ALBERT

Description: Objectives: Although very rare in children, chronic myeloid leukemia (CML) can be cured by hematopoietic stem cell transplantation (HSCT). However, the 5-year survival probability following allogeneic HSCT is only about 50–60% in childhood CML. With imatinib successful in CML patients, the impact of imatinib treatment on subsequent allogeneic hematopoietic stem cell transplantation (HSCT) remains unclear. Imatinib therapy prior to HSCT has not been reported in children. Herein we describe imatinib induction preceding HSCT in three children newly diagnosed. Methods: Imatinib was administered as induction in three boys, aged 11, 14, 15 years, newly diagnosed in the chronic phase. Between induction and cytogenetic remission, two patients received HSCT from matched unrelated donors (MUD) and one received HSCT from matched sibling donor. Busulphan and cyclophosphamide were utilized as a conditioning regimen. Cyclosphorin A and methotrexate were both used as graft versus host disease (GVHD) prophylaxis. Anti-thymocyte globulin was added for MUD cases. Results: Successful engraftment and neither severe liver toxicity nor acute GVHD were encountered. All three cases show cytogenetic remission, with chronic GVHD well controlled in one. We monitored each patient’s status for minimal residual disease by real-time quantitative polymerase chain reaction. Absence of detectable minimal residual disease was found in two patients at 28 and 63 months after HSCT. At present, all of these three patients have good quality of life. Conclusions: As first-line therapy, imatinib appears useful for providing a bridge to HSCT in children with CML. Further study is warranted to determine impact of imatinib prior transplantation on child CML.

Description: Diabetes is associated with hyperglycemia and increased thrombin generation. It is unknown whether high glucose (HG)/thrombin can modulate the expression of NAPDH oxidase (Nox) subtypes in human aortic endothelial cells (HAECs). Besides, we investigate whether miR-146a is involved in endothelial cell inflammation. We observed that HG (25 mmol/l) exerted a synergistic effect with thrombin (2 U/ml) for induction of Nox4 mRNA level in HAECs. The increased Nox4 mRNA was associated with increased Nox4 protein and ROS production. We also demonstrated that HG/thrombin treatment increased interleukin-8 and interleukin-6 protein levels. Besides, HG/thrombin treatment caused an 11.43-fold increase of THP-1 adhesion to HAECs. In Silico analysis identified homology between miR-146a and the 3’-UTR of the human Nox4 mRNA, suggesting a potential regulation of Nox4 by miR-146a. Furthermore, HG/thrombin treatment decreased miR146a expression to 58% of the control, indicating an impaired feedback restrain of HG/thrombin-induced endothelial inflammation. MiR-146a mimic transfection prevented HG/thrombin-induced upregulation of Nox4 mRNA, Nox4 protein, and ROS generation. In addition, inflammatory phenotypes were attenuated in miR-146a mimic-transfected HAECs. In conclusion, miR-146a is involved in the regulation of endothelial inflammation via modulation of Nox4 in an in-vitro milieu mimicking diabetic atherothrombosis. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Childhood acute lymphoblastic leukemia (ALL) is a heterogeneous disease including multiple subtypes, defined on the basis of cell lineage and chromosome anomalies. Previous genome-wide association (GWA) studies report that several ARID5B and IKZF1 single nucleotide polymorphisms (SNPs) are associated with the incidence of ALL. However, high resolution melting analysis (HRM) supplies a faster and more convenient technique to detect SNPs. We applied HRM to detect the SNPs in ARID5B and IKZF1 gene. Method Seventy-nine pediatric ALL patients and 80 healthy controls were enrolled. Polymorphic variants of IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs7073837, rs10740055, and rs7089424) were detected by HRM method. The SNPs were further analyzed the association with childhood ALL. Result We evaluated 6 mutations by PCR amplicons (81 -176 bp) using the 96-well Light Cycler system. The primer detail was listed in Table 1. The melting curve data showed the difference in normalized temperature and we easily and accurately extended application of HRM analysis to genotyping of IKZF1 (Figure 1) and ARID5B (Figure 2) gene variants. The genotype distribution and the allele frequencies of IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs7073837, rs10740055, and rs7089424) gene polymorphisms among cases and controls and their association with the risk of childhood ALL were shown in Table 2. The distribution of genotype rs7073837 in ARID5B was significantly different between the ALL group and the control group (P = 0.046), while the distributions for IKZF1 (rs6964823, rs4132601, and rs6944602) and ARID5B (rs10740055, and rs7089424) were not significantly different. In addition, we analyzed the association for those SNPs with B lineage ALL and found rs7073837 in ARID5B conferred a higher risk for B lineage ALL (odds ratio, OR = 1.70, 95% confidence interval, CI = 1.01-2.87, P = 0.049). Conclusion HRM was a practical method to detect SNPs in ARID5B and IKZF1 gene. We determined the rs7073837 in ARID5B was associated with the risk of childhood B lineage ALL. