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The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.

Current prognostic models for acute myeloid leukemia (AML) are inconsistent at predicting clinical outcomes for individual patients. Variability in the quality of specimens utilized for biomarker discovery and validation may contribute to this prognostic inconsistency.We evaluated the impact of sample heterogeneity on prognostic biomarkers and methods to mitigate any adverse effects of this heterogeneity in 240 cryopreserved bone marrow and peripheral blood specimens from AML patients enrolled on SWOG (Southwest Oncology Group) trials.Cryopreserved samples displayed a broad range in viability (37% with viabilities ≤60%) and nonleukemic cell contamination (13% with lymphocyte percentages20%). Specimen viability was impacted by transport time, AML immunophenotype, and, potentially, patients' age. The viability and cellular heterogeneity in unsorted samples significantly altered biomarker results. Enriching for viable AML blasts improved the RNA quality from specimens with poor viability and refined results for both DNA and RNA biomarkers. For example, FLT3-ITD allelic ratio, which is currently utilized to risk-stratify AML patients, was on average 1.49-fold higher in the viable AML blasts than in the unsorted specimens.To our knowledge, this is the first study to provide evidence that using cryopreserved specimens can introduce uncontrollable variables that may impact biomarker results and enrichment for viable AML blasts may mitigate this impact.

Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.

Contemporary risk stratification algorithms commonly use threshold-defined categories of clinically relevant risk factors. The Children's Oncology Group (COG) uses National Cancer Institute (NCI) risk group (RG), cytogenetics, and early response to therapy measured by minimal residual disease (MRD) using flow cytometry on day 8 peripheral blood (D8 PB) and day 29 bone marrow (D29 BM). However, it is unclear whether assigning different weights to individual risk factors, as well as using numerical values as continuous, rather than categorical, would more accurately predict relapse risk. Previous work (Loh, ASH 2020) described validation of a continuous prognostic index (PI) for risk of relapse published by UK investigators incorporating favorable and unfavorable genetics, white blood cell count (WBC), and D29 BM as continuous variables (O'Connor, JCO 2018; Enshaei, Blood 2020), and assessed the added value of D8 PB. We now extend this work by comparing patient outcomes with current COG risk classification to PI-derived risk classifications on the previously described population (Loh, ASH 2020). We first retrospectively classified patients (pts) (N=21,199 from prior COG trials AALL0331/0232 or AALL0932/1131 enrolled 2004-2019) in our analysis population using the COG risk stratification algorithm employed in the current generation of COG trials. Pts with Down syndrome or BCR/ABL1 were excluded. We classified our analysis population as SR-Favorable [SR-Fav, 24.5% (5-year relapse free survival (RFS) probability 0.97)], SR-Favorable/Average (not distinguishable because of missing D8 PB) [SR-Fav/Avg, 5.3% (.96)], SR-Avg [20.5% (0.93)], SR-High [12.5% (0.83)], HR-Fav [3.0% (0.96)], HR [29.6% (0.82)], and Very HR [VHR, 1.1% (0.54)] according to NCI RG, CNS status, cytogenetics, D8 PB where relevant, D29 BM, and EOC MRD. Ninety-seven percent of pts had sufficient data to be retrospectively classified and thus 20,176 pts were considered for subsequent analyses. We next developed a multivariable model for RFS using log transformed MRD (τ(MRD)). Temporal external validation was first employed by developing models considering AALL0932/1131 data (n=12,453) and then validating them with AALL0331/0232 data (n=7,723). Of the full cohort of 20,176 pts, 24.4% could not be classified by COG PI, primarily due to missing D8 PB MRD which was not assessed routinely in earlier studies; thus the model was developed on 11,151 pts and validated on 4,103 pts. The COG PI (PI COG) was calculated using the equation [τ(d8 MRD) x -0.036 + τ(d29 MRD) x -0.119 + CYTO-GR x -0.914 + CYTO-HR x 0.752 + WBC log x 0.178]. The UK PI (PI UK) was also calculated using published coefficients [τ(d29 MRD) x -0.218 + CYTO-GR x -0.440 + CYTO-HR x 1.066 + WBC log x 0.138] for comparison to assess the practical significance of adding D8 PB. In contrast to the UK method, we identified risk groups by selecting PI cutoffs that maximized the discrimination of the predictive model as quantified by the concordance probability estimator (CPE) (Barrio, SORT 2017). This objective method of cutpoint determination allows for risk group definition without investigator agreement on exact prespecified risk group characteristics; this method also defined four risk groups (Low, Standard, Intermediate, and High). Cutpoints derived from the two different indices, applied to the pts who could be classified by PI COG (n=15,254), resulted in different proportions of pts in each of the risk groups with generally similar RFS estimates for each group. Using cutpoints estimated for PI COG (-2.073, -1.307, and -0.857) 36.0% (RFS = 0.97) were classified as low, 29.6% (0.93) standard, 17.1% (0.88) intermediate, and 17.4% (0.73) high risk of relapse. For PI UK ( -2.916, -2.534, and -1.15), among those who were classifiable by PI COG, 33.4% (0.97) were classified as low, 26.3% (0.93) standard, 30% (0.87) intermediate, and 10.4% (0.69) high. Finally, we compared the COG risk stratification to PI CPE-defined risk stratification in the cohort. As shown in the table, PI COG improves discrimination among individuals by identifying groups with different relapse risk than expected. The PI COG can thus identify patients for whom therapeutic intensification may not result in significantly better outcomes while improving the discrimination of HR pts to allow randomized interventions with achievable hazard ratios. Figure 1 Figure 1. Disclosures Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz: Amgen, Blueprint Medicines: Honoraria. Zweidler-McKay: ImmunoGen: Current Employment. Mullighan: AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding. Hunger: Amgen: Current equity holder in publicly-traded company. Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member.

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that differ from adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values (TMM) from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the particular biology of pediatric CML might impact the treatment of those patients in the future. Disclosures Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.

Current risk stratification for COG ALL patients (pts) relies on National Cancer Institute (NCI) risk group (RG) at diagnosis, somatic genetics, and early response to therapy as measured by specific thresholds of minimal residual disease (MRD) using flow cytometry on day 8 peripheral blood (D8 PB) and day 29 bone marrow (D29 BM). NCI RG is defined as age 1-10 years (yrs) and white blood cell count (WBC) We first log transformed WBC, D8 and D29 MRD and displayed these by treatment protocol, NCI RG, and FRG/URG (separating out Ph-like independently). Age and WBC followed the normal expected distribution with the median age of SR pts 4.0 yrs (range 1-9) and HR 12 yrs (range 1-30). Transformed MRD was displayed as a variable t(MRD), corresponding to the negative log; max t(D29 MRD) was 13.82, corresponding to MRD We next conducted a univariate analysis for risk factors for relapse, including sex, age, WBClog, CNS status, protocol-defined rapid early response status, FRG, URG, t(D8 MRD), and t(D29 MRD); all variables except CNS status were significant p < 0.0001). Multivariable modeling showed that WBClog, FRG, URG, t(D8 MRD), t(D29 MRD) retained significance (p < 0.0001). Finally, we applied the UK Prognostic Index (PIUKALL) equation [t(d29 MRD) x -0.218 + CYTO-GR x -0.440 + CYTO-HR x 1.066 + WBClog x 0.138] to the COG data using protocol, NCI RG, FRG, URG, IRG, or Ph-like RG in the model and validated the trends for relapse-free survival (RFS), which were similar in our groups with an overall median PIUKALL of -2.63 (mean -2.32, SD -.90, min -3.54, max 1.79). We next added in t(D8 MRD) to define a PICOG and determined that D8 PB MRD added significantly to the model, mostly through discriminating between the hazard ratios of the FRG and the URG RGs. Importantly, the D8 PB MRD led to a qualitatively more distinctive group with a potentially lower predicted RFS in NCI SR pts, a group that has been more difficult to predict in the past, and yet comprises nearly half of all relapse events. Our analyses of 21,178 COG B-ALL pts confirm and extend the utility of integrating WBC and MRD as continuous rather than categorical values to refine risk stratification for patient treatment and trial design. Disclosures Loh: Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Zweidler-McKay:ImmunoGen, Inc.: Current Employment. Mattano:Melinta Therapeutics: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Hunger:Amgen: Current equity holder in publicly-traded company, Honoraria; Novartis: Consultancy. Raetz:Celgene/BMS: Other; Pfizer: Research Funding. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; AbbVie, Inc.: Research Funding.

