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Background The hepcidin-ferroportin axis is known to play the essential role on iron homeostasis in human. The binding of hepcidin to ferroportin (FPN) on the membranes of iron-exporting cells induces the ubiquitination of FPN and thereby decreases the delivery of iron to plasma. Low hepcidin with high FPN was well established in patients with ineffective erythropoiesis, e.g. beta-thalassemia major and myelodysplastic syndrome. On the other hand, studies showed increased hepcidin accompanied with decreased FPN in patients with some cancers. Further, anemia in multiple myeloma and Hodgkin's lymphoma were reported to be associated with high hepcidin levels (Blood 2008;112:4292). Here, we investigated the function of the hepcidin-ferroportin axis in childhood acute lymphoblastic leukemia (ALL) survivors who completed chemotherapy. Methods Thirty-three children, aged ≤18 years with ALL and completing TPOG-ALL-2013 protocol before June 30, 2020, were enrolled (ALL-off Gr). According to the treatment protocol, they had been classified as standard-risk (SR) in 20, high-risk (HR) in 12 and very-high-risk (VHR) in 1, respectively. Thirty-eight healthy siblings were assigned as the normal controls (normal Gr) after exclusion of acute illness and iron deficiency. Twenty newly-diagnosed ALL children (ALL-Dx Gr) who did not have sepsis at diagnosis (Dx), and 13 patients with beta-thalassemia major (TM Gr) who received regular transfusion were also enrolled. Serum iron, total iron-binding capacity, ferritin, hepcidin and FPN were collected at Dx in ALL-Dx Gr and at 6-month follow-up after completion of chemotherapy in ALL-off Gr. Selected biochemical assays were checked in normal Gr and TM Gr on the day of routine RBC transfusion. Hepcidin and FPN levels of peripheral blood were measured using ELISA kits (DRG, EIA-5782 for hepcidin & Elabscience, E-EL-H2355 for FPN, respectively). Results The clinical features and biochemical data of all patients are shown in Table 1. The hepcidin and FPN of normal Gr were comparable with those of reference papers. The low hepcidin, high FPN and relatively low hepcidin/ferritin of TM Gr were in consistent with those of published reports. There were no significant differences of age (P =.243) and gender (P =.899) between ALL-off Gr and normal Gr. The ferritin and transferrin saturation between ALL-Dx and ALL-off Grs showed statistical difference (P 300 ng/mL; median, 871.55; range, 351.6-959.0)(high-ferritin). MRI scans of liver and heart from the 6 high-ferritin patients did not show any evidence of iron overload. In Figure 2, hepcidin levels were significantly higher in ALL-off patients with high-ferritin or receiving cumulative RBC transfusions >10 units. And ALL-off patients with high-ferritin, RBC transfusions >10 units or receiving HR/VHR treatment regimens had significantly higher FPN levels. Multivariate analysis demonstrated only high-ferritin predicted significantly higher hepcidin (P =.02) and FPN (P Conclusions The dysregulation of hepcidin-ferroportin axis showed high hepcidin and uncontrolled FPN hyperactivity at Dx of childhood ALL which may point to the impaired down-regulation of FPN by hepcidin. Our findings imply the impaired mechanism may not be fully recovered in ALL survivors after completing chemotherapy, especially those with ferritin >300 ng/mL, HR/VHR groups or receiving higher RBC transfusions. The routine monitoring of the axis function could facilitates the early detection of iron overload in liver and heart. Disclosures No relevant conflicts of interest to declare.

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.

