the residual cells that can survive chemotherapy at remission. These patients carried common genetic mutations such as AML1-ETO, CBFB-MYH11 and FLT3-ITD, which represents genetically diverse WHO subtypes. To maximize the power to detect tumor cells that can survive chemotherapy, we used 54 unsorted total bone marrow (BM) and/or peripheral blood (PB) nucleated cells collected at diagnosis and Day 26 (D26) of the first cycle of chemotherapy for 10X genomics' scRNA-seq. Cells from each pAML were compiled into one UMAP together with normal reference using unsupervised clustering to distinguish tumor clusters from normal. Clusters were defined to be tumor if patient's diagnosis contributed at least 80% of the cells, and were confirmed by the presence of somatic mutations and/or known AML mRNA expression signature associated with chromosome translocations. By projecting onto the closest normal hematopoietic cells based on transcription features, the tumor cells were classified as one of the 12 cell types (HSC/MPP, LMPP, GMP, MEP, E/B/M, CLP, classical/nonclassical Monocyte, cDC, pDC, and pre/inmature Neutrophil like cells). Consistently with findings in adult AML, cell populations were heterogeneous at diagnosis with 5-9 distinct clusters. Interestingly, majority (9/11) of the patients had 1-6 tumor clusters detected (mean 52 cells per cluster) at D26 post-chemotherapy. These D26 residual tumor cells possessed mutations originally detected by genomic sequencing and/or known AML signatures, and consistently clustered with tumor cells from diagnosis. These residual tumor cells accounted for average 1.4% of total BM cells at D26, while the morphological examination and flow cytometry analysis of MRD showed average 4.0% and 0.71% of tumor cells. To further evaluate the chemo-resistant potential of identified residual tumor cells, we focused on three distinct features known to be associated with chemo-resistance in mouse models, including LSC activity, active oxidative phosphorylation (OXPHOS) or leukemic-regenerating cell (LRC) state. Among the total 18 residual tumor clusters detected at D26, 33.3% (6/18) exhibited expression signatures associated with at least one chemo-resistant features. The remaining clusters consisted more differentiated progenitor/monocyte-like cells. Specifically, three patients (1 PR and 2 CR) had the HSC/MPP or LMPP-like clusters possessing strong LSC and OXPHOS-associated signatures. The remaining one patient had cDC-like cluster expressing reported LRC signature. Importantly, all these four patients had either unfavorable cytogenetics or persistence of driver mutations detected by PCR. Taken together, these data showed that pediatric AMLs represented heterogeneous populations at both diagnosis and remission. Among the residual tumor clusters that survived chemotherapy, a small fraction (6/18) were HSC/MPP-like, LMPP-like and cDC-like cells with known chemo-resistant expression features. These findings provide the first in vivo characterization of cellular heterogeneity in chemo-treated pAML with complete remission. Further studies are needed to determine the molecular characteristics of these residual cells that may convey chemo-resistance and to determine whether the presence of these cells are associated with increased risk of relapse. Disclosures No relevant conflicts of interest to declare.

Pediatric acute myeloid leukemia (pAML) is a heterogeneous disease. The clinical outcome of pAML has improved significantly over the past four decades with current long-term survival rates of ~70%. This improvement is due to intensification of chemotherapeutic regimens, better risk-group stratifications, better salvage at relapse, and improved supportive care. Nevertheless, novel therapies are still needed as the cure rates for some subtypes of pAML remain unacceptably low. In addition, the profound marrow suppression caused by near-myeloablative induction therapy may lead to life-threatening complications, such as severe infections, which not only prolong the hospitalization but also cause significant morbidity and mortality during or after treatment. Moreover, intensive chemotherapy usually affects the patient's quality of life and they can experience low physical, emotional, or social function. To determine the impact of reducing the intensity of induction chemotherapy on the outcome of pAML, we recently performed a study using G-CSF priming plus low-dose chemotherapy (GLDC) to treat pAML, compared with standard chemotherapy (SDC) during induction. Our study showed that complete remission (CR), 4-year event free survival (EFS), overall survival (OS), and cumulative incidence of relapse (CIR) rates of 46 pAML patients, initially given a GLDC for the first induction remission, were comparable to those of 94 children admitted to the same hospital and given SDC. However, GLDC showed a much lower toxicity than SDC. After the first course of induction, the recovery of white blood cell (WBC) and platelet counts were significantly faster in patients receiving GLDC than SDC. Grade III or IV infectious complications occurred in 4 (8.7%) patients receiving GLDC and 23 (24.4%) patients receiving SDC, and 2 patients receiving SDC died of complications (Hu Y. et al, 2019). Even though the clinical efficacy of GLDC has been seen in multiple clinical trials, the detailed mechanisms remain unclear. Some clinical and pre-clinical studies showed that G-CSF could push AML cells into cell-cycle, accelerate WBC and platelet recovery even when used after induction chemotherapy, and reduce the viability of AML cells when co-cultured with BM stroma, but not cultured alone. However, all of these studies were not established with LDC. To determine the potential mechanism(s) of GLDC in pAML, we established Mll-Af9 transplanted chimeric mice to develop AML, and we assessed the clearance of leukemic cells and the impact on normal cells by GLDC or SDC. Based on previous studies, we used combined cytarabine (100mg/kg) and doxorubicin (3mg/kg) (DA) in mice as SDC, half dose of SDC as LDC, and G-CSF (300ug/kg) plus half dose of SDC as GLDC in our study design. The G-CSF priming times were inconsistent in previous studies. To establish the best priming effect, especially for the stem cells, we first analyzed the cell cycle stimulation by G-CSF to the SLAM hematopoietic stem cells (SLAM-HSCs) and the more quiescent stem-like HSC population (endothelial protein C receptor expressing, EPCR+ HSCs) in WT mice. We found that G-CSF can stimulate SLAM-HSCs and EPCR+ HSCs into cell-cycle as early as 12hrs and 24hrs post G-CSF injection, so we used G-CSF one day priming before the chemotherapy estimating that even the deeper quiescent stem cells have been stimulated into the cell-cycle at that time point. We then arranged the Mll-Af9 transplanted mice with similar disease burden into 5 groups to receive different treatments for 6 days: LDC, GLDC, SDC, and also the G-CSF or the PBS groups as control (G-CSF was given on D1-D6, DA was given on D2-D6). We found that GLDC and SDC had similar clearance to the leukemic cells (CD45.2+), which were better than LDC, as observed 2 days after the treatment. However, the recovery of normal cells (CD45.1+) was significantly faster in the GLDC group compared with LDC and SDC groups. We also found that G-CSF could simulate the differentiation of both leukemic cells and normal cells, shown by the increase of the CD11b+Ly6G+ population of both CD45.2+ and CD45.1+ cells in the G-CSF group compared to the PBS group. Studies to further elucidate the additional mechanisms of GLDC are still ongoing. In conclusion, our clinical and mouse modeling studies consistently suggest that GLDC promotes leukemia cells clearance and normal hematopoietic regeneration simultaneously, which may be a better regimen for pAML. Disclosures No relevant conflicts of interest to declare.

OBJECTIVE To determine whether dasatinib given at 80 mg/m2 is more effective than imatinib at 300 mg/m2 to improve event-free survival of children with Philadelphia chromosome-positive ALL, in the context of intensive chemotherapy without prophylactic cranial irradiation. DESIGN, SETTING, AND PARTICIPANTS This open-label phase III randomized study was conducted at 20 hospitals in China. Enrollment began in January 2015 and randomization was stopped in October 2018 when the early stopping criterion of the trial was met. Patients aged between 0 and 18 years were recruited. Of the 225 patients with the diagnosis, 35 declined and 1 died before treatment. INTERVENTIONS Patients were randomized to receive daily dasatinib (n=92) or imatinib (n=97) continuously for the entire duration of ALL therapy from the time of diagnosis made during remission induction to the end of continuation therapy. MAIN OUTCOMES AND MEASURES The primary outcome was event-free survival, analyzed by intent-to-treat. The secondary outcomes were relapse, toxic death, and overall survival. RESULTS With a median follow-up of 26.1 months (IQR 16.3-34.1), the 4-year event-free survival rate was 71.0% (95% CI 56.2-89.6) in the dasatinib group and 48.9% (95% CI 32.0-74.5, p=0.005) in the imatinib group (hazard ratio 2.36, 95% CI 1.27-4.40, p=0.007). The 4-year cumulative risk of any relapse was 19.8% (95% CI 4.2-35.4) in the dasatinib group and 34.4% (95% CI 15.6-53.2) in the imatinib group (p=0.01), while the 4-year cumulative risk of an isolated central-nervous-system relapse was 2.7% (95% CI 0.0-8.1) in the dasatinib group and 8.4% (95% CI -1.2-15.6) in the imatinib group (p=0.06). There were no significant differences in the frequency of severe toxicities between the two treatment groups. CONCLUSION AND RELEVANCE Intensive chemotherapy including dasatinib at 80 mg/m2 per day yielded superior results in the treatment of Philadelphia chromosome-positive ALL compared to imatinib at 300 mg/m2 per day and provided excellent control of central-nervous-system leukemia without the use of prophylactic cranial irradiation. Disclosures No relevant conflicts of interest to declare.

Introduction: The efficacy of extended duration of vincristine (VCR) and dexamethasone (DEX) pulses in the treatment of childhood acute lymphoblastic leukemia (ALL) is uncertain in the context of contemporary chemotherapy. By contrast, the long-term sequelae of vincristine-induced peripheral neuropathy and dexamethasone-related metabolic syndrome, osteonecrosis, and neurocognitive impairment are well recognized. We therefore conducted a randomized study in the CCCG-ALL-2015 protocol participated by 20 hospitals in China to compare the event-free survival (EFS) and overall survival (OS) between children with newly diagnosed ALL who did or did not receive extended duration of VCR+DEX pulses during maintenance treatment. Methods: After remission induction and consolidation treatment with high-dose methotrexate and mercaptopurine, all patients received antimetabolite-based maintenance treatment. During the initial 20-week of maintenance treatment, low-risk (LR) patients received two reinduction cycles and three pulses of VCR+DEX pulses, while intermediate-/high-risk (I/HR) patients received daily mercaptopurine interrupted every three weeks with PEG-asparaginase, mercaptopurine, daunorubicin, and VCR+DEX pulses for 5 courses followed by a reinduction treatment. During the subsequent maintenance treatment, all patients received VCR+DEX pulses every 4 weeks for 5 cycles. All Philadelphia chromosome (Ph)-negative patients were then stratified and randomized during the fifth cycle of maintenance course (between weeks 51 and 53) to or not to receive the VCR+DEX pulse every 8 weeks for 7 cycles between weeks 54 and 109; the remaining maintenance treatment between weeks 110 and 125 consisted of only mercaptopurine and methotrexate. Results: Between January 2015 and December 2018, 3985 eligible Ph-negative patients, including 2318 (58.2%) LR patients and 1667 (41.8%) I/HR patients, were enrolled in the study. With a median follow-up of 2.53 years (IQR 1.82-3.26), the 4-year event-free survival (EFS) and overall survival (OS) rates were 91.6% (95% CI 89.4-93.9) and 98.8% (95% CI 98.2-99.4) in the LR patients, and 85.9% (95% CI 83.2-88.7) and 95.4% (95% CI 93.8-96.9) in the I/HR patients, respectively. The 4-year OS were 99.0% (95%CI, 98.3%-99.8%) in the 1145 LR patients randomized to receive VCR+DEX pulse (LR-A) and 98.6% (95% CI, 97.7%-99.5%) in the 1173 LR patients not to receive the pulse (LR-B, hazard ratio 1.5, 95% CI 0.6-4.0; p=0.374, Fig 1A). The 4-year EFS were 91.1% (95% CI, 87.4%-95.1%) in LR-A group and 92.0% (95% CI, 89.6%-94.5%) in LR-B group (hazard ratio 1.3, 95% CI 0.8-1.9; P=0.27; Figure 1B). There were no significant differences between LR-A and LR-B group among patients with or without the t (12;21) (ETV6-RUNX1) in univariate analysis (Table1). Likewise, there was no significant difference in the 4-year OS between the 834 I/HR patients who did (I/HR-A) and the 833 I/HR patients who did not (I/HR-B) receive the pulse: 94.3% (95% CI, 91.7%-97.0%) versus 96.3% (95%CI 94.6%-98.0%, hazard ratio 0.8, 95% CI 0.4-1.4; P=0.421) (Fig 1C). The 4-year EFS were 83.6% (95%CI 79.2%-88.3%) for I/HR-A group and 88.1% (95%CI 85.1%-91.2%, P=0.372) for I/HR-B group (hazard ratio 0.9, 95% CI 0.6-1.2; P=0.37) (Figure 1D). There were no significant differences between I/HR-A and I/HR-B group among patients with B-ALL, T-ALL or t (1;19) (TCF3-PBX1) in the univariate analysis (Table1). Conclusion: The additional pulses of VCR+DEX pulse beyond one year of maintenance treatment had no impact on 4-year EFS or OS among LR or I/HR patients. If this finding remains unchanged with additional follow-up, the VCR+DEX pulses can be omitted after one year of maintenance therapy in childhood ALL. Disclosures No relevant conflicts of interest to declare.

