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Tisagenlecleucel is a CD19 chimeric antigen receptor (CAR) T-cell therapy approved for treatment of pediatric and young adult patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and adults with non-Hodgkin lymphoma (NHL). The initial experience with tisagenlecleucel in a real-world setting from a cellular therapy registry is presented here. As of January 2020, 511 patients were enrolled from 73 centers, and 410 patients had follow-up data reported (ALL, n = 255; NHL, n = 155), with a median follow-up of 13.4 and 11.9 months for ALL and NHL, respectively. Among patients with ALL, the initial complete remission (CR) rate was 85.5%. Twelve-month duration of response (DOR), event-free survival, and overall survival (OS) rates were 60.9%, 52.4%, and 77.2%, respectively. Among adults with NHL, the best overall response rate was 61.8%, including an initial CR rate of 39.5%. Six-month DOR, progression-free survival, and OS rates were 55.3%, 38.7%, and 70.7%, respectively. Grade ≥3 cytokine release syndrome and neurotoxicity were reported in 11.6% and 7.5% of all patients, respectively. Similar outcomes were observed in patients with in-specification and out-of-specification products as a result of viability

Background: Children with Acute Lymphoblastic Leukemia (ALL) in Low Middle income countries(LMIC) face major challenges including treatment abandonment and poor overall survival (OS) and event-free survival (EFS).The preliminary data and results of outcome of Pediatric ALL from our pediatric hematology-oncology center which follows BFM based ALLtreatment protocol has been published in 2015 in ASH forum.Due to non availability of Polymerase chain reaction (PCR)based Minimal residual disease(MRD)analysis, we use multiparametric flowcytometry (FCM) based MRD analysis for remission assessment and risk stratification in our patients. Within resource constraints, we present evidence that outcomes comparable with that seen in high income (HIC)and upper middle income countries(UMIC) can be achieved with a post remission therapy guided by a risk stratification incorporating FCM MRD. Methods: Following IRB approval,an ambidirectional cohort study was performed using clinical information and outcomes of all patients aged 1to 14 years treated for newly diagnosed B- or T-ALL between January 1,2015 and December 31, 2020. AIEOP BFM ALL 2009 protocol with modifications was followed as the institutional protocol in all patients.The treatment algorithm is mentioned in Figure 1. Patients who underwent multiparametric FCM MRD analysis at the end of Induction IA and whose 6 months follow up details were available were included in the analysis. Patients with Ph positive ALL and those who died during Induction IA were excluded. FCM MRD analysis was performed after Induction IA on Day 33.MRD level above 0.01% was considered positive.MRD assessment was repeated following Induction IB on Day 74 in patients who had MRD positivity on Day 33. At the end of Induction IB patients were risk stratified into High risk(HR) and non High risk(Non HR).HR features included Hypodiploidy( Results: Median follow-up time was 33.5(4-102) months . Study had 102(n=102) consecutive Ph negative patients who underwent induction therapy. Six patients (5.88%) died during induction IA;96(n=96) children who continued treatment were included in further analysis. The mean age at diagnosis was 6(1-14) years .Forty seven (48.95%)patients were male..B-ALL n=84(87.5%) and T ALL n=12.(12.5%). Four patients(4.16%) had CNS disease at diagnosis. Three children (3.13%) had high risk cytogenetics.Ten children (10.42%)in the cohort had poor prednisolone response. Five patients(5.20%) didnot achieve morphological remission on Day33. Fifteen (15.63%) patients were risk stratified as HR during IA. Four more (4.16%) were reallocated to HR in view of persistent MRD positivity after Induction IB. At median follow-up, the OS was 95.35%±2.65(95% CI 90.16-100.54) and the EFS 94.48%±2.74 (95% CI, 89.11%-99.84%).Female gender predicted better EFS (p=0.042).The EFS of patients without CNS disease at presentation was significantly better(93.5% Vs 23.2% p=0.000). The EFS at median follow up of patients in re HR cohort was 64.19% Vs 97.81% in non HR (p=0.010). EFS by risk stratification is shown in Figure II. Conclusion:Our study suggests that FCM MRD can be successfully incorporated into the treatment algorithm in a resource limited setting. With FCM MRD were able to identify an additional subset of HR patients who otherwise would have been stratified into non HR group. In a prospective cohort , FCM MRD could be tested at an earlier time point in IA induction to facilitate identification of early drug responsive patients with lowest risk of relapse. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background. Although the Event Free Survival for Childhood Acute lymphoblastic leukemia (ALL) reaches 85%, the remaining 15% of patients relapse, and 25-40% of them die. Novel molecular targets may increase the efficacy of therapy and reduce treatment toxicity. Among B-other ALL patients, JAK/STAT pathway alterations represent about 7% of the 'Philadelphia-like' cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis, cellular proliferation and survival. In last years, JAK2 mutations have been widely studied in leukemia and lymphoma, whereas JAK2 fusion genes are still poorly characterized. Aim. This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients and develop a target strategy in in vitro and in vivo preclinical models. Methods. A targeted capture RNA Next Generation Sequencing strategy was applied to discover JAK2 fusion genes in a large cohort of PCR-based MRD high risk (HR) BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. Primary patients' cells have been in vivo expanded in NSG mice. We performed ex vivo and in vivo drug treatments with JAK2 inhibitors; phosphoflow and apoptosis-viability assays were performed in patients' blasts in co-culture with human bone marrow stroma. Results. We identified 10 pediatric cases carrying a JAK2 fusion gene with different partners in single cases, such as ATF7IP, ZEB2, MPRIP, BCR, TLE4, GIT2 and RAB7, in addition to PAX5, which was the only recurrent in three cases. Cells were available from 3 cases, carrying PAX5-JAK2, ATF7IP-JAK2 and ZEB2-JAK2, respectively. After in vivo expansion, we demonstrated that the JAK2 signaling pathway was active at basal level, through phosphorylation on Y1007-1008 JAK2 residues inside the catalytic activation loop, compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.0355); a positive trend was also shown compared to primary cells with P2RY8-CRLF2 rearrangements and JAK2 mutation, as positive controls (+40% two-tailed P value 0.158). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated. We thus setup a JAK2 targeted drug treatment using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes of treatment, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5-JAK2, -22% in ATF7IP-JAK2 and -35% in ZEB2- JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%). After 48h monotherapy treatment by CHZ868, we detected apoptosis induction and cell viability decrease between 20- 75% at IC50. In combination with dexamethasone, we assessed a further decrease of viability between 10 to 95%. A biological variability among the three different patients was appreciated, according to the different partner genes. Exclusively for the PAX5-JAK2 fusion, we also performed treatments with the kinase inhibitor BIBF1120/Nintedanib, targeting LCK, which is activated downstream PAX5 fusions and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, we found that ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+ 45%, p Finally, we demonstrated the in vivo efficacy of CHZ868 in patient derived xenograft model in presence of PAX5-JAK2 fusion. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in bone marrow (p Conclusion. CHZ868 is a promising candidate for treatment of BCP-ALL carrying JAK2 fusions, showing high efficacy and specificity, both ex vivo and in vivo. Further studies will include combination with standard chemotherapy drugs with the aim to maintain its efficacy by reducing the intensity and toxicity of chemotherapy. Disclosures Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Colmmune: Honoraria; Amgen: Honoraria.