Introduction: IKZF1 deletion was firstly reported to be very frequent in Philadelphia (Ph) positive B-cell acute lymphoblastic leukemia (B-ALL), and continuously found in other B-ALL subtypes. IKZF1plus defined as the presence of any of CDKN2B, CDKN2A,PAX5 or PAR1 in the absence of ERG deletion and was reported to describe a new minimal residual disease (MRD)-dependent poor prognostic profile in B-ALL.1 We aimed to analyze the frequency and prognostic relevance of IKZF1 deletion with or without co-occurring gene alterations in Taiwanese children with B-ALL treated with TPOG-ALL-2002 protocol or MRD-directed TPOG-ALL-2013 protocol. Methods: Bone marrow samples at diagnosis from 561 children excluding Ph+ and infant ALL were analyzed. Detection of gene deletion was carried out with multiplex ligation dependent probe amplification (MLPA) kit (SALSA MLPA P335 and P327) and mutations of RAS pathway genes (NRAS, KRAS, and PTPN11) were assessed by Sanger sequencing. The outcome was analyzed on 259 patients treated with TPOG-ALL-2002 protocol and 158 patients treated with TPOG-ALL-2013 protocol. Results: IKZF1 deletions were present in 11.2% of 561 patients. DEL 4-7, DEL 1-8, and other variants were present in 50.8%, 14.3%, and 34.9%, respectively of 63 IKZF1-deleted patients. Co-existence of additional deletions with IKZF1 deletion was detected in 34.0% for CDKN2B, 37.3% for CDKN2A, 27.7% for PAX5, 2.1% forPAR1, and 8.0% for ERG. IKZF1plus comprised of 4.4% of 546 patients who had all the genes examined. Co-existed mutations of RAS pathway genes were detected in 5.0% for PTPN11, 13.3% for NRAS, and 10.0% for KRASin the IKZF1-deleted patients. The 5-year event-free survival (EFS) of IKZF1-undeleted patients was significantly better compared with IKZF1-deleted patients in TPOG-2002 cohort (82.5 ± 2.6% vs. 56.9 ± 9.1%, P = 0.002) as did TPOG- 2013 cohort (89.4 ± 3.9% vs. 59.1 ± 19.8%, P = 0.012). The 5-year overall survival (OS) of IKZF1-deleted patients was worse than that of patients with undeleted IKZF1 in TPOG-2013 cohort (58.3 ± 19.8% vs. 89.2 ± 4.2%, P = 0.004) but not in TPOG-2002 cohort (90.9 ± 5.0% vs. 86.9 ± 2.3%, P = 0.846). DEL 1-8 patients had an inferior 5-year EFS (33.3 ± 19.2%) compared with DEL 4-7 (63.3 ± 12.0%,) or DEL-others (60.0 ± 18.4 %) in TPOG- 2002 cohort (P = 0.003). No significant difference in OS was observed between patients among different IKZF1-deleted subtypes in TPOG-2002 cohort (P = 0.283) (Fig. 1A) but there was different in TPOG-2013 cohort (P = 0.003) (Fig. 1B). Patients with IKZF1 deletion alone had comparable 5-year EFS and 5-year OS compared with patients with IKZF1plus in TPOG-2002 cohort whereas patients with IKZF1 deletion alone had worse 5-year EFS (33.3 ± 27.2% vs. 80.0 ± 17.9%, P = 0.284) and 5-year OS (33.3 ± 27.2% vs. 80.0 ± 17.9%, P = 0.415) than that of KZF1plus in TPOG-2013 cohort. The prognostic impact of IKZF1 deletion with co-existed gene alterations in TPOG-2002 cohort was further analyzed as follows: IKZF1plus with ≧ 2 co-existed deletions had an inferior 10-year EFS (23.8 ± 20.3%) compared with patients with IKZF1 deletion alone (36.7 ± 17.5%) or IKZF1-undeleted patients (81.5 ± 2.7%) (P = 0.005) (Fig. 1C). Three patients carried both IKZF1 and ERG deletions had a superior 5-year EFS (100%) compared with IKZF1-deleted alone or IKZF1-undeleted patients (P = 0.005) (Fig. 1D). The 10-year EFS of patients with any gene mutation of RAS pathway was worse than that of patients with RAS wild-type genes (66.6% ± 6.4% vs. 83.7% ± 2.6%, P = 0.011). In multivariate analysis, in addition to the initial white blood count > 50 x 109/L and KMT2A-rearranged, IKZF1 deletion (HR=2.609, 95% CI: 1.363-5.034; P = 0.004) and RAS pathway gene mutations (HR=2.360, 95% CI: 1.336-4.168; P = 0.003) were independent genetic predictors for inferior EFS. Co-existence of IKZF1 deletion with RAS pathway mutations had worst 5-year EFS (20.0% ± 12.6%, P < 0.0001, Fig. 1E), and 10-year OS (53.3% ± 17.6%, P = 0.042, Fig. 1F). Conclusions: Our results showed that IKZF1 deletion alone was associated with an inferior OS in MRD-directed TPOG-2013 treated cohort but not in TPOG-2002 cohort in which it became significant when co-existed with RAS pathway mutations. Reference: 1. IKZF1plus defines a new minimal residual disease-dependent very-poor prognostic profile in pediatric B-cell precursor acute lymphoblastic leukemia. J Clin Oncol. 2018, 1240-1249. Figure 1 Disclosures No relevant conflicts of interest to declare.