Introduction: Septicemia is one of the common complications after chemotherapy for acute lymphoblastic leukemia (ALL) and is also an important cause of treatment related mortality and treatment failure. A multicenter study CCCG-ALL-2015 was conducted in China and factors associated with septicemia and mortality were studied. Methods: Patients participated in CCCG-ALL-2015 study from January 2015 to December 2017 were included in this study. Patients with documented septicemia were identified from the Database of Central Data Center. Additional data were then collected from participation centers, including associated co-morbidity, blood counts before septicemia and infection preventive measures. Results: A total of 4080 patients were recruited from 18 centers. There were 527 patients with septicemia identified (12.9%). The Intermediate risk (IR)/ High risk (HR) group(n=1930) had significantly higher incidence of septicemia as compared with Low risk (LR) group (n=2150), 17.15% vs 9.12% (p Conclusion: Overall the incidence and pattern of septicemia in this multicenter study in China was similar to reports of western countries. The septicemia related mortality rate was low. However there was marked variation in the incidence of septicemia among the centers, further studies are required to validate factors such as infection preventive measures which might influence the septicemia incidence. Disclosures No relevant conflicts of interest to declare.

Background: Acute megakaryocytic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) with a dismal outcome in children without Down syndrome. Refractory or relapsed disease accounts for treatment failure in most cases. Hematopoietic stem cell transplantation (HSCT) is recommended as post-remission therapy, although some patients have been cured with intensive chemotherapy alone. Genetic and genomic abnormalities are common in AMKL, but their prognostic significance is not known. In this study, we analyzed AMKL using high-throughput sequencing in addition to conventional morphology, immunophenotype, cytogenetic, and molecular (MICM) approaches. Methods: From February 2015 to April 2019, 19 cases of AMKL in non-Down syndrome patients were diagnosed according to conventional methods in our center. All patients, except one, were treated using a low-dose chemotherapy (LDC) regimen (cytarabine 10 mg/m2, subcutaneously every 12 h, 20 doses; mitoxantrone 5 mg/m2 days 1, 3 and 5, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) for induction remission followed by four courses of intensive chemotherapy or hematopoietic stem cell transplantation (HSCT) as post-remission consolidation[1]. Patients were treated with consolidation chemotherapy if parents refused HSCT. High-throughput mutational sequencing of leukemia cells obtained at diagnosis was performed. The average sequencing depth was 500-1000× and analyses were performed using mGATK, SAMTools, SIFT, PolyPhen2, LRT, and MutatioinTaster methods. Results: The average age at diagnosis of AMKL of the nine boys and 10 girls was 19.7 months (7-53 months). Eighty-four percent of the cases were younger than 2 years of age. The 19 patients with AMKL represent 10% of cases diagnosed with AML during the study period. The median initial white blood cell (WBC) count was 13×109/L(2.38-126×109/L)and the average blasts at initial bone marrow (BM) was 54.5% (13-85%). In all cases of AMKL, more than 25% of cells were positive for CD41. Sixty-three percent of patients had recurrent fusion genes, including NUP98-KDM5A, HOXA11-AS/ANGPT1, CBFA2T3-GLIS2, KMT2A-MLLT3, RBM15-MKL1. Also, we identified PICALM-MLL10, TNIP1-CSF1R, NUP98-TAF3, and CBFA2T3-GLIS1 fusion genes which had not been previously reported in AMKL. EVI-1 expression was evaluated in all cases and was overexpressed in two cases. Forty-seven percent of patients showed at least single-gene mutations, including JAK2, MPL, KRAS, NRAS, TP53, NTRK3, MYO16, PDGFB, FLT3, and others. The response rate after remission induction I (IND-I) were 84.2% with 63.1% (12/19) attaining complete remission (CR) and 21.1% (4/19) partial remission (PR). After IND-II, the CR, PR, and no response (NR) rates was 68.4%, 10.5%, and 21.1%, respectively. Four patients (21.1%) were refractory to induction chemotherapy. Seven patients (36.8%) relapsed, three during consolidation chemotherapy courses, and four after transplantation (Table 1). Currently, 75% (6/8) patients who received chemotherapy only for consolidation were alive (median follow-up time 27 months). Eleven patients underwent allo-HSCT, and 8 patients are alive, including 6 cases living without evidence of disease. Four patients relapsed after allo-HSCT, and two of them have died. One additional patient died of transplantation related mortality. The 4-year OS, RFS, and EFS in this cohort were 62.4%±13.9%, 54.1±13.0% and 40.5±11.5%, respectively (Figure 1).Conclusion: Non-Down syndrome AMKL is genetically heterogeneous with recurrent and new fusion genes. Patients treated with LDC for induction followed by intensive chemotherapy or HSCT for consolidation have outcome comparable to those treated with intensive induction regimens. New treatment strategies are needed to improve the outcome of pediatric AMKL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Promoting donor T cell tolerance to host non-hematopoietic tissues remains the ultimate therapeutic goal in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dendritic cells (DCs) play dual functions in regulating alloimmunity. DCs can elicit alloreactive T cell responses to mediate graft-versus-host disease (GVHD), but are also implicated in reducing GVHD. In patients, the depletion of plasmacytoid DCs (pDCs) from donor BM grafts resulted in GVHD acceleration. On the other hand, acute GVHD causes complete failure of donor pDC reconstitution after allo-HSCT, and low levels of donor pDC correlate with significantly increased GVHD severity. Thus, the impairment of pDC reconstitution by GVHD may be responsible for the dysfunctional immune regulation. Delineation of the mechanism involved may allow therapeutic intervention to reduce GVHD and improve the efficacy of allo-HSCT. In this study, we demonstrate that alloreactive T cells produce GM-CSF to impair reconstitution of donor pDCs by inhibiting Flt3 expression and its-regulated transcription programs in DC progenitor cells. Using murine GVHD model, we confirmed GVHD severely impaired reconstitution of both donor pDCs and conventional DCs (cDCs). Adoptive transfer of donor-type pDCs rather than cDCs prevented the occurrence of severe GVHD in mice, suggesting donor pDC reconstitution is important to restore tolerance of donor T cells against host tissues. Flt3 is required to induce pDC production through a successive differentiation pathway: HSC → multiple potential progenitors (MPP) → common DC progenitors (CDP) → precursor DCs (pre-DCs). GVHD mice produced significantly less MPP, CDP and pre-DCs compared to normal donor mice and allogeneic mice receiving T cell-depleted BM. Ex vivo culture with Flt3 ligand (Flt3L) showed that those MPP and CDP derived from GVHD mice dramatically decreased the capacity to produce pDCs. Thus, GVHD not only causes decreased numbers of MPP and CDP but also their intrinsic defect in producing pDCs. While both MPP and CDP gave rise to similar numbers of pDCs within 3 days of culture with Flt3L, MPP produced 40-fold more pDCs than CDP by day 9 of culture. This indicates the impairment in GVHD MPP may have much more profound long-term impact on pDC reconstitution than that in CDP. Based on surface expression of Flt3, normal MPP contained two subsets: CD135high MPP and CD135mod MPP. CD135high MPP produced 4-fold more pDCs than CD135mod MPP. As compared to CD135mod MPP, CD135high MPP expressed lower levels of Ink4 family genes, which are cyclin-dependent inhibitors restraining cell proliferation and survival, suggesting that CD135high MPP represent earlier stage differentiated progenitors with greater proliferative capacity. Intriguingly, although GVHD mice generated similar amount of CD135mod MPP as did normal mice, they failed to reconstitute highly proliferative CD135high MPP. Thus, the failure of donor pDC reconstitution may largely result from GVHD-mediated inhibition of CD135high MPP. Alloreactive T cells are known to produce high levels of effector molecules, such as IFN-γ, TNF-α, GM-CSF and other cytolytic molecules. We observed that GVHD effector T cells significantly reduced the production of pDCs from Flt3L-induced normal MPP. Blocking GM-CSF using neutralizing antibody but not other effector molecules markedly inhibited this repressive effect of GVHD T cells on pDC production. GM-CSF dose-dependently decreased the expression of Flt3 and its-regulated transcription factors Irf8 and Tcf4, which are important for development of functional pDCs. However, GM-CSF failed to inhibit the conversion of SiglecH+ pre-pDCs into pDCs. These data suggest that alloreactive T cells produce GM-CSF to block pDC reconstitution by targeting DC progenitors (e.g., MPP and CDP). Building on these findings, we established a novel optimized culture system to produce adequate numbers of SiglecH+ pre-pDCs. Adoptive transfer of these pre-pDCs prevented GVHD, leading to significantly improved overall survival of mice undergoing allo-HSCT. Our findings identify for the first time that selective restoration of donor pDCs early after allo-HSCT may represent an effective cellular therapy to prevent GVHD. Further delineation of the molecular pathway(s) involved in GVHD inhibition of DC progenitors may allow the development of novel approaches to circumvent mortality and morbidity associated with GVHD. Disclosures Zheng: Pfizer: Research Funding.

Graft-versus-host-disease (GVHD) remains a leading cause of transplant-related mortality (TRM) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients who do not respond to GVHD therapy are at high risk for death without relapse. Strategies that can predict the development of severe GVHD in transplant patients may improve risk stratification for steroids treatment refractory GVHD and TRM. Dendritic cells (DCs) play nonredundant roles in regulating both immunity and tolerance. Clinical and pre-clinical studies suggest that GVHD causes dysregulated development of functional DCs and impaired reconstitution of donor DCs is associated with increased risk of severe GVHD. We demonstrate that donor plasmacytoid DC (pDC) reconstitution around the engraftment time is a useful predictor for the development of severe GVHD in children after allo-HSCT. We examined reconstitution of donor DCs, including total DCs (lineage-negative (LIN-)HLADR+), pDCs (LIN-HLADR+CD123+CD1c-) and CD1c+DCs (LIN-HLADR+CD123-CD1c+), in peripheral blood of patients undergoing allo-HSCT, using age-matched healthy children as controls. The median time for engraftment was 14 days (range, 13.0~17 days), whereas clinical signs of GVHD occurred approximately 10 days later (median time, 24 days;range, 18~35 days). Thus, measuring the number and frequency of circulating blood DCs early at the engraftment time allowed us to evaluate the subsequent occurrence of GVHD. Transplant patients were followed up for 6 months after allo-HSCT. Of evaluable 45 transplant patients, 27 patients had no GVHD (grade 0) or mild GVHD (grade I), and 18 patients developed severe GVHD (grade II-IV GVHD). Patients who developed grade II-IV GVHD had no significant difference in frequency ( median, 0.2%; range, 0.04~0.35%) and number (median, 1.6 cell/ml; range, 1~3.8 cell/ml) of total DCs than patients with grade 0-I GVHD (median frequency, 0.41%; range, 0.15~0.63%, P Disclosures No relevant conflicts of interest to declare.