Introduction: Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood, with a current incidence of 3,000 cases annually. Incremental alterations in treatment over time have resulted in overall survival rates that now exceed 90%. Although minor differences exist between ALL treatment protocols from different cooperative groups, risk-stratified treatment for ALL is generally standard across the country. As more has been understood about ALL biology and outcomes, treatments have been further risk stratified to optimize outcomes for higher risk individuals while minimizing toxicities for those with more favorable disease characteristics. With the improvements in outcome for ALL, cost of care is a critical component and financial toxicity for patients and society requires evaluation. Methods: Using commercial insurance data from the OptumLabs ® Data Warehouse, a cohort of 1941 ALL patients was identified, aged 1-30 years and diagnosed between 2000 and 2017 in the United States. ALL diagnosis was confirmed based on ICD-9 and ICD-10 diagnostic codes, in combination with bone marrow and lumbar puncture procedure codes within 14 days of diagnosis. Individuals were followed up to 36 months from diagnosis. Mean monthly and cumulative costs were computed for the initial 8 months of therapy and for 36 months from diagnosis, as were mean number of inpatient and outpatient days. Costs were further broken down by the types of services provided. All results were stratified by age group (1-9 years, 10-12 years, ≥13 years) as a proxy for risk group, with the younger group most consistent with standard risk disease and the older groups indicating high risk and very high risk disease, respectively. Results: Among the 1941 identified ALL patients (1-9 years, 1233; 10-12 years, 162; ≥13 years, 546), 44% were female, 51% were white, median age at diagnosis was 6 years (interquartile range 3-14 years) and 941 of enrolled patients had continuous data through 36 months. Mean cost of care over the approximate 3 years of therapy was $537,000, with approximately 58% of costs incurred within the initial intensive 8 months of therapy. Mean cost was highest among the ≥13 year age group, both for the initial 8 months of therapy ($487,000, 95% CI $449,300-530,400)), as well as over the 36 months following diagnosis ($850,000, 95% CI $778,400-937,100) (Figure). This was reflective of the highest mean number of inpatient days among the ≥13 year age group (88.1 days over 36 months, 95% CI 82.1-94.9) compared to the 10-12 year group (73.2 days, 95% CI 64.3-85.1) and the 1-9 year group (47.5 days, 95% CI 45.2-50.9). Mean number of outpatient visits over 36 months was not significantly different between 1-9 and 10-12 year age groups but was significantly higher among the ≥13 year age group compared with the 1-9 year group. Thirty-six month survival varied by age group and was highest among the 1-9 year group (95.9%, 95% CI 94.6-97.2) compared with the 10-12 year group (90.5%, 95% CI 85-4-95.8) and the ≥13 year group (79.6%, 95% CI 75.8-83.6). Conclusions: This robust real-world cost analysis shows for the first time that the average cost of ALL care in the current treatment era, including inpatient and outpatient data, is more than $500,000. We showed that the cost of care is highest among the oldest pediatric age group, indicating increasing cost with increased therapeutic intensity. This study provides a valuable benchmark that can be used to analyze the financial toxicity of ALL therapy on patients and families and can be re-assessed over time as novel therapeutics are introduced into ALL therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common cancer in children, with a peak age of incidence of 2-5 years. In the majority of pediatric cases, B-ALL is initiated in utero, with full transformation from pre-leukemia to leukemia being driven by additional oncogenic events acquired in early childhood. Surprisingly, common in utero genetic events lead to the expansion of pre-leukemic clones in >1% of all newborns, yet only 1 in 100 of these infants go on to develop clinically overt leukemia in their lifetime. This suggests the presence of certain protective and/or non-protective mechanisms that influence the fate of pre-leukemic clones. While several epidemiological studies of early-life infection indicate a consequent increased risk for ALL, protective effects of infection exposure against ALL have also been reported. An infectious agent responsible for pro- or anti-leukemic effects has not been identified and a mechanistic explanation for the seemingly contradictory effects of early-life infection has not been defined. We have previously reported the ability of immune stimulation with infection-associated toll-like receptor (TLR) agonists to reduce pre-leukemic cell burden and delay leukemia onset in the Eμ-RET transgenic mouse model of hyperdiploid B-ALL. We observed that while TLR9 (CpG) and TLR7/8 (R848) ligands were effective at reducing viable pre-leukemic and leukemic cells, a TLR3 ligand (PolyI:C) appeared to have an opposite effect. In this study, we further investigate the potential pro-leukemic effect of TLR3 ligands (pathogenic dsRNA) on abnormal BCP cell behavior. Purified pre-leukemic or leukemic cells from Eu-ret mice were treated in vitro with TLR agonists and cell viability was assessed after 48 hours. In contrast to the reduced viability observed after exposure to TLR7/8 (R848) and TLR9 (CpG) ligands, a ~1.5-fold increase in viable cell recovery, compared to untreated cells, was observed after treatment with PolyI:C (p To assess the relevance of this TLR-induced activity for clinical B-ALL, we evaluated the responses of patient-derived B-ALL samples. A similar survival benefit was observed with primary B-ALL samples treated with TLR3, but not other TLR ligands. Consistent with observations from primary murine leukemia, CpG and R848 were able to induce in vitro killing of human leukemia cells, while PolyA:U promoted increased leukemia cell viability. As the overall outcome of early-life infection will also involve TLR-induce immune responses, we next assessed the effect of TLR3 agonists in the presence of immune effector cells. Bulk splenocytes from 1- to 12-week-old Eu-RET mice were cultured in the presence or absence of TLR agonists and viable cell recovery after 48 hours measured. In the presence of immune effector cells, the impact of pro-survival TLR3 signalling was diminished in an age-dependent manner, implicating neonatal immunity in the abnormal pro-leukemic response induced by TLR3 ligation. Overall, our results reveal previously unrecognized pro-survival signalling through TLR3 that could contribute to an early-life infection-driven progression of B-ALL. We are currently exploring the signaling pathway(s) responsible for this effect and evaluating the immune variables that influence pre-leukemia cell fate in vivo. This study will significantly enhance our mechanistic understanding of immune-mediated modification of B-ALL progression induced by early-life infection exposure. Disclosures No relevant conflicts of interest to declare.

Background Childhood acute lymphoblastic leukemia (ALL) has a very high cure rate, however, long-term survivors are at increased risk of chronic medical illnesses that are likely in part microbiome mediated. Previous studies comparing microbiota of pediatric ALL survivors to healthy siblings showed altered composition in survivors. Longitudinal microbiome changes through treatment leading to these alterations are unknown. Methods Children with ALL were enrolled and stool samples were collected at diagnosis and at the end of induction, consolidation, interim maintenance I, delayed intensification, interim maintenance II, as well as approximately 3 months and 6 months into maintenance. Stool samples from healthy siblings were used as controls. Clinical data were collected. DNA was extracted from stool samples and 16S rRNA was sequenced for analysis of hypervariable region V4. Differences in alpha and beta diversities and relative abundance of taxa were calculated between phases and with sibling controls. Results 35 ALL patients age 3 months-19 years were included. The diagnoses were 14 standard risk pre-B ALL, 14 high risk pre-B ALL, 6 T-ALL, and 1 relapsed pre-B ALL. Stool samples were sequenced from 19 healthy siblings. Statistically significant differences in alpha diversity (Shannon) were found between healthy siblings and ALL patients during the more intense chemotherapy phases before low dose maintenance (Figure 1A). Beta diversity (Bray-Curtis), was significantly different between microbiota of ALL patients at diagnosis and their siblings, as well as between ALL patients at diagnosis and at each of the subsequent treatment phases (Figure 1B). Longitudinal comparison using multivariate analysis showed that leukemia risk group (high risk vs standard risk) and antibiotic treatment were significant factors in beta diversity changes. The relative abundance of microbes showed that with treatment, ALL patients exhibit a significant decrease in the phylum Verrucomicrobiota, driven by the genus Akkermansia, which is beneficial to health and is associated with protection against obesity and other chronic inflammatory diseases (Figure 2). Proteobacteria and Fusobacteria, which include proinflammatory species, were increased. Conclusion Pediatric ALL patients have decreased diversity of gut microbes at diagnosis and during treatment. The types of gut microbes harbored in ALL patients are already different than their siblings at diagnosis and continue to change during treatment, but may begin to recover in low dose maintenance therapy. Taxa considered to be beneficial are depleted, while more pathogenic microbes become prominent. Microbiota changes are likely influenced by intensity of chemotherapy and antibiotic exposure. Continued longitudinal follow up is needed to determine whether these changes correlate with adverse long term health outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

B ackground Acute Lymphoblastic Leukemia (ALL) is the most common cancer seen in the pediatric age group. The treatment consists of an initial intensive phase of chemotherapy followed by a prolonged period of maintenance chemotherapy intended to reduce the risk of relapse. Children are commonly seen in clinic every 4-6 weeks for bloodwork and physical examination during the maintenance phase. The COVID-19 pandemic has prompted consideration of alternative means of providing medical care. The objective of this study was to determine the proportion of in-person clinic visits during ALL maintenance therapy for which the outcome of the physical examination resulted in a change of patient management. Methods A retrospective chart review of children diagnosed with precursor-B ALL between January 2017 and December 2018, and who were in maintenance therapy between September 2019 and February 2020, was conducted. All routine maintenance visits were reviewed to identify new physical examination findings and patient outcomes and classified as either "could be managed virtually" or "essential in-person visit". For the latter, a second classification was conducted to distinguish between visits necessitating a change of management versus not. Results Eighty-five children were diagnosed with precursor B ALL and continued to maintenance treatment during the study period. 10 children were excluded as not meeting the inclusion criteria or not evaluable. Of the remaining 75 children, 54 were male (72%) and 21 female (28%). The median age at diagnosis was 4.83 years (0.73 - 14.8 years). 39 patients (52%) had standard risk ALL, 35 patients (46.7%) had high risk ALL and one patient had Infant ALL (1.3%) A total of 240 routine maintenance visits were included in the final analysis. An abnormal physical exam finding was noted in 20 visits (8.3%) and of these, new findings were noted in 14 (5.8%). 6 visits were classified as essential in-person visits (2 for new bruising, 1 for new limp, 1 for new lymphadenopathy, 1 for acute otitis media, and 1 for new wheezing). Among the 14 visits with new exam findings, only 5 had an impact on patient management and of these, only 2 (0.8%) were classified as obligate in-person visit for requiring immediate management (acute otitis media and wheezing). Conclusion Our results demonstrate that most in-person visits can be provided as virtual visits without affecting patient outcomes. The results of this study provide the foundation for a prospective study that will evaluate the benefits, risks and families' preferences associated with virtual visits and delineate the optimal frequency and timing of in-person clinic visits during ALL maintenance therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcome. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood, beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP resistant and sensitive ALL cell lines (n = 7) as well as primary leukemia samples from newly diagnosed patients. We identified 2219 response genes (absolute log 2FC > 1.5, FDR p-value Disclosures Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Mullighan: Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Pfizer: Research Funding. Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee. Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months.

