ALBERT

Description: Our understanding of the pathogenesis of lymphoid malignancies has been transformed by next-generation sequencing. The studies in this review have used whole-genome, exome, and transcriptome sequencing to identify recurring structural genetic alterations and sequence mutations that target key cellular pathways in acute lymphoblastic leukemia (ALL) and the lymphomas. Although each tumor type is characterized by a unique genomic landscape, several cellular pathways are mutated in multiple tumor types—transcriptional regulation of differentiation, antigen receptor signaling, tyrosine kinase and Ras signaling, and epigenetic modifications—and individual genes are mutated in multiple tumors, notably TCF3, NOTCH1, MYD88, and BRAF. In addition to providing fundamental insights into tumorigenesis, these studies have also identified potential new markers for diagnosis, risk stratification, and therapeutic intervention. Several genetic alterations are intuitively “druggable” with existing agents, for example, kinase-activating lesions in high-risk B-cell ALL, NOTCH1 in both leukemia and lymphoma, and BRAF in hairy cell leukemia. Future sequencing efforts are required to comprehensively define the genetic basis of all lymphoid malignancies, examine the relative roles of germline and somatic variation, dissect the genetic basis of clonal heterogeneity, and chart a course for clinical sequencing and translation to improved therapeutic outcomes.

Description: Background: The Hyper-CVAD regimen is safe and effective in the frontline treatment of B-ALL. The addition of rituximab to the Hyper-CVAD regimen (HCVAD-R) improved the 3-year overall survival (OS) to 60% in pts with B-ALL. Ofatumumab is an anti-CD20 monoclonal antibody that binds to the small extracellular loop of the CD20 molecule and has greater in vitro potency and increased complement-mediated cell lysis compared to rituximab. We hypothesized that ofatumumab plus Hyper-CVAD may increase the rates of complete remission (CR) and measurable residual disease negativity (MRD-) and improve survival by decreasing relapse rates. Methods: Pts were eligible if they had newly diagnosed untreated or minimally treated (≤ 1 cycle) Philadelphia chromosome (Ph)-negative CD20+ B-ALL. CD20 positivity was defined as ≥ 1% positive B-ALL cells. Pts received 8 alternating cycles of Hyper-CVAD and high-dose methotrexate/cytarabine (MTX/AraC). Ofatumumab was administered on days 1 and 11 of cycles 1 and 3; and days 1 and 8 of cycles 2 and 4. Pts then received POMP maintenance on cycles 1-5, 8-17 and 20-30 and late intensifications on cycles 6-7 and 18-19 (Hyper-CVAD + ofatumumab followed by MTX + peg-asparaginase). Pts received a total of 8 intrathecal injections of MTX and AraC for CNS prophylaxis. The primary endpoint was relapse-free survival (RFS) and secondary endpoints include CR rates, MRD negativity rates and OS. On a subset of 27 patient samples, transcriptome sequencing (RNA-seq) was performed to identify translocations and RNA expression signature for Ph-like ALL. We also performed a comprehensive detection of fusions and mutations reported in Ph-like ALL on RNA from these 27 samples using a multiplex fusion and mutation detection assay (Archer® FusionPlex® ALL). Results: Between August 2011 and May 2017, 69 pts were enrolled, including 4 already in CR at baseline after receiving 1 cycle of chemotherapy. Pts characteristics are summarized in Table 1. The median age was 41 years (18-71) and 48% pts were in the adolescent and young adult (AYA) age category (18-39 year-old). 7 of the 27 pts (26%) who had RNA-seq had Ph-like ALL gene expression signature. Among the 7 pts; 5 had Ph-like ALL fusions identified by Archer and/or RNA-seq-based fusion detection, including 2 P2RY8-CRLF2, 1 IGH-CRLF2, 1 BCR-FGFR1, and 1 ATF71P-PDGFRB. One patient had high CRLF2 expression with an unknown fusion partner. The remaining case lacked a fusion by either platform. Pts with Ph-like ALL had a higher median WBC of 41 x 109/L (range, 2 - 184). 43 pts (62%) had CD20 expression on ≥20% of the leukemic cells. 10/44 tested pts (23%) had TP53 mutation and 10/37 (27%) had CRLF2 overexpression by flow cytometry (4/5 CRLF2 rearrangement confirmed by Archer). 