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The development of a second malignancy after the diagnosis of childhood acute lymphoblastic leukemia (ALL) is a rare event. Certain second malignancies have been linked with specific elements of leukemia therapy, yet the etiology of most second neoplasms remains obscure and their optimal management strategies are unclear. This is a first comprehensive report of non-Hodgkin lymphomas (NHLs) following pediatric ALL therapy, excluding stem-cell transplantation. We analyzed data of patients who developed NHL following ALL diagnosis and were enrolled in 12 collaborative pediatric ALL trials between 1980-2018. Eighty-five patients developed NHL, with mature B-cell lymphoproliferations as the dominant subtype (56 of 85 cases). Forty-six of these 56 cases (82%) occurred during or within 6 months of maintenance therapy. The majority exhibited histopathological characteristics associated with immunodeficiency (65%), predominantly evidence of Epstein-Barr virus–driven lymphoproliferation. We investigated 66 cases of post-ALL immunodeficiency-associated lymphoid neoplasms, 52 from our study and 14 additional cases from a literature search. With a median follow-up of 4.9 years, the 5-year overall survival for the 66 patients with immunodeficiency-associated lymphoid neoplasms was 67.4% (95% confidence interval [CI], 56-81). Five-year cumulative risks of lymphoid neoplasm– and leukemia-related mortality were 20% (95% CI, 10.2-30) and 12.4% (95% CI, 2.7-22), respectively. Concurrent hemophagocytic lymphohistiocytosis was associated with increased mortality (hazard ratio, 7.32; 95% CI, 1.62-32.98; P = .01). A large proportion of post-ALL lymphoid neoplasms are associated with an immunodeficient state, likely precipitated by ALL maintenance therapy. Awareness of this underrecognized entity and pertinent diagnostic tests are crucial for early diagnosis and optimal therapy.

Activating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

Introduction KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) is a heterogeneous genetic subgroup with a frequency of about 25% in children with AML. At the 62 nd ASH annual meeting last year, we reported on the differences in outcome of various KMT2A subgroups based on translocation partner and the significance of minimal residual disease (MRD) status during and after induction as a follow-up study of Balgobind et al., Blood 2009. The impact of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) and the presence of additional cytogenetic aberrations (ACAs) on prognosis have not yet been described for our cohort. Methods Data on allo-HSCT in CR1 and the presence of ACAs of 1256 KMT2A-r de novo pediatric AML patients from 15 AML study groups affiliated with the I-BFM Study Group, diagnosed between 2005 and 2016, were retrospectively collected and studied. Karyotypes were reviewed and classified by two of the authors (RW&CH). Based on translocation partners, patients were classified to the KMT2A high-risk subgroup (6q27, 10p11.2, 10p12, 4q21, and 19p13.3) or non-high-risk subgroup (9p22, 19p13, 19p13.1, 1q21, Xq24, 17q21, 1p32, and 17q12). These two categories have been used to estimate a Cox model. Patients with unknown translocation partners were excluded from these analyses (n=126). Flow cytometry MRD levels at the end of induction course 1 (EOI1) and 2 (EOI2) Results Of 1256 pediatric patients with KMT2A-r AML, data on HSCT in CR1 and ACAs were available for 1186 (94.4%) and 1204 patients (95.9%), respectively; 211 (17.8%) patients received HSCT in CR1 and ACAs were present in 601 (49.9%) patients. Compared with the KMT2A non-high-risk subgroup, patients in the KMT2A high-risk subgroup underwent HSCT in CR1 more often (23.8% vs 15.0%; P < .001). ACAs were borderline significantly more common in the KMT2A high-risk subgroup (54.1% vs 46.4%; P = .015). Univariate analysis of the probability of DFS (Table 1) showed that the KMT2A high-risk subgroup (HR 2.1; 95% CI, 1.7-2.5), age ≥10 years (HR 1.4; 95% CI, 1.2-1.7), and MRD ≥0.1 at EOI1 (HR 1.5; 95% CI, 1.1-1.9) were associated with DFS. HSCT in CR1 was a borderline significant prognostic factor (HR 0.7; 95% CI, 0.6-0.9). In a multivariate analysis for DFS (n=515) (Table 1), the KMT2A high-risk subgroup (HR 2.0; 95% CI, 1.6-2.6), MRD ≥0.1 at EOI1 (HR 1.7; 95% CI, 1.2-2.3), and HSCT in CR1 (HR 0.6; 95% CI, 0.4-0.9) were associated with DFS. Univariate analysis of the probability of OS (Table 1) showed that the KMT2A high-risk subgroup (HR 1.8; 95% CI, 1.5-2.2), age ≥10 years (HR 1.6; 95% CI 1.3-2.0), WBC ≥100 x10 9/L (HR 1.4; 95% CI, 1.1-1.7), the presence of ACAs (HR 1.4; 95% CI, 1.2-1.7), and MRD ≥0.1 at EOI1 (HR 2.1; 95% CI, 1.6-2.7) were associated with OS. HSCT in CR1 was not associated with OS. The effect of HSCT in CR1 was not significantly different between the KMT2A high-risk and non-high-risk subgroups. In a multivariate analysis for OS (n=557) (Table 1), the KMT2A high-risk subgroup (HR 1.9; 95% CI, 1.4-2.5), age ≥10 years (HR 1.5; 95% CI, 1.1-1.9), the presence of ACAs (HR 1.6; 95% CI, 1.2-2.1), and MRD positivity at EOI1 (HR 1.9; 95% CI, 1.4-2.5) were associated with OS. Conclusions In this cohort of KMT2A-r pediatric AML patients, the presence of ACAs at diagnosis was independently associated with inferior OS, but not with DFS. This may be due to the exclusion of refractory patients in DFS analysis, who were significantly more common in the group of patients with ACAs. Analysis has yet to be performed to distinguish karyotype complexity. In addition, allo-HSCT in CR1 was an independent predictor of improved DFS, but was not a prognostic factor for OS. Figure 1 Figure 1. Disclosures Abrahamsson: wedish Children´s Cancer Foundation. Research grants and 50% senior research position for clinical research on pediatric leukemia: Research Funding. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau.

