ALBERT

Description: Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Description: Acquisition of drug resistance in tumor cells in children with T-cell acute lymphoblastic leukemia (T-ALL) during chemotherapy results in relapse and poor outcome. T-ALL cell lines that have acquired resistance to chemotherapeutics are therefore critical tools for the study of acquired resistance, yet there is a paucity of cell lines available for study. In this study, we hypothesize that drug resistant T-ALL cells can be produced by prolonged exposure to chemotherapeutics and that microarray analysis can be employed to identify the gene products responsible for acquired drug resistance. By incrementally increasing the drug concentration in growth media, we have produced T-ALL cell lines (Jurkat and Sup T1) that grow well in the presence of therapeutic concentrations of L-asparaginase (ASNase) and daunorubicin (DNR). The genetic profiles of the drug-resistant cell lines were compared to their parental progenitors using the Affymetrix HG-U133Plus2 GeneChip platform, capable of hybridizing ~54,000 genes and ESTs/chip. Signal intensity was normalized using the robust multi-array average (RMA) technique in GeneSpring 7.2. The Sup T1 and Jurkat ASNase-resistant cell lines increased their IC50s 26-fold (0.044 IU/mL to 1.14 IU/mL) and 320-fold (0.003 IU/mL to 0.96 IU/mL), respectively. The IC50 of the Jurkat DNR resistant cell line increased 77-fold (30 nM to 2300 nM), and 4.0-fold, (0.46 nM to 1.85 nM), respectively. Notably, DNR resistant Jurkat cells were also resistant to therapeutic concentrations of vincristine and prednisolone, but not ASNase. In contrast, the ASNase resistant cell lines remained sensitive to DNR, vincristine, and prednisolone. Microarray data comparing DNR-resistant and parental cell lines showed 288 genes upregulated 〉1.5-fold in the resistant line. Two sets of genes were the most upregulated in the drug resistant cells in comparison to parental cells. ABCB1 (ABC transporter P-glycoprotein) was upregulated ~940-fold and genes coding for 6 different killer-cell immunoglobulin-like receptors (KIRs) were upregulated 〉6-fold. In the case of the ASNase-resistant cell lines, 96 genes were found to be upregulated 〉1.5-fold in both Jurkat and Sup T1 lines. The most highly upregulated gene in both cell lines was argininosuccinate synthase (ASS), 32-fold upregulated in Jurkat and 6.5-fold in Sup T1. All expression results were confirmed by qRT-PCR. These genes have previously been implicated in the acquisition of drug resistance: ASS is critical for responding to asparagine depletion caused by ASNase. ABCB1 acts as a molecular pump capable of lowering intracellular concentrations of substrate chemotherapeutics such as DNR, vincristine, and prednisolone, consistent with our observation of multi-drug resistance in that cell line. To our knowledge, this is the first description of DNR and ASNase resistant Jurkat and Sup T1 T-ALL cell lines. In addition, our results suggest that microarray technology is a valid method for elucidating the genetic nature of drug resistance in T-ALL cell lines, making it a productive approach to identify mechanisms of chemotherapy resistance. Finally, these cell lines will serve as useful tools for studying mechanisms of chemotherapeutic resistance in T-ALL.

Description: Precursor-B acute lymphoblastic leukemia (pre-B ALL) is the most common malignancy in children and can be cured in a majority of patients. However, cure remains elusive in approximately 20% of patients for reasons that are not well understood. Moxetumomab pasudotox is one of several CD22-targeting therapies currently under investigation in clinical trials for subjects with B-cell malignancies, including pediatric subjects with pre-B ALL. Moxetumomab pasudotox (MP) is a second-generation immunotoxin composed of disulfide-stabilized, affinity matured VL and VH regions of the mouse anti CD22 monoclonal antibody RFB4 fused to PE38, a truncated form of Pseudomonas exotoxin E. We evaluated in vitro activity of MP against six pre-B ALL cell lines (697, Nalm6, MHH-Call3, RS4;11, SupB15, REH), as well as freshly isolated patient blasts. We found the most sensitive cell line to be Reh, followed by 697, MHH-Call3, and the least sensitive cell lines to be Nalm6 and RS4;11. Toxicity of MP on ALL cell lines and patient blasts was not well-correlated with the number of the CD22 receptors present on the cell surface. However, we found that cleavage of MP by ALL cell lines into active toxin correlated with uptake and inhibition of protein synthesis. Using ALL cell lines, we also demonstrated that binding and internalization of MP/CD22 complexes was correlated with pre-B ALL cell line responses to MP. In addition, the Fv MP/CD22 complexes internalized more slowly than the parent RFB4 antibody/CD22 complexes. In addition to evaluating cell lines, we applied similar assays to 7 patient samples of bone marrow blasts, where we evaluated bound and internalized MP with cellular toxicity up to 72 hours. Our results suggest that some of the heterogeneity observed in in vitro responses to treatment with MP may be related to differences in internalization. Additional studies evaluating intracellular cleavage and trafficking/processing of MP in cell lines and patient blasts is ongoing. These studies, combined with planned in vivo studies evaluating MP antitumor efficacy using ALL cell lines and patient blasts, will provide a more comprehensive picture for differences in ALL response to treatment with MP. TLaVallee currently affiliated with Kolltan Pharmaceuticals, New Haven, CT, USA; work related to this abstract occurred at MedImmune. Disclosures: Burke: MedImmune: Employment. LaVallee:MedImmune: full employee when work was conducted Other.