4 pts (6%) had low-hypodiploidy / near triploidy (Ho-Tr) and 2 (3%) pts had complex karyotype (CK). All but 1 pt (98%) achieved CR (2 after 2 cycles); only 1 pt (2%) died during induction. The MRD- rate was 65% after cycle 1 and 93% overall. These rates were 14% and 71%, respectively for pts with Ph-like ALL. The median time to MRD- was 0.7 month (range, 0.4-8 months) overall and 3 months (range, 0.7-6.5 months) for pts with Ph-like ALL. A total of 13 pts (19%) underwent allogeneic stem cell transplantation for adverse-risk cytogenetics (CK or Ho-Tr), Ph-like ALL (n=1/7), or persistent MRD+. The most common non-hematologic grade 3-4 toxicity was infection which occurred in 56% and 81% of pts, during induction and consolidation, respectively. With a median follow-up of 44 months, 46 pts (64%) are alive, including 37 pts (54%) in CR1. The median RFS and OS were 52 months (95% CI, 43 - NR) and not reached (95% CI, 65 - NR), respectively. The estimated 4-yr RFS and OS rates were 60% (95% CI, 49 - 73%) and 68% (95% CI, 58 - 81%), respectively (Figure 1A-1B). For AYA pts, the 4-yr OS rate was 74% (95% CI, 60 - 91%) (Figure 2A). The 4-yr OS rates were 54% (95%, 26 - 100%) for pts with Ph-like ALL compared to 74% (95% CI, 57 - 97%) for pts without Ph-like ALL (Figure 2B). There was no difference in OS according to the CD20 expression level (20% cut-off; p = 0.31). Using historical control pts, there was a trend towards improved OS with HCVAD-O versus HCVAD-R for pts with CD20 ≥ 20% (4-yr OS rate 63% vs 49%, p = 0.16) and HCVAD-O versus HCVAD alone for pts with CD20 1-19% (4-yr OS rate 73% vs 62%, p = 0.46). Conclusion: HCVAD-O is a safe and highly effective regimen in pts with CD20+ Ph-negative B-ALL. This regimen achieves excellent outcomes in the AYA population. Disclosures Kantarjian: BMS: Research Funding; AbbVie: Honoraria, Research Funding; Takeda: Honoraria; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; Jazz Pharma: Research Funding; Immunogen: Research Funding; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Astex: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Konopleva:Agios: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; Kisoji: Consultancy, Honoraria; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Ascentage: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria. Ravandi:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel LTD: Research Funding; Menarini Ricerche: Research Funding; Xencor: Consultancy, Research Funding; Macrogenix: Consultancy, Research Funding; Selvita: Research Funding. Jain:AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Short:AstraZeneca: Consultancy; Amgen: Honoraria; Takeda Oncology: Consultancy, Research Funding. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Cortes:Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Immunogen: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Sun Pharma: Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BiolineRx: Consultancy. Sasaki:Otsuka: Honoraria; Pfizer: Consultancy. Kadia:Celgene: Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. DiNardo:celgene: Consultancy, Honoraria; medimmune: Honoraria; abbvie: Consultancy, Honoraria; jazz: Honoraria; syros: Honoraria; agios: Consultancy, Honoraria; daiichi sankyo: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees. Verstovsek:Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding. Mullighan: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; Amgen: Honoraria, Other: speaker, sponsored travel. O'Brien:AbbVie: Consultancy, Honoraria; Acerta: Research Funding; Alexion: Consultancy; Amgen: Consultancy; Astellas: Consultancy; Aptose Biosciences, Inc: Consultancy; Celgene: Consultancy; Kite: Research Funding; GlaxoSmithKline: Consultancy; Eisai: Consultancy; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Regeneron: Research Funding; Vaniam Group LLC: Consultancy; Verastem: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding. Jabbour:Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding. OffLabel Disclosure: Ofatumumab is not approved by the FDA for treatment of B-cell acute lymphoblastic leukemia.