Introduction Outcome of KMT2A-rearranged (KMT2A-r) pediatric AML (pAML) is in general poor with a 5-year probability of event-free survival (5y-pEFS) and overall survival (5y-pOS) of 44% and 56%, respectively (Balgobind et al., 2009). However, over the past decades, the heterogeneity of KMT2A-r pAML has emerged, showing differences in outcome between subgroups based on translocation partners. The predictive value of MRD in KMT2A-r pAML is undefined. This retrospective study aimed to confirm the outcome of pediatric KMT2A subgroups (Balgobind et al., 2009) in a more recent era and to study the significance of MRD status during and after induction. Methods Outcome and MRD data of 1257 KMT2A-rde novo pAML patients from 15 AML groups affiliated with the I-BFM-AML study group, diagnosed between 2005 and 2016 were retrospectively collected. Patients were assigned to KMT2A subgroups, or to the KMT2A-other group in case of unknown translocation partner. Flow cytometry MRD levels Results The 1257 patients were assigned to 13 KMT2A subgroups, or the KMT2A-other group. Two novel subgroups were identified: t(X;11)(q24;q23) (n=21, 2%) and t(1;11)(p32;q23) (n=12, 1%). The median age was 2.5 years (range, 0-18.9). The median WBC was 21.4 x 109/L (range, 0.2-727). Overall complete remission rate was 91%. The 5y-pEFS was 46% [SE, 2%] and the 5y-pOS was 62% [SE, 2%]. Differences across subgroups in 5y-pEFS (Figure 1) ranged from 24% [SE, 5%] to 76% [SE, 9%], and in 5y-pOS from 25% [SE, 13%] to 92% [SE, 8%] (both p The subgroups t(10;11)(p12;q23) (HR 1.7, p100 x 10^9/L (HR 1.3, p=.006), and age >10y (HR 1.3, p=.005) were revealed as independent predictors of poor EFS. These factors also predicted OS. MRD data after induction course one were available for n=635 (MRD-positivity (range, 0.1-94) n=126, 20%) and after course two for n=527 (MRD-positivity (range, 0.1-88) n=51, 10%). In the four KMT2A poor-risk subgroups, MRD-positivity was not significantly more common after induction course one (p=.0232) or two (p=.066), compared with the other subgroups. MRD-positivity was associated with inferior 5y-pDFS after both induction course one (36% [SE, 4%] vs 48% [SE, 2%]; p=.002) and course two (28% [SE, 6%] vs 49% [SE, 2%]; p10y (HR 1.5, p=.002) were revealed as independent predictors of poor DFS. Within the group of patients with MRD-negativity after induction course two, the subgroups t(10;11)(p12;q23) and t(10;11)(p11.2;q23) were independent predictors of poor EFS (5y-pEFS 35%, HR 1.7, p=.003 and 5y-pEFS 18%, HR 2.7, p=.004, respectively). Conclusion Outcome for KMT2A-r pAML patients has improved slightly, but similar subgroups were identified as poor risk (Balgobind et al., 2009), including t(10;11)(p12;q23), t(10;11)(p11.2;q23) and t(6;11)(q27;q23). In our study, t(4;11)(q21;q23) was poor risk as well. These subgroups should be considered for high-risk pAML therapy protocols. The favorable risk of t(1;11)(q21;q23) could not be confirmed in our cohort. MRD status is highly predictive of outcome within KMT2A subgroups. In MRD-negative patients after induction course two, both t(10;11) KMT2A subgroups were associated with poor outcome. Disclosures Guest: Syndax Pharmaceuticals: Consultancy. Locatelli:Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Rubnitz:AbbVie Inc.: Research Funding. Kaspers:Helsinn Healthcare: Ended employment in the past 24 months; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Ended employment in the past 24 months; AbbVie: Ended employment in the past 24 months.

Children with Down syndrome (DS) have a 20-fold increased risk of acute lymphoblastic leukemia (ALL) and distinct somatic features, including CRLF2 rearrangement in ∼50% of cases; however, the role of inherited genetic variation in DS-ALL susceptibility is unknown. We report the first genome-wide association study of DS-ALL, comprising a meta-analysis of 4 independent studies, with 542 DS-ALL cases and 1192 DS controls. We identified 4 susceptibility loci at genome-wide significance: rs58923657 near IKZF1 (odds ratio [OR], 2.02; P(meta) = 5.32 × 10(−15)), rs3731249 in CDKN2A (OR, 3.63; P(meta) = 3.91 × 10(−10)), rs7090445 in ARID5B (OR, 1.60; P(meta) = 8.44 × 10(−9)), and rs3781093 in GATA3 (OR, 1.73; P(meta) = 2.89 × 10(−8)). We performed DS-ALL vs non-DS ALL case-case analyses, comparing risk allele frequencies at these and other established susceptibility loci (BMI1, PIP4K2A, and CEBPE) and found significant association with DS status for CDKN2A (OR, 1.58; P(meta) = 4.1 × 10(−4)). This association was maintained in separate regression models, both adjusting for and stratifying on CRLF2 overexpression and other molecular subgroups, indicating an increased penetrance of CDKN2A risk alleles in children with DS. Finally, we investigated functional significance of the IKZF1 risk locus, and demonstrated mapping to a B-cell super-enhancer, and risk allele association with decreased enhancer activity and differential protein binding. IKZF1 knockdown resulted in significantly higher proliferation in DS than non-DS lymphoblastoid cell lines. Our findings demonstrate a higher penetrance of the CDKN2A risk locus in DS and serve as a basis for further biological insights into DS-ALL etiology.

Comprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age 50 chromosomes (n=44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): -7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1; 22)(p13; q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9; 11)(p22; q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk-7p abnormalities; poor risk-normal karyotypes, -7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, -13/13q-, and -15; and intermediate risk-others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

Pediatric acute promyelocytic leukemia (APL), a rare childhood neoplasm, can be cured with all-trans retinoic acid (ATRA) and anthracycline. However, most published trials to date have employed high cumulative doses of anthracyclines. Here, we report the outcome of patients with newly diagnosed APL enrolled into the International Consortium for Childhood APL (ICC-APL-01) trial, which reduced anthracycline exposure but extended that of ATRA. The study recruited 258 children/adolescents with molecularly/cytogenetically-proven APL. Patients were stratified into standard-risk (SR) and high-risk (HR) according to the baseline WBC count (< or ≥10x109/L); both groups received identical induction treatment with ATRA (25 mg/m2/day, for 30 consecutive days) and 3 doses of idarubicin (12 mg/m2/dose). Two or three blocks of consolidation therapy were administered to SR and HR patients, respectively, while maintenance therapy with low-dose chemotherapy and ATRA cycles was given to all patients for 2 years. The cumulative dose of daunorubicin-equivalent anthracyclines in SR and HR patients was lower than that of previous studies, being 355 mg/m2 and 405 mg/m2 in SR and HR patients, respectively. Hematologic remission was obtained in 97% of patients; 8 children died of intracranial hemorrhage in the first 2 weeks following diagnosis. The 5-year overall and event-free survival for the whole cohort were 94.6% and 79.9%, respectively; they were 98.4% and 89.4% in SR and 84.3% and 74.2% in HR patients (p=0.002 and p=0.043, respectively). These data demonstrate that extended use of ATRA coupled to a risk-adapted consolidation can achieve high cure rates in childhood APL and limit anthracycline exposure. The trial was registered at www.clinicaltrials.gov with the following identification number EudractCT 2008-002311-40.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Munster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19+ leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19+ ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19- and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.