Gene mutations involving epigenetic regulators recently have been described in adult acute myeloid leukemia (AML). Similar studies are limited in children. We analyzed gene mutations and cooperation in pediatric AML with special reference on mutated epigenetic regulators. Nineteen gene mutations, including 8 class I genes, 4 class II genes, WT1 and TP53 (class III), and 5 epigenetic regulator genes (class IV), were analyzed in 206 children with de novo AML. Mutational analysis was performed with polymerase chain reaction-based assay followed by direct sequencing. One hundred seventeen of 206 patients (56.8%) had at least one mutation: 51% class I, 13% class II, 6.8% class III, and 5.6% class IV. FLT3-internal tandem duplication was most frequent, and 29% of patients had more than one gene mutation. Two patients carried ASXL1 mutations, both with t(8;21), 2 had DNMT3A mutations, 2 had IDH1 mutations, 1 had IDH2 mutation, and 3 had TET2 mutations. Both patients with IDH1 mutations had AML-M0 subtype and MLL-partial tandem duplication. Cooperating mutations with mutated epigenetic regulators were observed in 8 of 10 patients. We conclude that mutated epigenetic regulators were much less than those in adult AML but with frequent cooperating mutations. ASXL1, TET2, and IDH1 mutations were associated with specific genetic subtypes.

Contemporary treatment of pediatric acute myeloid leukemia (AML) requires the assignment of patients to specific risk groups. To explore whether expression profiling of leukemic blasts could accurately distinguish between the known risk groups of AML, we analyzed 130 pediatric and 20 adult AML diagnostic bone marrow or peripheral blood samples using the Affymetrix U133A microarray. Class discriminating genes were identified for each of the major prognostic subtypes of pediatric AML, including t(15;17)[PML-RARα], t(8;21)[AML1-ETO], inv16 [CBFβ-MYH11], MLL chimeric fusion genes, and cases classified as FAB-M7. When subsets of these genes were used in supervised learning algorithms, an overall classification accuracy of more than 93% was achieved. Moreover, we were able to use the expression signatures generated from the pediatric samples to accurately classify adult de novo AMLs with the same genetic lesions. The class discriminating genes also provided novel insights into the molecular pathobiology of these leukemias. Finally, using a combined pediatric data set of 130 AMLs and 137 acute lymphoblastic leukemias, we identified an expression signature for cases with MLL chimeric fusion genes irrespective of lineage. Surprisingly, AMLs containing partial tandem duplications of MLL failed to cluster with MLL chimeric fusion gene cases, suggesting a significant difference in their underlying mechanism of transformation.

Contemporary treatment of pediatric acute lymphoblastic leukemia (ALL) requires the assignment of patients to specific risk groups. We have recently demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-cell lineage ALL (T-ALL), E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid karyotypes with more than 50 chromosomes. As the next step toward developing this methodology into a frontline diagnostic tool, we have now analyzed leukemic blasts from 132 diagnostic samples using higher density oligonucleotide arrays that allow the interrogation of most of the identified genes in the human genome. Nearly 60% of the newly identified subtype discriminating genes are novel markers not identified in our previous study, and thus should provide new insights into the altered biology underlying these leukemias. Moreover, a proportion of the newly selected genes are highly ranked as class discriminators, and when incorporated into class-predicting algorithms resulted in an overall diagnostic accuracy of 97%. The performance of an array containing the identified discriminating genes should now be assessed in frontline clinical trials in order to determine the accuracy, practicality, and cost effectiveness of this methodology in the clinical setting.