Purpose: Prognosis and therapeutic treatment of Fanconi anemia (FA) and acquired aplastic anemia (AA) are different. However, delayed and incorrect treatments are frequently occurred in FA because its symptoms are very similar to those of AA. This study aimed to elucidate the differences between FA and AA at the molecular level and to identify biomarkers and pathways of FA through quantitative proteomics and bioinformatics analyses. Methods: Samples were obtained from the bone marrow of patients with FA and AA followed the institutional guideline. Proteins were extracted, digested with Lys-C and trypsin, and chemically labeled with tandem mass tag (TMT) duplex reagents (TMT126 and 127) for mass spectrometric analyses. Proteins with fold changes of > 1.5 or < 0.667 were considered as differentially expressed proteins (DEPs). Gene ontology analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted using the ClueGO plug-in in Cytoscape. A DEP-associated protein-protein interaction (PPI) network was constructed using STRING and visualized in Cytoscape. The degree and topological index of these DEPs were calculated using CentiScape. The PPI network was subjected to modular analysis using the Molecular Complex Detection plug-in in Cytoscape. Results: A total of 114 DEPs, including 71 upregulated and 43 downregulated proteins, were discovered in the FA sample compared with those in the AA sample. The upregulated proteins were enriched in nucleosome assembly, cell or subcellular component movement, glycolysis, and gluconeogenesis pathway. The downregulated proteins were enriched in immune response, negative regulation of apoptosis, proteolysis, phagosome, and pantothenate and CoA biosynthesis. Five important modules were detected from the PPI network of DEPs. Eight hub proteins, a-enolase (ENO1), phosphoglycerate kinase 1 (PGK1), HSP90AA1, HSP90AB1, ACTC1, ACTBL2, EEF1A1, and CFL1, with a high degree of connectivity were obtained. Conclusions: Quantitative proteomics coupled with bioinformatics analyses are useful for the identification of protein biomarkers and pathways associated with FA and AA. Some critical DEPs, such as ENO1, PGK1, ACTC1, ACTBL2, EEF1A1, and CFL1, likely play important roles in FA and could be used as serologic markers for its early diagnosis and to distinguish FA from AA. Disclosures No relevant conflicts of interest to declare.

Objective: To investigate the incidence, clinical characteristics and prognosis of nervous system (NS) complications after hematopoietic stem cell transplantation (HSCT) in children. Methods: Three hundred children who received HSCT from October 2010 to June 2018 were retrospectively analyzed to identify NS complications in our center. Factors which may be related to NS complications were figured out. Median follow-up time is 3.9 years (range, 0.2- 7.7 years). Results: Among 300 cases with transplantation, 290 cases were allogeneic transplantation, 9 Autologous transplantation and one case with Isogenic transplantation. The median age was 8 years old (0.7-16 years old), and median weight was 25kg (7-88kg).47.33% of them were male(142/300). Of 290 allogeneic transplantation, 79 cases received sibling matched transplantation, 90 cases received non-relative cord blood transplantation, and 121 cases were haploididentical transplantation. Diseases distribution was as following: 48 cases with aplastic anemia (AA), 174 cases with acute leukemia(AL), 4 cases with chronic myelocytic leukemia (CML), 1 case with juvenile Myelomonocytic Leukemia (JMML),12 cases with myelodysplastic/myeloproliferative neoplasm(MDS/MPN), 28 cases with Wiskott-Aldrich syndrome(WAS), 5 cases with Fanconi anemia (FA), 6 cases with thalassemia, 5 cases with neuroblastoma, one case with neuroblastoma and acute myeloid leukemia, 16 cases with other congenital disorders. Twenty six children developed NS complications after HSCT which accounted for 8.67% (26/300). The diseases which developed NS complications from high to low were as following: FA (33.3%), MDS/MPN (19.2%), AL (7.3%), AA (7.0%), 3.1% in genetic immunodeficiency disease. The average age of children who developed NS complications was 8.3±3.8years old which was older than non- NS complications (6.5±4.0 years old) (p=0.02). Different types of NS complications included 23.1% of demyelinating encephalopathia, 30.8% of cerebral acute graft versus host disease(GVHD), 15.4% of hypertension encephalopathia,11.5% of cerebrovascular lesion, one case of transplantation associated thrombotic microangiopathy (TA-TMA),7.7% of drug therapy-related toxic encephacopathy,3.8% of metabolic encephalopathy,7.7% of peripheral neuropathy. The mortality of NS complications was 34.6%. Of 8 cases with cerebral GVHD, 5 cases died which had the highest mortality (62.5%). No patients with drug therapy-related toxic encephacopathy, metabolic encephalopathy, and peripheral neuropathy died. The overall survival in children with NS complications is lower than the children without NS complications after HSCT (65.4% vs 84.2%, p=0.015). Conclusions: The incidence of NS complications after HSCT was correlated with primary diseases and children's age. Patients with drug therapy-related toxic encephacopathy, metabolic encephalopathy and peripheral neuropathy had better prognosis than cerebral GVHD and TA-TMA patients. Reduction of NS complications may improve long term survival of children after HSCT. Disclosures No relevant conflicts of interest to declare.

Background: It is difficult for pediatric refractory and relapse acute myeloid leukemia (RR-AML) and MDS-RAEB/RAEBT patients to achieve complete remission (CR) and these patients develop recurrence and die of either disease progression or associated complications. The CAG regimen (cytarabine, aclarubicin and G-CSF) has been widely used in treating patients with MDS-EB and AML-MRC in Asia. Likely, Short term CAG derived regimens called low dose induction therapy, MAG regimen (Mitoxantrone for 3 doses, cytarabine and G-CSF for 10 days) also showed efficacy in De Novo AML. However, MAG regimen showed less efficacy in RR-AML and MDS-5(5q-). Purpose: To evaluate the clinical efficacy and safety of low-dose decitabine in combination with low-dose MAG regimen (D-MAG regimen) in the treatment of RR-AML and MDS-RAEB/RAEBT. Method A total of 17 patients with MDS-RAEB/RAEBT and RR-AML((2 cases of MDS-RAEB, 3 cases of MDS-RAEBT, 10 cases for refractory AML, and 2 cases for relapse AML) from June 2017 to April 2018 in our center were included in the retrospective study. All the patients were treated with decitabine of 20 mg/m2 for 5 days and followed by 10 days of MAG regimen (cytarabine of 10mg/m2 q12h for 10 days, mitoxantrone of 5 mg/ m2.d for 3 days, and G-CSF of 5μg/kg.d for 10 days),called D-CAG regimen. After two cycles of induction chemotherapy, the patients who obtained CR received consolidation chemotherapy or hematopoietic stem cell transplantation (HSCT). Results Among a total of 17 patients, the median age was the median age was 102 months (32-200 months) and 64.71 % of them were male. Characteristic features of these patients were illustrated in Table 1. Only 2 cases died of severe lung infection due to continuous agranulocytosis who had been received high dose induction therapy (Daunorubicin of 50mg/M2.d for 3 days, cytarabine of 100mg/m2 q12h for 10 days, and Etoposide of 150mg/m2.d for 5 days) for 2 cycles with poor physical condition before D-MAG. In the other 15 cases, 10 of them had complete remission (CR) after the first course of treatment, 4 cases were partial remission (PR), 1 case with a high blast cells at 25% indicated a poor response to D-MAG, and the effective rate was 93.3%. Among 4 children with PR, one case reached CR after the second courses of treatment. The most common adverse reactions in all children were hematological toxicity, grade III-IV. Liver damage was found in 2 cases, grade I and grade II respectively. There were 3 cases of oral side reactions, 1 case in grade I and 2 cases in grade II. The gastrointestinal reactions in all children were grade I - II. By July 2018, the median follow-up was 11 months (7-16months). Among 15 patients after D-MAG, 11 patients received HSCT. The twelve-month survival rate was 88.24% and the median leukemia-free survival (LFS) was 11 months. Conclusion The low-dose decitabine in combination with Low-dose MAG regimen improved CR rate for pediatric patients with MDS-RAEB and RR- AML, and is a promising treatment for pediatric patients with MDS/RR-AML. Disclosures No relevant conflicts of interest to declare.

Insulin-like growth factor binding proteins (IGFBPs) are secretory factors that play essential roles in regulation of insulin-like growth factors (IGFs) in tissue as well as in modulating IGF binding to its receptors. IGFBP7, known as IGFBP-related protein 1 (IGFBP-rP1), mac25/angiomodulin, function as a potential tumor suppressor in various human solid cancers, including breast, prostate, gastric and liver cancer. We have reported the overexpression of IGFBP7 in the context of acute myeloid leukemia (AML), showing that IGFBP7 expression level in AML patients is significantly increased compared with controls (P In order to study the role of IGFBP7 in AML, stable cell lines expressing IGFBP7 and control in AML cells were constructed using lentiviral packaging system. Expression microarray assay was carried out to analyze the global gene level changes driven by IGFBP7. MTT and transwell assays were performed to evaluate the effect of IGFBP7 on cell proliferation and migration. Bioinformatics results found that IGFBP7 appeared to utilize multiple cellular processes for its oncogenic roles, including adhesion, migration, and proliferation. Experimental data showed overexpression of IGFBP7 in K562 cells resulted in a 2-3 fold increase in migration in contrast to control cells. Moreover, enforced expression of IGFBP7 also led to phosphorylation of Akt and Erk, whose activities inactivation by pharmacologically inhibitors resulted in the loss of ability to migrating. Finally, knockdown of IGFBP7 in cells with high IGFBP7 level, their migration abilities were significantly decreased. To assess the role of IGFBP7 in leukemogenesis in vivo, the same numbers of K562/IGFBP7 and K562-Vector cells, U937-shIGFBP7 and U937-shNEG cells were injected into NOD-SCID mice by tail vein injection, respectively. About two weeks later, it was showed that mice of K562/IGFBP7 and U937 groups displayed higher white blood cell counts compared with mice of K562-Vector and U937-shIGFBP7 groups, respectively. Mice of K562/IGFBP7 and U937 groups had more severe splenomegaly and hepatomagaly compared with its corresponding control groups. We further characterized the molecular mechanism underlying leukemogenesis driven by IGFBP7 in AML cell lines. The global expression profiling and molecular biological experiments showed PI3K/AKT signaling was activated by overexpression of IGFBP7, and knockdown of IGFBP7 in AML cells led to a decrease of PI3K/AKT activity in PTEN-dependent manner. IGFBP7 promotes proliferation and migration of AML cells, the promotion could be suppressed by both RNA interference and pharmacological inhibition of PI3K/AKT pathway. Immuno-precipitation assay showed that IGFBP7 associated with AXAN2 and induced PTEN degradation. The expression of ANXA2 was significantly positive correlated with the expression ANXA2 in AML patients. The expression of IGFBP7 in AML, overexpression as well as knockdown of IGFBP7 in leukemia cells and in mice model, all suggest that IGFBP7 is a potential proto-oncogene. Collectively this work suggests that targeting IGFBP7 activity may be an effective therapeutic strategy for AML. Disclosures No relevant conflicts of interest to declare.