Background: Patients with Down syndrome (DS) have an approximately 10-fold increased risk of developing ALL, and the spectrum of genetic alterations differs from that of non-DS ALL. Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of DS ALL, compared to only 5-10% CRLF2+ cases in non-DS children and adolescents. JAK2 or JAK1 mutations co-occur in about half of CRLF2+ cases in both DS and non-DS ALL. The prognostic significance of CRLF2+ ALL also appears to differ in the limited data reported to date, with less adverse impact in patients with DS compared to non-DS ALL. Here, we report the clinical characteristics and prognostic significance of B-ALL with CRLF2 overexpression and JAK alterations in children and adolescents/young adults (AYA) with DS who were treated on Children's Oncology Group (COG) clinical trials from 2003-2016. Methods: We analyzed clinical, laboratory, and outcome data for 317 patients with DS B-ALL treated on standard risk (SR) trials AALL0331 and AALL0932 and high risk (HR) trials AALL0232 and AALL1131, for whom CRLF2 status and rearrangement partners (IGH or P2RY8) were ascertained by flow cytometric assessment of surface expression; fluorescence in situ hybridization; and/or polymerase chain reaction (PCR) testing. JAK mutations were ascertained in a subset by PCR and sequencing. Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at end of consolidation (EOC) for a subset of EOI MRD+ patients. Results: We identified 168/317 (53.0%) CRLF2+ cases, and among those assessed for CRLF2 partner, 17/73 (23.3%) were IGH-CRLF2 and 56/73 (76.7%) were P2RY8-CRLF2. In the subset of 165 cases tested for JAK mutations (85 CRLF2- and 80 CRLF2+), 42/165 (25.4%) had JAK mutations, all of which co-occurred in CRLF2+ cases. CRLF2 positivity was significantly associated with younger age at diagnosis: 140/168 (83.3%) of CRLF2+ cases were under 10 years old, versus 106/149 (71.1%) of CRLF2- cases, p Discussion: Whereas CRLF2 and JAK alterations are associated with higher MRD, poorer survival, and increased CIR in patients with high-risk ALL without DS, these alterations do not demonstrate strong adverse prognostic impact in children and AYAs with DS-ALL treated on recent frontline COG trials, although larger sample sizes are needed to adequately assess for possible poorer prognoses associated with the CRLF2+/JAK+ and IGH-CRLF2 subgroups. Regardless, given the frequency of these targetable lesions and the increased risk of relapse and chemotherapy-associated toxicities in patients with DS-ALL, targeted therapies currently under investigation for these genetic lesions may be beneficial to replace some intensive blocks of therapy in DS-ALL with CRLF2 and/or JAK alterations, both to enhance anti-leukemic efficacy and decrease intensive chemotherapy-associated toxicities. Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau. Raetz:Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria.

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) occurs in 5-30% of children and adolescents/young adults (AYAs) with B-ALL, is driven by genetic alterations that induce constitutive cytokine receptor and kinase signaling, and is associated with poor clinical outcomes across the older pediatric-to-adult age spectrum (Tasian Blood 2017c, Reshmi Blood 2017, Roberts Blood 2018). Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of Ph-like ALL cases with frequently co-occurring JAK2 or JAK1 point mutations or IL7R indel mutations. This study reports the clinical outcomes of children and AYAs with newly-diagnosed National Cancer Institute (NCI) standard-risk (SR) or high-risk (HR) CRLF2+ ALL without Down syndrome treated on four successive Children's Oncology Group (COG) phase 3 clinical trials from 2003 to 2018. Methods: We retrospectively assessed demographic characteristics, laboratory data, and clinical outcomes of 3757 patients with B-ALL treated on COG trials AALL0331 and AALL0932 (SR) and AALL0232 and AALL1131 (HR) whose diagnostic leukemia specimens were analysed by low-density microarray (LDA), fluorescence in situ hybridization, polymerase chain reaction (PCR), and/or anchored multiplex PCR testing (Harvey and Tasian Blood Advances 2020). Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at the end of consolidation for a subset of EOI MRD+ patients. Results: We identified 77/1541 (5.0%) SR and 244/2216 (11.0%) HR patients with CRLF2+ B-ALL in this cohort. Amongst those with diagnostic leukemia specimens analysed by LDA, 57/72 (79.2%) of SR CRLF2+ and 175/213 (82.2%) of HR CRLF2+ patients were positive for the Ph-like gene expression profile with an 8-gene score ≥0.5. P2RY8-CRLF2 fusions and IGH-CRLF2 translocations were detected in 64/77 (83.1%) and 10/77 (13.0%) of SR CRLF2+ patients and in 98/244 (40.2%) and 103/244 (42.2%) of HR CRLF2+ patients, respectively. CRLF2 rearrangements or F232C mutations were not found in the remaining 3 SR and 43 HR CRLF2+ patients, although other Ph-like alterations were discovered in some (n=3 IGH-EPOR fusions, 1 IL7R indel). Importantly, CRLF2+ vs non-CRLF2-overexpressing (CRLF2-) status was associated with older age (10.8 ±6.5 vs 7.8 ±5.8 years [mean ±SD], p Discussion: Patients with newly-diagnosed CRLF2+ B-ALL treated on frontline COG trials have higher rates of EOI MRD positivity, inferior survival, and increased CIR compared to their CRLF2- counterparts. EFS is especially poor in children and AYAs with NCI HR CRLF2+ ALL, particularly those with the Ph-like expression profile. Conversely, outcomes for children with NCI SR CRLF2+ ALL are reasonably favourable, irrespective of Ph-like status. Development of successful treatment strategies to decrease relapse and to improve survival remains a major therapeutic gap for NCI HR CRLF2+ ALL patients. Current clinical trials are studying the potential efficacy of kinase inhibitor addition to chemotherapy for children, adolescents, and adults with HR Ph-like ALL harboring CRLF2 rearrangements/other JAK pathway alterations or ABL class kinase fusions (NCT0240717, NCT02723994, NCT02883049, NCT03571321). Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria. Raetz:Pfizer: Research Funding; Celgene/BMS: Other. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Additional file 1: Table S1. Targeted Sequencing Details. Table S1A. Target regions for Wafergen Sequencing. Table S1B Primers and Amplicon Details for Wafergen Sequencing. Table S1C. Primers for CEBPA targeted MiSeq Assay. Table S2. Loci that Failed Quality Control. Table S3. Characteristics of selected and unselected SWOG patients. Fig. S1. Comparison of Performance Characteristics of selected and unselected SWOG patients. Table S4. Characteristics of SWOG patients in the discovery and validation cohorts. Fig. S2. Comparison of Performance Characteristics of SWOG patients in the discovery and validation cohorts. Table S5. Mutation distribution in discovery and validation cohorts. Fig. S3. Mutation Frequency (OncoPrint). Table S6. Expression fold change differences between MNCs and VLBs in discovery cohort. Table S7. Univariate analyses results, non-significant findings Models Details. Table S8. ELN2017 versus ELN2017-MOD risk assignment in MNCs and VLBs.

Hyperdiploidy with greater than 50 chromosomes is usually associated with favorable prognosis in pediatric acute lymphoblastic leukemia (ALL), whereas hypodiploidy with ≤43 chromosomes is associated with extremely poor prognosis. Sometimes, hypodiploidy is "masked" and patients do not have a karyotypically visible clone with ≤43 chromosomes. Instead, their abnormal karyotypes contain 50-78 or more chromosomes from doubling of previously hypodiploid cells. When the hypodiploid and doubled hyperdiploid clones are both present, patients can be identified by traditional test methods [karyotype, DNA Index (DI), fluorescence in situ hybridization (FISH)], but the incidence of masked hypodiploid cases in which only the doubled clone is visible is unknown. We analyzed 7013 patients with B-ALL enrolled in COG AALL03B1 (2003-2011) for whom chromosome studies were available. Of 115 patients with hypodiploidy (25-39 chromosomes), karyotypes of 40 showed only the hypodiploid clone, 47 showed mosaicism with both hypodiploid and hyperdiploid (doubled) karyotypes, and 28 with masked hypodiploidy showed only a hyperdiploid (doubled) clone. Unique karyotypic signatures were identified, and widespread loss of heterozygosity (LOH) was seen in the microsatellite panel for all patients with masked hypodiploidy. An increased awareness of the unusual karyotypic profile associated with a doubled hypodiploid clone and coordinated use of DI, FISH, and LOH studies when indicated can identify patients with masked hypodiploidy and allow appropriate treatment selection.

INTRODUCTION: ELN-2017 guideline is the gold standard for risk stratifying AML patients. However, clinical prognostic factors such as age are not incorporated into the guideline. Therefore, we examined if novel prognostic models incorporating clinical factors and expression of select transcripts can improve the current guideline. The prognostic models were developed utilizing molecular data from unsorted mononuclear cells (MNCs) and viable leukemic blasts (VLBs). METHODS: Specimens were obtained from untreated AML patients (N=383) who received intensive chemotherapy on SWOG trials S9031, S9333, S0106 and S0112. Patients were randomly separated into discovery (N=190) and validation (N=193) cohorts. Participants provided informed consent in compliance with the Declaration of Helsinki, SWOG, NCI, and Fred Hutch regulations. VLBs were isolated using fluorescence-activated cell sorting (FACS, Pogosova et al., 2018, Biopreserv Biobank 16;42-52). RNA and DNA were extracted from MNCs and VLBs. FLT3-ITD, NPM1, CEBPA, ASXL1, RUNX1 and TP53 mutations were identified using fragment analyses and NGS. Transcript expression for BAALC, CEBPA, CCNA1, CD34, ERG1, EVI1, FLT3, GATA2, IL3RA, JAG1, KIT, MN1, RUNX1, and WT1 was quantified using Q-RT/PCR assays. Specimen attributes across paired samples were compared using McNemar's test and the Wilcoxon signed rank test. Associations with overall survival (OS), relapse-free survival (RFS), and complete remission (CR) were assessed using the Kaplan-Meier method, Cox proportional hazards regression, and logistic regression. Models were built in the discovery cohort using a pre-defined 3-step process of variable selection and 5-fold cross validation of this process; final model performance was evaluated in the validation cohort. RESULTS: Mutations, transcripts and clinical variables . Mutation frequencies, FLT3-ITD AR, and clinical outcomes were not significantly different between discovery and validation cohorts. The percentage of patients with high FLT3-ITD AR (per ELN-2017 definition) was increased in the VLBs compared to MNCs (77% vs 66%, respectively); FLT3-ITD AR was reclassified for 19 patients. Transcript expression was significantly increased in VLBs relative to MNCs for 8 transcripts (P Model Development and Performance. Multivariable models for CR, OS, and RFS were fit using age, ELN-2017 risk group, PS, AML onset, immunophenotype, clinical trial, and combinations of these baseline variables as covariates. In the discovery cohort, the models with the best performance were obtained when clinical variables and transcript expression were integrated; however, these integrated models were not superior to AGE+ELN2017 models in the validation cohort (Table 1), whether utilizing molecular data from MNCs or VLBs. Quartile risk groups from the AGE+ELN2017 model displayed increased discrimination with respect to OS than the ELN risk groups (Figure 1A,B). The discriminative performances of the ELN2017 and AGE+ELN2017 models for OS were also examined in older adults (age ≥55). Given the small numbers of patients, risk scores for the AGE+ELN2017 model were divided into tertiles to identify 3 risk groups. The three risk groups derived from the AGE+ELN2017 scores showed increased ability to discriminate for projected OS relative to ELN2017 risk groups (Figure 1C, D). DISCUSSION: ELN2017 models were significantly associated with clinical outcomes, but AGE+ELN2017 models may perform slightly better than ELN2017 models. Models incorporating selected transcript expression did not improve the ELN2017 models, whether transcripts were examined in MNCs or VLBs. While ELN-2017 prognostic guideline remains a critical tool for risk stratifying AML patients, the findings highlight the need for additional prognostic factors, including age, to improve the accuracy of risk stratification. Disclosures Othus: Celgene: Other: Data Safety and Monitoring Committee; Glycomimetics: Other: Data Safety and Monitoring Committee. Erba:Agios, Amgen, Astellas Pharma, Daiichi Sankyo, ImmunoGen, Janssen, Jazz Pharmaceuticals, Juno, Millennium, Seattle Genetics: Research Funding; Celgene, Incyte, Novartis: Speakers Bureau; Amgen, Celgene, Daiichi Sankyo, ImmunoGen, Incyte, Jazz Pharmaceuticals, Millennium, Novartis, Ono, Pfizer, Seattle Genetics, Sunesis: Consultancy. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Radich:Novartis: Other: RNA Sequencing; TwinStrand Biosciences: Research Funding. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding.

Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with myeloid leukemia and 2 to 9% of all pediatric leukemias. Prior to the discovery of tyrosine kinase inhibitors (TKI) such as imatinib, stem cell transplantation was the only curative treatment for both adults and children with CML. However, due to the small numbers of patients, standardized treatment approaches for pediatric CML have not been established. There are several unique characteristics of CML diagnosed in children and adolescents, and young adults (AYA; 16-29 years), compared to adults. Children and AYA with CML present with a higher white blood count and have larger spleens, higher peripheral blast counts, and lower hemoglobin levels, suggesting that the biology of pediatric CML is different than adult CML. In addition, potential side effects of TKIs unique to pediatric CML patients include impaired bone growth, fertility and immune function, however none have been extensively studied. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that are absent in adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated from pediatric CML (n=7), adult CML (n=8), pediatric normal (n=2) and adult normal (n=3) bone marrow samples. Total RNA was isolated from cells, and then cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. We aligned reads using the HISAT2 alignment software, and mapped to genes with HT-Seq. We removed genes that had zero reads across all the samples, resulting in a set of 4,696 genes that were detected in one or more samples. In case of technical replicates, we used mean of replicates. We performed three differential expression comparisons with edgeR: (1) Pediatric CML vs Adult CML, (2) Adult CML vs Adult Normal, and (3) Pediatric CML vs Pediatric Normal. We used a False Discovery Rate (FDR) of £ 20% and absolute log2 fold-change ³ 1 for selecting differentially expressed genes in each comparison. We used Fisher's exact test to identify significant KEGG pathways for the differentially expressed genes in each comparison. Results Pediatric CML vs Adult CML We found 24 differentially expressed genes (15 over- and 9 under-expressed). Though no pathway was found to be significant at the false discovery rate (FDR) £ 20%, we identified a number of sub-pathways that are relevant. For example, the Chemokine Signaling pathway shows at the top of the list (ordered by raw p-value) because of two genes, XCR1 and HCK, associated with VEGF and MAPK pathways involved in cell proliferation, angiogenesis, DNA repair, and cancer pathogenesis. Adult CML vs Adult Normal We found 60 genes (30 over- and 30 under-expressed) differentially expressed when comparing adult CML patients to normal adults. Ten genes overlapped with 24 genes we identified when comparing pediatric and adult CML patients. We found 11 pathways as significant at FDR £ 10%. Multiple pathways, including Cell adhesion, allograft rejection, Graft versus Host Disease, and Type I diabetes pathways, showed downregulation of MHC, with subsequent downstream reduction in expression of apoptosis-related genes. The IL-17 pathway makes sense, as MAPK, well-known to be associated with various cancers, is down-regulated. Lastly, in the NK pathway the gene DAP12 is up-regulated. This gene is known as a tyrosine kinase binding protein, and although tyrosine kinase inhibitors are the standard treatment for CML, the role of DAP12 in relation to leukemia has not yet been described. Pediatric CML vs Pediatric Normal We found 509 genes (350 over- and 159 under-expressed) differentially expressed in pediatric CML patients compared to normal. Interestingly, transcriptional regulators are differentially enriched in the hematopoietic stem cell differentiation function group including GATA1, GATA2, KLF1 and KLF2. RFC is down-regulated. RFC is a mismatch repair gene known to be involved in colorectal cancer. Many of the significant pathways are involved in glucose and fatty acid metabolism. Our pilot study identified novel molecular features of pediatric CML bone marrow stem cells, providing new insights into the novel biomarkers and pathogenesis of pediatric CML. Disclosures Gotlib: Blueprint Medicines: Consultancy, Honoraria, Research Funding; Promedior: Research Funding; Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.

Introduction: Incorporating cytogenetics into risk stratification for the treatment of childhood ALL has contributed to increased survival rates. However approximately 25% of patients, the B-other subgroup, harbour none of the known major chromosomal abnormalities. Within this group, Philadelphia-like (Ph-like) patients show a similar gene expression profile to those with the BCR-ABL1 fusion and share the same high risk of relapse. Ph-like ALL is genetically heterogeneous with some patients harbouring tyrosine kinase activating gene fusions, such as EBF1-PDGFRB, or activation of the JAK-STAT signalling pathway, making them amenable to targeted therapy with kinase inhibitors. Methods: We studied patients with B-other ALL entered to the MRC UKALL2003 treatment trial using a panel of break-apart FISH probes to identify rearrangements of PDGFRB/CSF1R , ABL1 , ABL2 and JAK2. Rearrangements of the CRLF2 gene were identified using break-apart FISH probes for CRLF2, P2RY8 and IGH and/or MLPA using the P335-ALL-IKZF1 kit (MRC Holland, The Netherlands). Where possible, partner genes were identified by cytogenetics and FISH. RNA samples from 135 of the B-other cohort were screened using a Taqman low density array (TLDA) to detect the Ph-like gene expression signature and P2RY8-CRLF2 gene fusion. The overlap between the two cohorts is shown in Figure 1. Results: Within this B-other cohort, rearrangement of CRLF2 was the most prevalent abnormality, occurring in 13% of patients (n=65/503), with two-thirds of them showing deletion of the PAR1 region, resulting in P2RY8-CRLF2 fusion (n=43) and the remaining third showing IGH-CRLF2 (n=22). Rearrangements of PDGFRB/CSF1R occurred in 3.4% of the cohort(n=17/491), while ABL1 (n=4/485), ABL2 (n=1/454) and JAK2 (n=2/481) rearrangements were rare, each occurring in less than 1% of B-other patients. Partner genes were identified in 17 cases with kinase rearrangements, with EBF1-PDGFRB being the most common (n=10). PAX5-JAK2 was identified in 2 patients and ATF7IP-PDGFRB, MEF2D-CSF1R, SSBP2-CSF1R, ETV6-ABL1 and ZC3HAV1-ABL2 in single cases. By TDLA, 33/135 (24%) patients tested positive for a Ph-like signature. In 9 patients the arrays indicated high expression of CRLF2 with 7 of them having a P2RY8-CRLF2 transcript by TLDA, which was confirmed by FISH in 5 cases and MLPA in another case. In the remaining patients, the mechanism of CRLF2 over-expression remains unknown as no P2RY8-CRLF2 transcript was detectedby TDLA and no material was available for FISH or MLPA. A single case negative for Ph-like signature tested positive for P2RY8-CRLF2 transcript. Seven patients showed an ABL-class rearrangement by cytogenetics and FISH (EBF1-PDGFRB, n=5; ETV6-ABL1, n=1; ABL1 rearrangement partner unknown, n=1). The genetics underlying the remaining cases is being further investigated by whole genome and transcriptome sequencing. Of the 102 patients negative for a Ph-like signature, 71 of them were tested by FISH for kinase fusions. In a single case positive for EBF1-PDGFRB by FISH, expression of the EBF1-PDGFRB transcript was confirmed by RT-PCR. The 5-year event free survival (EFS) of the entire B-other cohort was 82% with a relapse rate (RR) of 12%. Patients with rearrangements of PDGFRB/CSF1R (n=17)and those testing positive for a Ph-like expression signature (n=33) showed an inferior outcome, with EFS rates of 31% and 65% and RR of 64% and 28%, respectively (p Conclusion: This is the largest unbiased cohort of paediatric B-other ALL screened for ABL1, ABL2, PDGFRB/CSF1R and JAK2 fusions. We demonstrate that FISH can reliably detect the full spectrum of these genetic changes, most of which are cryptic by cytogenetic analysis. These abnormalities were mutually exclusive with an overall frequency in B-other ALL of ~15%. Patients with rearrangements of PDGFRB/CSF1R and those testing positive for a Ph-like gene expression had high rates of relapse. Given the efficacy of tyrosine kinase inhibitors in treatment of these patients, testing for these abnormalities should be integrated into future clinical trials. Disclosures Mullighan: Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding.