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: Deferiprone (L1) has been suggested to be an effective oral chelation therapy for regular transfusion thalassemia major patients. The purpose of this study was to test the efficacy either of L1 or DFO alone, or combination use, and also monitor the safety of L1 in Taiwan. MATERIALS AND METHODS: From April 1999 to December 2005, 114 thalassemic patients from 5 treatment centers were enrolled in this program. L1 at the standard dose of 75mg/kg was given to 57 patients. The mean administered dose of L1 was 72.5mg/kg body weight divided into three doses per day. DFO at the standard dose of 30–50mg/kg/day at least 5 days/week was given to 26 patients, and the mean DFO dose was 46.5mg/kg/day 5 or more days/week. Combined therapy of daily L1 with subcutaneous DFO was given 2 to 6 days each week to the other 31 patients. The mean administered dose of DFO was 45.7mg/kg body weight 2 to 6 days per week, and the mean L1 dose was 71.5mg/kg/day divided into 3 doses per day. The therapeutic efficacy and potential side effects on cardiac and/or hepatic systems of these patients were assessed by left ventricular ejection fraction, T2-weighted magnetic resonance imaging (T2-MRI) and biochemical parameters and liver biopsies. RESULTS: No significant liver function impairment, neutropenia or arthropathy ever occurred. Only one patient was found to have transient leukopenia (he had a coincidental viral infection at that time). Three patients had temporary G-I upset but no one required discontinuation of L1. The serum ferritin level was significantly lower in 9 patients receiving L1 for more than 6 years (p=0.04), 22 patients receiving L1 for 2–3 years (p

Description: Background: Discontinuation of E. coli- asparaginase in patients with acute lymphoblastic leukemia (ALL) upon severe allergic reactions is unavoidable. We aimed to examine the outcomes following E.coli- asparaginase discontinuation upon severe allergic reactions in ALL. Patients and methods : In Taiwan Pediatric Oncology Group (TPOG)-2002-ALL protocol (enrolled 2002-2012), intramuscular E. coli- asparaginase (Kyowa Hakko, Japan) was given at 5000 IU/m2 per dose thrice weekly for 3 weeks during the remission induction therapy of high-risk (HR) and very-high-risk (VHR) groups. During the first 20 weeks of continuation therapy, HR patients received weekly intramuscular E. coli- asparaginase 10,000 IU/m2 per dose every week. They also received two reinduction treatments during which E.coli- asparaginase was given at 5000 IU/m2 per dose thrice weekly for 3 weeks. Patients in VHR groups received one reinduction treatment during which E. coli- asparaginase was given at 5000 IU/m2 per dose thrice weekly for 2 weeks. Patients in standard-risk (SR) group, received no E. coli- asparaginase in induction, but were randomized to receive one or two reinduction phases, during which E. coli- asparaginase was given at 5000 IU/m2 per dose thrice weekly for 2 weeks. The scheduled cumulative doses of E.coli- asparaginase in each risk group were 30,000 IU/m2 or 60,000 IU/m2 in SR, 265,000 IU/m2 in HR and 75,000 IU/m2 in VHR group. We evaluated outcome of children enrolled in TPOG-2002-ALL protocol who had E. coli- asparaginase discontinued due to severe allergic reactions (marked swelling and redness at the injection site or anaphylaxis) between 2002 and 2012, and compared outcomes between those who with Erwinase continued and those who without after the discontinuation of E. coli- asparaginase. The distributions of the Kaplan-Meier estimates of event-free survival (EFS) and overall survival (OS) were compared using log-rank test. Chi-square test was used to compare each parameter between groups. Results: In 700 patients from 10 hospitals retrospectively studied, 52 patients had E. coli- asparaginase treatment discontinued due to the development of severe pancreatitis in 17 patients, severe thrombosis in 2, and severe allergic reactions in 33. In Taiwan, Erwinase has been available since 2012, and could be purchased from foreign countries before. PEG-asparaginase is not available in Taiwan. In the 33 patients had E. coli- asparaginase discontinued due to severe allergic reactions, 17 continued Erwinase and 16 did not. The parameters between these two groups were similar. They were of more HR group reflecting that HR patients received more E. coli- asparaginase than other patients. The 5-year OS did not differ significantly among the 648 patients without discontinuation (81±1.6%, mean±S.E.), the 17 with allergic reactions and continued with Erwinase (88±7.8%) and the 16 with allergic reactions not treated with Erwinase (87±8.6%). The P value for the difference between the latter two groups was 0.96. In the 16 patients who did not receive Erwinase, all the 10 patients, who had received 〉= 50% scheduled dose of E.coli- asparaginase before discontinuation, survived without events. Conclusions: We suggest that patients who had received 50% or more of the scheduled doses of E. coli- asparaginase before the development of severe allergic reactions do not need to continue treatment with Erwinase. This may be helpful in those who cannot afford Erwinase or in the vast majority of the world where Erwinase is not available. It also raises a question on that how much asparaginase is enough for the treatment of childhood ALL. Disclosures No relevant conflicts of interest to declare.