Introduction: Despite the achievement of progressively higher cure rates, acute lymphoblastic leukemia (ALL) remains the main cause of cancer-related deaths in children, adolescents, and young adults, particularly in low- and middle-income countries. According to SEER data, the 3-year survival for ALL ages Objectives: To describe the treatment lines, protocols and fatality rates in children, adolescents and young adults up to the age of 25 years diagnosed with ALL (PedALL) in Brazil, based on a public healthcare national database. Methods: The "DataSUS" is an anonymized open-access database of all patients treated in Brazilian public healthcare centers, divided in three datasets: outpatient, inpatient, and death reports. Citizens have a unique number and all diagnoses, diagnostic procedures and medical interventions are included. This national registry is used to track public expenses. DataSUS has information regarding ALL (ICD-10, C91.0) including protocols, treatment lines, and survival. We performed a descriptive cross-sectional study of PedALL included in the database between Jan/2013 and Dec/2018 to understand the burden of the disease in the public healthcare system. Then, we selected a cohort of patients with PedALL included in DataSUS as "First-line chemotherapy for pediatric leukemia" between Jan/2014 and Dec/2015, and followed these patients until Dec/2018, to describe the management and outcomes of PedALL in our country. Results: Between 2013 and 2018, 17,658 patients ages 0-25 years (86% < 18 years) had the underlying diagnosis of ALL, a mean of 6,045 patients per year: 2,725 newly diagnosed, and the remaining, continuation therapy. The male/female ratio was 1.40. The patients underwent a total of 86,332 procedures in this timeframe including diagnostic procedures, chemotherapy, multiple treatment lines, hematopoietic stem cell transplant, transplant-related exams, and the management of disease and treatment-related complications. Treatment protocols most frequently reported were BFM and two national protocols, GBTLI-LLA and RE-LLA. A total of 1,266 patients died between 2013-2018, a mean of 211 patients each year, 79.3% of them younger than 18 years of age. In the cohort diagnosed between Jan/2014 and Dec/2015, 2,368 patients 0-18 years of age and 91 ages 19-25 years were first included in DataSUS system as "First-line chemotherapy for pediatric leukemia". The male:female ratio was 1.44 and 1.84 for the 0-18 and 19-25 years of age, respectively. The median time from diagnosis to treatment initiation was 2.3 months. Within the three year-follow-up, a 2 nd line chemotherapy was registered in 142 patients (6%), 3 rd line in 11 patients (0.46%) and a 4 th line in a single patient (0.04%). The mean duration of each treatment line was 16.4, 8.3, and 4 months, respectively, for the 1 st, 2 nd, and 3 rd line. Patients were hospitalized for a mean total of 65.3 days: 9.7 admissions per patient, for a mean of 6.7 days each. The absolute death count was 303 (0-18) and 13 (19-25) patients, which would represent a 3-year fatality rate of 12.8% (0-18) to 14.3% (19-25); 61% of the deaths occurred in the first year of treatment. Conclusion: The burden of ALL for the public health care system is large, with over 80 thousand procedures every year. This has a high emotional and socio-economic impact to the families and to the society. More than 200 children and young adults with ALL die every year in Brazil, but fatality rate is very similar to SEER data. The two-month delay between diagnosis and treatment initiation can be an explanation for the early mortality observed in our database and must certainly be appointed as a great opportunity for improvement. Standardization of the national PedALL protocol and new treatment modalities may greatly improve this scenario. Disclosures Queiroz Hazarbassanov: Novartis: Current Employment.

Background In pediatric hematologic malignancy, the incidence of invasive fungal infections (IFI) is ~ 5-10% and leads to significant morbidity and mortality. Acute lymphoblastic leukemia (ALL) accounts for the largest group at risk and has the largest absolute number of IFI. Antifungal prophylaxis has the potential to mitigate risk of invasive infections in ALL but is not currently standard of care due to the paucity of data in ALL subgroups. A recent systematic review showed a significant reduction in proven/probable IFI and fungal infection-related mortality in pediatric patients when using a mold active agent compared with fluconazole, therefore an echinocandin was selected for systemic antifungal prophylaxis in our institution. In this study we investigate the incidence of IFI in patients with ALL in a highly endemic area (Ohio River Valley) and describe the impact of echinocandin prophylaxis in this population. Methods We conducted a retrospective cohort study of consecutive patients Results Demographics and patient characteristics are summarized in Table 2. Between 2015 to 2020 we identified 100 patients with ALL. Mean age at diagnosis was 7.2 years and 43% were female. We identified 14 unique cases of IFI in 13 patients with ALL who did not receive prophylaxis with caspofungin during 2015 to 2020 (14%). Prior to the implementation of prophylaxis, IFI occurred in 0% (0/43) of the SR B-ALL group, 18.2% (6/33) in HR B-ALL group and 35% (8/23) in T-ALL group. IFI incidence was highest in induction and consolidation phases (71.4%) and implicated species during these phases included Aspergillus, Candida, Fusarium, and Papulasopora. From April 2020 to July 2021, there were 22 newly diagnosed patients with ALL, 11 (50%) of whom received inpatient prophylaxis with caspofungin. There was 1 case (4.5%) of reported IFI during delayed intensification (no prophylaxis at the time of infection). As seen in Figure 1, patients with HR B-ALL and T-ALL who were hospitalized and received caspofungin during induction saw a notable decrease in the cumulative incidence of IFI, from 18.3% to 0% at 6 months into treatment before reaching similar levels in the patients who did not receive prophylaxis. Conclusion In our pediatric population, patients with T-ALL and HR B-ALL were more likely to develop IFI than those with SR B-ALL. Prophylaxis with caspofungin and inpatient hospitalization during induction were effective strategies for reducing the incidence of IFI in induction and consolidation for our high-risk leukemia population. This approach should be validated in larger studies with special consideration being given to patients in fungal endemic areas. Figure 1 Figure 1. Disclosures Raj: Terumo Medical Corporation: Honoraria, Speakers Bureau; Forma therapeutics: Consultancy; Global biotherapeutics: Speakers Bureau.

PURPOSE Asparaginase is a key component of acute lymphoblastic leukemia (ALL) therapy. Hypersensitivity reactions challenge its use and occur frequently (30-75%) after native Escherichia. Coli (E.coli) asparaginase (Appel et al., 2008; Muller et al., 2001; Panosyan et al., 2004; Silverman et al., 2001). The international ALL Ponte di Legno Toxicity Work Group (PTWG) classifies hypersensitivity to asparaginase as (i) allergy in case of symptoms of allergy (always associated with undetectable asparaginase activity levels), (ii) allergic-like reactions in case of symptoms without inactivation, and (iii) silent inactivation (SI) with inactivation of asparaginase activity, but without hypersensitivity symptoms (Schmiegelow et al., 2016). Allergic-like reactions and SI can only be diagnosed with monitoring of asparaginase activity levels. A meta-analysis was performed based on data from the PTWG to estimate the incidence of hypersensitivity and risk factors for hypersensitivity to asparaginase in ALL protocols using pegylated E.coli asparaginase (PEGasparaginase) as first line of treatment. PATIENTS AND METHODS Questionnaires were sent to all members of the PTWG. Information on protocol level regarding PEGasparaginase dose, dosing regimen (e.g. dosing frequency, total number of doses, PEGasparaginase-free intervals(s)), administration route, total induction and post-induction hypersensitivity rates per protocol and per risk group and use of therapeutic drug monitoring (TDM). To facilitate comparison between protocols with and without TDM, we defined allergic reactions as the sum of allergies and allergic-like reactions. Silent inactivation was analyzed separately. RESULTS A total of 5880 patients with newly diagnosed ALL, aged 1 to 24 years old, were enrolled in seven different upfront ALL protocols using PEGasparaginase as first-line treatment. The overall incidence of allergic reactions along with 95% confidence interval (CI) were 9% [6%; 13%], 2% [1%; 3%] and 8% [5%; 11%] in the overall protocol, induction and post-induction, respectively (Figure 1). Severity of allergic reactions were described, according to the CTCAE version 3.0 or 4.03, per protocol. 47% of the reactions were classified as grade 3/4. Univariate meta-regression analysis showed a positive association between the incidence of allergic reactions and number of PEGasparaginase-free intervals (P=0.005). High risk group stratification (P Two out of seven study groups reported an incidence of allergic reactions of 1.6-2.0%, which was 9-16% of all hypersensitivity. Three out of seven study groups reported an incidence of SI of 3.7-4.1%, which was 23-29% of all hypersensitivity reactions. All protocols prescribed a switch to Erwinia asparaginase in case of clinical hypersensitivity and/or SI. 308 out of 348 (89%) of the patients with hypersensitivity to PEGasparaginase received Erwinia asparaginase. 19 out of these 308 (6%) exposed patients had an allergic reaction to Erwinia asparaginase, of which 7 out of 19 (37%) were grade 3/4. CONCLUSION The incidence of allergic reactions is lower in protocols using PEGasparaginase as first-line treatment compared to that reported for native E.coli asparaginase or PEGasparaginase after native E.coli asparaginase. Post-induction phase, a higher number of PEGasparaginase-free intervals, and initiation of PEGasparaginase in post-induction phase are risk factors for allergic reactions. These results are important for planning of PEGasparaginase administrations in future frontline therapy. Figure 1 Figure 1. Disclosures Albertsen: Erytech: Honoraria, Speakers Bureau; Servier: Speakers Bureau; BKA: Other: Sponsor of the investigator-initiated trial NOR-GRASPALL2016.