Background:Ph-like acute lymphoblastic leukemia (ALL) is a high-risk subtype of ALL in children. There are limited and conflicted data on the incidence and prognosis of Ph-like ALL in adults. Methods:Patients with newly-diagnosed B-ALL who received frontline chemotherapy at MD Anderson Cancer Center underwent gene expression profiling of leukemic cells to identify Ph-like ALL. Gene expression profiling was performed on 148 RNA samples using either U133 Plus 2.0 microarrays, or a customized Taqman low density array (LDA) card to identify patients with the Ph-like ALL gene signature (Roberts et al. NEJM 2014). An additional 7 previously untreated patients were found to have CRLF2 overexpression by multicolor flow-cytometry (MFC), and received induction chemotherapy at MDACC were included in the outcome analysis (but not for subtype frequency calculation). We performed targeted sequencing of 303 recurrently mutated genes (L300 panel, MDACC) in 40 patients with CRFL2 rearrangements (15 with matched germline control). Minimal residual disease (MRD) was assessed by MFC, with a sensitivity of 0.01%. Results:Of 148 patients, 49 (33.1%) were Ph-like, 46 patients (31.1%) were Ph+, and 53 patients (35.8%) were of other B-ALL subtypes (B-other). The median age of Ph-like cohort was 33.5 years (range, 15-71), Ph+ cohort was 49 years (range, 22-84), and B-other was 38 years (range, 15-79). Within the Ph-like ALL cohort, 61% had overexpression of CRLF2. Patients received hyper-CVAD (80%) or an augmented-BFM regimen (20%). The rate of CR/CRp was similar in the 3 disease subgroups (Ph-like ALL 89%, Ph+ ALL 93%, B-other 94%, p = 0.57). However, patients with Ph-like ALL were significantly less likely to achieve MRD-negative remission (30% vs. 56% for Ph+ ALL vs. 87% for B-other, p The most common mutations by L300 sequencing of 40 patients with CRLF2 were JAK2 (n=19, 47.5%), KRAS (n=10, 25%), CRLF2 (n=7, 17.5%), NRAS (n=5, 12.5%), PAX5 (n=5, 12.5%), JAK1 (n=4, 10%) (Figure 2). The CRLF2 F232C mutation, noted in 7 (17.5%) patients in this study, appears more frequent than in pediatric patients (3/134, 2.2%, Chen et al. Blood 2012), and in range with a smaller adult series (3/14, 21.4%, Yoda et al. PNAS 2010). CRLF2 F232C mutations were mutually exclusive with JAK2/JAK1 mutations (except in one patient). Conclusions:Our findings show a high frequency of Ph-like ALL in adults; an increased frequency of Ph-like ALL in adults with Hispanic ethnicity; significantly inferior outcomes of adult patients with Ph-like ALL; and significantly worse outcomes in Ph-like ALL patients with CRLF2 overexpression. The frequency of CRLF2 F232C mutation appears to be higher in adult patients with B-ALL than in the children. Ph-like ALL represents a high-risk disease subtype of adult B-ALL. Novel strategies are needed to improve the outcome of these patients. Disclosures Jain: Pharmacyclics: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Incyte: Research Funding; BMS: Research Funding; Abbvie: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Infinity: Research Funding; Novimmune: Consultancy, Honoraria. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

INTRODUCTION. Most studies of AML biomarkers have examined cryopreserved mononuclear cells (MNCs) obtained from repositories. These MNCs typically include non-leukemic cells (e.g., lymphocytes) and heterogeneous populations of viable and nonviable AML blasts at various stages of differentiation. We hypothesize that these variables negatively impact the prognostic power of current biomarkers. In an attempt to improve the prognostic power of such biomarkers, we examined enriched populations of viable AML blasts from 190 randomly selected AML patients on SWOG trials. To our knowledge, this study represents the largest examination of the quality of cryopreserved AML blasts and the potential prognostic benefit of enriching for viable leukemic blasts. METHODS. Cryopreserved bone marrow (BM, N=124) and peripheral blood (PB, N=116) samples from 190 AML patients on SWOG trials (SWOG-9031, SWOG-9333, S0106, and S0112) were randomly selected. The samples were thawed and sorted for viable AML blasts using a combination of fluorochrome-conjugated antibodies and DAPI. DNA and RNA were extracted and evaluated from both the unsorted AML samples (A-MNCs) and viable AML blasts (A-Blasts). FLT3-ITD allelic ratio (AR) was quantified by fragment analyses. Expression of 13 potentially prognostic transcripts was quantified via quantitative RT/PCR: BAALC, CCNA1, CEBPA, ERG1, EVI1, FLT3, GATA2, IL3RA, JAG1, KIT, MN1, RUNX1, and WT1. Fold expression differences were computed by the comparative CT method. Associations between quantitative biomarkers (FLT3-ITD AR and transcript expression) and survival (overall, OS and relapse-free, RFS) were analyzed using Cox proportional hazards regression, with interaction terms for biomarker by cell population (A-MNCs vs. A-Blasts) or CD34 expression (separately for A-MNCs and A-Blasts). A predetermined FLT3-ITD AR cutoff of 0.5 was utilized based on results by Schneider et al. (Blood, 2012) and others. For paired samples, BM was used. RESULTS. Lymphocyte percentage varied (median 7.1%, range 0.4 - 70.1%) and was correlated with patients' pre-treatment blast percentages (rs = -0.29, P = 0.0002). Viability by DAPI varied widely (median 66.5%, range 5.2 - 95.6%), and AML blasts displayed inter- and intra-sample immunophenotypic heterogeneity, with the slight majority expressing CD34 (54%) using a predetermined immunofluorescence cut-off > 104. For OS, there was no significant difference (P=0.67) in the effect of FLT3-ITD AR between A-Blasts (HR=0.91, P=0.78) and A-MNCs (HR=1.05, P=0.88). However, for RFS, the effect of FLT3/ITD AR differed significantly (P=0.025) between A-Blasts (HR=1.93, P=0.14) and A-MNCs (HR=0.87, P=0.73). Kaplan-Meier curves of RFS by FLT3-ITD AR for A-MNCs (Figure 1A) and A-Blasts (Figure 1B) also suggest this trend. A similar, but nonsignificant, trend towards higher HRs in A-Blasts was displayed in analyses restricted to NPM1 mutated patients (Table 1). Similar analyses did not show such a striking interaction between cell population and transcript biomarkers, but we found that most transcripts displayed significant associations with immunophenotype (data not shown). Therefore, we examined if CD34 expression might impact the prognostic value of transcripts, separately by cell population. The expression of two genes (CCNA1 and GATA2) displayed significant interactions with CD34 expression in relation to clinical outcomes (Table 1). Moreover, the interaction between biomarker and CD34 expressions differed depending on which cell population was examined, such that the interaction was significant in the A-Blasts but not A-MNCs. CONCLUSION. Cryopreserved samples vary widely in percentages of non-leukemic, dying cells and differentiation stage of leukemic blasts. These factors impact the measurement of quantitative biomarkers and may also impact the significance and prognostic value of these biomarkers. Future studies must consider the effects that sample viability, composition, and differentiation stage may have on quantitative biomarkers. In addition, we are examining the potential impact that mutations in other genes (e.g., ASXL1, DNMT3A, RUNX1, etc.) may have on our results. ACKNOWLEDGEMENT. The authors wish to gratefully acknowledge the important contributions of the late Dr. Stephen H. Petersdorf to SWOG and to study S0106. SUPPORT. NIH/NCI grants CA160872,CA180819, CA180828, and CA180888. Disclosures Wood: Seattle Genetics: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement. Erba:Jannsen: Consultancy, Research Funding; Sunesis: Consultancy; Gylcomimetics: Other: DSMB; Agios: Research Funding; Celgene: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Incyte: Consultancy, DSMB, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; Astellas: Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; Celator: Research Funding; Juno: Research Funding. Othus:Celgene: Consultancy; Glycomimetics: Consultancy. Radich:Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy; Gilliad: Consultancy.

Mutations in the all-trans retinoic acid (ATRA)-targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse.Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD + were also associated with reduced postrelapse survival. © 2012 by The American Society of Hematology.