In this issue of Blood, Roberts et al describe a preclinical mouse model of acute lymphoblastic leukemia (ALL) caused by the ETV6-NTRK3 fusion gene.1

ABL-class fusions other than BCR-ABL1 (or Ph+) are found in 2-3% of precursor B-cell acute lymphoblastic leukemia (pB-ALL) in children and adolescents. Occasional reports suggest that this rare ALL subtype has a poor prognosis and patients can benefit from treatment with tyrosine kinase inhibitors (TKIs). Aim of this retrospective study is to investigate the presenting features, treatment response and outcome in ABL-class fusion positive cases identified within large cohorts of patients treated in AIEOP-BFM ALL trials. This retrospective survey of ABL-class fusion positive pB-ALL other than Ph+ ALL was performed in patients aged 1-17 years at the diagnosis, treated from October 2000 to August 2018 according to the AIEOP-BFM (Associazione-Italiana- di- Ematologia-Oncologia Pediatrica-Berlin-Frankfurt-Münster) ALL 2000 and 2009 protocols in Austria, Australia, Czech Republic, Germany, Israel, Italy and Switzerland. While ABL-class fusions screening was not required by protocols, it was performed in some patients, according to centers' policies, usually after poor early treatment response. Overall, 46 ABL-class fusion positive cases with ABL1 fusions (N=15), ABL2 fusions (N=5), CSF1R fusions (N=3) and PDGFRB rearrangements (N=23) were identified. Compared with other pB-ALL children and adolescents, the ABL-class fusion positive cases presented with higher proportions of patients aged 10 years or older (52.2 vs. 22.2, P< .0001), hyperleukocytosis (WBC ≥100x109/l, 41.3 vs. 6.3, P< .0001), or poor minimal residual disease (MRD) response (>5x10-4 levels were observed in 65.2% vs. 18%, P< .0001 of patients after induction treatment phase IA and in 45.7% vs. 4.8%, P< .0001 after consolidation phase IB). For the entire cohort of 46 cases, the 5-year probability of event-free survival (EFS) was 49.1+8.9% and that of overall survival (OS) 69.6+7.8%; the cumulative incidence of relapse (CI) was 25.6+8.2% and treatment-related mortality 20.8+6.8%. Although not prescribed by the protocols, 13 patients received a TKI during different phases of treatment (TKI group), by choice of treating physicians, generally due to poor early treatment response. Eight TKI patients with high MRD levels at the end of induction phase IA received the TKI during consolidation phase IB, and six of them achieved either a low positive or negative MRD level at the end of consolidation phase IB. Nine of the 13 patients treated with TKIs underwent hematopoietic stem cell transplantation (HSCT) and only 1/9 (TKI+HSCT) relapsed. Thirty-three cases did not receive any TKI (no-TKI group) and eight of them relapsed; 6/17 patients treated with chemotherapy only, versus only 2/16 who underwent HSCT. Overall, 25 patients underwent HSCT, and of them 3 relapsed and 6 died of treatment-related complications. In patients with a WBC higher or lower than 100x109/L, the 5-year EFS was 36.8+12.7% vs. 59.9+11.6%, respectively (P= .21), and the 5-year OS was significantly lower in patients with a high WBC (48.8+12.9% vs. 87.4+6.8%, P= .036). This difference was more pronounced in the no-TKI group with a 5-year EFS 27.8+13.6% vs 61.8+12.7%, (P= .07), and an OS of 36.7+14.6% vs 94.4+5.4%, respectively (P= .0015). Presenting features, treatment response and outcome in this cohort of ABL-class fusion positive patients are markedly similar to those of patients with Ph+ ALL included in the EsPhALL studies. Our results suggest that TKIs and HSCT may be beneficial in reducing the risk of relapse. Thus there is an urgent need for large international cooperative controlled studies to investigate the impact of TKI, in combination with an appropriate chemotherapy backbone and the role of HSCT. To this purpose, an early identification of patients with ABL-class fusion positive acute lymphoblastic leukemia will be necessary. Disclosures Izraeli: sightdx: Consultancy; novartis: Honoraria; prime oncology: Speakers Bureau. Locatelli:Miltenyi: Honoraria; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Schrappe:Together with study group from SHIRE, JazzPharma, Servier, SigmaTau, Amgen, and Novartis.: Research Funding; SHIRE, Servier, and JazzPharma: Honoraria.

Background and aim: The AIEOP-BFM ALL 2009 trial (https://www.clinicaltrials.gov/NCT01117441) is an international collaborative treatment protocol for children and adolescents with acute lymphoblastic leukemia (ALL) wherein PEG-ASP (Oncaspar®, Servier) was used as the front-line ASP product. Since HSR represent the most relevant limitation to the completion of ASP treatment schedules in the context of any study protocol, we evaluated the cumulative incidence of HSR following PEG-ASP treatment for better understanding the impact of this product on the HSR phenomenon in the context of the treatment strategy outlined in the AIEOP-BFM ALL 2009 study protocol. Patients and Methods: Children with newly diagnosed ALL were enrolled in the AIEOP-BFM ALL 2009 protocol and stratified as Standard (SR), Intermediate (IR) or High-Risk (HR) according to their presenting and response criteria. A first exposure to PEG-ASP, administered I.V. at 2,500 IU/sqm throughout the whole protocol, was scheduled in all patients during induction (phase IA, 2 doses, on day 12 and 26), whilst second or further exposures were scheduled as follows: a. in SR and MR patients during the reintensification (protocol II, 1 dose); and b. in HR patients during each of the three intensive blocks and of the three protocols III (1 dose in each of these phases). In addition, MR patients were randomized (RMR) at the start of reintensification (protocol II) to receive 0 (Standard Arm, SA) vs 9 (Experimental Arm, EA) additional biweekly PEG-ASP doses throughout protocol II and continuation therapy. Therefore, all the SR patients and the MR patients randomized to SA overall received 3 PEG-ASP doses with an 18-week-long interval between doses #1+#2 (induction, first exposure) and dose #3 (reintensification, second exposure) whilst the MR patients randomized to the EA overall received 12 PEG-ASP doses with an 18-week-long interval between doses #1+#2 (induction, first exposure) and doses #3 to #12 (reintensification and continuation therapy, second exposure). HR patients were randomized (RHR) at the start of the consolidation phase (protocol IB) to receive 0 (SA) vs 4 (EA) additional weekly PEG-ASP doses. Therefore, HR patients randomized to the EA received overall 12 PEG-ASP doses (first exposure: #1+#2 in IA; further exposures: #3 to #6 in IB, #7 to #9 in each of the 3 blocks and #10 to #12 in each of the protocols III) without relevant time-intervals among the various doses, whilst HR patients randomized to the SA received overall 8 doses with an 8-week-long interval between the doses #1+#2 (IA, first exposure) and the second and further exposures (3 blocks and 3 protocols III, doses #3 to #8). HSR were recorded by treating physicians and collected in the trial database and cumulative incidence (CI-HSR) was estimated adjusting for competing risks (death for any reasons and leuekemia recurrence). Results: Between June 01, 2010 and February 28, 2017, 6136 patients were eligible and evaluable in the study and are here reported. An HSR occurred in 468 patients, with a CI-HSR of 7.6% (SE 0.3). In particular, 121 (2.0%) occurred in IA, 20 (0.3%) in IB, 140 (2.3%) in Protocol II, 183 (3.0%) in blocks, 4 (0.07%) in Protocols III and maintenance. According to the stratification group, CI-HSR was 4.9%(0.5) in SR, 4.9% (0.4) in MR and 16.8% (1.0) in HR groups. In the RMR study, the CI-HSR was 3.1% (0.6) vs 4.3% (0.8) in the SA vs the EA, respectively (p-value=0.26), whilst in the RHR study the CI-HSR was 18.3% (1.9) vs 6.1% (1.3) in the SA vs the EA, respectively (p-value Conclusions: In the AIEOP-BFM ALL 2009 trial, the incidence of HSR was much lower with respect of historical experiences of previous AIEOP-BFM ALL protocols wherein the native E.Coli ASP product was used I.V. as first-line product. No difference in terms of HSR was found between patients belonging to the EA and SA of the RMR. As expected, due to the higher number of PEG-ASP doses administered, the cumulative incidence of HSR was much higher in the HR group. Interestingly and in keeping with the concept that continuous and prolonged exposures to PEG-ASP reduce the probability of developing an HSR, patients randomized to receive in the RHR the SA (long interval between the first and subsequent exposures) showed an incidence of HSR much higher (18.3%) than that observed in the EA control arm (6.1%). Disclosures Rizzari: Together with study group from Shire, medac , Sigma-Tau, Baxalta, Shire and Servier: Research Funding; JazzPharma, Shire, medac , Sigma-Tau, Baxalta, Shire and Servier: Consultancy, Honoraria. Locatelli:Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; BluebirdBio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Boos:medac GmbH: Membership on an entity's Board of Directors or advisory committees, Other: Safety Boards, invited lectures, drug monitoring program; Eusa Pharma: Other: invited lectures, drug monitoring program; Jazz Pharmaceuticals: Other: invited lectures, drug monitoring program; Baxalta: Other: invited lectures, drug monitoring program; Shire: Other: invited lectures, drug monitoring program; Sigma-Tau: Other: invited lectures, drug monitoring program. Zucchetti:Together with study group from medac , Sigma-Tau, Baxalta, Shire and Servier: Research Funding. Schrappe:Together with study group from SHIRE, JazzPharma, Servier, SigmaTau, Amgen, and Novartis.: Research Funding; SHIRE, Servier, and JazzPharma: Honoraria.