Description: Congenital dyserythropoietic anemia (CDAs) is a group of hereditary disorders characterized by ineffective erythropoiesis and distinct morphological abnormalities of erythroblasts in the bone marrow. Diagnosis of CDA is based primarily on the morphology of bone marrow erythroblasts but genetic tests might have more and more important roles recently. Here, we describe a 31-year old female with atypical CDA under regular blood transfusions. The results of blood tests were as followed: RBC: 3080000/µL, Hb: 9.9 g/dL, Hct: 26.8%, MCV: 87.0 fl, MCH:32.2 pg, and MCHC: 36.9 g/L. In this study, analysis of CDA-related genes, including SEC23B, CDAN1, KLF1, and C15orf41, were analyzed by exome sequencing but no pathogenic variant was found. In additional, we analyzed RBC-related genes and a novel variant in the PKLR p.A468G (NP_000289) was found which was also confirmed by Sanger sequence. Variant of PKLR gene has been reported as the cause of pyruvate kinase (PK) deficiency anemia. PK deficiency is the most cause of congenital nonspherocytic hemolytic anemia with GMAF (global minor allele frequency) of 0.0001. The clinical features of PK-deficient patients are highly variable degree of chronic hemolysis with severe neonatal jaundice and fetal anemia at birth. In this case, PKLR p.A468G heterozygous variant was detected in a patient with PK deficiency, as an example of precision diagnosis by exome sequencing. Disclosures No relevant conflicts of interest to declare.

Description: Backgrounds and Purposes Minimal residual disease (MRD) monitoring has been proved to be the most important prognostic predictor in childhood acute lymphoblastic leukemia (ALL). The nationwide TPOG-ALL-2013 protocol (TPOG-2013), adapted from the St. Jude Total Therapy XV Study and Total Therapy XVI Study, was launched since January 2013. This is the first MRD-directed protocol for treatment of childhood ALL in Taiwan. Here, we report the improved treatment outcomes and the impacts of adherence to MRD time points. Patients and Methods Totally, 402 patients aged between 1-18 years and diagnosed before December 31, 2018, who had MRD monitoring at the major central laboratory (Chang Gung Memorial Hospital-Linkou), were enrolled with the last follow-up on June 30, 2019. According to TPOG-2013, two MRD measurements were scheduled on days 15-19 of induction (MRD1 time point, TP1) and days 35-42, end of induction (MRD2 time point, TP2) to make the definitive risk stratification to guide subsequent therapy. The methodologies of MRD measurement included multicolor flow cytometry for leukemia-associated immunophenotypes (LAIP) (82.3% of TPOG-2013 cohort), qPCR assay for clonally rearranged antigen-receptor genes (Ig/TCR) if no LAIP (12.5%). Since January 2018, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) was applied to patients carrying fusion transcripts (5.2%) of TCF3-PBX1, ETV6-RUNX1, BCR-ABL1, KMT2A-AFF1 (AF4) and KMT2A-MLLT3. The clinical features and outcomes of patients treated with TPOG-2013 were compared with those of 1,300 patients treated with the previous TPOG-ALL-2002 protocol (TPOG-2002), which did not integrate the MRD monitoring. Results The median follow-up time of the 402 patients of TPOG-2013 cohort was 32.5 months (range, 1.0-79.2 months). There were no significant differences in gender, age, WBC counts, and lineage at diagnosis between the patients treated with TPOG-2002 and TPOG-2013. However, based on the MRD data, the percentages of patients assigned to each risk group of TPOG-2013 was statistically differed from those of TPOG-2002 (P〈 0.0001). The 5-year event-free survival (EFS) (% ± SE) was significantly improved from 78.1 ± 1.2 of TPOG-2002 to 85.4 ± 2.5 of TPOG-2013 (P〈 0.0001). Further, the cumulative incidences (% ± SE) of isolated CNS relapse and any CNS relapse significantly decreased