Introduction The nationwide TPOG-ALL-2002 protocol for treating children with acute lymphoblastic leukemia (ALL) was activated in January 2002 in Taiwan. To eliminate cranial radiation (CrRT)-related sequelae and minimize the adverse prognostic impact of traumatic lumbar puncture with blasts (TLP), we conducted a prospective study beginning in 1999 to modify CNS-directed therapy with delayed first triple intrathecal therapy (TIT) and omission of CrRT for all risk groups of newly diagnosed ALL. This approach improved CNS control at the single-institution study (J Clin Oncol, 2014) and has been used in whole TPOG since January 2009. Patients with non-CNS-1 status (i.e., CNS-2, CNS-3 and TLP) have higher risk of CNS relapse and need intensive CNS therapy. On behalf of TPOG members, we reported the outcomes of these non-CNS-1 patients in the two eras (2002-2008 and 2009-2012) with or without the modified CNS therapy. Methods From 2002 to 2012, all newly diagnosed ALL children in Taiwan were enrolled in TPOG-ALL-2002 protocol (Leukemia, 2010). B-precursor ALLs were stratified into standard risk (SR), high risk (HR) and very high risk (VHR) groups, according to age, WBC count, cytogenetics and molecular analysis at diagnosis. T-cell ALL was designated as VHR. Since 2009, patients had been treated with TIT alone without CrRT. The first TIT was performed until the disappearance of blasts from peripheral blood (PB), no later than 10 days. SR patient with detectable PB blasts on day 8 was upgraded to HR group. SR with CNS-1 received 14 doses of TIT, and those with CNS-2, or TLP got 2 additional TITs during induction and 3 additional TITs during maintenance therapy. Totally, SR with CNS-2 or TLP received 19 doses of TIT. And SR patients with CNS-3 or cranial nerve palsy at diagnosis were classified as HR. For HR with CNS-1, 17 doses of TIT were given. If HR groups with CNS-2, CNS-3, or TLP, they will receive total 25 doses of TIT including 2 additional ones during induction and 6 additional ones during maintenance therapy. Before 2009, only SR and HR with refractory CNS leukemia (failure to clear CSF blasts after 3 consecutive TITs) and those with CNS relapse will receive CNS radiation. For VHR group before 2009, CrRT of 18 Gy was given to all patients at the end of remission induction. Instead, TIT was given for age < 2 years. After 2009, CrRT was totally omitted. VHR with CNS-1 received 19 doses of TIT. For VHR patients with CNS-2, CNS-3 or TLP at diagnosis, TIT was administered once/week until achieving CSF remission and then once 4 weeks during maintenance therapy. Results Since January 2009, all patients received TIT alone, irrespective of risk groups. The two cohorts, totally 1,366 patients, 909 treated with CrRT (2002-2008) and 457 without CrRT (2009-2012) were balanced in presenting features and distribution of CNS status. There were no significant differences between two eras in the event-free survival (EFS), overall survival (OS), incidences of isolated CNS relapse and any CNS relapse (Pediatr Blood Cancer, accepted June 2016). The number of patients with CNS-2, CNS-3 and TLP were 35, 26 and 19 before 2009; and 18, 10 and 9 after 2009, respectively. Patients with younger age (< 1 year), higher WBC counts (> 100 X 109/L), HR/VHR or MLL rearrangement had significantly more non-CNS-1 status in the era of 2002-2008. And the significant factors prone to be non-CNS-1 were male, T-lineage and HR/VHR with the first TIT delaying. There were no significant differences between the two eras in the 5-year EFS and OS. Though the incidence of 5-year any CNS relapse was not significantly different, fewer relapses were observed in the era of 2002-2008 (Table 1). Before 2009, eleven CNS relapses experienced between 1 month and 3.1 years (median, 10 months) after remission. Two relapses after 2009 occurred on 1 month and 7 months, respectively. Conclusions Delayed first TIT until the clearance of circulating blasts and total omission of CrRT did not compromise the survivals and CNS control of childhood ALL patients with CNS-2, CNS-3 and traumatic lumbar puncture with blasts. Moreover, this approach prevents the adverse sequelae caused by CrRT. Furthermore, a skillful TIT is mandatory to improve results. Disclosures No relevant conflicts of interest to declare.

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.

