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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.

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.

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.