Background and Objectives: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency with microthrombocytopenia. Hematopoietic stem cell transplantation is a curative treatment for WAS. Unrelated cord blood transplantation (UCBT) become a more successive alternative donor transplantation for WAS with the improvement of high resolution HLA-typing method. However, graft rejection is the primary cause for mortality of WAS after UCBT. We hypothesized that the addition of fludarabine to a myeloablative conditioning regimen with antithymocyte globulin (ATG) would support engraftment for these children. Methods: Seventeen Children with WAS underwent UBCT matched 6-10/10 at higher resolution (HLA-A, -B, -C, -DRB1and DQB1) in our center between 2013 and 2018. The conditioning regimen consisted of fludarabine 160 mg/m2 , busulfan (BU)12.8 mg/kg, cyclophosphamide (CY)120 mg/kg, ATG 7.5 mg/kg, and cyclosporine and mycophenolate for GVHD prophylaxis. Median follow-up time is 2.5 years (range, 0.2 to 3.5 years). Results: All children had sustained donor cell engraftment and are stable engraftment. Twelve (71%) patients had cytomegalovirus (CMV) antigenemia and no one developed into CMV disease. Six (35%) children underwent acute Graft-versus-host disease (GVHD) (grade II-IV) and four (24%) children developed into chronic GVHD (cGVHD). Among these cases, only one child with HLA matched 6/10 died of kidney failure because of cGVHD. He experienced aGVHD with skin bullosa. Sixteen (94%) children are survival. Conclusion: Conditioning regimen consisted of Fludarabine combined BUCY and ATG was able to reduce graft rejection in the WAS children after UCBT without increasing infection related mortality. Future efforts will focus on further reducing rates of acute GVHD and extensive cGVHD. Disclosures No relevant conflicts of interest to declare.

Plasmacytoid dendritic cells (pDCs) derived either from adoptive transfer from the donor graft or stem cell reconstitution can attenuate and prevent graft-versus-host disease (GVHD) in both pre-clinical and clinical settings. However, the reconstitution of donor pDCs is severely impaired during GVHD via an unknown mechanism. Here we demonstrate that the histone methyltransferase Dot1l, which specifically catalyzes methylation of histone H3 at lysine 79 (H3K79me), is critical for regulating the commitment and differentiation of pDCs from hematopoietic stem cells (HSCs) and we observed its function was severely impaired in GVHD mice. We have previously demonstrated that Flt3L-induced DCs can program allogeneic T cells to reduce their GVHD toxicity (Blood 2016). Using this platform, we explored candidate histone methyltransferase(s) that affected pDC development. We found the inhibitor specific to Dot1l dramatically decreased the frequency and number of pDCs in cultures compared to other chemical probes that inhibit Ezh2, MLL1, G9a and Jmjd3, respectively. Under steady-state condition, pDCs develop from HSCs through successive steps of lineage commitment and differentiation: multiple potent progenitors (MPP) → macrophage and DC progenitors (MDP) → common DC progenitors (CDP). Upon culturing in the presence of Flt3L+SCF, MPP produced 2-fold and 5-fold more pDCs than MDP and HSCs, respectively, over 6 days' incubation. For this reason, we focused on evaluating the effect of Dot1l deletion on pDC development from MPP and CDP, which represent the early and later stage of pDC progenitors, respectively. To examine the specific role of Dot1l in DC development, we crossed Dot1l conditional knock mice (Dot1lf/f) to ER-Cre B6 mice to generate ER-Cre.Dot1lf/f B6 mice. We administered tamoxifen to ER-Cre.Dot1lf/f B6 mice at day 0 and day +1 to delete Dot1l, highly purified MPP and CDP, and cultured them in the presence of Flt3L+SCF. Deletion of Dot1l led to significant decrease of (3- to 5-fold) pDCs from MPP but marginally decreased CDP production of pDCs. These results suggest that Dot1l is required for the initial commitment and differentiation of MPP into pDCs or the expansion of selected pDCs. To test it, we added Dot1l inhibitor SGC to the cultures containing wild-type (WT) mouse-derived MPP, CDP and pDCs. Inhibiting Dot1l with SGC decreased both the frequency (3-fold) and number (5-fold) of pDCs in the MPP culture compared to control, however, it did not affect the generation of pDCs from the cultures of either CDP or pDCs. Thus, Dot1l regulates the commitment and differentiation of pDCs during the MPP stage. To understand the molecular mechanism by which Dot1l regulates pDC commitment and differentiation, we examined the expression of transcription factor Tcf4, which promotes pDC development, and Id2, which antagonizes Tcf4 effects on pDCs. Dot1l inhibition led to 2-fold reduction of Tcf4 expression and 5-fold increase of Id2 in Flt3L-induced bone marrow cells. ChIP assays confirmed the presence of high amount of Dot1l-catalyzed H3K79me2 at the promoter regions of Id2 and Tcf4 loci. Retroviral introduction of Tcf4 into hematopoietic progenitors lacking Dot1l restored their capacity to produce pDCs in cultures. These findings clearly establish that Tcf4 is the down-stream effector of Dot1l to control pDC development. Building on these observations, we finally examined whether GVHD may impair the reconstitution of donor pDCs through a mechanism of reducing Dot1l expression and function. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we confirmed the loss of donor pDCs in GVHD recipients at day 14 and day 21 after transplantation and, observed that these GVHD mice had 3- to 12-fold fewer MPP and CDP compared to normal donor mice. Additionally, both HSCs and MPP derived from GVHD mice showed significant reduction of H3K79me2 compared to their normal counterparts. Finally, adoptive transfer of donor BM-derived pDCs attenuated the incidence and severity of GVHD in Balb/c mice receiving B6 T cells. Collectively, these data suggest that GVHD-mediated inflammation profoundly decreases Dot1l function in engrafted donor DC progenitors, leading to damaged reconstitution of donor pDCs. Novel strategies that target Dot1l and its-regulated transcriptional programs may improve the reconstitution of donor pDCs to inhibit GVHD while also promoting anti-leukemia activity in recipients undergoing allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Objective: To describe the epidemiological profile, cytogenetic and molecular aberrations and the survival rate of patients with acute myeloid leukemia (AML) in a province reference pediatric hospital and explore their clinical features and prognosis. Patients and Methods: This is aretrospective Clinical-epidemiological study. The cohort of this study included cases of newly-diagnosed pediatric patients with non-M3- AML between 2010 and 2015, with the age younger than 14 years. The clinical characteristics such as gender, age, subtype of FAB, blood routine, bone marrow blast at the first visit, cytogenetics and molecular markers were analyzed in correlation with their prognosis between different characteristics groups. Survival analyses were calculated by Kaplan-Meier survival curves and the log rank test. Multivariate analyses on categorized data were performed using Cox proportional hazards model. Results: Of the 165 patients studied, 42.4% were females and 57.6%, males, with a age younger than 1 year in 4.8%, from 1 to 10 years in 73.4%, and older than 10 years in 21.8% ( median age 6.8 years ). According to FAB subtype, the majority subtypes were M2, M4 and M5, for 42%, 21.3% , and 26% respectively. 40.6% of patients presented a WBC count below 10 X 109/L at diagnosis while 12.7% of patients higher than 100 X 109/L. 77.0% of patients had less than 90g/L hemoglobin, and 47.9% of patients had less than 50 X 109/L platelets. In 70.0% of patients, the percentage of blasts in bone marrow was higher than 50% at diagnosis. The most common cytogenetic abnormalities in these children, including t(8;21)(q22;q22), inv(16)(p13.1q22) and 11q23/MLL-rearranged abnormalities were detected, with the occurrence of 34.1% , 12.2% and 14.0% respectively. The recurrent gene mutations rates are as the follows: 2.0% FLT3-ITD negative and NPM1 mutated, 4.9% FLT3-ITD positive and NPM1 wild type, 2.0% FLT3-ITDpositive and NPM1mutated, 3.9% FLT3-TKDpositive, 18.6% c-KIT mutation, and 2.9% PTPN11 mutation. Among the 165 cases of non-M3-AML patients, 114 cases achieved complete remission (CR) (69.1%) after one course of chemotherapy, 36 cases were relapsed, and 51 patients accepted hematopoietic stem cell transplantation (HSCT). The 3-year relapse-free survival (RFS) and overall survival (OS) rates were (62.5±3.5)% and (70.6%4±4.6)%, respectively. We failed to correlate the OS with the clinical parameters, such as gender, age, WBC, hemoglobin, blasts in BM, cytogenetic abnormalities except MLL-rearrangements, and gene mutations except FLT3-ITD and PTPN11 mutations . The patients with high platelet count, MLL -rearrangements, FLT3-ITD or PTPN11 mutation exhibited a significantly low OS rate (P£¼£¼(Table 1). Meanwhile, the RFS rate was correlated with the platelet count, FLT3-ITDand HSCT. respectively (Table 1). Multivariate analysis demonstrated that higher platelet count at diagnosis, MLL-rearrangements, FLT3-ITD and PTPN11 mutation were risk prognostic factors in childhood AML, while HSCT was a favorable factor. Conclusion:Higher platelet count at diagnosis, MLL-rearranged abnormalities, FLT3-ITD and PTPN11 mutation were poor prognostic markers in pediatric AML. HSCT could effectively improve the clinical outcome of AML patients. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

Administration of near-myeloablative courses of chemotherapy followed by hematopoietic stem cell transplantation (HSCT) is considered the standard of care for children with acute myeloid leukemia (AML). However, this approach is associated with severe impairment of hematopoiesis and immune response, high rate of toxic death, and a high economic burden for families. Recent genomic studies show that the failure associated with treatment is due to the selection of chemotherapy-resistant clones present at diagnosis. In this study, we (1) report the outcomes of children with AML who received low-intensity induction chemotherapy, and (2) evaluate the quality of remission by using next-generation sequencing. From July 2012 to May 2016, 90 Chinese children with AML who had white blood cell counts less than 50.0×109/L were treated at a single center. Of them, 31 (Group I) received dose-reduced induction chemotherapy because they had an active infection or had socioeconomic constraints. The remaining 59 children (Group II) received standard-dose chemotherapy. The induction regimen in Group I consisted of homoharringtonine 1 mg/m2, i.v., once daily, 7 doses; cytarabine 10 mg/m2, i.v., q12 h, 20 doses; and G-CSF 5 µg/kg, i.v., once daily, 10 doses (HAG). On day 28 of induction, Group I patients with Whole-exome deep sequencing was performed for 20 patients who achieved morphologic remission. The variant allele frequency (VAF) of non-synonymous mutations (median, 5 mutations; range 2-11 mutations) was analyzed at day 28 of each induction course. After induction I, the average reduction in VAF for all mutations was 76.3% and 99.0% in Group I and II patients, respectively. After induction II, of 16 patients analyzed, all mutations cleared under the VAF threshold of 2.5% in 7 of 8 patients in Group I and 6 of 8 patients in Group II. 77.4% and 69.4% of mutations were undetectable in Group I and II patients, respectively. At the molecular level, both regimens achieved similar clearance efficiencies, but patients given the low-intensity regimen had a slower clearance than those given the high-intensity regimen. Interestingly, exome sequencing of the primary and relapsed tumors from 1 patient in each cohort revealed that the dominant clone remained the same as that at diagnosis in the patient treated with the low-intensity regimen but changed in the patient treated with the high-intensity regimen. Our preliminary results suggest that children treated with HAG/MAG for induction, followed by conventional post-remission strategies, have similar outcomes as those treated with the high-intensity induction regimen. Although patients with active infections and a high financial burden were recruited to Group I, our clinical and genomic analyses suggest that HAG/MAG represents a valuable alternative to conventional chemotherapy and can be used in the general pediatric population with AML. The molecular signature of patients responsive to HAG/MAG and its prognostic implications remain to be determined. Disclosures No relevant conflicts of interest to declare.