Background and purpose: The Mixed-lineage leukemia (MLL) gene located at 11q23 can be fused to a variety of partner genes through chromosomal translocations (MLL-T) in acute leukemia. The co-existence of commonly known mutated genes has not been comprehensively studied in MLL-T AML. We aimed to determine (1) the prevalence and clinical relevance of gene mutations at diagnosis, and (2) the genetic evolution profile at relapse in de novo AML with MLL-T. Materials and methods: Bone marrow samples from 80 de novo AML with MLL-T patients were analyzed on 20 gene mutations involving activating signaling pathway (class I), transcription pathway (class II), epigenetic regulators (class III), and tumor suppressors, TP53 and WT1 (class IV). MLL-T was all confirmed by FISH analysis. The common MLL fusion transcripts were detected by RT-PCR, including 26 MLLT3, 13 MLLT10, 12 MLLT4, 11 ELL, 3 MLLT1, 3 AFF1, and 2 EPS1. Another 6 infrequent or rare partner genes (one each of MLLT6,CBL, ARHGEF12, TET1, SEPT6, and SEPT9) were identified by cDNA panhandle PCR. In the remaining 4 cases, the partner genes were not identified. Mutational analyses were performed with PCR-based assays followed by direct sequencing. Twenty-two of 26 patients who relapsed had relapse samples for comparative analysis. Results: The frequencies of gene mutations of class I, II, III, and IV, in 80 de novo MLL-T AML patients were 49.4%, 3.8%, 10.3% and 1.3%, respectively. Together, 53.8% of patients with MLL-T had at least one mutated gene. KRAS (17.7%), FLT3-TKD (11.5%), and NRAS (11.4%) mutations occurred most frequently. Among the epigenetic regulators, 7.9% of patients had DNMT3A, 2.7% TET2, 1.4% ASXL1, and none had IDH1/2 or EZH2 mutations. Three patients had 3 co-existing mutations and 9 patients had two mutations, with 6 of the 12 carrying FLT3-TKD mutations. Gene mutations of transcription pathway and tumor suppressors rarely occurred, only involving NPM1 in two cases and one each for RUNX1 and WT1 mutation. The patients with DNMT3A mutations were significantly associated with older age (P=0.005), FAB AML-M4 (P Conclusions: Our results showed that co-existing mutations of RAS and FLT3-TKD were most frequently detected at diagnosis in de novo MLL-T AML. Gene mutations, except FLT3-ITD, had no impact on outcomes in MLL-T AML. FLT3-TKD mutations were absent in all relapse samples while clonal evolution of WT1, TP53 or KRAS mutations might contribute to the relapse of leukemia in a subset of MLL-T AML patients. Grant support: NHRI-EX93-9011SL, NHRI-EX96-9434SI, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3, MMH-E-101-09 and OMRPG3C0021. Disclosures No relevant conflicts of interest to declare.

Abstract 3564 Background The introduction of CNS-directed therapy had successfully improved the outcome of children with acute lymphoblastic leukemia (ALL). Pui et al. (N Engl J Med, 2009) demonstrated that with effective risk-adjusted systemic chemotherapy and intrathecal therapy initiated at diagnosis (Dx), cranial irradiation can be safely omitted from CNS-directed treatment in all children with newly diagnosed ALL. Manabe et al. (J Clin Oncol, 2001) reported a delay of diagnostic lumbar puncture (LP) and triple intrathecal therapy (TIT) after 1 week of prednisolone monotherapy. Starting 1999, in order to eliminate the adverse prognostic impact of traumatic LP with blasts, we conducted a prospective study of delayed TIT after the disappearance of blasts from peripheral blood (PB) on around 10 days of multi-drug induction therapy. Methods Since June 1999, children with ALL were treated with TIT alone without cranial irradiation, and chemotherapy with TPOG-ALL-93 or TPOG-ALL-2002 (activated in January 2002) protocols. These protocols had been reported previously (Leukemia, 2010). B-precursor ALL patients were stratified into standard risk (SR), high risk (HR) and very high risk (VHR) groups, according to age, WBC count, cytogenetics and molecular analysis at Dx. T-cell ALL was designated as VHR. The first TIT was performed until the disappearance of blasts from PB. If patients with persistent PB blasts on the treatment day 10 (D10), the TIT was planned. Furthermore, SR patient with detectable blasts on D10, the treatment was upgraded to HR group. Cranial irradiation was totally omitted in all patients. For patients with CNS-1 status, SR group received TIT 20 times in TPOG-ALL-93 and 14 times in TPOG-ALL-2002 protocols, respectively. HR patients with CNS-1 status received 23 and 17 times of TIT in TPOG-ALL-93 and 2002 protocols, respectively. In VHR group, TIT was administered 19 times for CNS-1. Additional TIT doses were given if patients with CNS-2 or CNS-3. Results From June 1999 to August 2010, a total of 158 ALL patients were consecutively diagnosed at our hospital. There were 86 patients in SR group, 41 in HR and 31 in VHR, respectively. Six patients were excluded: 2 refusals to further treatment and 4 protocol violations. There were 86 boys and 66 girls enrolled. The median age at Dx was 4.1 years (range, 0.3–17.7 years) with 7 infants. The median WBC count at Dx was 12,100/μL (range, 800–596,000/μL). Fourteen had counts >100,000/μL. There were 141 patients of B-precursor ALL and 11 of T-cell ALL. At Dx, 17 patients (16 in SR and 1 in VHR) did not have detectable blasts on PB. In the remaining 135 patients, the blasts of 101 patients (74.8%) can be eliminated from PB after 10-day induction therapy, those included 77.9% of SR, 75.6% of HR and 65.4% of VHR, respectively. Six patients of SR group were upgraded to HR protocol since the detection of PB blasts on D10. Bleeding into the CSF at the time of LP was apparent in 4 patients (2.6%), but no leukemic blasts were identified in any patients. As of July 31, 2012, the follow-up duration of 133 surviving patients ranged from 24 to 153 months (median, 88 months). The 7-year event-free survival and overall survival rates were 83.0 ± 3.2% (± SE) and 86.5 ± 3.1%, respectively. No isolated CNS relapse occurred. One infant with MLL rearrangement experienced combined CNS, BM and testicular relapses and one child with t(12;21) had combined CNS and molecular BM relapses. The cumulative risk of any CNS relapse was 1.5 ± 1.0% at 7 years. Conclusions A delay of TIT after disappearance of PB blasts with multi-drug induction therapy can effectively prevent CNS disease in children with ALL. Cranial irradiation can be completely omitted for CNS prophylaxis in our treatment protocols. Disclosures: No relevant conflicts of interest to declare.