Background: Inotuzumab ozogamicin (InO) is a CD22-directed monoclonal antibody linked to calicheamicin. It is approved for adults with relapsed/refractory (R/R) CD22+ B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at the dose of 1.8 mg/m2/course fractionated at day 1, 8 and 15 (Kantarjian, NEJM, 2016). The pediatric experience with InO is still limited. Patients (pts) We collected cases of infants and young children diagnosed with R/R ALL and treated with InO via a compassionate access program. Methods: Participating international pediatric oncology centers submitted retrospective demographic, outcome, and toxicity data on infants (age Results: Between 2015 and 2019, 9 infants and 3 children (1.0-2.9 years of age) were diagnosed with ALL and later on received 1-3 cycles of InO due to R/R disease. At the time of InO treatment 1 pt was At the start of InO, 8 (67%) pts had an M3 bone marrow status, 2 (17%) an M2 and 2 an M1 with MRD positivity. CD22 expression was reported in 7/12 pts: in 3 cases >90% of blasts expressed CD22, the others ranging from 52% to 80%. In total 21 courses were administered (60 doses; 3 courses not completed), median administered dose of InO per course 1.74 mg/m2 (divided in 3 weekly doses, with the loading dose at D1 of cycle 1): 6 pts received 1.8 mg/m2 (adult approved dose), 4 intermediate dosages as 1.5 or 1.6 mg/m2, 1 1.4 mg/m2 and 1 1.2 mg/m2. The dose was based on BSA; 0.07 mg/Kg/course was the median pro-Kg dose administered (higher than in adults). 6 pts (50%) achieved a complete remission (CR) (3 after 1 course and 3 after 2-3 courses), 1 additional MRD-positive pt. turned MRD-negative ( Only limited data about toxicity were reported in our records, although hematologic toxicity was the most common (thrombocytopenia in 33% of pts). 1 case of neurotoxicity described as toxic leukoencephalopathy was reported after 1 course of InO, causing death in CR. 1 pt. developed severe veno-occlusive disease (VOD) after HSCT performed in PR, which was fatal. Conclusions: Single agent InO in infants and younger children had a CR rate of 58% in this cohort of heavily pretreated pts. Even with a median dose close to the adult recommended starting dose, InO appeared to be well tolerated, allowing a subsequent HSCT in 5 pts. While taking into account the lower CD22 expression showed by MLL-r pts, further investigation with InO treatment might be valuable in this subgroup of ALL pts with a particular dismal prognosis. Disclosures Pieters: medac: Consultancy; jazz farmaceuticals: Consultancy. Zwaan:Celgene: Consultancy, Research Funding; Roche: Consultancy; Sanofi: Consultancy; Novartis: Consultancy; Daiichi Sankyo: Consultancy; Servier: Consultancy; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Jazz pharmaceuticals: Other: Travel support; Incyte: Consultancy. OffLabel Disclosure: Inotuzumab ozogamicin (InO) is a CD22-directed monoclonal antibody linked to calicheamicin. It is approved for adults with relapsed/refractory (R/R) CD22+ B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at the dose of 1.8 mg/m2/course fractionated at day 1, 8 and 15 (Kantarjian, NEJM, 2016). The pediatric experience with InO is still limited. Patients (pts)