Introduction. Prior studies have described a subset of B-progenitor ALL cases with a distinct gene expression profile and/or deletions involving ERG (encoding the ETS family member v-ets avian erythroblastosis virus E26 oncogene), however the relationship of these alterations and their role in leukemogenesis are poorly understood. We performed integrated genomic and epigenetic analyses, biochemical studies and leukemogenesis assays to define the genetic basis of this form of ALL. Methods. We studied 1674 childhood, adolescent and young adult B-progenitor ALL cases with microarray gene expression profiling and/or RNA-sequencing data to enable the identification of ERG ALL by unsupervised clustering and predictive analysis of microarrays. Detailed genomic analysis was performed for 144 ERG ALL cases, including whole genome (N=38), exome (n=46) and/or RNA-sequencing (n=57) cases, and single nucleotide polymorphism array analysis. Epigenetic profiling, including whole genome bisulfite sequencing, chromatin immunoprecipitation and sequencing for ERG and histone modifications and ATAC-sequencing were performed for a subset of 8 xenografted ERG tumors and reference cell lines. ERG transcript expression was measured by analysis of RNA-seq analysis and quantitative RT-PCR assays, and by interrogation of TCGA and PCGP RNA-seq data. The function of ERG isoforms was evaluated by EMSA and transcriptional reporter assays, immunofluoresence, colony forming assays and retroviral bone marrow transplant assays. Results. One hundred and forty four cases (8.6%) of B-ALL cases exhibited a distinct gene expression profile and lacked known chromosomal rearrangements (ERG ALL). Such cases had favorable outcome. Eighty cases (55.6%) had focal deletions of ERG with no evidence of oncogenic or chimeric ERG fusions. The deletions were most commonly heterozygous and involving exons 3-7 (n=27) or 3-9 (n=22) of 10 coding exons, and less commonly involving exon 1, or a larger region of the gene. No ERG deletions were identified in non-ERG ALL. Two cases harbored missense mutations in the ETS domain. Analysis of whole genome and exome sequencing data of 71 cases identified a high frequency of alterations of lymphoid transcription factors (46.5%; IKZF1 36.7%, PAX5 11.3%); mutation of transcription factors otherwise uncommon in ALL (21%; MYC, MYCBP2, MGA, ZEB2, GATA3); activation of signaling pathways, most commonly NRAS or KRAS (35.2%); cell cycle regulation (22.5%); and epigenetic modifiers (56.3%), most commonly KMT2D, SETD2, ARID2 and NCOR1. Notably, the five year event-free survival of ERG ALL cases with IKZF1 alterations exceeded 85% in both St Jude and Children's Oncology Group cohorts. We observed striking transcriptional deregulation at the ERG locus. Most (51/56) ERG- deleted cases expressed an ERG isoform encoded by a novel exon in intron 6 that splices in frame to distal exons, resulting in expression of a truncated C-terminal ERG protein that lacks the pointed and central regulatory domains, but retains the ETS and transactivation domain (ERGalt). ERGalt was also present in most (36/44) cases lacking an ERG deletion, and was strongly associated with presence of ERGalt protein in leukemic cells. We also identified expression of an Antisense Long non-coding RNA associated with the ERG locus (ALE) in ERG ALL. ERGalt and ALE were absent, or uncommonly expressed at very low levels in non-ERG ALL. ERGalt was absent, and ALE rarely expressed in non-ALL PCGP and TCGA samples. ERGalt and point mutant ERG were retained in the nucleus, bound DNA targets and acted as competitive inhibitors of wild type (WT) ERG in transcriptional reporter assays. Lineage-negative Arf -null bone marrow cells transduced with ERG WT induced an aggressive erythro-megakaryoblastic leukemia; in contrast ERGalt induced an immature lymphoid progenitor leukemia. Conclusions. Genomic alterations drive aberrant transcription of ERG, resulting on expression of a truncated, C-terminal oncogenic ERG protein. This represents a novel mechanism of transcription factor deregulation in leukemia. As a subset of ERG ALL cases lack ERG deletion, and as IKZF1 alterations are not associated with inferior outcome in this form of ALL, diagnostic approaches must incorporate gene expression profiling in addition to identification of ERG and IKZF1 alterations to accurately identify this form of leukemia. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Stock:Gilead: Membership on an entity's Board of Directors or advisory committees. Voorhees:Oncopeptides: Consultancy; Onyx Pharmaceuticals: Research Funding; GSK: Consultancy; Oncopeptides: Research Funding; Janssen: Research Funding; A Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy; Millennium Pharmaceuticals: Consultancy, Research Funding; Acetylon Pharmaceuticals, Inc.: Research Funding; Novartis: Consultancy; Array BioPharma: Consultancy; GSK: Research Funding; Celgene: Research Funding. Hunger:Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership. Mullighan:Incyte: Consultancy; Amgen: Honoraria.

Background. Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with genomic alterations that activate JAK/STAT and PI3K/Akt/mTOR signal transduction and with poor clinical outcomes. Therapeutic disruption of PI3K pathway signaling in Ph-like ALL has been minimally investigated to date, however. We hypothesized that PI3K isoform-selective or dual PI3K pathway protein inhibition would robustly inhibit Ph-like ALL proliferation in vivoand abrogate aberrant signaling. Methods. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were engrafted with primary CRLF2/JAK-mutant or ABL/PDGFR-mutant Ph-like ALL specimens (Table 1) and treated with inhibitors of PI3K? (BYL719), PI3K? (idelalisib), PI3K/mTOR (gedatolisib), TORC1/TORC2 (AZD2014) or with vehicle. Treated patient-derived xenograft (PDX) models were assessed for pharmacodynamic inhibition of signal transduction phosphoproteins at 72 hours by phosphoflow cytometry and for residual ALL in murine spleens after 3-4 weeks of inhibitor or vehicle treatment by quantitative flow cytometry. Subsequent studies tested the efficacy of gedatolisib with the JAK1/2 inhibitor ruxolitinib (CRLF2/JAK-mutant models) or gedatolisib with the SRC/ABL inhibitor dasatinib (ABL/PDGFR-mutant models). Table 1. Genomic characteristics of Ph-like ALL specimens utilized for PDX studies. USI Disease status CRLF2/JAK alterations ABL/PDGFR alterations PALTWS D IGH@-CRLF2* PAMDKS D IGH@-CRLF2, JAK2R683G PAMDRM D IGH@-CRLF2,JAK2GPinsR683 ALL121 R IGH@-CRLF2, JAK2R683G ALL4364 R P2RY8-CRLF2, JAK2R683G PAKMVD D JAK1 S646F PAKYEP D BCR-JAK2 PAKKCA D EBF1-PDGFRB PAKVKK D NUP214-ABL1 PANSFD D ETV6-ABL1 USI = unique specimen identifier. D = de novo, R = relapse. * non-Ph-like by prediction analysis of microarrays. Results. All tested PDX models demonstrated inhibition of leukemia proliferation and abrogation of activated signaling with PI3K pathway inhibition. Gedatolisib treatment resulted in near-eradication of leukemia in CRLF2/JAK-mutant models (n=7) with mean 92.2% (range 86.0-99.4%) leukemia reduction vs vehicle treatment (p Conclusions. PI3K pathway inhibition is a biochemically relevant therapeutic approach for Ph-like ALL. Dual PI3K/mTOR inhibition with gedatolisib monotherapy potently inhibited leukemia proliferation and demonstrated additive or synergistic activity in combination with JAK or SRC/ABL inhibition in JAK-mutant or ABL/PDGFR-mutant Ph-like ALL, respectively. These data provide compelling rationale for testing combinations of signal transduction inhibitors without or with cytotoxic chemotherapy in children and adults with Ph-like ALL. Disclosures Off Label Use:preclinical testing of signal transaction inhibitors in Ph-like ALL models. Teachey:Novartis:Research Funding. Maude:Novartis:Consultancy, Research Funding. Perl:Actinium Pharmaceuticals:Consultancy; Asana Biosciences:Consultancy; Arog Pharmaceuticals:Consultancy; Ambit/Daichi Sankyo:Consultancy; Astellas US Pharma Inc.:Consultancy. Hunger:Sigma Tau:Consultancy; Jazz Pharmaceuticals:Consultancy; Spectrum Pharmaceuticals:Consultancy; Merck:Equity Ownership. Grupp:Novartis:Consultancy, Research Funding.

Acute leukemias are the most commonly occurring cancers among children, adolescents and young adults. Approximately 15% of newly-diagnosed patients present with T-lineage acute lymphoblastic leukemia (T-ALL). In contrast to B-precursor ALL, molecular lesions that deregulate homeobox (HOX) genes appear to be widely prevalent in T-ALL, but their impact on outcome is unclear. We hypothesized that the molecular lesions associated with HOXA -deregulated T-ALL might identify patients with high-risk disease. We performed gene expression profiling (GEP) on a cohort of 213 T-ALL diagnostic samples obtained from children and young adults enrolled on Children's Oncology Group (COG) treatment studies 9404 (n=50) and AALL0434 (n=163). We identified a cluster of 54 cases (25%) characterized by increased expression of HOXA3, 5, 7, 9, and 10 (FDR ≤ 0.05; fold change cut off 3). We screened our database for the presence of MLL-R, AF10-R (MLLT10) and other HOXA deregulating lesions using an analysis of cytogenetics, FISH, LDI-PCR and RNA sequencing. We identified 15 cases with MLL-R, including MLL-AF6 (n = 5), del3'MLL (n = 3), MLL-ENL (n = 5), MLL-AF17 (n = 1), and PICALM-MLL (n = 1). Five cases were confirmed to have PICALM-AF10 fusions and two cases showed DDX3X-AF10 lesions. Two cases harbored NUP98 fusions, two cases had inv(7)(p15q34), and one case each was identified for HOXA10-(3'UTR)TRBC, STAG2-LMO2, LOC338817-CCDC91. We could not identify fusion transcripts in 3 cases, but MLL -R and AF10-Rwere confidently excluded with RNA sequencing. Almost 90% of cases identified to have MLL-R or AF10-R had ≥ 8-fold over-expression of HOXA9/10. Because HOXA-deregulated T-ALL is characterized by a high degree of molecular heterogeneity, we hypothesized that subset analyses might identify lesions that were more likely to be associated with an inferior outcome. We found that chromosomal abnormalities involving the MLL gene, but not AF10, were associated with induction failure (IF) in T-ALL (P = 0.02, OR = 5.34). Since MLL- and AF10 -R leukemias also demonstrate features of undifferentiated leukemias, we discriminated early T-cell precursor (ETP) from non-ETP cases using a GEP developed by Coustan-Smith et al. (Lancet Oncol, 2009). Among the 26 cases that showed ETP features, eleven (42%) also co-expressed HOXA9/10 (≥ 8-fold increase over the median). We found an association between ETP-ALL and early treatment failure (P = 0.01, OR = 4.37), and next assessed whether ETP cases are enriched with translocations harboring MLL or AF10 genes. We found overlap between MLL-R and the ETP cases (P = 0.03, OR = 4.14). We confirmed that ETP-ALL and MLL-R are risk features for IF (P = 0.026, OR = 4.37), and that cases with MLL-R (n = 11) had an inferior EFS compared to those that did not (N = 89) (P = 0.0158). We extended these observations to assess the impact of ETP/MLL-R (n = 6 vs. non-MLL/non-ETP, n = 69) on EFS, and found a significant association with treatment failure (P = 0.0007). For T-ALL, MRD has emerged as a prognostic indicator of high-risk disease. While MLL-ENL (n = 5)cases did not fail therapy regardless of Day 29 MRD levels, all patients with Day 29 MRD > 0.1 and MLL-AF6 (n = 5) or FISH-identified del3'MLL (n = 3) either failed induction or relapsed. Patients with AF10 -R (N = 7) have been reported to have inferior EFS, but we observed that only patients with Day 29 MRD ≥10% failed treatment. Our findings show a heterogeneity of outcomes related to MLL-R, but those with MLL-AF6 and del3'MLL should be considered high-risk. We propose that cytogenetic testing including specific FISH should be performed on all T-ALL patients at diagnosis and relapse. The current development of epigenetic modifying therapies targeted against HOXA-deregulating lesions warrants further study in T-ALL. Disclosures Aplan: NIH Office of Technology Transfer: Patents & Royalties. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Hunger:Merck: Equity Ownership; Sigma Tau: Consultancy; Jazz Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Consultancy.