Background Approximately 15% of children newly diagnosed for acute lymphoblastic leukemia (ALL) present with the T cell lineage (T-ALL), which historically has had inferior outcomes compared to the children with precursor-B cell ALL. In this study, we analyzed the efficacy of the Chinese Children Leukemia Group 2008 (CCLG-ALL 2008) protocol on pediatric T-ALL patients enrolled from 4 centers located at north, east, and southwest of China, to identify specific prognostic factors. Patients and methods Patients enrolled on the CCLG-ALL 2008 Protocol came from four hospitals in China: two hospitals in the southwest, one in the east, and one in the north, with 33, 37, 34 and 48 patients respectively. The study was approved by the hospitals' Ethical Committee, with informed consent obtained from patients' patents or legal guardians. Definitions: Failures of relapse-free survival (RFS) included relapse and death of any cause. M1: Blast 25%. Results Outcome disparity is observed across the four hospitals (Table 1 and Figure 1). Response to prednisone window treatment (Good vs. Poor) was highly prognostic and its prognostic value held universally across the centers despite the outcome disparity across the centers and abandonment: log-rank test stratified by centers (hospitals) P value = 0.0311, and by abandonment (Y vs. N) P = 0.0004. Multivariable Cox regression analysis showed that response to prednisone window treatment was an independent early (before end of remission induction) prognostic factor (Table 2). The hazard ratio of prednisone response (Poor vs. Good) was 2.6 (95% CI 1.49 to 4.57) after accounting for RFS disparities across centers and abandonment. When further adjusted for post-induction prognostic markers by Day-35 and Month-three bone marrow status (M1, M2 or M3), response to predisone window remained boarder-line significant (P=0.0683). Prednisone response was only significantly associated with Day-15 bone-marrow status, with poor responders tended to likely have M2 and M3 marrow status at Day 15 (Table 3). Day-15 and Day-35 marrow status was not significantly associated with RFS, but patients with Day-35 M2 and M3 status tended to fail early. Marrow status before intensification (Month 3) seemed highly associated with RFS, with all 4 M3 patients failed within 1 year. The percentage of abandonment in the southwest 1, the southwest 2, the east, and the north hospital was 21.6%, 33.3%, 8.8% and 25% respectively; the difference was not significant (P=0.1078). Nonetheless the east hospital seemed to have distinctly lower percentage of abandonment. The percentage of non-urban patients in the southwest1, the southwest2, the east and the north is 52.8%, 75.8%, 61.8% and 68.8% respectively; this difference was not significant (P=0.2171), although the percentage seemed higher in southwest. Patients from urban area tended to fair better in RFS but the difference was not statistically significant (P=0.1785). There was a weak trend that abandonment rate tended to be higher among non-urban patients, but not statistically significant (P=0.3361). Patients' Day-35 bone marrow status (with M2 and M3 marrow related to higher likelihood of abandonment), abandonment and socio-economic status might be relevant factors for the inter-center disparity in RFS. With the current data however, we are unable to identify any strong factor that can explain the inter-center disparity of treatment outcome. Conclusion Response to prednisone window treatment is a universal early prognostic marker in Chinese children with T lineage ALL despite outcome disparity. Disclosures No relevant conflicts of interest to declare.

Objective: To investigate the effect and mechanisms of KPT-330 on cell apoptosis of human leukemia cells. Methods: CCK-8 assay was used to quantify the growth inhibition of cells after exposure to KPT-330 for 24 hours. Fluorescence microscope was used to observe the morphological change of leukemia cell when treated with KPT330 for 24 hours. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the expression level of CRM1 and the activation of apoptosis related protein Caspase-9、Caspase-3 and PARP level. LncRNA microarray was used to analyze the differentially expressed genes from the cluster analyses. Results: The inhibition of cell proliferation of leukemia cells was in a dose-dependent manner and KPT-330 treatment induced expression of CRM1 down-regulated .Abnormal cells were observed under fluorescence microscopy. Cell apoptosis, cell cycle and activation of apoptosis markers demonstrates that KPT-330 induced apoptosis in leukemia cells. LncRNA microarray analysis showed differently expressed mRNAs and lncRNAs in KPT-330 treated HL-60 cells compared with control group. Molecular function analysis showed that KPT-330 induced apoptosis in leukemia cells partially related with Glutathione metabolism and Toll-like receptor signaling pathway. Conclusion: In this study, KPT-330 treatment resulted in inhibition of cell proliferation and induction of apoptosis in leukemia cells. LncRNA microarray analysis showed differentially expressed genes and lncRNAs in KPT-330 treated HL-60 cells and we demonstrated that KPT-330 induced apoptosis in leukemia cells partially related with Glutathione metabolism and Toll-like receptor signaling pathway. These results may provide new clue about molecular mechanism of KPT-330 induced apoptosis; however, underlying details will be required to determine further. Taken together, our findings suggest that for the first time that KPT-330 may act as new candidate's drug for leukemia. Disclosures No relevant conflicts of interest to declare.

The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation. Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people. In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. Disclosures No relevant conflicts of interest to declare.

Objective and Purpose: Despite improvements in medical management, both engraftment syndrome (ES) and pre-engraftment syndrome (pre-ES) which were named as peri-engraftment (peri-ES)remain associated with severe morbidity and decreased the survival following hematopoietic stem cell transplantation (HSCT). Though many studies on peri-EShave been published in recent years, there is no report on the incidence of peri-ES and related factors in pediatric HSCT, Meanwhile, the intervention with MP on peri-ES remains controvertial. Methods and patients: We retrospectively analyzed the data of 34 cases of pediatric allo-HSCT patients and the effect of methylprednisolone (MP) on the outcome of children with peri-ES transplanted between Nov 2010 and Dec 2013. The stem cell sources came from bone marrow alone [n=7], combining with peripheral blood [n=10], and cord blood alone [n=10], combining with bone marrow anf peripheral blood (n=7). Clinical characteristics and HSCT type were illustrated in Table 1 and 2. The incidence rate of peri-ES in cord blood transplantation (CBT), haploid transplants and sibling matched donor were 88.24%, 87.570% and 11.11%, respectively. All patients, who received either CBT or Hapolidentical SCT in conjunction of cord blood as the third part donor,developed peri-ES. We also identified that the peri-ES was highly associated with HLA disparity and mismatched ABO and aGvHD (Table 3 and 4).The median time of onset of peri-ES was 9 days after allo-HSCT. The most common symptoms of the peri-ES was eruthrodermous rash, followed by fever (Table 5). Twenty three children with peri-HSCT received intravenous MP at three doses of 0.5mg/kg, 1mg/kg, and 2mg/kg, respectively, based on the organs involved and the severity of peri-ES (Table 6). An excellent outcome was observed with relieving peri-ES in every patientand without influencingthe outcome of acute graft versus host disease (aGvHD), chronicgraft versus host disease (cGvHD), cytomegalovirus (CMV) infection, relapse, and overall survival (OS) with median follow up of xx months. (Table 4 and Figure 1 and 2). Conclusion: Peri-ES is closely associated with the stem cell source with the sequence of CB, PB and BM. Meanwhile, disparity of HLA type and blood type mismatch also contributed to peri-ES. peri-ES caneasily proceeded into aGvHD. MP efficiently relieved the process of peri-ES without any significant adverse event or affecting theoutcome of HSCT and can be recommended to control peri-ES in this patient population.Table 1.The clinical and laboratory characteristics of HSCT patientsViable NumberAge (year)Median, range9(1-16)SexMale/female20/14Primary diseaseAcute myeloid leukemia17Acute lymphoblastic leukemia4Chronic myelogenous leukemia2Aplastic anemia8Myelodysplastic syndrome (monosome 7)1Juvenile myelomonocytic leukemia2Number of infused nuclear cellsCB Median (range), 107/kg4.8(1.2-9.6)Haplo Median (range), 108/kg10.65(7.2-14.39)Sibling Median (range), 108/kg9.6(6.48-18.66)Number of infused CD34+ cellsCB Median (range), 106/kg0.32(0.047-0.52)Haplo Median (range), 106/kg4.6(1.92-8.36)Sibling Median (range), 106/kg4.4(2.5-8.37)HLA matching(low resolution) of A, B, DR6/6(sibling or CBT)175/6(Haplo or CBT)74/6(Haplo or CBT)63/6(Haplo or CBT)4Table 2.Risk factors for peri-ESRisk factorsperi-ES group(n=23 )Non peri-ES group (n= 11)Totalperi-ES /Total(%) SourceBM0440BM+PB15616.67BM+PB+CB707100CB1521788.24 Transplantation typesibling18911.11unrelated1521788.24Haploid71887.50 sexMale1282060.00Female1131478.57 ABO compatibilitymatched991850.00mismatched1421687.50 HLA disparitymatched891747.06mismatched1521788.24Neutrophilengraftmentmedian+14+13.5STR(2W)median96.8%95.7%Table 3.The Effect of MP on HSCT complicationsOutcome 0.5mg/kg1mg/kg2mg/kgNon peri-ESP 1P 2Neutrophil engraftment(median day)+16+15+13.513.50.5320.478aGVHD4/85/76/82/110.5290.010cGVHD1/82/72/82/110.7251.000CMV infection4/85/76/87/110.5291.000Relapse1/80/70/82/110.4980.239 BM, bone marrow; CB, cord blood; PB, peripheral blood; HLA, human leukocyte antigen; STR on second week. Note: P1: the comparison among three different doses of MP; P2: A comparison between peri-ES group and non-peri-ES group. Figure 1 Overall survival of pediatric allo-HSCT with and without peri-ES Figure 1. Overall survival of pediatric allo-HSCT with and without peri-ES Disclosures No relevant conflicts of interest to declare.

Objective Multidrug resistance-associated proteins 1 to 6 have been reported involved in a large number of tumors and have a close correlation with tumor multi-drug resistance. In this work, we detect the MRP2-6 genes expression in childhood acute lymphoblastic leukemia (ALL) by Q-RT-PCR and explore their clinical significance. Methods 156 patients at different stages of ALL were enrolled in this study and treated by the protocol (CCLG-2008) during 2012 to 2013, including 67 cases at initial stage, 70 cases at complete remission, and 9 cases at relapse, 10 patients diagnosed as idiopathic thrombocytopenic purpura (ITP) as control. MRP1-6 genes’ expressions were detected using real-time quantitative PCR (QRT-PCR)and their clinical significance was analyzed by the SPSS software 16.0. P value below 0.05 was regarded as statistic significance. Results The median expression of MRP1 was 5.82 and 8.49 for initial and relapse group,respectively, which was statistic higher than that at complete remission, the latter was 1.99. MRP1 expression level had close correlation with ALL risk, the median of MRP1 expression was 4.28, 5.62 and 7.56 for standard-risk group (SR), intermediate-risk group (IR) and high-risk group (HR), respectively. Initial ALL children were divided into two groups including high expression group and low expression group by the median expression, the rate of sensitivity of blast cells to prednisone on 7th day was 70.6% in high expression group (n=34), which was statistic lower than that in low expression group which was 90.9% (n=33, P=0.035). The rate of complete remission on 33th day in high expression group was 64.7%, while 87.9% in low expression group, which showed a significant difference between them (P=0.026). The rate of complete remission on 15th day in high expression group was 68.8%, and 69.7% in low expression group, which showed no significance between them (P=0.664). The transcription level of MRP1 in initial group of T-ALL (median=7.71) was statistic higher than that in B-ALL (median=5.18) (P=0.007). Correlation analysis indicated that mRNA expression level of MRP1 didn’t show any relationship with gender, age, WBC count, hemoglobin, platelet and blast percentage in bone narrow and peripheral blood at diagnosis. The median expression of MRP2 at initial stage was highest, higher than that at relapse and complete remission, but did not reach statistic significance. However, the median expression of MRP3 at initial stage was highest and statistic higher than complete remission. MRP4 and MRP5 showed a similar pattern in their expression, namely, high expression for relapse, intermediate expression for initial stage and low expression at complete remission which reached statistic significance. The median expression of MRP6 for relapse was 2.003, which was higher than initial and complete remission group, but the differences among them were not significant. The median of MRP2 and MRP5 expression for intermediate-risk group (IR) (Median MRP2=4.622, Median MRP5=1.712) was higher than standard- risk group (SR) (Median MRP2=3.279, Median MRP5=1.277) and high risk group (HR) (Median MRP2=2.145, Median MRP5=1.673). However, there was no statistical significance among them. The median of MRP4 expression was increase continually with the risk of ALL, but did not reach statistical significance among them; the median of MRP6 expression for high-risk group (HR) was higher than standard-risk group (0.8812 vs 0.6205) and intermediate-risk group (0.8812 vs 0.4053), but the differences among them were not significant either. Initial ALL children were divided into two group including high expression group and low expression group by median expression, and evaluated their prediction on the treatment response on 7th, 15th and 33th day. The results revealed no significant difference between them, neither between B-ALL and T-ALL. Single factor analysis showed that MRP2 has relationship with platelet count at diagnosis and MRP4 has relationship with gender. Conclusions In children ALL, the expression of MRP1 is closely related with immunophenotyping, treatment response, hazard level and disease relapse which was a poor biomarker for ALL prognosis. MRP2-6 has a different expression pattern. MRP4 and MRP6 mRNA expression showed a close relation with relapse. Disclosures No relevant conflicts of interest to declare.