Mucormycosis has emerged as an increasingly important infection in patients with hematological malignancies, and is associated with considerable morbidity and mortality. However, contemporary data concerning epidemiology and outcome in children is lacking. Here we report a nationwide multicenter study of mucormycosis among children with hematological malignancies. Between the years 2004-2017, 1136 Israeli children aged ≤18 years with acute leukemias (acute lymphoblastic leukemia (ALL)-941; acute myeloid leukemia (AML)-195) were prospectively enrolled on a centralized clinical registry. Fungal infections, including mucormycosis, were prospectively and retrospectively captured. In addition, all 7 pediatric hematology centers were required to search hospital medical records, microbiology databases, and pathology information systems for cases of mucormycosis. Following central revision, 39 cases of mucormycosis were identified. Patient characteristics: Median age at mucor diagnosis: 13.5 years. Underlying diseases: ALL-27, AML-9, other-3. In 24 patients mucormycosis occurred during frontline therapy of their disease, and in 15 patients at relapse. Known risk factors included stem cell transplantation (SCT)-13 (33%), severe neutropenia-33 (85%) and corticosteroids-26 (67%). Among 20 patients on frontline ALL therapy, mucormycosis was diagnosed during induction in 13 cases, reinduction-6 cases, consolidation-1 case. Twenty-five patients with mucormycosis were enrolled on the national acute leukemia registry (not included: other malignancies, non-Israeli patients, relapsed leukemia with primary diagnosis preceding registry initiation, secondary AML). An analysis of registry cases demonstrated that the incidence of mucormycosis did not significantly increase during the study period. There was no seasonal clustering. Mucormycosis was significantly more common among patients ≥10 years old (4% vs 1%, p=0.004). Among patients on frontline treatment, mucormycosis was significantly more common in high-risk (HR) ALL patients (6%) than in non-HR (1%) or AML (1%) patients (p=0.001). Mucormycosis: Patterns of infection included sinusitis/sino-orbital-16 (41%), rhinocerebral-8 (21%), pulmonary-3 (8%), mandibular -4 (10%), gastrointestinal-1, cutaneous-1, otomastoiditis-1, disseminated-6 (15%). The majority (46%) were caused by Rhizopus spp. EORTC criteria: proven-30, probable-7, possible-2 (pathognomonic radiological findings and molecular diagnosis). Treatment: Nineteen patients were treated with amphotericin B formulations only and 19 received combined antifungal treatment. Nine patients received step-down treatment with posaconazole/ isavuconazole. In addition, 33 patients underwent surgical interventions, 26 underwent 2 procedures or more (range 2-14). Median time from presentation to antifungal treatment-3 days. Outcome: Twenty-one patients died, 15 of mucormycosis, 3 of other toxicities, 3 of leukemia. Factors significantly associated with mortality from mucormycosis were preceding SCT (p=0.01), less than 2 surgical interventions (p=0.0003) and relapsed disease (p=0.006). Eight-year OS of the whole cohort, those on frontline therapy and relapsed patients was 42% (±21%), 70%(±13%), 13%(±66%), respectively. Eight-year cumulative incidence of death from mucormycosis (CIDM) in those subgroups was 40%(±8), 21%(±8), 67% (±13), respectively. To our knowledge, this is the largest population-based study of mucormycosis in children with hematological malignancies. Our study demonstrates that mucormycosis is an age-dependent toxicity, significantly more common in patients ≥10 years of age. A striking finding was the high incidence of mucormycosis among patients on frontline high-risk ALL therapy, a fact which may reflect the increasing intensity of childhood ALL treatment. In a cohort of 24 patients who developed mucormycosis on frontline therapy, including 2 patients with AML and 13 with HR-ALL, 8y-OS was 70% and 8y-CIDM was 21%.The majority of patients in this cohort were salvageable through control of underlying disease, rapid instigation of antifungal treatment, and surgical debridement procedures, albeit in some cases at a price of mutilation and long-term sequelae. Future research should focus on the complex interactions between fungi and host, and on developing novel therapeutic strategies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor. Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases. Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations. In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases. Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

Acute leukemia (AL) of ambiguous lineage (AMBI-L) comprises up to 5% of AL cases in both children and adults. Although several definitions exist, a general treatment guideline has been missing. Single country studies usually report fewer than 50 cases of children or adults. Accordingly, the international iBFM AMBI2012 Study/Registry collected 275 AMBI-L cases in patients In total, 275 patients were included in the study. Of these, 240 fulfilled the definitions of biphenotypic/mixed phenotype AL, partially overlapping with cases in whom two clones had been identified (n=68) and 15 cases presented with undifferentiated AL. Most patients started their treatment with an ALL-type protocol (n=161), 79 with AML therapy, 27 with a combined regimen, including the Interfant protocols, 2 patients were not treated, 2 received other treatment, and in 4 patients such information was missing. The 5yEFS of the entire cohort was 56±3.7% and 5y overall survival was 67±3.3%. Patients treated by ALL-type protocols had superior 5 year event free survival (5yEFS) (70±4.6%, n=158) compared to those who started AML-type treatment (5yEFS: 40±6.4%, n=78) or hybrid ALL/AML treatment (5yEFS: 50±11%, n=27). Although protocol selection was likely biased, we recommend ALL treatment, when diagnostic findings, including molecular genetics, fail to indicate AML therapy. Although myeloperoxidase (MPO) has been used as the ultimate marker of myeloid lineage, patients who started with ALL-type treatment demonstrated a better prognosis even among cases classified as MPOpos/part pos (Fig. 1). These differences by initial choice of treatment are most prominent when CD19pos/part pos cases are analyzed regardless of the overall lineage (Fig. 2). This shows that at least for CD19pos/part pos cases in the absence of RUNX1/RUNX1T1 fusion, treatment should not start with current AML-type protocols. Until week 12, patients with higher leukemia burden were slightly overrepresented compared to non-AMBI ALL patients (data not shown). In addition, patients with higher residual disease had a much poorer prognosis. Thus, Prednisone poor and good responders (based on day 8 blood blast counts) had a 5yEFS of 50±9.7%, n=38 and 81±5.8%, n=82, respectively (p=0.005). By day 15 bone marrow (BM), only cutoffs of 10-4 and 10-3 were analyzed and neither showed significant associations with EFS. At the end of induction, patients with BM residual disease ≥10-3 had a 5yEFS of 51±10%, n=49 compared to 90±4.3% for those with lower levels, n=75 (p=0.0002). Especially higher residual disease at week 12 was associated with an extremely poor EFS (Fig. 3). Early identification of patients with inadequate response and designing alternative treatment for them is our important challenge. No overall benefit of transplantation was seen in patients who started on ALL treatment or hybrid ALL/AML treatment. Again, this may be caused by a biased selection of more severe cases for transplant. In patients who started with AML treatment, transplant appeared to improve prognosis (Fig. 4). This study provides the basis for improved treatment of future patients with AMBI-L, with more accurate diagnostics. OH, AL, IJ, EM and JS were supported by Czech Health Research Council 15-28525A. Disclosures Bleckmann: JazzPharma: Other: financial support of travel costs. Moricke:JazzPharma: Honoraria, Other: financial support of travel costs. Inaba:Arog: Research Funding. Kattamis:Novartis: Honoraria, Research Funding; ApoPharma: Honoraria. Reinhardt:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Jazz Pharma: Other: Travel Accomodation; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Introduction After treatment response, cytogenetics and molecular aberrations are the most important prognostic factors in children with de novo acute myeloid leukemia (AML). However, little is known about the impact of cytogenetics at relapse. This international retrospective study aimed to provide insight into the prognostic impact of cytogenetic profiles and the role of karyotype changes from diagnosis to relapse in pediatric patients with relapsed AML. Methods Cytogenetic reports from patients registered to the Relapsed AML 2001/01 Study and diagnosed between 2001 and 2010 were centrally reviewed and classified by two independent researchers plus a cytogenetic expert. Patients with refractory or relapsed AML and available cytogenetics at relapse were included in order to assess the prognostic impact of different cytogenetic subgroups at relapse. Patients with karyotypes available at both diagnosis and relapse were included in order to study the impact of karyotype changes. Recurrent cytogenetic aberrations present in ≥5 patients defined the subgroups. Changes at relapse were categorized as: no change, gain, loss, both gain and loss, or structural other aberration(s). Primary endpoints were the probabilities of event-free survival (pEFS) and overall survival (pOS). Univariate analyses were conducted using chi-square tests, binary univariate logistic regression or Kaplan Meier estimates with a log-rank test. Multivariable Cox regression analyses were conducted to evaluate the independent impact of cytogenetic profiles and karyotype changes at relapse. For these analyses, cytogenetic subgroups were regrouped into good risk (GR) cytogenetics [t(8;21)(q22;q22) or inv(16)/t(16;16)(p13.1;q22)] or "other". Results Of the 569 registered patients, 402 patients (71%) had available cytogenetic information at relapse. Frequently detected aberrations at relapse were t(8;21) (n=60, 15%) and inv(16)/t(16;16) (n=24, 6%), both indicating a relatively good prognosis. Although patient numbers were small (n=5), t(6;9)(p23;q34) also had a relatively good outcome. Monosomy 7/7q-, t(9;11)(p22;q23), t(10;11)(p12;q23) and complex karyotypes had a relatively poor outcome. Figure 1 shows the Kaplan Meier curves of the investigated cytogenetic subgroups with corresponding patient numbers. In total, 306 patients (54%) with available karyotypes at both diagnosis and relapse were included to study cytogenetic changes. Patients with any change (n=148, 48%) had inferior outcome compared to patients without changes (3-year pEFS 21% [SE, 3.4%] versus 39% [SE, 3.9%]; overall P After multivariable adjustment (final models including cytogenetics at relapse, time to relapse GR cytogenetics at relapse were significantly associated with better early treatment response. Subsequently, response to treatment at day 28 (Creutzig et al. Haematologica 2014) was an important mediator. If this prognostic parameter was included in the models, the effect of changes diminished, but GR cytogenetics at relapse remained an important prognostic factor associated with superior outcome (pEFS: HR 0.53 [95% CI 0.35-0.81], pOS: HR 0.51 [95% CI 0.32-0.80]). Conclusion Together with early treatment response, the cytogenetic profile at relapse is an important prognostic factor. In particular t(8;21) and inv(16)/t(16;16) at relapse were associated with a favorable outcome. Furthermore, cytogenetic changes between diagnosis and relapse were associated with inferior outcome. Future studies should explore the mechanism(s) of these changes, being either clonal evolution or clonal selection. Interpretation of our results is hampered by the retrospective design of the study, small subgroup numbers and missing cytogenetic and molecular data. Nonetheless, these findings suggest that assessing cytogenetics at time of relapse is of high importance, and can be used for risk group adapted treatment. Disclosures Reinhardt: Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding.