Abstract 2713 Background. The cooperation of gene mutations, especially their impacts on survivals of childhood acute myeloid leukemia (AML) has not been well known. Aims. Our aims were (1) to study the frequency of each gene mutation in childhood AML, (2) to study the impact of each gene mutation on the treatment outcome, and (3) to examine the cooperativity of gene mutations. Materials and Methods. From Feb. 1996 to Jan. 2010, bone marrow samples at diagnosis from 198 children with AML at Chang Gung Children's Hospital, Taoyuan and Mackay Memorial Hospital, Taipei, were analyzed for gene mutations including FLT3-ITD, FLT3-TKD (D835), c-KIT, cFMS, JAK 2V617F, NRAS, KRAS, PTPN11 (Class I mutations), RUNX1, CEBPα, NPM1 (Class II mutations), WT1 and P53 (tumor suppressor genes). The subtypes included: t(8;21) 19.9%, inv(16) 8.9%, t(15;17) 8.4%, t(9;11) 5.2%, t(10;11) 2.6%, trisomy 21 4.2%, intermediate-risk group 40.3% (including 13 patients with other MLL translocations), and poor-risk group 11.0% (including 7 patients with complex chromosomal abnormalities and 4 patients with MLL-PTD). Results. FLT3-ITD occurred in 15.0% of patients, FLT3-TKD 7.2%, c-KIT 11.5%, c-FMS 2.9%, JAK2V617F 3.3%, NRAS 9.1%, KRAS 7.7%, PTPN11 3.3%, RUNX1 2.7%, CEBPα 7.9%, NPM1 4.1%, WT1 3.9% and P53 1.7%. Taken together, 52.5% of patients had Class I gene mutations, 13.1% had Class II gene mutations, and 5.1% had WT1 or P53 mutations. In all, 59.1% of patients had Class I, Class II or tumor suppressor gene mutations. Only one patient (0.5 %) had gene mutations involving all Class I, Class II and tumor suppressor genes. Ninety-eight patients, who were treated with Taiwan Pediatric Oncology Group (TPOG) APL protocols (for acute promyelocytic leukemia) and TPOG 97A protocol (for other AML) (Liang et al, Leukemia 2006), were analyzed for survivals. In patients with t(8;21), the 5-year event-free survival (EFS) was 66±12%; 71±17% for patients with c-KIT mutations and 50±35% for the 2 patients with JAK2V617F. In patients with inv(16), the EFS of 70±15% seemed to be compromised (60±22%) for those with c-KIT mutations. In patients with t(15;17), the EFS of 78±11% was not compromised by FLT3-ITD or FLT3-TKD mutations. In patients with t(9;11), the EFS of 64% seemed to be compromised (50±35%) in the 2 patients with FLT3-TKD mutations. In 3 patients with t(10;11), no gene mutations were found. In trisomy 21, the EFS of 75±22% seemed to be compromised (50±35%) in the 2 patients with CEBPα mutations. Of the 5 patients with complex chromosomal abnormalities, the only one patient carrying RUNX1 survived. Of the 3 patients with MLL-PTD having an EFS of 33±27%, one each patient with c-FMS or WT1 mutation died. The only one patient who had all Class I, Class II and tumor suppressor gene mutations (FLT3-TKD+ CEBPα+ WT1) died in induction therapy. Two of the other 4 patients who had 3 mutations acrossing 2 classes had EFS of 6 and 10 months, respectively. Conclusions. Our study on a large cohort of pediatric AML patients revealed that 59.1% patients had at least one gene mutation. That 3 of 5 patients who had 3 gene mutations soon failed suggested that gene mutations, especially in 3 combinations, might compromise the survival. Further study on more patients is warranted to explore more of the prognostic significance of cooperating gene mutations in pediatric AML. (Supported by grants MMH-E-98009, NSC 96–2314-B-195-006-MY3, NHRI-EX-96-9434SI and DOH99-TD-C-111-006.) Disclosures: No relevant conflicts of interest to declare.