Description: Introduction: Salvage therapies in children and young adults with relapsed T-ALL are successful in

Description: Several γ secretase inhibitors (GSIs) were tested for the ability to induce apoptosis in precursor B acute lymphoblastic leukemia (pre-B ALL) cells. Of five GSI’s tested, treatment with two compounds resulted in effective killing of both pre-B lymphoblasts and cells from multiple pre-B ALL lines. Since Notch receptors represent an important group of γ secretase targets, we evaluated expression and activation status of Notch receptors in CD19+ lymphoblasts from pediatric pre-B ALL patients, as well as cultured pre-B ALL cells. We found that, unlike T-ALL where activating mutations are common, pre-B ALL cells appear to drive constitutive Notch signaling through autocrine signals. Blasts from 11 patients expressed 3 Notch receptors and multiple Notch counter-ligands. Expression of Notch pathway genes was also confirmed by microarray analysis of genes expressed in 207 children with high risk B precursor ALL. GSI treatment of pre-B ALL cells led to dephosphorylation of AKT and Foxo3, Bim expression and caspase activation. GSI treatment also blocked cleavage of Notch 1 and 2 to their active forms and inhibited expression of Notch targets, Hey2 and Myc. In contrast, increased expression of Hes1 and Hey1 was correlated with GSI-induced loss of the co-repressor, SMRT. GSI treatment appears to induce precursor B cell death by disrupting the balance between constitutive Notch signaling and repression.

Description: Acquired drug resistance eventually leads to treatment failure in T-cell acute lymphoblastic leukemia (T-ALL). To define mechanisms of vincristine (VCR)-resistance, and to address whether inducible resistance in T-ALL might be mechanistically-dependent on ABC drug transporter expression, we developed VCR-resistant Sup T1 and Jurkat by incrementally growing cells in increasing drug concentrations. Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) are arrested at “mature” and “cortical” stages of T-cell development and, respectively, showed 1190-fold (0.0006 μM to 0.714 μM) and 790-fold (0.0006 μM to 0.474 μM) increased resistance to VCR. Microarray analysis showed that expression of the ATP Binding Cassette C1 (ABCC1; Multidrug Resistance Protein, MDR2) was increased more than 29-fold in VCR-resistant cells as compared to the parental controls; fold increases in mRNA of ABCC1 were confirmed by RT-PCR, and cell surface expression by flow cytometric analysis. We then employed a flow cytometry-based assay that measures the ability of ABC pumps to extrude fluorescent dyes (Calcein AM). We confirmed that VCR-resistant Jurkat and Sup T1 actively extrude Calcein AM; drug extrusion was also effectively blocked by verapamil and other known ABCB1 antagonists. In contrast to our previous observations for ABCB1-mediated multidrug resistance (Estes, et al., British Journal of Haematology, in press), ABCC1-mediated drug resistance conferred a more narrow spectrum of multidrug resistance for the chemotherapeutic agents commonly used in T-ALL induction. To investigate whether ABC expression might impact patient outcome, we next examined ABCC1 and ABCB1 expression (〉1.5-fold) in 80 T-ALL patients treated on COG studies 8704 and 9404. Different from our earlier findings that ABCB1 expression in diagnostic marrows conferred a 100% risk for on-therapy relapse, we found that ABCC1 expression only predicted treatment failure in approximately 25% of affected patients. These results suggest that ABC transporters are differentially regulated in Sup T1 and Jurkat, and that substrate-specific ABC protein regulation results in unique multidrug resistance phenotypes in T-ALL. This study contributes to the repertoire of substrate-specific, drug resistant T-ALL cells lines that are available for modeling treatment failure, provides mechanistically-based insights into relapse, and establishes an experimental means for investigating compounds that reverse ABCC1 mediated drug resistance.