The combined treatment of all-trans retinoic acid (ATRA) and anthracyclines at high cumulative doses (>500-600 mg/m2) is associated with good outcomes in adults and children with acute promyelocytic leukemia (APL). However, high cumulative anthracyclines doses carry a significant risk of late cardiotoxicity, especially in children. In addition, children are more susceptible to ATRA-related side effects (particularly pseudo-tumor cerebri). This suggests that children should receive a lower dose of ATRA. We therefore established an international consortium on childhood APL, ICC APL, and developed the first multinational, multicenter childhood APL study, ICC APL Study 01, with the aim of investigating the safety and efficacy of a regimen with reduced anthracycline cumulative doses and modified ATRA. In some countries the protocol treatment was delivered out as a treatment guideline. Eligible patients had either typical cytogenetics (t(15;17)) or ATRA-sensitive molecular aberration (predominantly PML-RARα). Treatment was stratified by risk group (standard-SR or high-HR, based on white blood cell (WBC) count at diagnosis < or > 10x109/L) and minimal residual disease (MRD) level during post-remissional treatment. SR patients received induction (ATRA plus idarubicin on days 3-5-7) followed by two consolidation courses (I: ATRA, intermediate-dose cytarabine, mitoxantrone; II: ATRA plus idarubicin). HR patients received ATRA plus idarubicin (days 1-3-5) in induction, followed by 3 consolidation courses (I and II identical to SR patients, III: ATRA, intermediate-dose cytarabine, idarubicin). This therapy was followed by 2 years of maintenance in all patients with ATRA given every 3 months, methotrexate weekly and mercaptopurine daily. The daunorubicin-equivalent dose of anthracyclines was 355 mg/m2 and 405 mg/m2 for SR and HR patients, respectively. The cytarabine dose was 6,000 mg/m2 and 12,000 mg/m2 for SR and HR patients, respectively. ATRA was given at a dose of 25mg/m2. Treatment was guided by MRD bone marrow monitoring at the end of consolidation and 3 monthly during and for one year after maintenance completion. SR patients with persistent molecular disease after consolidation received a third course of treatment identical to that given to HR patients. Children with persistent molecular disease after 3 courses, and those with molecular or frank relapse during or after treatment, were eligible for salvage arsenic-trioxide (ATO) treatment. A total of 227 patients were enrolled into the study between January 2008 and May 2015; median age was 10,7 years (range, 0 - 19,9) and 53% of patients were female. FAB type was M3 in 81% and M3 variant in 16% (data N/A for 3% of patients). One hundred and twenty-nine (57%) and 98 (43%) were assigned to the SR and HR groups, respectively. Median WBC and platelet count at diagnosis were 6.1x109/L (range, 0.59-454) and 26x109/L (range, 2-262), respectively. PML/RARα fusion transcript was bcr 1 in 100, 2 in 6, 3 in 88 and not known in 33 patients. Clinical response and MRD data are available for 179/227 patients (79%). Early death rate was 3.1%. MRD positivity decreased over time; 80% of patients MRD were positive after induction, 31% after consolidation course I, 11% after consolidation course II and 4% after consolidation course III. A frank or molecular relapse occurred in 6 (all HR) and 8 (4 HR; 4 SR) patients, respectively; salvage ATO treatment was effective in 12 of these patients. One patient died of therapy-related leukemia. The 3-year probability of overall survival was 95% in the whole cohort of patients (96.5% and 93% in SR and HR patients, respectively; p 0.17). The 3-year probability of event-free survival was 83% for all patients (87% and 79% in SR and HR patients, respectively; p 0.09). In summary, the ICC APL study 01 produced an outcome comparable to that of children treated with AIDA 2000 protocol, which employed higher anthracyclines doses (650 mg/m2 daunorubicin-equivalent) (Testi et al. ASH 2010). These results confirm those reported by Creutzig et al. (BrJHaematol 2010), delivering reduced anthracyclines (cumulative dose 350 mg/m2) with extended ATRA and cytarabine. Data from a randomised study published by Lo Coco et al. (NEJM 2013) urged our consortium to develop ICC APL Study 02, in which SR patients will be treated with ATRA plus ATO and HR patients with ATRA, ATO and gemtuzumab ozogamicin; a front-line treatment without conventional chemotherapy. Disclosures Kaspers: Janssen-Cilag: Research Funding.