Introduction: BCR-ABL1-like, or Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), is characterized by a gene expression profile similar to BCR-ABL1-positive ALL, with a broad range of genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We previously reported a rearrangement of EPOR, encoding the erythropoietin receptor, into the immunoglobulin heavy chain locus (IGH). The aims of this study were to define the frequency and genomic architecture of EPOR rearrangements in B-ALL and to examine their role in kinase signaling and lymphoid transformation. Methods: Whole genome and/or transcriptome sequencing was performed on 154 Ph-like ALL cases. Sanger sequencing and fluorescent in situ hybridization were used to confirm and map the EPOR rearrangements. Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of erythropoietin (EPO). EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Epor-/- fetal liver cells were transduced with empty vector, EPOR wild-type or rearranged alleles and used for erythroid colony forming unit (CFU-E) and erythroid burst-forming unit (BFU-E) assays. Luciferase-marked xenografts of human EPOR-rearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases (5.2% of Ph-like ALL) harbored rearrangements of EPOR into either the IGH or immunoglobulin kappa light chain (IGK) loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR. Such rearrangements resulted in overexpression of a C-terminal truncated receptor that retained the phosphorylation site required for STAT5 activation, but lacked multiple intracytoplasmic tyrosine residues whose phosphorylation is required for normal negative regulation of the receptor. Notably, the locations of the truncation sites overlap with those arising from inherited mutations in primary familial congenital polycythemia, in which frameshift and nonsense mutations truncate the receptor. A real-time quantitative PCR assay was established to provide a diagnostic tool and to confirm that primary leukemia cells with these EPOR rearrangements overexpress N-terminal exons but lack expression of C-terminal truncated exon eight. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/- mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Expression of wild-type or truncated EPOR in Epor-/- fetal liver cells promoted erythroid differentiation with formation of CFU-E and BFU-E colonies, indicating that truncated receptors sustain erythroid development. Xenografted EPOR-rearranged leukemic cells exhibited high levels of mutant EPOR on the cell surface, constitutive STAT5 phosphorylation and sensitivity to the JAK2 inhibitor ruxolitinib ex vivo and in vivo. Conclusions: We have identified a subset of Ph-like ALL cases characterized by rearrangements of truncated EPOR into the IGH/IGK chain loci. This represents an entirely new mechanism of EPOR deregulation and unexpectedly implicates EPOR signaling as an important factor influencing B-lymphoid malignancies that are amenable to JAK-STAT5 inhibition. Clinical trials testing ruxolitinib in ALL patients with EPOR rearrangements are warranted. Disclosures No relevant conflicts of interest to declare.

The genetic basis underlying inferior outcome of adolescent and young adult acute lymphoblastic leukemia (AYA ALL) as compared to childhood cases is largely unknown. To comprehensively characterize the genetic landscape of AYA ALL we studied 423 adolescent (16-21 yrs; median 17.7±1.3 yrs) and 250 young adult (21-39 yrs; median 28.3±7.0 yrs) samples from the Children's Oncology Group high-risk trial AALL0232, St Jude Children's Research Hospital Total XV and XVI, Eastern Cooperative Oncology Group E2993, MD Anderson Cancer Center and the Alliance - CALGB trials. Single nucleotide polymorphism (SNP) microarray analysis and gene expression profiling were performed to identify copy number alterations and distinct genetic subgroups. Samples were also sub classified using hierarchical clustering, ROSE outlier and PAM analysis of gene expression profiling data. Sequence mutation analysis was performed on candidate genes known to be mutated in pediatric ALL (including IKZF1, PAX5, JAK1/2, NRAS, KRAS, FLT3, IL7R, SH2B3, TP53 and CREBBP), and mRNA-seq was performed on selected BCR-ABL1-like cases (n=41). The genetic subgroups were divided into ETV6-RUNX1, TCF3-PBX1, hyperdiploid (>50 chromosomes), MLL rearrangements, BCR-ABL1, BCR-ABL1-like, ERG and other (cases with no known lesions). As expected, ETV6-RUNX1 and hyperdiploid ALL were less frequent in adolescents (4% and 11%, respectively) and adults (2% for both) than in childhood ALL ( Notably, BCR-ABL1 and BCR-ABL1-like ALL patients presented with higher white blood counts at diagnosis compared to non BCR-ABL1-like ALL patients in both adolescents (117.6 and 76.8 vs 21.9 x109/L, p We then sought to characterize the alterations activating kinase signaling in AYA BCR-ABL1-like ALL cases. As observed in pediatric ALL, approximately 55% of these cases harbored CRLF2 rearrangements. Using mRNA-seq we identified a variety of additional rearrangements involving the tyrosine kinase or cytokine receptor genes ABL1, ABL2, CSF1R, JAK2, EPOR or PDGFRB, with a marked enrichment of fusions involving JAK2 (6 different fusions in 9/20 cases sequenced), thus providing a rationale for the investigation of targeted therapies directed against these alterations. Collectively, the kinase-activating BCR-ABL1 and BCR-ABL1-like subtypes are associated with poor outcome and make up ∼25% of adolescent and ∼50% of young adult ALL patients. The identification of these patients at diagnosis will provide an opportunity to incorporate tyrosine kinase inhibitor treatment to current chemotherapeutic regimens, and significantly improve the treatment outcome for AYA ALL. Disclosures: Hunger: Bristol Myers Squibb: Consultancy.

Introduction Infant acute lymphoblastic leukemia (ALL) is an orphan disease with unmet need for safe effective therapies. This is an urgent problem because conventional chemotherapies are ineffective and have life-threatening toxicities in infants. Although the MLL rearrangements occurring in 75% of cases are associated with poor outcome, survival is inferior whether MLL is rearranged or not. We recently reported that infant ALL proved sensitive to obatoclax mesylate (GeminX Pharmaceuticals; now an indirect, wholly owned subsidiary of Teva Pharmaceutical Industries Ltd.) in vitro regardless of poor prognostic features including MLL gene rearrangement. Moreover, we showed that the leukemia cell killing by obatoclax involved apoptosis, necroptosis and autophagy (Urtishak et al., Blood 2013). Therefore, the recent pharmaceutical abandonment of obatoclax led us to search for similarly acting drugs, the Results of which identified the well-known antipsychotic thioridazine as a candidate for potential repurposing. Methods Correlative analyses were performed between basal gene expression profiles at leukemia diagnosis and single agent obatoclax EC50 values from MTT assays in 47 cases of infant ALL from the Children's Oncology Group P9407 trial (25 MLL-AF4; 8 MLL-ENL; 7 other MLL-rearranged; 7 MLL-germline) in order to find a priori determinants of obatoclax sensitivity; significant genes were further studied by Ingenuity Pathway Analysis (IPA). A search for similarly acting compounds was conducted by Connectivity Map analysis of gene expression profiles of MLL-AF4 ALL cell lines after obatoclax treatment. MTT assays without and with cell death pathway inhibition, Western blot and flow cytometric cell death assays, and phosphoflow cytometric signaling analyses were utilized to investigate activity and target modulation by potential candidates. Results IPA identified significant correlations between basal gene expression of the mTOR and downstream intersecting eIF4/p70S6K signaling programs and obatoclax EC50 in all 47 primary cases of infant ALL, as well as in the subset of the 25 cases with MLL-AF4 rearrangements. Consistent with the relevance of this pathway in leukemia cell killing that was suggested by the basal gene expression profiles in the primary cases, the Connectivity Map analysis of obatoclax-treated cell lines for compound matching returned a number of highly ranked PI3K/AKT/mTOR signal transduction inhibitors as potential obatoclax substitutes. Three of the compounds (LY294002, wortmannin, thioridazine) were not only cytotoxic in MLL-AF4 ALL cell lines, but also they abrogated PI3K/AKT/mTOR signaling as indicated by robust inhibition of phosphorylated S6. Of these compounds, the phenothiazine derivative thioridazine, which has been used clinically for decades as a neuroleptic, was of high interest because of potential advantages of drug repurposing for more rapid drug advancement. Moreover, detailed flow cytometric and Western blot analyses, and MTT assays of thioridazine in the presence of cell death pathway inhibitors validated activation of all three cell death mechanisms in the MLL-AF4 ALL cell lines similarly to obatoclax. Conclusions Thioridazine is a well-known antipsychotic drug that also has recently recognized properties as a PI3K/AKT/mTOR signaling inhibitor and as an inhibitor of other pathways relevant to cancer. In MLL-AF4 ALL cell lines characterized by the most common chromosomal translocation in infant ALL, single-agent thioridazine is highly cytotoxic, robustly inhibits PI3K/AKT/mTOR signaling and, moreover, like obatoclax, demonstrates activity as a multi-cell-death pathway agonist. Further preclinical studies now are warranted to determine the extent to which thioridazine inhibits PI3K/AKT/mTOR signaling and causes leukemia cell killing in primary infant ALL cells in vitro and in vivo. The repurposing strategy that this drug may allow could have promise to streamline drug development in infant ALL where the need for new therapies is so urgent. Disclosures: No relevant conflicts of interest to declare.