Description: Abstract 2505 The 11q23 MLL gene is commonly rearranged by a diverse range of chromosomal translocations in B-progenitor acute lymphoblastic leukemia (ALL), acute myeloid leukemia and, in particular, in infants less than one year of age. These genomic lesions, commonly identified as MLL (MLL-R) rearrangements via fluorescence in situ hybridization (FISH), are highly variable but consistently encode for MLL fusion proteins that function as transcriptional deregulators of HOX genes, leading to sustained high expression of MEIS1 and many HOXA gene family members. The incidence of MLL-R in T-ALL has not been studied extensively, but has been reported to occur in approximately 1–3% of cases. Using the Affymetrix U133 Plus 2.0 microarray to assess gene expression, we hypothesized that over-expression of MEIS1, HOXA9 and HOXA10 could be use to screen for MLL-R in a cohort of 214 T-ALL cases from recent Children's Oncology Group (COG) studies. Fifty cases and 164 cases, respectively, were obtained from COG studies P9000/9404 and AALL03B1/AALL08B1/AALL0434; all cases underwent nucleic acid extraction, hybridization and profiling as previously described. Following RMA normalization and ROSE analysis for expression outliers, 27 cases (12.6%) showed ≥4-fold increase over the median expression of MEIS1 and 58 cases (27.0%) had ≥4-fold increase over the median expression of HOXA9/10. Interestingly, of the 27 cases with high MEIS1, 25 (92.6%) also had high expression of HOXA9/10 (Fisher's exact test P

Description: The biology and treatment of acute lymphoblastic leukemia (ALL) continues to attract attention as an area for which targeted therapies offer great promise. However, the integration of targeted therapies remains a challenge, both in identifying targetable genomic alterations, and the practical challenge of how best to integrate targeted therapies into clinical practice. To address these challenges in children and young adults, we developed a feasibility study to assess the prevalence of targetable genomic alterations in our population. We hypothesized that within our patient population, a spectrum of novel genomic alterations could be identified and characterized early in the treatment course. In cases where actionable alterations were identified, we further hypothesized that inclusion of novel therapies into standardized regimens might prevent relapse. In accordance with the Declaration of Helsinki, we obtained written, informed consent from eligible study subjects to perform genome sequencing in newly diagnosed patients. We enrolled 8 participants; 4 males and 4 females, ranging in age from 2 to 22 years (Table). Samples underwent deep sequencing of more than 400 cancer relevant genes (Ion Ampliseq Comprehensive Cancer Panel, Thermo Fisher) and unbiased RNA sequencing. In accordance with our institutional practices, all 8 participants underwent standardized diagnostic testing to ascertain CNS status, cytogenetics and induction responses before assignment/randomization onto a Children's Oncology Group (COG)-sponsored therapeutic study. Seven subjects had B-ALL and one had T-ALL. Genomic and molecular aberrations were identified in all patients. Using NCI risk factors, karyotypic features and Day 29 response metrics, 2 were treated with standard-risk B-ALL therapies (COG AALL0932), 1 with T-ALL (COG AALL0434) and 5 with high-risk B-ALL (COG AALL1131). Two patients started with standard-risk therapy, but were escalated to high-risk treatment based on elevated day 8 peripheral blood MRD. Genomic testing revealed that two patients harbored mutations in JAK2; one with a known mutation site and one with a novel F694L mutation. While all subjects entered into morphologic remission, our 17-year old patient with a novel JAK2 F694L mutation had high persistent MRD, suggesting she was at risk for an on-therapy relapse. With the novel JAK2 mutation confirmed in a CLIA setting, we integrated ruxolitinib into her post-induction therapy. Over the course of three month's time, her MRD diminished from 10% to 0.007%. Following a related, allogeneic bone marrow transplant, she remains in CR1. The second patient with a JAK2 R683G mutation relapsed after 20 months amid concerns of non-compliance with his maintenance therapy. Genomic testing on the relapse clone identified the same JAK2 R683G mutation that was identified at diagnosis. Our results show that prospective testing and therapy modifications can be achieved in real-time, allowing patients who might have suffered relapse to achieve adequate disease control for high-risk acute lymphoblastic leukemia. Table Table. Disclosures No relevant conflicts of interest to declare.