Abstract 3064 Poster Board III-1 Both ETV6-RUNX1 (TEL-AML1)fusion and hyperdiploidy (>50 chromosomes) of lymphoblasts are favorable outcome predictors in childhood acute lymphoblastic leukemia (ALL). In 433 children with ALL diagnosed at our hospitals between 1997 and 2007 in Taiwan, the frequency of ETV6-RUNX1 fusion was 15.8%, and the frequency of hyperdiploidy (>50 chromosomes) was 14.1%, both were lower than those of the West. While ETV6-RUNX1 fusion had borderline favorable impact on outcome (p=0.053-0.061), hyperdiploidy showed significant favorable impact on event-free survival (91.1% vs 76.6 %, p= 0.016) in our patients. A meta-analysis from literature enrolled reports in which the case numbers and frequency of ETV6-RUNX1 fusion or hyperdiploidy in childhood ALL were described. It revealed that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East (Japan, Korea, Hong Kong, Chinese in Singapore, and Taiwan) was 14.2% (127/893, range 10-17%), significantly lower than 21.8% (152/697, range 19-27%) in the West (USA, Germany, Italy, France and Chile) (p < 0.0001). The frequency of hyperdiploidy in Japan and Taiwan was 15.2% (140/921, range 13-20%), significantly lower than 31.6% in the West (977/3,158, range 19-34%) (USA, UK and Germany) (p < 0.0001). So far as we know, there were several articles, including ours, addressing that the frequency of ETV6-RUNX1 fusion in childhood ALL was lower in a Far East country. This is the first meta-analysis to demonstrate that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East was lower than that in the West. There was no report on that the frequency of hyperdiploidy in Far East was lower than that in the West. This is also the first meta-analysis to demonstrate that the frequency of hyperdiploidy in childhood ALL in Far East is significantly lower than that in the West. The nature of these differences, probably due to racial, needs further study. In Far East, with both a lower frequency of ETV6-RUNX1 fusion, and a lower frequency of hyperdiploidy, it warrants renewed effort to cure a higher proportion of children with ALL. Disclosures No relevant conflicts of interest to declare.

Background. Two-hit model of leukemogenesis has been proposed for AML; class I mutations that drive proliferation and survival, and class II mutations that block differentiation. Core-binding factor (CBF) AML consists of AML with AML1-ETO and AML with CBFβ-MYH11, that are class II mutations. Aim. We sought to determine the frequencies of cooperating mutations of class I including receptor tyrosine kinases (RTK)/Ras/JAK2 signaling pathways in CBF-AML, and to compare the patterns of cooperating mutations between AML with AML1-ETO and AML with CBFβ-MYH11. Patients and methods. By RT-PCR analysis, 130 adult and 45 children were identified to have CBF-AML, 129 with AML1-ETO and 46 with CBFβ-MYH11. Bone marrow samples at diagnosis were analyzed for FLT3-LM, FLT3-TKD, c-KIT, c-FMS, N-ras, K-ras and JAK2 mutations. Results. Sixty-six of 129 patients (51.2%) with AML1-ETO had RTK/Ras/JAK2 mutations compared with 30 of 46 patients (65.2%) with CBFβ-MYH11 (p=0.121). The frequencies of RTK/Ras/JAK2 mutations in 129 AML1-ETO AML were 3.9% (n=5) for FLT3-LM, 6.2% (n=8) for FLT3-TKD, 2.3% (n=3) for N-ras, 3.9% (n=5) for K-ras, 35.7% (n=46) for c-KIT, and1.6% (n=2) for JAK2 mutation. The frequencies of RTK/Ras/JAK2 mutations in 46 CBFβ-MYH11 AML were 2.2% (n=1) for FLT3-LM, 19.6% (n=9) for FLT3-TKD, 21.7% (n=10) for N-ras, 23.9% (n=11) for c-KIT, and none for K-ras or JAK2 mutations. No c-FMS mutations were detected in both subtypes of CBF-AML. All RTK/Ras/JAK2 mutations were mutually exclusive except three, one each with N-ras and K-ras mutations, FLT3-TKD and c-KIT mutations, c-KIT and JAK2 mutations, respectively. Patients with CBFβ-MYH11 had a significantly higher frequency of FLT3-TKD and N-ras mutations than patients with AML1-ETO (p=0.017 for FLT3-TKD, and p Conclusion. Our results showed that occurrence of cooperating mutations of RTK/Ras/JAK2 pathways are common in patients with CBF-AML, but the patterns of mutations were different between AML1-ETO and CBFβ-MYH11 subtypes.