Using gene expression profiling, we and others identified a novel subgroup of B-precursor acute lymphoblastic leukemia (B-ALL) with a gene expression signature similar to Philadelphia (Ph) chromosome (BCR-ABL1)-positive ALL. Termed “Ph-like” or “BCR-ABL1-like” ALL, this subgroup constitutes 10-15% of pediatric and 25% of adolescent/young adult ALL cases and is associated with a very poor clinical outcome. Using next generation sequencing, we have shown that Ph-like ALL is characterized by a highly heterogeneous spectrum of activating mutations or gene fusions targeting genes regulating cytokine receptor and tyrosine kinase signaling (JAK1/2, ABL1/2, PDGFRB, EPOR, CSF1R, AKT2, STAT5B, CRLF2, IL7R, SH2B3). As Ph-like ALLs may be sensitive to tyrosine kinase inhibitors (TKIs) in vivo, incorporating TKIs into therapy may significantly improve clinical outcomes. Here we report the development and validation of a robust gene expression classifier that can prospectively identify Ph-like ALL patients for therapeutic intervention. Methods Supervised learning methods were applied to gene expression profiles (Affymetrix U133_Plus_2.0; RMA normalized) generated from pre-treatment leukemic samples from 811 B-ALL patients accrued to COG High-Risk ALL Trials P9906 and AALL0232. Patients were partitioned into a training (P9906: n=207; AALL0232: n=278) and an independent test set (AALL0232: n=325). Next generation sequencing was used to identify Ph-like ALL-associated genomic lesions in these cohorts. The 54,675 Affymetrix probe sets were evaluated using Prediction Analysis of Microarrays (PAM), applying the method of nearest shrunken centroids to identify those probe sets best distinguishing Ph-like ALL. These probe sets were then distilled by 100 iterations of 10-fold cross-validation using three optimization criteria (overall error, average error, and ROC accuracy), leading to the identification of the 64 most predictive probe sets (derived from 38 unique genes). Quantitative RT-PCR assays were developed for each of the 38 genes by selecting optimized primer/probe sets and assays were run on 384-well Low Density Microarray (LDA) cards; 780/811 cases had residual material for LDA testing. LDA data were remodeled in the training set using double loop cross validation, resulting in a best and final predictive model and statistical algorithm containing 15 of the 38 genes (IGJ, SPATS2L, MUC4, CRLF2, CA6, NRXN3, BMPR1B, GPR110, CHN2, SEMA6A, PON2, SLC2A5, S100Z, TP53INP1, IFITM1). The sensitivity and specificity of the predictor was then evaluated in the independent test set. Results The 15 gene LDA classifier was able to predict Ph or Ph-like ALL in the test set with a high degree of sensitivity (93.0%) and specificity (89.7%) and identified the heterogeneous genomic lesions associated with Ph-like ALL with very high frequency (Table 1). When compared to non-Ph-like ALL, Ph-like cases had a significantly poorer event-free survival (HR 3.58; p Conclusions We have developed and validated a highly robust gene expression classifier for the prospective identification of Ph-like ALL. Rapidly screened patients will then undergo targeted sequencing to confirm the presence of specific genomic lesions. This approach will facilitate the therapeutic targeting of Ph-like ALL patients to novel clinical trials, hopefully leading to improved outcomes. Disclosures: No relevant conflicts of interest to declare.

TEL/ABL-positive (TEL/ABL+) acute lymphoblastic leukemia (ALL) is a very rare prognostically unfavorable disease. Its pathogenesis is similar to the BCR/ABL-positive (BCR/ABL+) ALL. TEL/ABL kinase activity can be inhibited by tyrosine kinase inhibitors (TKI), which may be used in the therapy of TEL/ABL+ ALL. Thanks to the rareness of TEL/ABL+ ALL there is a lack of knowledge on potential development of TKI resistance and its mechanisms. We have used previously established TEL/ABL+ ALL cell line (Baeumler et al., Cancer Genetics and Cytogenetics, 2008) to create a TKI resistant daughter cell line and study the kinetic and mechanism of its resistance. Resistance to IM was acquired after a very long term (16 months) exposure to slowly increasing doses of imatinib mesylate (IM). We have tested the sensitivity of the newly established resistant cell line against a panel of 7 kinase inhibitors and we found that the cell line acquired resistance not only to IM but also to dasatinib, nilotinib and ponatinib, while it remained sensitive to bosutinib, sunitinib and sorafenib. The resistance to IM remained stable even after the withdrawal of IM from the growth medium and was not abrogated by the co-treatment with valproic acid, which has potential to reverse imatinib resistance in BCR/ABL-positive leukemia (Morotti et al., Cancer, 2006). These findings imply that the cells acquired de novo resistance rather than that primarily resistant subclone was selected, and that the acquired resistance has a genetic background. We have studied several potential mechanism of IM-resistance described in BCR/ABL+ leukemia - mutations in ABL kinase domain, genomic amplification and enhanced expression/autophosphorylation of chimeric protein - but we did not find any of them to be acquired by our resistant cell line. To screen for other potential causes of IM resistance we have performed whole genome genomic profiling by high density SNP arrays, whole genome gene expression profiling and whole exome sequencing. Genomic profiling and subsequent FISH analysis of parental versus resistant cell line revealed 3 discordant aberrations: amplification of 8q24.13-24.3, deletion of 10q26.13-26.3 and deletion of 13q12.12-12.13. Gene expression profiling showed broad changes in expression pattern with at least 2 fold change of expression between parental and resistant cell line in 566 probes representing 469 genes. Interestingly, this 469-gene set contained 5/27 genes from a published gene set which expression correlates with survival in BCR/ABL-positive ALL (Juric et al, Journal of Clinical Oncology, 2007) - the changes in their expression in the resistant versus parental cell line correspond to the changes between the patients with the worse versus better survival. Functional annotation of these 469 differentially expressed genes covers spectrum of diverse biological processes and pathways. Of interest, 2/5 top down-regulated genes in resistant cell line are pro-apoptotic protein PAWR and negative regulator of cytokine signaling SOCS2, both involved in pathways with potential biological relevance for the studied resistance. The data from exome sequencing are currently being analyzed. To summarize, we have successfully established multi-resistant TEL/ABL+ ALL cell line model to study the kinetics and mechanism of TKI resistance. We have demonstrated a very slow kinetics of the IM-resistance induction and excluded several resistance mechanisms typically found in BCR/ABL+ leukemia such as ABL kinase domain mutations, genomic amplification or enhanced expression/phosphorylation of the chimeric protein. We found 3 genomic and multiple gene expression changes to be associated with the resistant phenotype, their contribution to the mechanism of the resistance will be subjected to future studies. Grant support: IGA MZ NT/2121167; UNCE 204012; RVO-FNM64203; RVO-VFN64165 Disclosures: No relevant conflicts of interest to declare.

Abstract 1038 The relationships of inherent disease-related characteristics and of treatment to the nature of APL cell clones that emerge at disease relapse are poorly understood. We studied these relationships in 45 patients who relapsed after ATRA/chemotherapy treatment on the non-arsenic trixoxide arm of intergroup phase III trial C9710 (Powell, et al. Blood, Epub). Four variably-expressed APL cell clonal markers were assessed: PML-RARA ligand binding domain mutations (LBD-M), FLT3 internal tandem duplication mutations (ITD), FLT3 receptor tyrosine kinase mutations (D835; RTK-M) and additional chromosome abnormalities (ACA), i.e., in addition to the hallmark t(15;17). The methods for mutation analysis of PML-RARA and FLT3 have been reported and were RNA transcript-initiated supplemented by selected quantitative FLT3 mutation analyses initiated from DNA. Karyotype data were derived by standard cytogenetic methods. Four patients were excluded from analysis because the sample level of PML-RARA, as determined by quantitative RT-PCR, was insufficient to exclude possible false-negative mutation results. In 41 evaluable patients, the marker incidences were: LBD-M, 44%; ITD, 37%; RTK-M, 12%; ACA (22 tested), 59%. At presentation, the corresponding incidences were: LBD-M, 0% (by high-sensitivity testing a minor subclone of the relapse mutation was found in 2/7 patients tested); ITD, 43% (37 tested); RTK-M, 22% (37 tested); ACA, 24% (34 tested). The low frequency of RTK-M impeded further analysis. The other markers, using relapse determinations, were assessed for potential associations between the markers and with the following parameters: age, sex, presenting WBC count, PML-RARA type, time to relapse, relapse on or off ATRA treatment (on = taking or within 30 days of discontinuing) and post-relapse survival. The most essential positive findings are summarized below:Association of Marker with Parameter or Other MarkerCases with ParameterCases without Parameterp-valuePretreatment parameters: LBD-M with WBC count < 5,000/uL12/18 (67%)7/23 (30%)0.030 ITD with WBC count >10,000/uL10/15 (67%)5/26 (19%)0.006 ITD with S-form PML-RARA15/15 (100%)10/26 (38%) The results confirm reports, which tested pretreatment samples, of a strong association between FLT3ITD mutations present at relapse and high WBC count and S-form PML-RARA. At relapse, there was, also, divergent selection of ITD-harboring vs ACA-harboring clones. LBD-M occurred more frequently in patients with low presenting WBC counts. They, also, occurred in patients who relapsed while on ATRA, consistent with an ATRA selective role for clones harboring LBD mutations. A significant segregation of patients with LBD-M or ITD was observed after relapse off ATRA treatment (p = 0.019), while no segregation was observed in overall relapse patients (p = 0.346) or after relapse on ATRA (p = 0.316). In 2 patients who relapsed on ATRA, quantitative analysis indicated that LBD-M and ITD mutations must be present in the same APL cell clone. There were no significant survival differences related to any clonal marker (post-relapse survival = 66%). Although the small number of cases is cautionary, the overall data suggest that LBD-M may have an overriding, dominant APL clone selection role in relapse that occurs on ATRA therapy. Other inherent clinicopathologic characteristics of APL may predispose to divergent molecular pathways of disease progression and relapse in patients alternatively harboring clones with LBD-M or ITD after the termination of ATRA selection pressure. Disclosures: No relevant conflicts of interest to declare.