from 4.0 ± 0.5 to 0.3 ± 0.3 (P= 0.001) and from 5.8 ± 0.7 to 1.2 ± 0.9 (P= 0.001), respectively. The issue of non-adherence to MRD monitoring emerged since the implementation of MRD-directed TPOG-2013. For further analysis, 321 (80%) patients with exact adherence (EA) to both TPs were assigned as MRD EA group; 80 (20%) patients who were non-adherence (NA) to either one of TPs as MRD NA group; and one patient died between the two TPs was excluded for the comparative outcome analysis. The rate of non-adherence decreased significantly from 26.5% in 2013 to 2.4% in 2018. The major causes of non-adherence for both TPs were delaying MRD monitoring due to neutropenic fever and documented infections. In MRD EA group, 12.5% of patients were upgraded to higher-risk treatment groups based on their MRD results. The MRD NA group had older age (≥ 10 years), lower standard-risk and lower incidence of ETV6-RUNX1 compared with MRD EA group. There were significant differences in outcomes between MRD EA and MRD NA groups: the 5-year EFS were 89.4 ± 2.4 and 71.9 ± 7.4, respectively (P= 0.0005), overall survival (OS) were 90.9 ± 2.1 and 75.6 ± 5.8, respectively (P= 0.0003), and the cumulative incidence of isolated CNS relapse were 0 and 1.4 ± 1.3, respectively (P= 0.048) (Figure 1). In multivariate analysis, older age (≥ 10 years), higher WBC count (≥ 50 × 109/L) at diagnosis and MRD non-adherence were independent predictors for inferior EFS. In addition to these three factors, a higher-risk classification also predicted an inferior OS (Figure 2). Conclusions Contemporary MRD-directed therapy has improved the treatment outcomes of childhood ALL in Taiwan. The adherence to MRD time points remains a significantly prognostic predictor in the era of MRD-guided treatment. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The primary effector of renin-angiotensin system (RAS) system is angiotensin II. RAS activation causes many detrimental effects via AT1 receptor. Angiotensin-(1-7)/ angiotensin-converting enzyme 2/Mas axis is a newly identified counter-regulatory pathway against RAS system. Thrombin plays a critical role in coagulation and inflammation processes in vascular endothelium. Although RAS activation is associated with thrombotic complications, it is unknown whether angiotensin-(1-7) can modulate the pleotropic effects of thrombin. In this study, we investigate the proteomic changes of angiotensin-(1-7) effects on thrombin-stimulated human aortic endothelial cells (HAECs). Materials and methods: HAECs were pretreated with 10-7M anigotenion-(1-7) for 1 h and stimulated with 2 units/mL thrombin for additional 5 h. Their proteomes were investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and MetaCoreTMsoftware. Results: A total of 653, 717 and 801 proteins were identified in triplicated iTRAQ analyses. MetaCoreTM pathway analysis identified that iTRAQ data showed the consistent pathway alterations (70%) in triplicated experiments. The same altered pathways included "Cytoskeleton remodeling_Cytoskeleton remodeling", "Cell adhesion_Integrin-mediated cell adhesion and migration", "Cell adhesion_Chemokines and adhesion" , "Cytoskeleton remodeling _ Regulation of actin cytoskeleton by Rho GTPases", "LRRK2 in neurons in Parkinson's disease", "Cytoskeleton remodeling_Fibronectin-binding integrins in cell motility", "Cytoskeleton remodeling _TGF, WNT and cytoskeletal remodeling" were among the top 10 statistically significant pathways. Additional experiments validated the phenotypes of angiotensin-(1-7) effects in thrombin-stimulated HAECs. Conclusions: Several regulatory pathways are altered by angiotensin-(1-7) in thrombin-stimulated HAECs, with cytoskeleton remodeling, cell adhesion and cell migration (motility) as the dominant altered phenotypes. Disclosures No relevant conflicts of interest to declare.