BACKGROUND. Ras mutations are thought to drive proliferation of leukemic cells. We sought to determine the frequency of N-Ras and K-Ras mutations in a large cohort of childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) with special reference to the presence or absence of MLL gene rearrangements. METHODS. Bone marrow samples from 300 children with B-precursor ALL, and 132 children with de novo AML were studied at initial diagnosis. Southern blot analysis was performed to detect MLL rearrangement. RT-PCR was used to detect common MLL fusion transcripts. cDNA panhandle PCR technology was used to identify the infrequent or unknown MLL partner genes. DNA PCR or RT-PCR followed by direct sequencing for each PCR product was performed to detect mutations at codons 12, 13 and 61 of N-Ras and K-Ras genes. RESULTS. Of the 300 patients with B-precursor ALL, 20 had MLL(+), the MLL fusion transcripts included 10 MLL-AF4, 7 MLL-ENL, 2 MLL-AF9, and 1 MLL-AF10, all had pro-B subtype (with CD10 negative). Of the 132 patients with de novo AML, 16 had MLL(+), including 4 MLL-AF9, 5 MLL-AF10, 2 MLL-ENL, one each of MLL-AF1, MLL-AF4, MLL-ELL, MLL-SEPT6 and MLL-PTD. N-Ras mutations were detected in 2 of 20 MLL(+) ALL and in 26 of 274 MLL(−) ALL patients (P=1.000). N-Ras mutations were detected in 2 of 16 MLL(+) AML and in 14 of 116 MLL(−) AML patients (P=1.000). K-Ras mutations were detected in 8 of 20 MLL(+) ALL patients compared with 29 of 280 MLL(−) ALL patients (P=0.001). K-Ras mutations were detected in 2 of 16 MLL(+) AML patients compared with 6 of 114 MLL(−) AML patients (P=0.256). Taken together, 10 of 20 MLL(+) ALL children harbored Ras mutations: 4 Gly12Asp, 2 Gly12Val, 1 Gly12Ala and 1 Gly13Asp for K-Ras, and 2 Gly12Asp for N-Ras mutations. The 4 Ras mutations in MLL(+) AML consisted of one each of Gly12Ala and Gln61Pro for N-Ras and one each of Gly12Ala and Gly13Asp for K-Ras mutations. The frequency of Ras mutations was higher in MLL(+) ALL than that in MLL(+) AML, but the difference did not reach statistical significance (P=0.176). Twenty-six of 274 MLL(−) ALL patients harbored N-Ras mutations: 6 Gly12Asp, one each of Gly12Cys and Gly12Ala, 6 Gly13Asp, 5 Gln61Leu, 3 Gln61Lys, 2 Gln61Arg, 1 Gln61His, and one with both Gly12Asp and Gly13Asp. Four of MLL(−) ALL patients had both N-Ras and K-Ras mutations. Fourteen of 116 MLL(−) AML patients harbored N-Ras mutations: 2 Gly12Asp, 2 Gly12Cys, 1 Gly12Ser, 2 Gly13Cys, one each of Gly13Asp and Gly13Arg, 2 Gln61His, 2 Gln61Leu, and 1 Gln61Arg. Six MLL(−) AML patients had K-Ras mutations: 2 Gly12Cys, and one each of Gly12Ala, Gly12Asp, Gly12Ser and Gly13Asp but no mutation at codon 61. One MLL(−) AML patient had both N-Ras and K-Ras mutations. For B-precursor ALL, the frequency of Ras mutations in MLL(+) patients was significantly higher than that of MLL(−) ALL (50% vs. 18.6%, P=0.002). There was no difference in the frequency of Ras mutations between MLL(+) AML and MLL(−) AML (P=0.485). CONCLUSIONS. Ras mutations were detected in 20.7% of children with B-precursor ALL and in 17.7% of childhood AML. MLL(+) B-precursor ALL was highly associated with Ras mutations (50%), especially with K-Ras mutations (40%), while MLL(+) AML was not.