Abstract 2752 Introduction: We recently identified a poor prognostic subgroup of pediatric BCR-ABL1 negative ALL patients characterized by deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) and a gene expression signature similar to BCR-ABL1 positive ALL, raising the possibility of activated tyrosine kinase signaling within this leukemia subtype. Targeted sequencing revealed activating sequence mutations in the Janus tyrosine kinases (JAK1 (N=3), JAK2 (N=17) and JAK3 (N=1)) in 21 of 187 (11.2%) BCR-ABL1 negative, high-risk pediatric ALL cases. All 21 cases with JAK mutations had the BCR-ABL1-like expression profile, accounting for about 50% of the cases with this phenotype, suggesting that mutations in JAK kinases account for some, but not all, cases with this distinctive profile. To determine whether mutations in other kinases might also be associated with this distinctive gene expression profile, we sequenced 126 genes encoding tyrosine kinases and mediators of kinase signaling in an additional 46 high-risk ALL cases with a BCR-ABL1-like expression profile. The genes sequenced included the entire tyrosine kinome. Methods: The 46 leukemia specimens studied were from patients enrolled on COG clinical trials for high risk ALL (P9906, n=23 and AALL0232, n=23), with risk defined primarily by elevated WBC and/or age > 10 years. All 46 cases had a BCR-ABL1 like expression profile. The 23 P9906 cases all lacked JAK mutations, while 3 of the 23 AALL0232 cases were found to have activating JAK mutations (JAK1 (N=1), JAK2 (N=2)). The entire coding region and UTRs of each gene was amplified by PCR of whole genome amplified genomic DNA, and subjected to Sanger sequencing. A CEPH sample (NA19085) was also included as a normal control DNA. Results: A total of 1,149,117 bases were sequenced bi-directionally for each sample; 96% of the targeted bases were covered with high-quality sequencing data. We identified a total of 2,302 variations predicted to change protein sequences, 173 of which are novel, putative variations after removing germline variations found in dbSNP, The Cancer Genome Atlas Project (TCGA) and the normal CEPH sample NA19085 in this study. For each novel variation, the tumor DNA was resequenced and matching normal DNA was sequenced to validate the original observation and to distinguish somatic from inherited variants. The results show that 105 variations are germline, 20 are false positives while the remaining markers failed in validation assay. Aside from 1 FLT3 mutation (23aainsN609), there are no confirmed somatic mutations in any other tyrosine kinase genes. Conclusion: Aside from JAK mutations, somatically acquired sequence mutations in tyrosine kinase genes are rare in children with high risk ALL and BCR-ABL1 like gene expression profiles. We are pursuing the identification of alternative mechanisms for kinase activation that might explain the distinctive expression profile observed in these cases. Disclosures: Relling: St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding. Hunger:bristol myers squibb: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 2757 Introduction: The outcome of infants with acute lymphoblastic leukemia (ALL) remains poor because of the association of frequently occurring MLL translocations with drug resistance and vulnerability of the very young to treatment complications. The two most common MLL partner genes in infant ALL, AF4 (AFF1) and ENL (MLLT1), are associated with particularly poor survival. Better therapies are urgent. One candidate is obatoclax (GeminX Biotechnologies, Inc.), which targets interactions of pan-anti-apoptotic BCL-2 family proteins with BH3 proteins and is now in a Phase I trial for relapsed/refractory pediatric cancers (COG ADVL0816). Previously we showed potent single agent in vitro activity of obatoclax against MLL-rearranged infant ALL (Zhang ASH 2008). Here we evaluate correlations of obatoclax activity with MLL translocation status and gene expression profiles in a large number of cases of infant ALL to define molecular determinants of sensitivity. Methods: Bone marrow, peripheral blood or apheresis samples from the time of diagnosis in 54 infants (age 1–365 d, median 168 d; WBC count 15–1230×103/μL, median 445×103/μL) with ALL (n=52) or bilineal acute leukemia (n=2) were examined, 48 of which were from the COG P9407 trial. By molecular/cytogenetic classification, the cases were MLL-AF4+ (n= 28), MLL-ENL+ (n= 11), other MLL rearrangement positive (other MLL+) (n= 8) or MLL germline (MLL-) (n= 7). Single agent IC50 values from MTT assays after 72 h obatoclax exposures were determined in all cases (including 13 previously tested; Zhang ASH 2008) by plotting the surviving fractions. IC50s in the MLL-AF4+ group were compared to those in each of the other 3 molecular/cytogenetic groups by Wilcoxon's test. Gene expression profiling was performed on Affymetrix HG_U133 Plus2.0 arrays in 47 of the 48 COG P9407 cases. Spearman test was used to identify correlation between log2 expression levels for each probeset and IC50 values across subjects. A heatmap of significant probesets (p≤0.001) was generated by transforming expression levels to z-scores and ordering rows and columns by complete linkage hierarchical clustering. Ingenuity pathway analysis was applied to all probesets with p≤0.01 to identify pathways significantly correlated with IC50. Additional MTT assays were initiated to test sensitivity to agents targeting these pathways. Results: Even though most cases in all 4 groups were sensitive to obatoclax as indicated by IC50s within a clinically achievable range, MLL translocation status still had a significant effect on IC50. MLL-AF4+ cases were least sensitive and MLL-ENL+ cases were most sensitive to obatoclax. Respective IC50 ranges across all 54 cases were: MLL-AF4+, 26–918 nM; MLL-ENL+, 13–294 nM; other MLL+ 10–356 nM; MLL−, 31–488. Compared to MLL-AF4+, the IC50s in MLL-ENL+ cases were significantly lower (p=0.047), IC50s in other MLL+ cases were lower but the difference did not achieve significance (p=0.10), and IC50s in MLL- cases were not significantly different (p=0.64). In the 47 COG P9407 cases studied by MTT assay and gene expression profiling, 450 probesets defined a cluster of 16 cases with higher IC50s, which were predominantly MLL-AF4+ (68.7%). Ingenuity analysis identified significant correlations of the following canonical pathways with the IC50 in the same 47 cases: glycolysis/gluconeogenesis, mTOR signaling, regulation of eIF4 and p70S6K signaling, EIF2 signaling, and fructose and mannose metabolism. In preliminary analyses, cell lines with t(4;11) exhibited time and dose-dependant sensitivity to the eIF4e inhibitor ribavirin. Conclusions: In infant ALL, obatoclax has broad-spectrum activity and there is pan-sensitivity across MLL translocation subtypes and MLL− cases. Still specific MLL partner genes have a strong effect on obatoclax IC50 and there is exquisite sensitivity in MLL-ENL+ cases. This result is important because MLL-ENL is associated with particularly poor survival when conventional therapies are used. The association of differentially expressed genes in canonical cell signaling and metabolism pathways with differences in obatoclax sensitivity forms the basis to combine obatoclax with targeted agents directed at restoring these pathways to enhance responsiveness even further. Disclosures: Felix: None: Patent not licensed.

Improved outcomes for children with acute lympboblastic leukemia (ALL) have been achieved, in part, from adaptation of risk-stratified therapy. The Children’s Oncology Group (COG) has implemented a real-time risk classification system (AALL03B1) using a combination of NCI-Rome risk criteria, blast cell genetic features, and early treatment response to determine the intensity of post-induction therapy. Between December 29, 2003 and June 1, 2007, more than 4,000 children over 1 year of age with B-precursor ALL were enrolled on AALL03B1, including 2293 (62%) with NCI Standard Risk (SR) and 1406 (38%) with NCI High Risk (HR) features who were subsequently enrolled on companion clinical trials. The most favorable genetic features used in AALL03B1 were identified in legacy COG studies and included TEL/AML1(TEL) or triple trisomies of chromosomes 4, 10, and 17 (TT). Unfavorable genetic features included the presence of BCR/ABL, MLL rearrangements, or extreme hypodiploidy (DNA index 0.1% at day 29 were defined as slow early responders (SER). Among the favorable cytogenetic subsets, patterns of early response differed. The presence of TEL was significantly associated with an RER to induction therapy in both NCI SR and HR groups (p< 0.0001), while the presence of TT was not (p=0.058). For NCI SR patients, the presence of TEL was significantly associated with the achievement of an M1 bone marrow by day 8 (50.9% of TEL+ pts vs. 41.2% of TEL- pts, p< 0.0001). Patients with an M1 or M2 BM on day 29 who had MRD >1% received extended induction (EI) for two weeks followed by an additional evaluation of BM morphology and MRD at day 43 of induction. One hundred and nineteen patients received EI, with 40% having NCI SR features at diagnosis. Of the patients who received EI, 63% achieved an M1 marrow with MRD < 1% by day 43 and were eligible to continue on protocol therapy. This was more likely to occur in NCI SR patients (77% vs. 55%, p 220 COG institutions.