Background: Relapse from acute lymphoblastic leukemia (ALL) is one of the most common causes of pediatric cancer-related death. Early relapse of ALL is associated with recurrent mutations of histone methyltransferase NSD2 (nuclear receptor binding SET domain protein 2), which is specific for H3K36me2, suggesting a link to therapy resistance or other mechanisms underlying relapse such as central neural system (CNS) infiltration. NSD2 p.E1099K affects gene expression through disturbing the balance of H3K36me2/H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines and PDX cells, we found that NSD2 p.E1099K drives oncogenic programming, CNS infiltration and glucocorticoid (GC) resistance. However, the molecular mechanisms underlying the relapse of this subtype of ALL are still under investigation. Aim: To elucidate the epigenetic landscape underlying the mechanism of the relapse of pediatric ALL with NSD2 mutation. Methods: We performed in vivo experiments to observe tumor burden, leukemia cell infiltration and survival of the NOD/SCID mice injected with a NSD2 p.E1099K mutation knock-out SEM cell line and knock-in CEM cell line. We determined transcriptome (RNA-Seq), chromatin accessibility (ATAC-Seq) in isogenic RCH-ACV, SEM, RPMI-8402 and CEM cell lines, transcription factor binding and histone modification (ChIP-Seq) and 3D organization (Hi-C) in RCH-ACV cells. Finally, we integrated analysis of RNA-Seq, ATAC-Seq, ChIP-Seq and Hi-C to comprehensively disclose the epigenetic landscape in ALL with NSD2 mutation. Results: NOD/SCID mice xenografted with NSD2 mutant cells developed high tumor burden and infiltration to spleen, liver and brain while the mice injected with WT cells accumulated significant less tumor cells and had extended survival. RNA-Seq analysis showed that reversion of NSD2 mutation to WT caused more genes downregulated while insertion of NSD2 mutation to WT cells led to more genes upregulated (Mutant vs WT: RCH-ACV 838 vs 494, SEM 1567 vs 1158, RPMI-8402 1922 vs 1745, CEM 1809 vs 1031). 50 upregulated genes and 3 downregulated genes were in common in B-ALL and T-ALL with NSD2 mutation. Most of the upregulated genes correlated with neural development and adhesion which might contribute to CNS infiltration (e.g., NCAM1 and NEO1). A few genes were associated with GC resistance such as decreased NR3C1 and increased NR3C2. Accordingly, ATAC-Seq showed that NSD2 mutant cells had more open chromatin peaks than those of WT (RCH-ACV 4853 vs 3212, SEM 10052 vs 7595, RPMI-8402 20392 vs 12133, CEM 10155 vs 6437). ChIP-Seq revealed general large gains of H3K36me2 in intergenic regions in NSD2 mutant cells. Importantly, genes upregulated with NSD2 mutation (e.g., NCAM1 and NEO1) lost H3K27me3 at promoters but gained H3K36me2 at promoters and whole gene bodies, accompanied with increased H3K27ac at enhancers. Conversely, a small subset of genes gained H3K27me3 and lost H3K36me2 in their promoters. Concentrated H3K36me2 in gene bodies diffused and broadened was less prominent and H3K27me3 accumulation became dominant. This for example was associated with repression of NR3C1 to drive GC resistance of NSD2 mutant cells. Genes upregulated in NSD2 mutant cells were enriched for binding sites for lymphoid transcriptional activators such as EBF1 and IRF2. The promoters of the downregulated genes had motifs for transcription factors poorly expressed in lymphoid cells and were enriched for binding sites for the BCL6 transcriptional repressor. Hi-C analysis revealed 430 topologically associated domains (TADs) with increased loop interactions while 136 TADs with decreased interactions were in NSD2 mutant cells compared to WT cells. Overall, 491 regions switched from compartment A to B and 444 regions switched from B to A in NSD2 mutant cells compared to WT cells. Compartment switching from inactive B to active A correlated with upregulated gene expression levels while the reverse was true for switching from A to B. Increased intra-TAD interactions were linked to upregulated genes while decreased intra-TAD interactions were linked to downregulated genes. Conclusions: The NSD2 mutation led to increased tumor burden, CNS infiltration and glucocorticoid resistance due to dysregulation of epigenetic patterns and 3D chromatin architecture, indicating mechanisms underlying the relapse of pediatric ALL and potential therapeutic targets in ALL with NSD2 mutation. Disclosures Licht: Epizyme: Research Funding.

Background: Recent evidence supports the role of cytomegalovirus (CMV) in the development of childhood ALL. The underlying mechanism and CMV's role in the leukemic cell phenotype is unknown, but CMV typically interacts with the host immune system allowing the virus to survive in a latent state; it may be that this immune dysregulation affects the risk of ALL. This study aims to explore the association of CMV and ALL at the time of leukemia diagnosis, using AML cases as a control and further, to determine whether CMV affects certain subgroups of ALL patients such as specific ethnicities, age groups, or cytogenetic subtypes. Methods: Pediatric diagnostic leukemia bone marrow samples obtained from the California Childhood Leukemia Study were screened for the presence of CMV DNA using a custom-designed droplet digital PCR assay. A total of 869 cases were analyzed including 125 AML cases and 744 ALL cases. Demographic and clinical features were compared between patients found to be CMV positive (cases with any detectable CMV positive droplets) and those who were CMV negative (cases with no detectable CMV positive droplets). The effect of the level of CMV viral DNA load was also assessed. For a subset of cases (n=61), Affymetrix Array gene expression data were available and differential gene expression performed to compare CMV positive cases with high viral load to CMV negative cases. Odds ratios and confidence intervals were estimated using logistic regression. Results: ALL cases were more likely to be CMV positive compared to AML (OR: 2.50; CI 1.00, 5.47, p = 0.039 for CMV highest quintile vs. CMV negative). Within ALL cases, B-cell ALL (B-ALL) was significantly associated with CMV positivity compared to T-cell ALL (T-ALL) (OR: 2.93; CI: 1.01, 8.52, p = 0.048 for CMV highest tertile vs. CMV negative). Further subtype analysis of B-ALL cases revealed CMV positivity to be significantly associated with high hyperdiploidy, one of the most common ALL subtypes, when compared to ETV6-RUNX1 (OR: 2.52; CI:1.34, 4.73, p = 0.004 for highest CMV tertile vs. CMV negative). CMV positive B-ALL cases were also more likely to harbor deletions of EBF1, a B-cell development gene, compared to CMV negative cases (OR: 6.10; CI: 1.09, 34.06, p = 0.04 for CMV 2 nd tertile vs. CMV negative; OR: 5.54; CI: 1.13, 27.18, p = 0.03 for CMV highest tertile vs. CMV negative). Differential gene expression analysis revealed 830 genes to be significantly differentially expressed between the highest quintile of CMV positive cases and CMV negative cases, and gene ontology analysis revealed upregulation of processes involved in viral infection and replication. Specifically, cytokine signaling pathways including IL-1, IL-8, and IL-7 were upregulated in CMV positive cases while Th1 and the pathway facilitating crosstalk between dendritic cells and natural killer cells were downregulated. Interestingly, B-cell receptor signaling was also upregulated in CMV positive cases. Conclusion: Our results support the hypothesis that CMV plays an enhanced role in leukemia development in specific subtypes of ALL, and not in AML development. The ability of CMV to interact with the host immune system, highlights immune dysregulation as a potential mechanism by which CMV contributes to risk of ALL. Gene expression analysis on a subset of cases revealed differentially expressed genes to be enriched in pathways involved in immune response, suggesting a potential role for active CMV infection in the leukemic phenotype. The patterns of up- and downregulation in these pathways were consistent with the host response to CMV. Additionally, acute CMV infection has been shown to promote B-cell activation and proliferation. Further, CMV seropositive individuals have been reported to have altered immune responses even with the virus in a latent state highlighting the virus's effect on B-cells. In our study we also found B-cell receptor signaling to be upregulated in CMV positive cases. This is consistent with the known effects of CMV in a typical host, but in patients with leukemia it provides an interesting potential link between CMV infection and development of pediatric ALL. These intriguing results require validation and warrant continued investigation of the role of CMV in pediatric leukemia development. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

[Figure 1.][1] Figure 1. Five-year event-free survival for pediatric ALL increased from 37% to 56%. ![Figure 2.][1] Figure 2. Three-year overall survival for pediatric ALL is 70% at the GHT compared with 50% reported in Mexico. ![Figure][1] ![Figure][1]

Introduction Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and represents 75-80% of leukemia cases. Despite its recognized role in the initiation of the disease, the bone marrow (BM) microenvironment is not well understood especially in its non-cellular component. High-throughput protein detection represent an opportunity to unveil new biomarkers for leukemia diagnosis, prognosis, monitoring, and to potentially identify biological targets. Methods In the present exploratory study, we evaluated the soluble compartment of BM and peripheral blood (PB) in 16 ALL pediatric patients by the means of the SOMAscan assay, a highly multiplexed, affinity proteomics platform able to measure 1305 proteins in as little as 65µl of sample using modified protein-binding single-stranded DNA aptamers called SOMAmers, revealed on a high dimension oligo-probe array. The raw hybridization data were normalized using hybridization controls and were log2-transformed. Transformed HybNormalized BM and PB plasma data were compared using GraphPad PRISM 8. Multiple comparisons were set with an FDR threshold of 0.01%. Results The comparative analysis between plasma samples from BM and PB showed that 228 proteins are differentially expressed in BM and PB plasma irrespective of the nature of disease (fig1). A more detailed mix-effect analysis of protein expression in the different patient categories and compartment defined in Table 1 revealed that out of these 228 proteins, 92 showed a significant differential expression when performing a mixed-effect analysis between the different groups. Among the top 20 proteins differentially expressed between BM and PB, 18 show differential expression between patient groups. The patient group effect is dominated by the differences between BM and PB plasma in ALL Common patients , which account for 17 of the observed differences in protein levels. In spite of the limited sample size, the analysis revealed a difference in NOTC2 level between ALL Common PB and ALL Pro-B PB. Conclusion In our study we applied a quantitative large-scale proteomic analysis on BM and PB plasma in children suffering from ALL, identifying differential protein expression between the two compartments. In the last few years, SomaScan has emerged as a very attractive method due to its high throughput capacity, faster discovery mode compared to other proteomic platforms and small sample size required, which make it ideal for the identification of novel biomarkers. In spite of the limiting samples sizes, some differences in protein expression were revealed. We are taking this analysis further by analyzing more patients in different groups and including normal donor samples. Table 1 Table Disclosures No relevant conflicts of interest to declare.

Improved personalized adjustment of primary therapy to the perceived risk of relapse by using new prognostic markers for treatment stratification may be beneficial to patients with acute lymphoblastic leukemia (ALL). Here, we review the advances that have shed light on the role of IKZF1 aberration as prognostic factor in pediatric ALL and summarize emerging concepts in this field. Continued research on the interplay of disease biology with exposure and response to treatment will be key to further improve treatment strategies.