Up to 5% of patients with acute leukemia (AL) are diagnosed as AL of ambiguous lineage. The ambiguous lineage ALs consist of mixed phenotype AL (MPAL, or biphenotypic AL, BAL), bilineal AL, switching AL and rare, undifferentiated ALs. From a molecular genetic point of view, they overlap with several molecular genetic subsets such as AL with MLL rearrangements or early T precursor AL. As no general treatment strategy exists, these patients have been variably treated with lymphoblastic (ALL)- , myeloid (AML)- or combined (hybrid) therapy, with or without stem cell transplant. They are often unreported as they are excluded from standard protocols. So far, attempts to shed more light on these patients has largely focused on definitions of ambiguous lineage AL. Only limited therapeutic observations have been possible in studies on this AL subset, usually reporting 50 or fewer pediatric/adult patients. In order to facilitate more detailed analyses, we have created an international study "iBFM AMBI2012 Study/Registry". In this study, patients under 18 years at diagnosis are eligible. Each center/country was asked to report all consecutive patients with ambiguous lineage AL, from a 2- to 13-year period ending May 31, 2015. The definitions included those with WHO and EGIL criteria and remained unchanged throughout the study. Complete information on type of treatment, follow up and immunophenotype was requested. Where available (n=101 at the time of this abstract uploading), raw cytometric FCS data files were stored centrally for review. Apart from the study itself, the central database served also as a basis for consulting individual patients during the diagnostic workup. Furthermore, data on fusion genes, cytogenetics, treatment response and availability of specimens for collateral studies were also collected. In total, 247 patients from Australia, Austria, Brazil, Czechia, Germany, Greece, Israel, Netherlands, NOPHO (Denmark, Estonia, Finland, Norway, Sweden, Iceland and Lithuania), PINDA (Chile), Poland, SAHOP (Argentina), Slovakia, St. Jude Children's Research Hospital (USA), Ukraine and United Kingdom are reported. Among those, 222 fulfilled the definitions of MPAL/BAL, partially overlapping with cases in whom two clones had been identified (n=47) and 14 cases presented with undifferentiated AL. Most of them, consistent with our general treatment guideline (Figure 1), started their treatment with an ALL type of protocol (n=150), 60 patients started on AML therapy, 8 patients received a combined regimen including the Interfant protocols, 2 patients were not treated, 13 received other treatment, and this information is missing in 9 patients (additional 5 pts. started on ALL treatment but their follow up information is incomplete). The 5 year event free survival of the entire cohort was 55±4% and its separation by first type of treatment is shown in Figure 2. In a collateral study, we set up a qPCR array on 90 genes that are characteristic of the lymphoid or myeloid lineages and/or are thought to be involved in their regulation. Using this array, sorted cells of granulocytic, monocytic, T, and B lineages at various stages of development (17 stages total) were analyzed and compared to samples of AL including 6 samples of MPAL of precursor B/myeloid phenotype. Although this array did not show a general deregulation in the MPAL genome compared to that in AL or healthy cells, subtle changes were seen such as decrease of CEBPE and LILRA2 gene expression, in comparison to classical B precursor ALL. Overall, our data shows that the general treatment guideline (Figure 1), which favors ALL treatment is justified by the outcome. However, although this study is larger than those published, caution is needed during its interpretation due to variations in diagnostics and treatment among the participating countries. Therefore, our data should be viewed as a basis for non-ambiguous treatment guidelines that will direct each patient to either ALL or AML treatment. These guidelines will be tested prospectively. In addition, the framework of this study is being used as a basis of consulting new AL cases with diagnostic uncertainties. Furthermore, it serves as a data resource for biologic studies. Supported by AZV 15-28525A, UNCE 204012, NT/14534-3, NT/13462-4, P302/12/G101. Disclosures Kattamis: Novartis: Research Funding, Speakers Bureau; ApoPharma: Speakers Bureau.

Acute megakaryoblastic leukemia (AMKL) comprises up to 10% of childhood acute myeloid leukemia (AML) cases. However, no large-scale studies have comprehensively evaluated the clinical characteristics and outcomes of patients with AMKL. We performed a large-scale international retrospective study of pediatric patients (diagnosed at age ≤18 years) with de novoAMKL without Down syndrome treated from 1989 to 2009. The study included 490 patients with AMKL, which comprises 7.9% of the pediatric AML patients treated by 19 members of the I-BFMSG. At diagnosis, the median age of patients was 1.5 years (range, 0.0–16.5 years), median white blood cell count was 12.0×109/L (range, 0.6–188.0×109/L), 23 (4.7%) patients showed central nervous system involvement, and both sexes were equally represented. Complete remission (CR) was achieved in 417 (85.1%) patients, and 5-year event-free (EFS) and overall survival (OS) rates were 43.7%±2.7% and 49.0%±2.7%, respectively. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed in 206 (42.0%) patients. The 5-year disease-free survival and OS rates for patients who received allo-HSCT in first CR (n=112, 56.3%±5.2%, and 57.7%±5.2%, respectively) and for those who did not receive transplantation in first CR (n=298, 50.2%±3.6% and 55.2%±3.5%) were not significantly different (P=0.12 and P=0.57). Complete cytogenetic data were available for 372 (75.9%) patients: diploid (n=49, 13.2%), hypodiploid (n=18, 4.8%), pseudodiploid (n=119, 32.0%), 47–50 chromosomes (n=142, 38.2%), and >50 chromosomes (n=44, 11.8%). Chromosome gain occurred in 195 (52.4%) patients: +21 was the most common (n=106, 28.5%), followed by +19 (n=93, 25.0%), and +8 (n=77, 20.7%). Losses occurred in 65 cases (17.5%), in the decreasing order of –7 (n=13, 3.5%), –9 (n=9, 2.4%), –13, and –15 (n=7 each, 1.9%). Structural chromosomal aberrations were observed in 278 (74.7%) patients, most commonly t(1;22) (n=51, 13.7%) and 11q23 rearrangements (n=38, 10.2%) with t(9;11) in 21 patients. Other abnormalities included 7p (n=43, 11.6%) and 13q (n=31, 8.3%; 16 with deletions) breakpoints. Patients with t(1;22) were significantly younger at diagnosis (P Multiple regression analysis for EFS and OS with clinical and cytogenetic features showed that treatment period (1989–1999 vs. 2000–2009), normal cytogenetics, –7, t(9;11), 13q-, and –15 were associated with significantly worse outcomes than those for other subgroups, whereas abnormalities in 7p were associated with better outcomes (Table). Patients with –13 and 9p abnormalities other than t(9;11) had a poorer EFS and OS, respectively. Patients with +21 or t(1;22) did not show a significant difference in survival rates. This international study on the largest cohort of AMKL patients analyzed to date shows heterogeneity in cytogenetic findings and identifies some subgroups with a particularly dismal outcome. The advent of newer methods to evaluate genetic lesions can help identify therapeutic targets for improving the outcome in this subgroup of patients. Disclosures No relevant conflicts of interest to declare.