Background: B cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. The t(1;19) translocation leukemia accounts for a quarter of pre-B ALL and up to 5% of all ALL patients, in which the transcriptional activator E2A and homeobox pre-B-cell leukemia transcription factor 1 (PBX1) fuses, resulting in expression of the chimeric transcription factor E2A/PBX1. E2A/PBX1 has been proved to be an oncogene and could induce malignant transformation. Methods: (1) Childhood B-ALL patients were collected and the stem/progenitor cells (CD34+CD38-) and leukemia cells (CD19+) were sorted with BD FACS Aria III. The autophagy level in these cells was measured by real-time Q-PCR, including gene expression of Beclin1, Atg7, Atg5, LC3 and p62. Normal bone marrow cells from healthy donors were used as control. (2) E2A/PBX1 fusion gene positive pre-B ALL 697 cells were used to establish leukemia mouse model and the autophagy activity in the mice was enhanced by administration of rapamycin. Mice were sacrificed three weeks post treatment, leukemia phenotype was then identified and E2A/PBX1 oncoprotein of liver was detected by western blotting. (3) Autophagy and ubiquitination were manipulated with inhibitors or starvation in 697 cells and the degradation mechanism of E2A/PBX1 was explored. Co-localization of E2A/PBX1-LC3 and E2A/PBX1-Ub was observed by confocal microscopy and quantified by Amnis image flow cytometry. Results: (1) B-ALL primary cells from childhood patients show down-regulated level of autophagy. (2) The NOD-SCID mouse model study shows that activating autophagy of mice by rapamycin improved the survival of leukemia animals, prevented leukemiagenesis by inhibition on the transplanted leukemia cells (examined by blood cell counting, liver HE staining and expression of CD 45, 10, 19 from transplanted human 697 cells by flow cytometry), promoted the degradation of oncoprotein E2A/PBX1 (by Western blotting)), and more importantly, restored hematopoietic stem cells (LSKCD34- cell number detected by flow cytometry). (3) The ALL 697 cell line study shows that activation of autophagy by rapamycin and starvation could down-regulate E2A/PBX1 expression detected by flow cytometry and western blotting. The confocal microscopic results show co-localization of E2A/PBX1 with autophagy marker GFP-LC3 in both rapamycin and starvation treatment groups. To confirm the degradation mechanism, autophagy inhibitor (3-MA or Baf-A1) and ubiquitin-proteasome inhibitor (MG132) were used to treat 697 cells. The results show that inhibition of autophagy in the early stage by 3-MA fails to degrade E2A/PBX1 in 697 cells, but ubiquitination also contributes to the degradation of E2A/PBX1. Quantitative analysis shows increased co-localization percentage of E2A/PBX1-LC3 in rapamycin and starvation treatment groups and increased co-localization of E2A/PBX1 with Ubiquitin in starvation group; but MG132 treatment inhibited the co-localization of E2A/PBX1-Ub induced by starvation, indicating a collaborative role between autophagy and ubiquitination in the degradation of E2A/PBX1. Conclusions: B-ALL primary cells from patients show low autophagy activity; Autophagy activation fights against B-ALL by inhibition on transplanted leukemia cells, degradation of oncoprotein E2A/Pbx1 and restoration of hematopoietic stem cells in the NOD-SCID B-ALL mouse model; autophagy collaborates with ubiquitination in the degradation of E2A/PBX1 in the 697 cells, thereby proposing a novel strategy for targeted therapy on childhood B-ALL. Disclosures No relevant conflicts of interest to declare.

Lysosomal-associated protein transmembrane 5 (LAPTM5) encodes a transmembrane protein in lysosomal, which is specifically expressed in hematopoietic system. Autophagy, a lysosomal degradation pathway, is now emerging as an importance prosurvival or prodeath factor in response to some chemotherapy. In our previously study, we found the expression of LAPTM5 is closely related with acute lymphocytic leukemia, but exactly role of this gene in acute leukemia(AL) was unknown. To investigate the association of LAPTM5 with autophagy, we overexpressed LAPTM5 in K562 cells, and the results showed LAPTM5 significantly decreased autophagy activity, which measured by testing the conversion of LC3-I to LC3-II and LC3-II dots using western blotting and imaging flow cytometry, respectively(Figure 1 and 2). Moreover, PCR array was performed to detect the changes of 92 genes regulating autophagic activity in LAPTM5 overexpressed cells, and 73 autophagy-related genes were found downregulated. Finally, the overexpression of LAPTM5 significantly reduced the pH value in lysosomal, which was very essential for autophagy-mediated degradation activity (Figure 3). Taken together, these results suggest that LAPTM5 decreased autophagy activity though downregulation of pH value in lysosomal. Therefore, LAPTM5 might represent a potential target modulating autophagy activity to increase sensitivity to chemotherapy in treatment of AL. Figure 1 LAPTM5 gene suppressed K562 cells' autophagy activity. K562 cells transfected with LAPTM5(L) and empty vector (P) were cultured in HBSS conditional media for 6 hrs. On the time point of 0hrs, 3hrs and 6hrs, transfected cells were collected and run Westernblot with the antibody of LC3-II and LC3-I. The ratio of LC3-II/LC3I which represented the extent of autophagy activity decreased in K562/LAPTM5 (L). Figure 1. LAPTM5 gene suppressed K562 cells' autophagy activity. K562 cells transfected with LAPTM5(L) and empty vector (P) were cultured in HBSS conditional media for 6 hrs. On the time point of 0hrs, 3hrs and 6hrs, transfected cells were collected and run Westernblot with the antibody of LC3-II and LC3-I. The ratio of LC3-II/LC3I which represented the extent of autophagy activity decreased in K562/LAPTM5 (L). Figure 2 Autophagy activity of K562 transfectants were detected by Flow. K562 cells transfected with LAPTM5(L) had less fluorescent spots than that of K562 transfected with empty vector (P). Figure 2. Autophagy activity of K562 transfectants were detected by Flow. K562 cells transfected with LAPTM5(L) had less fluorescent spots than that of K562 transfected with empty vector (P). Figure 3 Lysosomal acid decreased in K562 cells transducted with LAPTM5(L). Lysosomal was marked by LysoSensor™ Green DND-189£¨50 nM£©and run Flow cytometry. Fluorescent intensity represented pH value. More intensity means lower pH value. Figure 3. Lysosomal acid decreased in K562 cells transducted with LAPTM5(L). Lysosomal was marked by LysoSensor™ Green DND-189£¨50 nM£©and run Flow cytometry. Fluorescent intensity represented pH value. More intensity means lower pH value. Disclosures No relevant conflicts of interest to declare.

Background and purpose The effect of very high WBC over 100 or 200*109/L at initial stage on the outcome of pediatric acute lymphoblastic leukemia (ALL) was much argued by different clinical trials. CCLG-2008 clinical trial has been carried out on pediatric ALL for 5 years in China and reports on the evaluation of very high WBC in pediatric ALL are still lacking. In this study, we assessed the clinical features, complications and outcome of ALL patients with hyperleukocytosis treated by CCLG-2008. Methods Between March 2009 and June 2013, 399 ALL patients were enrolled in this clinical trial in the Children's Hospital of Soochow University and were treated with the protocol on risk-based principle. 121 patients who had an initial WBC count above 50*109/L were further divided into four groups as 50-100*109/L, 100-200*109/L, 200-300*109/L, 300*109/L. The clinical features, complications, the rate of relapse, event free survival (EFS) and overall survival (OS) within the four groups were analyzed by using SPSS 18.0. Continuous variables were defined as median values if they were not normally distributed and Mann-Whitney test was used in these dates. Groups for categorical variables were analyzed by Chi-square or Fisher's exact tests. Relapses, EFS, and OS were estimated for various patient subgroups by using the Kaplan-Meier method, and the log-rank test was used to compare survival curves between groups. Cox regression analysis was performed to evaluate predictors of adverse events. P-values £¼0.05 were considered statistically significant. Results High WBC patients count for 30.33% for all patients (121/399), among which 38 cases (31.4%) with initial leukocyte counts of 100-200*109/L, 8 cases (6.61%) with a leukocyte count of 200-300*109/L, and 19 patients (15.70%) with a leukocyte count of 300*109/L. The data indicated that less than 1 year at diagnosis, T-cell immune phenotype, massive hepatomegaly, SIL-TAL1 fusion gene, serum levels of lactic dehydrogenase (LDH), and serum levels of α-Hydroxybutyrate (α-HBDH) were positively correlated to super-hyperleukocytosis, while fibrinogen(Fib) was negatively correlated to high WBC. Early complications occurred in 42 of 121 patients (34.7%). Of which, 17 patients had respiratory events,11 patients suffered a tumor lysis syndrome (TLS), 4 cases diagnosed as intracranial hemorrhage, 5 cases accompanied with disseminated intravascular coagulation (DIC) and 5 cases died at their early stage (ED). TLS, cerebral hemorrhage, DIC and ED occurred significantly more frequently in patients with extreme hyperleukocytosis. For 121 patients with high WBC counts, 101 patients completed the treatment with CCLG-2008 protocol, 20 cases gave up due to early complications, early death or economic status. The complete remission (CR) rate was 86.1% (87/101). No significant effect of a high WBC was found on MRD on day 33. 24 /87 (27.6%) patients relapsed with an initial WBC of 50-100*109/L in 3 cases, 100-200*109/L in 9 cases, and 300*109/L (50%) in 12 cases, respectively. No case with WBC of 200-300*109/L relapsed probably due to the smallest number of patient in this category. Bone marrow was the most common site of relapse involved either alone or in combination (20 cases, 83.8%). 13 patients (54.2%) relapsed at early or very early time after CR. The treatment related death (TRD) occurred in 18 /121patients (14.9%) with high WBC with a distribution as WBC 50-100*109/L in 4 cases, 100-200*109/L in 2 cases, 200-300*109/L in 4 cases, and 300*109/L in 8 cases. Patients with WBC ¡Ý 300*109/L had a significantly shorter EFS (P=.003), higher relapse (P=.019), higher ED (P=.001) and higher TRD (P=.036) (Figure 1 and 2). However, the WBC count seemed not to significantly influence the OS (Figure 2). Conclusion In conclusion, WBC300*109/L confers a poorer prognosis on pediatric ALL patients due to increasing adverse events such as higher relapse, higher ED and TRD which resulted in lower EFS. More efficient protocols need to reduce early complications, early relapse and improve their OS for these patients Figure 1 TRD was close related with super-high WBC (P=.036) Figure 1. TRD was close related with super-high WBC (P=.036) Figure 2 5 years EFS and OS of ALL patients with high WBC Figure 2. 5 years EFS and OS of ALL patients with high WBC Disclosures No relevant conflicts of interest to declare.