Pediatric acute lymphoblastic leukemia (pALL) has excellent outcomes in high-income countries, with 5-year survival approaching 90%. This success has not been duplicated in low-income countries such as Belize because of multiple barriers such as lack of access to diagnostic testing and lack of

Background: The development of B-lymphocyte is controlled by the coordinated action of transcription factors networks, such as IKZF1, TCF3, EBF1 or PAX5. It is increasingly recognized that germline variants of those genes which disrupt their function are responsible for inborn errors of humoral immunity. Meanwhile, it is well understood genetic alterations of those molecules are frequently altered in B-lineage acute lymphoblastic leukemia (ALL) and are associated with leukemogenesis. However, those involvements are usually attributed to somatic mutation. Germline variations may also trigger cancer development in some cases. For instance, genome-wide association studies (GWAS) have identified B-cell precursor (BCP-) ALL susceptibility variants within IKZF1. Furthermore, germline variants of IKZF1, TCF3, and PAX5 are reported to be associated with B-ALL development. Therefore, we hypothesize that attenuated function due to germline variation in those genes may predispose to ALL development in Japanese population to some extent. Method: Saliva DNA from 583 pediatric ALL patients in remission registered in Tokyo Children's Cancer Study Group (TCCSG) were analyzed in this study. Targeted sequencing for exons and exon-intron boundaries of IKZF1, TCF3, and PAX5 was performed using next-generation sequencer. Frequency of the detected variant alleles was compared to data from the Exome Aggregation Consortium (ExAC) East Asian Cohort (n=4327), Tohoku Medical Megabank Organization (ToMMo) cohort (n=3554), and the Human Genetic Variation Database (HGVD) cohort (n=1208) by chi-square test or Fisher's exact test. Results: In total, 113 variants (28 nonsynonymous, 19 synonymous, 56 intronic, 8 untranslated regions, and 2 deletions) were detected. We could not detect any nonsynonymous variations, insertion or deletion in IKZF1. On the other hand, we detected 8 rare variations in TCF3 and 7 of them are found to be deleterious by in silico analysis. Among them, 1 variant, TCF3 p.S514L, was significantly more common in ALL patients (0.26% in patients vs 0% in ExAC, 0.01% in ToMMO and 0% in HGVD, p=0.0017, 0.0115 and 0.0343 respectively). In PAX5, we found 6 rare variations and 3 of them are found to be deleterious by in silico analysis, However, we could not identify a statistically significant difference in frequency among these variants between TCCSG and normal cohorts. On the other hand, a patient who harbored heterozygous PAX5 p.V26G, which had been reported as a somatic mutation in B-ALL, was diagnosed with primary antibody deficiency after treatment of B-ALL. He is receiving immunoglobulin replacement therapy without relapse of ALL for 15 years. Discussion: Our study implied TCF3 p.S514L is associated with ALL development. Besides, we identified a case of heterozygous PAX5 p.V26G variant who exhibited antibody deficiency and B-ALL. These findings suggest those rare variants impair the normal B-cell development and may predispose to ALL development. GWAS is effective in evaluating common genetic variants. However, it is hard to identify the genes involved Mendelian inheritance or functionally defective rare variants associated with disease susceptibility. Although it is rare, our study suggests that rare variants of genes involved in B cell development could be associated with ALL susceptibility and immunodeficiency, and we revealed the frequency of functionally defective rare variants in Japanese childhood ALL cohort. Disclosures No relevant conflicts of interest to declare.

Introduction: Hepatotoxicity is a frequent and challenging adverse event in children with acute lymphoblastic leukemia (ALL), but patient factors that are predictive of hepatotoxicity are not well understood. We leveraged a data repository jointly developed by two pediatric oncology centers within the Leukemia Electronic Abstraction of Records [LEARN] Consortium to assess the landscape and determinants of liver dysfunction throughout ALL therapy in patients who were risk-stratified to receive either standard- or high-intensity treatment blocks. Methods: The subjects were children ages 1-21 years who were treated for ALL between 2006-2014 at either Children's Hospital of Philadelphia or Texas Children's Hospital. Demographics, disease-related data, and every laboratory value collected during treatment were obtained by targeted manual abstraction and extensive semi-automated extraction of patient electronic medical record (EMR) data. To reduce cohort heterogeneity, we excluded patients who received non-standard ALL therapies. Patients were categorized as receiving either standard-intensity or high-intensity treatment for their first three blocks of therapy (Induction, Consolidation, Interim Maintenance 1 [IM1]) based on chemotherapeutic agents delivered in those blocks. Differences in laboratory value-determined hepatoxicity were then analyzed based on this categorization for all remaining phases of therapy. Hepatic lab values (AST [SGOT], ALT [SGPT], total bilirubin [t. bili], and conjugated bilirubin [c. bili]) were first normalized to the age-based upper limit of normal (ULN), and the median value was then determined. A multivariate mixed-effects linear regression model with random effects was used to identify differences in the treatment group medians and the following covariates: age, race/ethnicity, sex, BMI, and ALL immunophenotype. Laboratory values were classified by the CTCAE v5.0 grading system, with grade ≥ 3 considered 'elevated.' Results: 805 pediatric ALL patients were included in the analysis, representing 114,095 hepatic lab values (Table 1). Less than 10% of patients had elevated lab values at diagnosis. Throughout treatment, the majority of lab values fell within 1-2x ULN for age for both standard- and high-intensity treatment groups (Fig. 1a-d). The median hepatic lab values for the high-intensity group were slightly higher than the standard risk group across all treatment phases, and this difference was most consistently significant in Consolidation and Delayed Intensification. Among the four hepatic labs that were assessed, ALT had the most significant deviation above normal (up to 30x ULN, Fig 1a). Patients were more likely to have elevated transaminases during maintenance than prior to maintenance (Fig. 1e). Similarly, but to a lesser degree, patients were more likely to have elevated t. or c. bili during maintenance than prior to maintenance. Age, race, and BMI were correlated with elevated hepatic labs, with Hispanic and/or overweight patients more likely to have elevations in 3 or more phases of therapy (Table 2). However, no hepatic lab abnormalities were correlated with either overall or relapse-free survival. Conclusions: This is the first comprehensive study of measures of hepatotoxicity in a large and uniformly-treated cohort of pediatric ALL. While significantly elevated hepatic labs are rare at diagnosis, they are common during ALL treatment and are seen more commonly in maintenance than in prior phases. Patients who are overweight and/or Hispanic are more likely to experience grade 3 or higher hepatoxicity. We observed no relationship between hepatotoxicity and relapse or survival. Further studies are ongoing to delineate the temporal correlation of liver function and chemotherapy dosing and administration. Disclosures No relevant conflicts of interest to declare.

Background: Minimal residual disease (MRD) obtained at the end of Induction (EOI) is a powerful prognostic indicator for assessing relapse risk in pediatric acute lymphoblastic leukemia (ALL). We have shown that low absolute lymphocyte count at the end of induction (EOI-ALC) is a significant and independent adverse prognostic factor in childhood ALL that further refines MRD-based risk stratification algorithms (Gramatges and Rabin, 2011). However, the mechanisms underlying the relationship between EOI-ALC, MRD, and prognosis have not yet been determined. Here, we investigated associations between clinical and biological host factors and EOI-ALC. Given that hematopoiesis is highly sensitive to telomere shortening and that inflammation further contributes to cellular stress, we hypothesized that shortened lymphocyte telomere length and evidence for systemic inflammation at EOI would be associated with a lower EOI-ALC. The relationship between EOI-ALC and risk for microbiologically documented bacterial infections (MDBI) during the first three months of leukemia therapy was also assessed. Methods: Children between the ages of 1 and 21 years with newly diagnosed B- or T-cell acute lymphoblastic leukemia (ALL) and enrolled to the Reducing Ethnic Disparities in Acute Leukemia (REDIAL) study were included. Blood samples were collected at EOI, and patient demographics and relevant clinical information including EOI MRD, ALC, and MDBI within the first 90 days of treatment were abstracted from the medical record. EOI lymphocyte telomere length was measured with telomere flow fluorescence in situ hybridization, and age-based percentiles assigned based on population norms (Repeat Diagnostics). Mean fluorescence intensity (MFI) of cytokines, including interferon-γ, interleukin (IL)-1ra, IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-8, tumor necrosis factor (TNF)-α, and TNF-β was measured in plasma using a MILLIPLEX® MAP kit (EMD Millipore) on Luminex® equipment. Samples with at least one analyte with detectable MFI above normal range was considered evidence of systemic inflammation. Each sample was assessed in triplicate and analyzed with Belysa™ software. Clinical factors, systemic inflammation, lymphocyte telomere length ≤10th percentile for age, and number of MDBIs in the first 90 days of treatment were then compared between subjects with high or low EOI-ALC using Fisher's Exact Test. The EOI-ALC cutoff applied for this analysis was > or < 1500/µl (EOI-ALC-high and EOI-ALC-low, respectively), as applied in our prior study. All research activities were conducted under local IRB-approved protocols. Results: Forty subjects were enrolled, and their clinical, demographic, and biological characteristics are reported in Table 1. Of these subjects, 23 had EOI-ALC-low and 17 had EOI-ALC-high. Subjects with EOI-ALC-low were 9 times more likely to report Hispanic ethnicity (18/21 EOI-ALC-low subjects, vs. 5/15 EOI-ALC-high subjects, p=0.01). No significant differences in age, sex, or induction therapy regimen were noted between the EOI-ALC groups. Although there was no association between Hispanic ethnicity and MRD status (p=0.26), those with EOI-ALC-low were ~3 times more likely to have positive EOI MRD (p=0.17). We observed no relationship between EOI lymphocyte telomere length, evidence for systemic inflammation, and EOI-ALC. There was also no relationship observed between EOI-ALC and MDBIs in the first 90 days of treatment. Conclusion: In addition to EOI MRD, EOI-ALC is a low-cost, clinically relevant prognostic indicator in pediatric ALL. The relationship between ALC and MRD noted in this study was consistent with our prior observations, albeit not significant due to the relatively small sample size of this cohort. Our results suggest that Hispanic ethnicity is a primary host factor determinant of EOI-ALC, rather than other demographic, treatment, or biological factors. Given that a number of studies have demonstrated poorer survival among Hispanic children diagnosed with ALL, further work is needed to investigate whether genetic ancestry-associated determinants of host immunity may contribute to outcome disparities. Disclosures Aubert: Repeat Diagnostics: Employment.

Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy characterized by a heterogeneous genomic landscape. Copy number aberrations (CNA) emerge during the development, progression and treatment resistance of ALL, and can serve as genomic markers for prognostic classification of patients or for scrutinizing clonal evolution associated with relapse. While identification of distinct CNAs with well-characterized prognostic significance has its own value, uncovering the co-segregation of driver aberrations in individual patient samples could allow for a more personalized risk assessment and treatment response prediction. Methods: Disease-relevant CNAs were profiled in children with B- or T-cell precursor ALL using a next-generation sequencing based digital multiplex ligation-dependent probe amplification (digitalMLPA) assay containing 598 probes specific for 54 genes with key relevance in ALL. Besides the diagnostic samples of 91 patients treated according to the BFM protocols, 14 matching samples drawn at the time of first or second relapse were comparatively analyzed. Clonal relationship between B-cell precursor cell populations prevailing at different time points during the disease course was also investigated by screening immunoglobulin heavy-chain gene rearrangements in matching diagnostic and relapse samples using Illumina deep-sequencing with >20,000x coverage. Results: Whole chromosome gains and losses, subchromosomal CNAs as well as alterations conferring intrachromosomal gene fusions were simultaneously identified by digitalMLPA with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples and increased numbers of CNAs were detected in individual samples at the time of relapse as compared to diagnosis. DigitalMLPA results were successfully validated by conventional MLPA, FISH and PCR data. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients harboring CNAs revealed three different patterns of clonal relationships with (i) one patient displaying identical CNA profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse and (iii) four patients displaying conserved as well as lost or gained CNAs at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. Time between diagnosis and first relapse of T-ALL patients displaying altered CNA profiles suggested a prolonged time requirement of clonal evolution, and of the development of manifest leukemia from an ancestral clone compared to the quick return of an identical clone at the time of relapse. Comparison of the IGH gene rearrangements identified at diagnoses and relapse revealed identical compositions of the most abundant clonotypes in all but one B-ALL patients analyzed; hence, IGH repertoire did not reveal an additional depth of clonal history in our cohort, e.g. by demonstrating the presence of an ancestral clone as the major source of clonal expansion at disease progression in a patient with altered CNA profiles suggesting direct clonal evolution between diagnosis and relapse. Copy number profiles acquired by digitalMLPA were used for determining CNA-based risk groups (Table 1) which were combined with karyotyping and molecular cytogenetic data in order to establish an extended prognostic classifier for patients with B-cell precursor ALL. This novel classifier distinguished four combined genetic risk groups showing significantly different 5-year survival rates (GR: 97%, IR: 84%, IHR: 63% and PR: 13%). Conclusions: DigitalMLPA allows for a rapid, scalable and highly optimized copy number profiling of genomic regions recurrently altered by driver aberrations in pediatric ALL. Based on the comparison of CNA profiles at diagnosis and relapse, clonal evolution and emergence of relapse from an ancestral clone are the predominant driving mechanisms of disease progression. Comprehensive copy number profiling by digitalMLPA identifies distinct prognostic groups for risk assessment in B-cell precursor ALL. Supporting grants : LP95021, K_16 #119950, NVKP_16-1-2016-0004, KH17-126718, BO/00320/18/5, FK_19 #131476, ÚNKP-19-4-SE-77 Disclosures Benard-Slagter: MRC Holland: Employment. de Groot:MRC Holland: Employment. Savola:MRC Holland: Employment.

Increased understanding of pediatric acute lymphoblastic leukemia (ALL) pathobiology has led to dramatic improvements in patient survival. However, there is still a need to develop targeted therapies to enable reduced chemotherapy intensity and to treat relapsed patients. The interleukin-7 receptor α (IL-7Rα) signaling pathways are prime therapeutic targets because these pathways harbor genetic aberrations in both T-cell ALL and B-cell precursor ALL. Therapeutic targeting of the IL-7Rα signaling pathways may lead to improved outcomes in a subset of patients.

As controversy exists regarding the prognostic significance of genomic rearrangements of CRLF2 in pediatric B-precursor acute lymphoblastic leukemia (ALL) classified as standard/intermediate-risk (SR) or high-risk (HR), we assessed the prognostic significance of CRLF2 mRNA expression, CRLF2 genomic lesions (IGH@-CRLF2, P2RY8-CRLF2, CRLF2 F232C), deletion/mutation in genes frequently associated with high CRLF2 expression (IKZF1, JAK, IL7R), and minimal residual disease (MRD) in 1061 pediatric ALL patients (499 HR and 562 SR) on COG Trials P9905/P9906. Whereas very high CRLF2 expression was found in 17.5% of cases, only 51.4% of high CRLF2 expressors had CRLF2 genomic lesions. The mechanism underlying elevated CRLF2 expression in cases lacking known genomic lesions remains to be determined. All CRLF2 genomic lesions and virtually all JAK mutations were found in high CRLF2 expressors, whereas IKZF1 deletions/mutations were distributed across the full cohort. In multivariate analyses, NCI risk group, MRD, high CRLF2 expression, and IKZF1 lesions were associated with relapse-free survival. Within HR ALL, only MRD and CRLF2 expression predicted a poorer relapse-free survival; no difference was seen between cases with or without CRLF2 genomic lesions. Thus, high CRLF2 expression is associated with a very poor outcome in high-risk, but not standard-risk, ALL. This study is registered at www.clinicaltrials.gov as NCT00005596 and NCT00005603.

Killer cell immunoglobulin-like receptors (KIRs), via interaction with their cognate HLA class I ligands, play a crucial role in the development and activity of natural killer cells. Following recent reports of KIR gene associations in childhood acute lymphoblastic leukemia (ALL), we present a more in-depth investigation of KIR genes and their cognate HLA ligands on childhood ALL risk. Genotyping of 16 KIR genes, along with HLA class I groups C1/C2 and Bw4 supertype ligands, was carried out in 212 childhood ALL cases and 231 healthy controls. Frequencies of KIR genes, KIR haplotypes, and combinations of KIR-HLA ligands were tested for disease association using logistic regression analyses. KIR A/A genotype frequency was significantly increased in cases (33.5%) compared with controls (24.2%) (odds ratio [OR] = 1.57; 95% confidence interval [CI], 1.04-2.39). Stratifying analysis by ethnicity, a significant difference in KIR genotype frequency was demonstrated in Hispanic cases (34.2%) compared with controls (21.9%) (OR = 1.86; 95% CI, 1.05-3.31). Homozygosity for the HLA-Bw4 allele was strongly associated with increased ALL risk exclusively in non-Hispanic white children (OR = 3.93; 95% CI, 1.44-12.64). Our findings suggest a role for KIR genes and their HLA ligands in childhood ALL etiology that may vary among ethnic groups.

Background. In 2016 WHO classification of myeloid neoplasms and acute leukemias a new provisional entity 'B-lymphoblastic leukemia/lymphoma, BCR-ABL1-like' has been introduced. Two current strategies of BCR-ABL1-like ALL detection are based on gene expression profile revealed either by microarray ('BCR-ABL-like') or by NGS / patented TaqMan low-density array (TLDA) ('Ph-like'). Although both techniques and not widely applicable. Aims. To find out whether expression profile based on combined expression data of 5 genes assessed by real-time PCR can be used for the identification of BCR-ABL1-like pediatric ALL patients. Methods. The study was done on initial bone marrow samples of 147 primary pediatric BCP-ALL patients. Positive cohort included 10 BCR-ABL1-positive ALL patients, negative cohort consisted of 59 cases with known structural or numerical aberrations. Among them there were 21 patients with high hyperdiploidy, 1 low hypodploid, 1 near tetraploid and 1 iAMP21 cases, 23 patients with translocation t(12;21)(p13;q22), 5 cases with t(1;19)(q23;p13), 1 case with t(9;11)(p22;q23) and 6 Down syndrome (DS-ALL) patients. The rest 78 cases were called 'B-others' ALL and designated as training cohort. According to the previously published data (R. Harvey et al. ASH 2013 abs #826.) we picked 5 genes (IGJ, SPATS2L, MUC4, CRLF2, CA6) those expressions were estimated by real-time PCR. ΔCt method was applied in relation to ABL1 expression. In case of having expression results similar to BCR-ABL1-positive cases, patients with BCR-ABL1-negative ALL were attributed as BCR-ABL1-like ALL. In 113 patients we evaluated IKZF1 status by MLPA using P335 kit (MRC-Holland). Presence of ABL1, ABL2, CRLF2, IgH, JAK2, PDGFRb/CSFR1 gene rearrangements were done by FISH. Prognostic significance of BCR-ABL-like profile was estimated in 66 'B-others' patients uniformly treated according to the ALL-MB 2008 protocol. Informed consent was obtained in all cases. Results. Hierarchical cluster analysis and principal component analysis showed that 16 examined samples were clustered together with 9 BCR-ABL1-positive ones. Among them there were 3 DS patients, 1 iAMP21, 1 t(12;21) cases and 11 patients from 'B-other' group. IKZF1 deletions and high CRLF2 expression were more frequent in BCR-ABL1-like group in comparison to non-BCR-ABL1-like (67% vs 11%, and 40% vs 6%, correspondingly; p0.1%) together with BCR-ABL1-like profile identified a group of patients with dismal outcome (EFS 0.00 vs 0.88±0.11, p Disclosures No relevant conflicts of interest to declare.