Aim: microRNAs (miRs) have been implicated in many malignancies. Our aim was to identify specific miRNAs that can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated on BFM protocols, better than the current risk group stratification. Currently, the main method for risk group stratification is based on the amount of minimal residual disease (MRD) assessed at specific time points by real time quantitative PCR (RQ-PCR). Material and methods: Following microRNA expression analysis performed on 48 bone marrow samples at diagnosis, we focused on several miRs that correlated with at least 3 of the established prognostic markers in ALL to be validated in a cohort of 138 B-lineage ALL samples. Of the miRs studied, down regulated miR-151-5p and miR-451 and upregulated miR-1290 significantly correlated with outcome. Results: Low expression of miR-151-5p, miR-451 or of both together resulted in significantly worse relapse free survival (RFS) (56%, 63% and 47%, respectively) compared to RFS rates when either or both miRs were highly expressed (80%, 78% and 78%, respectively) (p=0.007, 0.042 and 0.002, respectively). High expression of miR-1290 resulted in worse RFS compared to those that expressed low levels (54% versus 81%; p=0.014). When combining the 3 miRs, a patient expressing low levels of both miRs and/or high levels of miR-1290 had a RFS of 54% (p=0.004). Furthermore, the expression of the 3 miRs could distinguish within the TEL-AML1 negative cohort two groups; one with 80% and the other with 44% RFS (p=0.004). Multivariate Cox regression analysis identified low expression of both miRs and/or high expression of miR-1290 as an independent prognostic marker in the PCR-MRD non-high risk cohort. Patients expressing abnormal levels of the 3 miRs had a 4.47-fold increased risk for relapse (p=0.037). Deletion of IKZF1 gene is a known adverse prognostic marker in B-lineage ALL. In 85 patients, IKZF1 status was analyzed and 9 patients were found to harbor the deletion. The expression of the 3 miRs could still identify an adverse group of patients with 69% RFS within the group with no deletion (p=0.004). When adding IKZF1 and TEL-AML1 status to the Multivariate analysis in addition to PCR-MRD, the expression of the 3 miRs remained a significant independent marker with a 24 fold increased risk for relapse (p=0.011). Figure 1 Figure 1. Conclusion: Our results identify the combination of miR-151-5p, miR-451 and miR-1290 as a novel biomarker for outcome in pediatric B-lineage ALL patients, already at time of diagnosis. This may lead to improved risk group stratification and enable early therapeutic intervention that may result in better RFS. Disclosures No relevant conflicts of interest to declare.

Pediatric B cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is characterized by recurrent chromosomal aneuploidies and translocations, which are important for diagnosis and risk stratification. Classical cytogenetic and FISH are routinely used for the diagnosis of most of these alterations, particularly those involving balanced translocations, and numerical changes. Recently, genome wide analysis, such as aCGH and SNP technologies have identified copy number alterations (CNA) in genes involved in lymphoid differentiation: IKZF1, PAX5, EBF1 BTG1; and in cell cycle control: CDNK2A/B, RB1 and ETV6. Multiplex ligation–dependent probe amplification (MLPA) technique has been used for the identification of those focal genetic aberrations in a single assay but it cannot reliably detect aberrations present in less than 50% of cells. Cytoscan HD SNP array (Affymetrix) can overcome this limitation and is the only array which enables independent confirmation of CNA with SNP allelic information. Thus minor genetic subclones missed by other methods, can be detected. The aim of this work was to compare between SNP array and MLPA platforms in a routine diagnostic setting. Samples of 39 pediatric BCP-ALL patients well characterized by classical cytogenetic and FISH at diagnosis, were studied by MLPA P335 (MRCHolland) and SNP array (Affymetrix). In total 273 genomic loci were evaluated, 13 (5%) were not informative, of them 7 were not covered by the MLPA kit and 6 gave not evaluable MLPA results. 231/260 (89%) of the loci showed concordance, 39/260 (15%) with CNA and 192/260 (74%) normal. A total of 68 CNA were detected, 29 were discordant and 39 concordant. IKZF1 deletion was identified by both methods in 5 cases (13%). However, a deletion was identified in 3 additional samples, only by cytoscan. In these cases, the deletion was present in a minor subpopulation (in 45% or less of the cells), while the main cytogenetic aberration was present in the majority of cells (60%-90%). We conclude that although MLPA is a useful technique, it is not sensitive enough to detect small populations. In this small cohort, 3 IKZF1 deletions were missed since they were present only in a minor subclone. Interestingly, one of these patients relapsed, and the relapse clone harbored the IKZF1 deletion. Conclusion Genome-wide sensitive methods identify different subpopulations in each patient, thus detecting previously unrecognized genetic heterogeneities. Disclosures: No relevant conflicts of interest to declare.

Abstract 2507 Aim: microRNAs (miRNAs) have been implicated in many malignancies. Our aim was to identify specific miRNAs that can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated according to BFM protocols already at diagnosis. The current risk group stratification is based on the amount of minimal residual disease (MRD) assessed at specific time points by real time quantitative PCR (RQ-PCR). Material and methods: Following miRNA expression analysis, we decided to focus on miR-151-5p and miR-451 that significantly correlated with known prognostic factors in ALL. Validation was performed by measuring the expression levels of miR-151-5p and miR-451 by RQ-PCR on bone marrow samples at diagnosis of 101 B-lineage ALL patients excluding Philadelphia positive patients. Results: Low expression of miR-151-5p, miR-451 or of both together resulted in significantly worse relapse free survival (RFS) (43%, 58% and 38%, respectively) compared to RFS rates when either or both miRNAs were highly expressed (81%, 75% and 75%, respectively) (p=0.001, 0.044 and 0.001, respectively). Moreover, following PCR-MRD stratification, low expression of miR-451 or both miRNAs remained significant within the PCR-MRD medium risk group (p=0.00001) and within the standard group for both miRNAs (p=0.024). Multivariate Cox regression analysis identified low expression of both miRNAs as an independent prognostic marker with 11.7 fold increased risk for relapse (p=0.002). After excluding patients harboring the adverse genetic markers Ikaros deletion and P2RY8-CRLF2 rearrangement, a patient expressing low levels of both miRs had a 35 fold risk to relapse (p=0.005). Analyzing a non-BFM treated B-lineage ALL cohort from The Netherlands, both miRNAs significantly correlated with outcome (p=0.003). Conclusion: Our results identify miR-151-5p and miR-451 as novel biomarkers for outcome in pediatric B-lineage ALL patients, regardless of treatment protocol. This may allow earlier and improved risk group stratification already at diagnosis, enabling exploration of early therapeutic interventions. Disclosures: No relevant conflicts of interest to declare.