Objective: MLL gene rearrangement in pediatric acute lymphoblastic leukemia (ALL) was regarded as high risk factor because of the poorer outcome of overall survival(OS). Some reports further proved that patients with MLL/AF4 had the worse outcome than MLL rearranged with other partners. CCLG-ALL2008 protocol has been carried out in China for more than 5 years. However, there was no reports to evaluate its efficiency on pediatric ALL patients with MLL rearrangement. In this study, we analyzed the data of ALL patients to compare the outcome of patients with MLL rearrangement positive and negative treated by CCLG-2008 protocol. Methods During the period from 2009 to 2013, 379 patients were enrolled in this protocol, of which 19 cases were MLL rearrangement positive and treated with CCLG-ALL2008 protocol for high-risk (HR) group. The treatment efficiency was evaluated on the time points of day 7, day15, day 33 and 12th week after treatment, respectively. OS and treatment-related mortality (TRD) was calculated within high risk groups of MLL positive and negative. Results Patients with MLL rearrangement positive accounted for 5.01% of all patients. The characteristics and response to the treatment were illustrated in Table 1. Cases younger than 2 years old, with initial white blood cell (WBC) 50*109/L , or MRD more than 10-2 on day 33 had a lower OS (P0.05). Compared with the male, female patients had a better 5 year EFS (100% versus 50%±17%, P Conclusion Factors including female, older than 2 years of age, sensitivity to prednisone, are good prognostic index in predicting the outcome of 5 years of OS and EFS in childhood ALL with MLL gene rearrangement. Table 1 Clinical and laboratory characteristics of ALL patients with MLL positive Table 1. Clinical and laboratory characteristics of ALL patients with MLL positive Figure 1 Gender exerts influence on 5 years EFS in MLL rearrangement group Figure 1. Gender exerts influence on 5 years EFS in MLL rearrangement group Figure 2 Figure 2. Age influenced 5 years EFS in MLL rearrangement group Disclosures No relevant conflicts of interest to declare.

Background and purpose CCLG-2008 protocol has been carried out for more than 5 years in most parts of China. This retrospective cohort study analyzed the clinical features and the role of prognosis index on the outcomes of patients with childhood acute lymphoblastic leukemia (ALL) treated by CCLG-2008 protocol in the Children's Hospital of Soochow University, Suzhou, China. Procedure From 2009 to 2013, 379 evaluable patients were enrolled in this protocol. ALL diagnosis was made by MICM and early prognosis index including age, gender, white blood cell (WBC), immunotype, molecular findings, karyotype and prednisone response were evaluated as predictors of adverse events by using SPSS 16.0. P-values Results The overall relapse treated with CCLG-2008 protocol was 17.4%, with 16.9% and 23.3% in B-ALL and T-ALL. Bone marrow, central nervous system and testis relapse counts for 95.4%, 3% and 1.52% respectively. Comparing with B-ALL (n=349), T-ALL (n=30) was associated with a high WBC (>100*109/L, P120 months, P=0.018), more adverse karyotype distribution, a poor prednisone response (P100*109/L) or increased age (>120 months) showed higher disease-relapse risk than other groups (P=0.003, 100*109/L. Patients with WBC < 50*109/L displayed the best survival while those with WBC higher than 100*109/L had the worst survival (Figure 1). The results of molecular alterations detected by multiple RT-PCR suggested that the MLL gene related and BCR-ABL positive patients exhibited the worst trend for EFS and OS (Figure 2a). Those with favorable karyotypes exhibited prolonged EFS and OS (Figure 2b). 44 cases who were not responsive well to prednisone displayed a shorter EFS and OS (Figure 3). Cox regression analysis indicated that WBC count, age, karyotype and prednisone response are independent factors as prognosis index for pediatric B-ALL treated by CCLG-2008 protocol . Conclusions Patients with T-ALL subtype often had higher risk factors, such as high WBC counts, increased age, adverse genes and karyotypes. It is more prone to resistance to prednisone and disease relapse, and has a poor survival outcome. WBC count, age, karyotype and prednisone response are independent factors in predicting prognosis at initial stage of B-ALL. Patients with either very high WBC ¡Ý100*109/L or older than 120 months have short survival and need more intensive therapy or novel drug combination regimen to improve their EFS and OS. Figure 1 EFS and OS of patients with different number of WBC. Patients with WBC >100*109/L have the shortest survival. Figure 1. EFS and OS of patients with different number of WBC. Patients with WBC >100*109/L have the shortest survival. Figure 2 a EFS and OS of B-ALL patients with fusion gene positive or negative. Figure 2. a EFS and OS of B-ALL patients with fusion gene positive or negative. Figure 2b EFS and OS of B-ALL patients with different risk of karyotype. Figure 3 EFS and OS of patients with different response to prednisone. Figure 3. EFS and OS of patients with different response to prednisone. Disclosures No relevant conflicts of interest to declare.

S100A8 and S100A9 are two members of the S100 calcium-binding protein family, preferentially form functional heterodimers of S100A8/S100A9, and have been increasingly recognized as biomarkers in malignancies. Recent proteomic studies revealed that S100A8 and S100A9 played pivotal roles in hematologic malignancies and elevated expression of S100A8/S100A9 implicated in glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia (ALL). In addition, S100A8 proteomic expression in leukemic cells was reported to predict survival in AML patients. However, information on the gene expression level of S100A8 and S100A9 and their clinical correlation in acute myeloid leukemia (AML) is lacking. Using the real-time quantitative RT-PCR, we analyze the transcription levels of S100A8 and S100A9 in AML patient leukemic specimens underwent pre-planned induction chemotherapy. A total of 189 patient cases at different stages of AML (excluding acute promyelocytic leukemia [APL]), including 91 newly diagnosed AML, 64 patient remission marrow specimens and 34 patient specimens as well as 20 controls without leukemia were included in the study collected from the period between 2007 and 2011. Among the cohort of 91 newly diagnosed AML, the over all median OS was 20 months, and the median follow-up for survivors was 24 months (range: 17 to 60 months). There were significant positive correlations of transcription levels between S100A8 and S100A9 in AML patients from different stages. The expression levels of S100A8 and S100A9 in newly diagnosed and relapsed AML patients revealed no significant difference, but were both lower than those in complete remission and control group. Patients with high transcription level of S100A8 and S100A9 were predominantly in AML with myelo-monocytic differentiation (M4, M5) whereas those with low transcription level of S100A8 and S100A9 often showed more immature cytomorphology (M0, M1), erythrocytic or megakaryocytic differentiation. The subgroup of patient with high transcription level of S100A8 could be a predictor for inferior overall survival (OS) (P = 0.0012). High levels of transcription for both S100A8 and S100A9 in de novo AML patients could predict shorter OS than those with low levels after adjustment on their ages at diagnosis (P = 0.003). In a multivariate analysis for OS, high S100A8 transcription was a significant prognostic factor (P =0.001) after analysis adjustment for age (P = 0.019), bone marrow blast percentage (P = 0.04) and cytogenetic classification (P = 0.05) at diagnosis. Using a combination of S100A8 transcription level and cytogenetic risk classification, survival analysis gave result that the new stratification was highly correlated with the OS (P < 0.0001). With significantly different OS, patients from the intermediate-risk group can be divided into two subgroups (IH = cytogenetically intermediate-risk with S100A8 high transcription and IL = cytogenetically intermediate-risk with S100A8 low transcription). Patients from group IL emerged with a probability of OS similar to the cytogenetically favorable-risk group, whereas the survival curve of IH subgroup was close to the unfavorable-risk group. (Figure)FigureRisk stratification of de novo AML patients by a combination of age and cytogenetic characteristics with S100A8 expression levels. 1H = cytogenetically favorable-risk with S100A8 high expression, 1L = cytogenetically favorable-risk with S100A8 low expression, 2H = cytogenetically intermediate-risk with S100A8 high expression, 2L = cytogenetically intermediate-risk with S100A8 low expression, 3H = cytogenetically unfavorable-risk with S100A8 high expression, 3L = cytogenetically unfavorable-risk with S100A8 low expression.Figure. Risk stratification of de novo AML patients by a combination of age and cytogenetic characteristics with S100A8 expression levels. 1H = cytogenetically favorable-risk with S100A8 high expression, 1L = cytogenetically favorable-risk with S100A8 low expression, 2H = cytogenetically intermediate-risk with S100A8 high expression, 2L = cytogenetically intermediate-risk with S100A8 low expression, 3H = cytogenetically unfavorable-risk with S100A8 high expression, 3L = cytogenetically unfavorable-risk with S100A8 low expression. In conclusion, the transcription levels of S100A8 and S100A9 were significantly associated with development and prognosis of AML. Both S100A8 and S100A9 expression levels provided useful clinical information, and more importantly, S100A8 expression level significantly correlates with prognosis in addition to well-known cytogenetic risk factors, and could potentially further refine current stratification of de novo AML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 1447 Insulin-like growth factor binding protein 7 (IGFBP7) has been ascribed properties of both tumor suppressor and enhancer of cell proliferation. In solid tumors the important role of IGFBP7 as a tumor suppressor was revealed in several studies. In acute T-lymphoblastic leukemia (T-ALL), high IGFBP7 expression is associated with a more immature phenotype of early T-ALL, inferior survival, and predicts primary chemotherapy resistance. In a separate study, IGFBP7 acts as a positive regulator of ALL and bone marrow stromal cells growth, and significantly enhances in-vitro resistance to asparaginase. Higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (P=0.003) in precursor B-cell Ph negative ALL patients (n=147) treated with a contemporary polychemotherapy protocol. In acute myeloid leukemia, the role of IGFBP7 is largely unknown. In our previous published study [Hu et al, 2011], we demonstrated that IGFBP7 overexpressed in majority of childhood AML (n=66) at diagnosis and upon relapsed, but not at remission stage. We now further explore its mechanism in promoting AML cells proliferation. Compared with control, transfection of full length IGFBP7 in K562 cells [V-BP7] resulted in 23% increased of proliferation in 48 hours. Cell cycle analysis by flow cytometry showed decreased G0/G1 phase and increased S phase in V-BP7 comparing with control, suggesting enhanced cell cycle progression. While transfection of IGFPB7 siRNA produced an opposite effect of reducing the cell growth in K562 cells. In consistent with the nature of a secretory protein, the extracellular IGFBP7 level in the condition media from v-BP7 was significantly higher than that from vector control or parental K562 cells measured by ELISA. Incubation parental K562 cells in V-BP7 derived conditioned medium resulted in significant growth enhancement. Gene expression profiling (GEP) was performed on V-BP7 in contrast to parental K562 cells. Genes which were up-regulated or down-regulated more than 2 folds were regarded as significant difference. Among 10 verified genes, AKT3 showed the highest extent of up-regulation and IGFBP7 siRNA transfection reduced its expression. Cyclin D1 (CCND1) expression was also significantly up-regulated and validated by RT-PCR and Western blot. V-BP7 treated with an AKT inhibitor (Triciribine) at 2.5μM for 72 hours showed 50% reduction of cell viability. The cell cycle analysis indicated that triciribine reversed cell cycle progression in V-BP7, by increasing cells in G0/G1 phase and reducing cells in S phase. Western blot demonstrated that both phospho-AKT3 and CCND1 were down regulated after treatment with triciribine. Using real time RT-PCR, we further identified that IGFBP7 and AKT3 expression were significantly correlated (p=0.001; r=0.255) in 39 newly diagnosed childhood AML. Conclusions IGFBP7 aberrantly overexpressed in majority of childhood AML. IGFBP7 promotes proliferation of K562 cells and AML via overexpression/activation of AKT3 and CCND1. Disclosures: No relevant conflicts of interest to declare.