In this issue of Blood, Khaw et al show that in contrast to the impressive antileukemic activity achieved by sole BCL-2 inhibition in chronic lymphocytic leukemia (CLL), optimal antileukemic activity in pediatric acute lymphoblastic leukemia (ALL) xenografts required concurrent inhibition of both BCL-2 and BCL-XL.1

Survival for childhood acute lymphoblastic leukemia (ALL) now approaches 90% with risk adapted therapy based on National Cancer Institute risk group (NCI RG) at diagnosis, somatic lymphoblast genetics, and early response to therapy as measured by minimal residual disease (MRD). Recent studies have identified multiple somatic genetic mutations in ALL, some of which confer increased risk of relapse or identify opportunities for additional targeted therapies. However, there are few genome sequencing analyses of representative cohorts of childhood ALL treated on contemporary regimens. To define the mutational landscape of childhood B-ALL, we performed whole exome sequencing (WES) on diagnostic tumor and remission samples of 192 patients with B-ALL consecutively enrolled between 4/1/06-9/8/06 on the Children's Oncology Group AALL03B1 classification trial that enrolled 11,145 patients up to age 30, which included patients enrolled on clinical trials for standard-risk (SR) B-ALL (N=5226, age 1-10 years and white blood cell count (WBC) < 50,000/uL) and high-risk (HR) B-ALL (N=2907; age ≥10 years or WBC ≥50,000/uL). An 8-gene expression low density array card identified Ph-like patients, and single nucleotide polymorphism arrays and multiplex ligation assays were used to determine DNA copy number alterations. Approximately 2/3 NCI SR and 1/3 NCI HR patients with sufficient banked samples were selected to reflect a population-based cohort (Table 1). Comparison of those selected vs. those not revealed significantly more NCI SR patients with a higher WBC and MRD, and fewer with double trisomy 4 and 10. Selected NCI HR patients were younger, had a higher WBC, and had more ETV6/RUNX1. There were a total of 3576 non-silent mutations with a median of 15 mutations/case (range 1-134). One case had 102 non-silent mutations and a germline mutation in MSH3. The additional mutations clustered in 11 pathways (Table 2), several of which were novel including cell-matrix interaction (e.g. SSPO, FAT1) and intracellular trafficking/cytoplasmic transport (e.g. DYNC2H1, ANK3, UNC13C). Most commonly altered were B-cell development (49.4%), transcription factors (45.8%), tumor suppressor genes (32.7%), cytoplasmic transport (27.3%), and Ras signaling (26.7%). Several pathways Ras signaling, Jak/STAT, and transcription factor mutations/deletions were associated with genetic lesions commonly used for risk stratification. Other pathways (epigenetic, B-cell development, or tumor suppressor) occurred in all subtypes. The most commonly identified genetic mutations were NRAS (n=33), KRAS (n=26), FLT3 (n=13), PAX5 (n=10), CREBBP (n=10), XBP1 (n=9), WHSC1 (n=7), and UBA2 (n=7). XBP1 and UBA2 mutations were novel. XBP1 (X-Box binding Protein 1) encodes a transcription factor that regulates the unfolded protein response. UBA2 (ubiquitin like modifier activating enzyme 2) encodes a protein involved in sumoylation to regulate protein structure and intracellular localization. Univariable analysis revealed no significant associations with any of these pathways or mutations with an increased risk of relapse with the exception of IKZF1 mutations or deletions (n=36; p=0.0087). Multivariable analysis modeling including IKZF1, age, presenting WBC, gender, NCI RG, ETV6/RUNX1, BCR/ABL1, Ph-like, white race, and MRD revealed only BCR/ABL1 and MRD positivity being significantly predictive of relapse. However, the risk of relapse was significantly increased based on the number of mutations identified in any one single patient (HR 1.02, 95% CI 1.01-1.023, p < 0.0004). Of note, only 18 patients (NCI SR, n=8, NCI HR, n=10) were Ph-like in this cohort, likely explaining the lack of significance between Ph-like status and an increased risk of relapse in this analysis. In summary, WES of a consecutively enrolled cohort of NCI SR and HR patients revealed a large number of novel genetic mutations that could be broadly assigned to 11 classes. Outcomes for patients overall were not influenced by any one of these classes, demonstrating that sentinel genetic alterations currently used in risk stratification are of paramount importance in directing therapy intensification for ALL. These data provide important information about pathways commonly mutated in childhood ALL, identifying classes of drugs that can be considered for clinical testing to further improve outcome. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Hunger:Amgen: Equity Ownership; Pfizer: Equity Ownership; Merck: Equity Ownership; Jazz Pharmaceuticals: Honoraria; Sigma Tau Pharmaceuticals: Honoraria; Erytech: Honoraria; Patent: Patents & Royalties: Dr. Hunger is a co-inventor of a patent (#8658,964) for the identification of novel subgroups in high risk B-ALL and outcome correlations and diagnostic methods related to the same; Spectrum Pharmaceuticals: Honoraria.

Background: Infections pose a considerable threat for children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) and although severe neutropenia is the most important predisposing factor, neutrophil dysfunction may contribute to this process. Neutrophil extracellular traps (NETs) bind and kill bacteria and fungi by providing a high local concentration of antimicrobial molecules. A crucial step in NET formation is the hydrogen peroxide-dependent translocation of neutrophil elastase (NE) from azurophilic granules to the nucleus. Myeloperoxidase (MPO) follows NE to the nucleus where both promote chromatin decondensation. Data on the production of NETs in leukemia patients are scant. Patients and Methods: All consecutive male and female pediatric leukemia patients treated in our Pediatric Hemato-Oncology Department between 01/03/14-31/09/15 were initially recruited to this prospective study. Phagocytosis, hydrogen peroxide production, NE and MPO enzymatic activity and NETs production were studied at the end of induction and the occurrence of neutropenia, fever and infections during the first 2 months of intensive chemotherapy recorded. The study was approved by the local IRB. All parents signed an informed consent form. Results: Of 22 consecutive male and female pediatric leukemia patients initially recruited, excluded were 1 AML patient with a subsequent diagnosis of shortened telomeres that could affect neutrophil function, 1 Philadelphia chromosome-positive ALL patient that received imatinib in combination with intensive chemotherapy and 3 AML patients that had partial results that did not include NETs. Therefore, the final study group (n=17) comprised 10 ALL patients (7 primary, 2 secondary and 1 relapsed) and 7 AML patients (5 primary and 2 relapsed). There were 10 males and 7 females with a median age [Q1, Q3] of 7.6 years [1.9 years and 11.2 years]. All patients entered complete remission after induction. On the day of examination, patients with AML had a higher median absolute neutrophil count than those with ALL (2800 vs 750 cells/μL) that approached statistical significance (P = .06). Nevertheless, NE activity and NETs were significantly lower among patients with AML than those with ALL (median [Q1, Q3] of 40.5% [36%, 43%] vs 90% [57%, 115%], P = .005 and 51% [9%, 56%] vs 93.5% [74%, 100%], P = .008, respectively), while there were no differences in the other neutrophil functions studied. Patients with AML had more episodes of febrile neutropenia (100% vs 40%, AML vs ALL, respectively, P = .011) and a trend for more invasive bacterial and fungal infection. Interestingly, there was no difference in the number of days with neutropenia (absolute neutrophil count ≤500) during the first 2 months of chemotherapy between the 2 groups [median [Q1, Q3] of 32 days [30.5, 36.5] in AML vs 30 days [25, 43] in ALL, P = 0.87) (Tables 1 and 2, Figure 1). Discussion: We have shown for the first time that NE activity and NETs formation are significantly decreased after induction chemotherapy in pediatric AML but not in pediatric ALL. This phenomenon in AML might be related to immaturity of the NE in neutrophils released from a recovering bone marrow after induction or to down-regulation of NE activity by a yet unknown cellular factor and might contribute to an increased rate of infections among this vulnerable population. Further studies are needed to elucidate on this topic. Disclosures No relevant conflicts of interest to declare.

Introduction: Our previous findings showed that the microRNA (miR) expression profile of miR-151, miR-1290 and miR-451 measured in bone marrow (BM) aspirations taken at diagnosis can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated according to BFM and DCOG protocols (Avigad et al., Genes, Chromosomes & Cancer; 55:328-339, 2016). The development of a minimally invasive blood test for predicting relapse in pediatric B-ALL patients can greatly benefit patients. Therefore, the aim of this current retrospective exploratory study was to investigate the predictive ability of miR-151-5p, miR-1290 and miR-451 measured in peripheral blood lymphocytes (PBL) at diagnosis. Methods: BM (n=67) and PBL (n=22) samples were collected at Schneider Children's Medical Center of Israel between 1995 to 2008. Twenty cases had paired BM and PBL samples. The expression levels of miR-151-5p, miR-1290 and miR-451 were measured in both BM and PBL samples from B-ALL patients (mean age = 6.6 years, median 5.5 years, range 1.3 to 19 years) taken at diagnosis. Pearson correlation (n=20) was applied for assessing the correlation between BM and PBL following logarithmic transformation. Kaplan Meier analysis was performed for testing relapse free survival (RFS). The cutoff value used for survival analysis for each miR, was determined by the highest Youden index (J=Sensitivity + Specificity - 1). Results: Similar to our previous study all three miRs measured in BM samples (n=67) were able to predict relapse risk (p=0.027). The expression levels measured in BM and PBL samples of all three miRs were significantly correlated (miR-151-5p, r=0.764, p Conclusions: Expression levels of miR-151 and miR-1290 in PBL are useful for predicting relapse risk. Results from this exploratory investigation established the feasibility for continuing research and performing a larger prospective clinical trial using blood samples. Moreover, we will investigate the utility of the miRs for long-term monitoring of B-ALL patients during follow-up. The development of a blood test based on miR-151 and miR-1290 for monitoring B-ALL patients risk for relapse will be pursued in parallel to the development of the bone marrow test. These findings should be interpreted with caution due to the small cohort size. Partly supported by Curewize Health Ltd. Figure 1. Figure 1. Disclosures Avigad: Curewize Health: Consultancy, Patents & Royalties: Patent Applicant of Curewize IP. Shichrur:Curewize Health: Employment, Patents & Royalties: Patent Applicant of Curewize IP. Dotan:Curewize Health: Employment, Other: Options Holder in Curewize Health. Yarden:Curewize Health: Employment, Equity Ownership. Yaniv:Curewize Health: Consultancy, Patents & Royalties: Patent Applicant of Curewize IP.

Improved supportive care, more precise risk stratification, and personalized chemotherapy based on the characteristics of leukemic cells and hosts (eg, pharmacokinetics and pharmacogenetics) have pushed the cure rate of childhood acute lymphoblastic leukemia to near 90%. Further increase in cure rate can be expected from the discovery of additional recurrent molecular lesions, coupled with the development of novel targeted treatment through high-throughput genomics and innovative drug-screening systems. We discuss specific areas of research that promise to further refine current treatment and to improve the cure rate and quality of life of the patients.

Recently , Carter et al[1][1] reported the prevalence of p16INK4a gene deletions and their prognostic value in a cohort of 45 children with initial acute lymphoblastic leukemia (ALL). Using a real-time quantitative duplex polymerase chain reaction (PCR) assay, the prevalence of homozygous and

With achievable survival rates of above 90%, non-leukemic events gather a high proportion of treatment failure causes with infections forming the majority. We analysed the incidence and outcome of reported invasive fungal infections as serious adverse events (SAE according to NIH criteria) in study ALL-BFM 2000 with the aim to characterize risk factors to optimize antifungal prophylaxis and treatment. A total of 4867 patients with ALL were enrolled from August 1999 to May 2010. Analysis of (mandatory) SAE reporting revealed 144 cases (3%). Fungal infections related to hematopoietic stem cell transplantation were excluded. According to the EORTC/MSG classification 65 (1.3%) were proven, 53 (1.1%) probable/possible and 26 (0.5%) not classifiable. There were 19 (0.4%) deaths and 41 (0.8%) events were classified as life threatening according to FDA definition. Positive mycological results were available in 98 cases: Aspergillus ssp. in 72, Candida ssp. in 21, Mucor in 3, Rhizopus and Fusarium in 1 case each. Fatality occurred in 16 cases with Aspergillus (22%) and in 1 each of the other species. Out of 41 life-threatening infections 30 were due to Aspergillus, 5 to Candida and 4 to the other species. Regarding all fungal infections, there was a significantly rising risk with increasing age (7.2% >15 y, 4.8% 10-1-< 10y; p Disclosures No relevant conflicts of interest to declare.

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

Genome-wide studies reveal somatically acquired regions of uniparental isodisomy (UPD) in 25% of pediatric acute lymphoblastic leukemia (ALL). These regions probably contain tumor-suppressor genes involved in pathogenesis.

Introduction Presence of malignant cells in cerebrospinal fluid (CSF) is a risk factor in pediatric acute lymphoblastic leukemia (ALL). Consequently, these patients receive extra intrathecal treatment. We evaluated the concordance between morphological and flow-cytometric (FCM) results, both on freshly analyzed and on stabilized, overnight transported samples. Methods Diagnostic CSF samples of 61 newly diagnosed pediatric ALL patients were divided in two aliquots. One was sent to the local laboratory and processed within a few hours after sampling (lab1). A second aliquot was 1:1 diluted with sterile medium and sent to the reference laboratory (lab2). For all samples, a MGG (May-Grünwald-Giemsa) stained cytocentrifuged slide was morphologically evaluated and two 6-color FCM stainings were performed. Samples were considered positive (CNS2) by MGG if at least one blast was seen and by immunophenotyping if a cluster (≥ 10 events) of ALL cells was detected. Results Comparison of morphological data between both laboratories showed concordance in 33 samples (53%). In 20 of the 28 discordant samples only 1 or 2 blasts were reported by a single laboratory. Comparison of FCM data between laboratories was concordant in 58 samples (95%). Three samples (tumor load 15%-18%) were reported positive by one laboratory only; in two of these Conclusions This study shows that morphological analysis of CSF samples is non-reproducible if only 1 or 2 blasts are suspected. In contrast, FCM analysis of CSF is highly reproducible between fresh and stabilized samples. Although immunophenotyping still seems less sensitive than morphology, discordant samples generally have only 1 or 2 blasts reported by MGG, which is highly irreproducible. Nevertheless, by using a single 8-color tube higher cell numbers may be acquired and the FCM sensitivity may be increased. Disclosures: No relevant conflicts of interest to declare.

Abstract 3239 We sought to define factors identifying children at increased risk for mortality during the induction phase of chemotherapy for ALL. We identified a cohort of 11,145 pediatric patients with newly diagnosed ALL using PHIS data from 1999–2009. The PHIS database contains de-identified records of all admissions from 42 children's hospitals across the United States, representing 85% of pediatric admissions to freestanding children's hospitals. Data are internally validated by PHIS, and participating hospitals must have an error rate of Table 1: Demographic characteristics of PHIS ALL cohort, 1999–2009 Patient characteristic N (%) Age at diagnosis, median 5.7 years Table 2: Risk of mortality by demographics and need for intervention Risk factor Mortality rate, N (%) RR (95% CI) Overall 172 (1.54) – Age at diagnosis Disclosures: No relevant conflicts of interest to declare.

Abstract 983 Poster Board I-5 Introduction: Tailored, intensified therapy has led to improved survival of pediatric acute lymphoblastic leukemia (ALL). However, intensified therapy leads to more infectious morbidity. The aim of this study is to investigate to what extent less intensive therapy in good risk patients decreases infectious morbidity. Patients and Methods: 203 children newly diagnosed with ALL between 1 and 19 years old were included in all 8 University Medical Centers in the Netherlands between 2004 and 2007 and followed prospectively until end of treatment. All patients kept a diary by which admittances for infection were reported. All these admittances were systematically reviewed by the investigators in the clinical files. Infections occurring during hospital stay for other indications (e.g. chemotherapy) or present at the time of diagnosis of ALL were also recorded but not included in this analysis. Patients were stratified into 3 arms identical to BFM-2000 criteria with a reduced treatment for good risk patients: standard risk (SR), an intensified reinduction treatment for medium risk patients (MR) and series of intensive courses of chemotherapy in most cases combined with allogeneic stem cell transplantation for high risk patients (HR). Statistics: All variables were corrected for patient time in study (standard 104 weeks). After logarithmic transformation data was evaluated using ANOVA. Results: 55 children were stratified to SR group, 123 to MR group and 21 to HR group. SR patients were followed for a mean of 102 weeks, MR 100 weeks and HR 47 weeks. A total of 507 admittances for infection were recorded. SR patients were admitted 1.1 times on average, versus 3.4 for MR and 6.9 for HR patients (p Conclusions: Less intensive treatment for SR pediatric ALL patients significantly decreases infectious morbidity for this group. The frequency, duration and severity of admittances for infection are all significantly reduced underlining the benefits of reduced therapy for good risk ALL patients. In addition, reduced therapy for SR patients leads to less chemotherapy interruption and thus compliance with the chemotherapy schedule is more easily achieved. Disclosures: No relevant conflicts of interest to declare.

Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Over the past four decades, the outcome for pediatric ALL has rapidly improved secondary to the participation of children on clinical trials, resulting in standardization of the treatment. Some have argued that the best outcome results from participation in a pediatric clinical trial. To address the potential benefits and barriers to participation in a clinical trial, we analyzed clinical trial participation in pediatric ALL at a large single institution. Methods: We evaluated 322 ALL patients < 22 years old at diagnosis who receive their initial therapy at Seattle Children’s Hospital Regional Medical Center (S-CHRMC) between January 1997 and December 2005. Using a retrospective chart review, we analyzed the following variables: study participation (SP) or non-participation (NP) in an ALL therapeutic study, gender, race, patient immunophenotype, risk group (standard risk (SR), high risk (HR) or infant), home state, and distance of primary residence from S-CHRMC. S-CHRMC is the largest pediatric cancer center in the Pacific Northwest, with referrals from Washington, Alaska, Montana, Idaho and Wyoming. Events were defined as relapse or death from any cause. Results: The overall 5 year event free survival (EFS) was 78% (+/− 2.5%). 157 patients participated in a treatment research study (49%). Only risk group was associated with EFS. SP and NP had similar EFS. Gender, race, immunophenotype, home state, or distance from primary residence were not associated with outcome. There was no difference in study participation by gender, race, home state, or distance of primary residence. There were trends to increased participation in SR vs HR (54% vs 35%, p value 0.15) and B lineage vs T lineage (50% vs 35%, p value 0.11). Variable n 5 year EFS p value Study participation No 165 76 (+/− 3.9) 0.47 Yes 157 81 (+/− 3.3) Risk group SR 175 85 (+/− 3.0) HR 132 73 (+/− 4.2) (0.038 to SR) Infant 15 59 (+/−12.9) (0.011 to SR) Discussion: Participation in a research study for treatment of pediatric ALL was not associated with improved outcome in our large single institution series. Study participation was not different by clinical features, including distance to the primary residence. Strict standardization of treatment for all patients may contribute to the similar outcome for SP and NP.