Abstract 1435 Abstract ABT-737 is a small molecule antagonist of BCL-2, BCL-XL, and BCL-w currently under evaluation in clinical trials in the oral form of ABT-263 (Navitoclax). Acquired resistance to BCL-2 inhibitors will inevitably emerge. Published pre-clinical data showed increased levels of BFL-1 and/or MCL-1 proteins in lymphoma, and concurrent up-regulation of BCL-XL and BCL2A1 in chronic lymphocytic leukemia, which are not targeted by ABT-737. To investigate potential mechanisms of resistance to BCL-2 inhibitors in acute myeloid leukemia, we developed resistant cell lines by long-term culture of HL-60 and MV4-11 cells with ABT-737, designated as HL-60R and MV4-11R. Parental lines, HL-60 and MV4-11 which were highly sensitive to ABT-737 with IC50 values of 30 nM and 90nM respectively, whereas those for HL-60R and MV4-11R were 10μM and 4.7μM respectively. Annexin V binding assay revealed that both resistant lines were resistant to ABT-737 induced apoptosis as compared to the parental HL-60 and MV4-11 cells. To explore the common pathway mediating acquired resistance to ABT-737, genes differentially expressed 2-fold or greater between parental and resistant lines on HL-60 and MV4-11 were studied. Interestingly, significant changes for BCL-2 family members were not found, suggesting that non-BCL2 family genes could play important roles in mediating the drug resistance to ABT-737. Among them, C3aR1 [Complement component 3a receptor 1] was significantly over-expressed in both resistant cell lines when compared to parental lines and verified with real-time RT-PCR. C3aR1 inhibitor, SB 290157, showed preferential cytotoxicity to both HL-60R and MV4-11R in a dose-dependent way and spared parental cells during 48 hrs in vitro MTT assay. Our findings suggest C3aR1 plays a key role in the resistance phenotype to Bcl-2 inhibitor and blocking C3aR1 revert the resistance. C3aR1 encodes a G-protein coupled seven trans-membrane receptor for C3a, an important inflammatory mediator. The C3a-C3aR axis modulated SDF-1–CXCR4 axis-dependent responses and regulated the homing of hematopoietic stem/progenitor cells into bone marrow. C3aR1 over expression has been associated with FLT3 and D835/I836 mutation cytogenetically normal acute myeloid leukemia, renal cancer and melanoma. We further investigate the role of C3aR1 in 138 cases of AML including 50 cases in initial diagnosis, 68 cases in remission and 20 cases in relapse with real-time RT-PCR. The result showed that C3aR1 expression is highest in initial stage and lowest in remission (p=0.006), there was no significant difference between initial and relapse stages (p=0.384). [Table 1] For newly diagnosed AML patients, high C3aR1 is statistically associated with younger age [median: 34 vs 43 years old], higher presenting WBC [median: 39K vs 27K], higher marrow % blasts [70 vs 55%], but not related to efficacy of first induction treatment, FAB classification or conventional chromosome risk groups.Table 1C3aR1 expression in AML clinical casesAML casesAge (year)C3AR1malefemalemedianrangeM-estimatorrangepInitial2822367-79199.777.49-2514.6P1 = 0.006Remission37313412-7092.2523.45-2286P2 = 0.046Relapse11944.513-54142.7512.4-2372.04P3 = 0.384Note: M-estimator was used for evaluating the differential expression of AML samples and the value was amplified by 104p1: initial vs remission; p2: relapse vs remission; p3 initial vs relapse The biological significant of C3a-C3aR axis in AML and resistance phenotype is intriguing and may suggest a novel mechanism of evading the apoptotic regulation by Bcl-2 gene family as suggested by our current study. Ongoing studies will further elucidate relationship of C3a-C3aR axis in AML. Disclosures: No relevant conflicts of interest to declare.

IGFBP7 (insulin-like growth factor binding protein 7) is a newly-identified protein which can bind with insulin-like growth factors(IGFs), with 45%~65% homology to other IGF binding proteins. Several studies reported that IGFBP7 may be involved in the development of meningioma, breast cancer and prostate cancer, while its correlation with human leukemia had not been reported yet. We have observed an induced expression of IGFBP7 in human leukemia K562 cells which were transfected with and overexpressed RbAp46 gene. Besides, the expression level of WT1, RbAp46 and IGFBP7 was coordinately regulated during the induced differentiation of NB4 acute promyelocytic leukemia cells (data not shown), suggesting a role of IGFBP7 gene in human leukemia.. To investigate the expression level of IGFBP7(previous known as Insulin-like growth factor binding protein related protein 1)in bone marrow (BM) of leukemia patients and its correlation with other leukemia biomarkers, Real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR) method was established for detecting IGFBP7 expression levels in BM of 127 patients with acute leukemia (AL), 24 with chronic myelogenous leukemia in chronic phase (CML-CP), 9 with CML in blast crisis (CML-BC) and 27 with non-leukemias. The M-Estimators of RbAp46N in 63 patients with newly diagnosed AMLs were higher than those of 27 with ALLs and 27 non-leukemic controls and 10 with AMLs in complete remission(CR)(193.80 v.s 88.84, 81.30 and 67.82, respectively). Although IGFBP7N was 101.13 in CML-BC and 168.77 in CML-CP, difference between them was not statistically significant. Difference in IGFBP7N values among CML-BC, AML, ALL and control groups was also not significant. The M-Estimators of IGFBP7N were lowest in M4 and highest in M5 among initial acute leukemia subtypes, and there was statistical differences between M4 and M5, and between M4 and M2. IGFBP7N expression level was not correlated to the expression of fusion genes BCR/ABL and AML1/ETO, but it was positively correlated with the expression level of PML/RARαfusion gene, the WT1 and RbAp46 gene. The correlation coefficiency was 0.71, 0.31 and 0.52, respectively. Our results suggested that IGFBP7 might act in WT1 mediating pathway to participate in acute myelocytic leukemias.

The methylation at CpG island in the promoter region of a gene is one of the important epigenetic mechanism which regulates the gene activity. To study the DNA methylation pattern of WT1 gene promoter region within hematologic neoplastic cell lines and its correlation with WT1 gene expression by using the PCR-based methods. RT-PCR and Methylation-specific PCR were performed to study the WT1 gene expression in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines and the DNA methylation status in promoter region of WT1 gene. After treatment of U937 cell line by 5-aza-CdR, a demethylation inducing agent, the changes of WT1 gene expression level and the methylation status in its promter region in U937 cells was determined. Our Results showed that HL-60, K562, KG-1, NB4, SHI-1 cell lines demonstrated higher level of WT1 expression, while extremely low level was found in 8226, Jurkat, Raji, U266 and U937. The DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cell lines. The WT1 gene expression in U937 was markedly enhanced after treatment with 5-aza-CdR in company with the decrease of methylated level and the increase of unmethylated level in its promoter region. These results indicate that modulation of the DNA methylation in WT1 promoter region is one of the epigenetic mechanisms to regulate its expression.

WT1, a tumor suppressor gene originally found to be responsible for the development of childhood kidney tumor, is now thought to be also involved in the pathogenesis of human leukemia. Since WT1 is reported to be expressed at much higher level in bone marrow cells from MDS and all subtypes of leukemia comparing to the normal blood cells, it is proposed to be a “panleukemic marker” for the diagnosis of leukemia. The expression level of WT1 may also have significance in predicting prognosis and monitoring relapse. However, this notion on the usage of WT1 is somehow still remaining controversial because WT1 is also expressed to a certain level in a small population of CD34+cells. To measure more accurately the expression level of WT1 in bone marrow cells of acute leukemia patients (ALs). Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH expression levels in BM of 108 ALs and 23 non-leukemias patients by LightCycler. Besides, BM cells of 15 AL patients, including a total of 111 BM specimen taken during the follow-up after allo-BMT were also subject to RQ-RT-PCR. Normalized WT1 expression level (WT1N) was determined as a ratio between WT1 and GAPDH times 104. The expression levels of WT1N in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those of 23 ALs with complete remission(CR) and 23 non-leukemic controls (108.50±137.08 and 135.00±107.87 vs 3.88±3.09 and 2.23±3.19). Statistical differences were found neither between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, WT1N expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias, but there were no statistic differences among the M1, M2, M3 and ALL subtypes. Furthermore, the WT1N levels were not correlated to WBC counts in peripheral blood, the blast ratio in the BM and the expression of MDR-1 at presentation, but do correlated to the leukemia cell chromosome karyotypes. The dynamic changes of WT1N levels in 2 patients during treatment suggested that WT1 expression levels could be used to monitor the disease outcome and predict relapse. In gereral, the dynamic curves of WT1 level following allo-BMT were consistemt with the tendnecy of changes in expression levels of corresponding fusion genefor MRD monitoring. A re-increment of WT1 expression level during follow-up could be detected 40 to 180 days earlier to hematological relapse. These studies have provided strong evidence suggesting that WT1 expression levels in cells of AL patients were markedly higher than an undetectable or a marginal level expressed in non-leukemias patients. WT1 can indeed be a marker for evaluating therapy efficacy in leukemias. WT1 expression levels in leukemia patients following allo-BMT measured by real time RT-PCR can be a useful tool for monitoring MRD and warning the clinical relapse during follow-up.

It has been well known that WT1 gene is overexpressed in leukemia cells regardless subtypes comparing to normal hematopoietic cells. The precise effect of WT1, whether an oncogenic or tumor suppressive effect, in leukemogenesis remains controversial. Isoforms of WT1 protein products caused by alternative splicing may exert different biological function. To investigate the role of WT1 product composed of four major isoforms in certain ratios in hematopoietic cells, we have established a leukemia cell line NB4 which stably expressed exogenous WT1 gene isoforms, then studied their effects on cell biologic behaviors including proliferation, apoptosis and differentiation and its possible molecular mechanisms. The eukaryotic expression recombinant vectors (pCB6+/WT1) containing 4 clones of full-length human WT1 isoforms (WTA: −17aa/-KTS, WTB:+17aa/-KTS, WTC: −17aa/+KTS, WTD: +17aa/+KTS) cDNA were transduced into the leukemia cell line NB4 by electroporation.The positive stable cell clones (NB4/WT1) were obtained. The integration and expresion of WT1 gene isoforms in NB4 cells were confirmed by PCR. RT-PCR and western blotting. We then mainly concentrated on the effect of WT1 isoform WTA (−17aa/-KTS) since this transgene will markedly change the WT1 isoform ratio in NB4 cells from +17aa/+KTS dominant to −17aa/-KTS dominant. The proliferation ability was measured by trypan blue exclusion assay, MTT assay, colony forming assay and cell cycles analysis. Morphology, NBT reduction and CD11b expression were examined to access the cell differentiation. AnnexinV binding tested by FCM and agarose gel electrophoresis were performed to access the susceptibility to action of apoptosis inducing agents. Expressions of PML/RARα, RbAP46, P21, P53, Bcl-2, Bcl-XL and C-myc genes in NB4/WTA cells were determined by semi-quantitative RT-PCR DNA microarray was used to explore the alteration of gene expression profiles in NB4/WTA cells. The proliferation rate of NB4/WTA significantly decreased as measured by growth curves and colony forming ability, while the NB4/WTA cells arrested in S stage increased. NB4/WTA cells treated with ATRA 0.5μM for 2 days were induce to partially differentiate compared to a much higher morphological differentiation rate and CD11b expression level in the negative control cells in same condition. After exposure to As2O3 at 0.8μM for 48 hours, the NB4/WTA cells,but not the control cells, exhibited features of apoptosis RT-PCR have showed increasing level of PML/RARα, RbAP46, P21 and C-myc gene expression, a decreased level of Bcl-2 and a relative constant expression of P53,Bcl-XL, VEGF, CyclinD1 and CyclinD2 in NB4/WTA cells. The gene expression profiles were found changed in transfected NB4 cells. 89 of 4096(2.17%) genes were found to have a differential expression pattern, most of which (nearly 88.7%) were down-regulated. Our results indicated that overexpression of exogenous WT1 isoform (−17aa/-KTS) gene inhibited the proliferation of leukemia cells by delaying the progression of S into G2/M stage in cell cycle and inducing cell apoptosis by down-regulating the Bcl-2 gene expression. WTA gene could also partially inhibit the differentiation of NB4 cells. The introduction and expression of exogenous WT1 isoform gene can change the gene expression profiles in NB4 cells, leading to inhibition of cell growth, retardation of differentiation and more sensitive to apoptosis inducing agents.