Description: Key Points Defective Ras oncoproteins initiate T-ALL. Murine T-ALLs lacking PTEN have gene expression profiles similar to human early T-cell precursor ALL and are resistant to MEK inhibition.

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: CD19-directed chimeric antigen receptor T cell (CAR-T) therapy has shown impressive results in children and adults with relapsed or refractory B-ALL or diffuse large B-cell lymphoma. However, 30 - 70% of initial responders will eventually relapse with CD19 antigen loss (CD19Neg) (Maude SL, et al. N Engl J Med. 2018). To avoid CD19Neg relapse, patients may undergo a hematopoietic stem cell transplant (HSCT). HSCT is an expensive and often morbid procedure that many physicians would prefer to avoid. The development of tools to accurately predict which patients are at risk for CD19Neg relapse would guide treatment decisions regarding HSCT or alternative therapies. Since CD19Neg relapses also occur in patients treated with other CD19-directed immunotherapies, like blinatumomab (Mejstríková E, et al. Blood Cancer J. 2017), a predictive model to detect patients at risk of CD19Neg relapse would have broader therapeutic impact. To address this problem, we performed CyTOF and RNA-seq analysis from paired patient samples collected before CD19-directed CAR-T administration and after CD19Neg relapse. High dimensional phenotyping by CyTOF clustered patient samples based on their mechanism of CD19 expression loss (frameshift mutation versus expression of intracellular isoforms), even before CAR-T administration. In addition, we identified identical immunoglobulin heavy and light chain RNA sequences before CAR-T administration and after CD19Neg relapse, suggesting that the clones destined to cause relapse are present at the time of CAR-T administration. Altogether, these results support our hypothesis that resistant tumor cells are present before CAR-T administration and could be discovered and interrogated for CD19Neg relapse prediction. To identify cell subpopulations responsible for driving CD19Neg relapse, we used the B cell developmental classifier previously developed in our lab (Good Z, et al. Nat Med. 2018). We observed a significant increase in the Early-non-BI population (CD38Pos CD24Pos CD19Neg CD20Neg CD3Neg CD16Neg CD61Neg cells) after CD19Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. Since our classifier relies on CD19 to classify cells, we compared the resulting classification of cells when CD19 was included or excluded in the classifier. This change had minimal impact in cell classification from healthy bone marrow controls. However, when applied to the samples collected before CAR-T administration, we found a subpopulation of CD19Pos Pro-B cells that classified as Early-non-BI cells when CD19 was excluded from the classification. We hypothesize that these Pro-B "discordant" cells are those that lose CD19 expression to escape the immune pressure exerted by the CD19-directed CAR-T and mediate CD19Neg relapse. Further, we found Pro-B "discordant" cells in 77% of independent cohort of 22 B-ALL samples collected at the time of diagnosis, suggesting these cells exist in de novo B-ALL. We likewise identified a CD19Neg IgMPos Early-non-BI subpopulation in 4 healthy bone marrow and further studies are ongoing to characterize these cells. We continue to interrogate this candidate population as that responsible for CD19Neg relapse after CAR-T cell therapy. In addition, we performed differential expression analysis between paired samples collected before (CD19Pos) and after (CD19Neg) CAR-T therapy. Through the application of the developmental classifier, we identified that CD19 loss is associated with upregulation of key B cell transcription factors IKAROS, PAX5 and glucocorticoid receptor in the pre-pro-B to Pre-B stages. Moreover, after CD19 loss, there are also increases in levels of phosphorylated proteins pSYK, pSRC and pSTAT5, involved in IL7 receptor and pre-BCR signaling pathways, essential for B cell development. This suggests that CD19Neg cells activate unique tumorigenic pathways that may provide novel therapeutic opportunities. Exploration and validation of these therapeutic targets could significantly improve clinical outcome and care of patients with CD19Neg B-ALL. In conclusion, these results support the feasibility to predict patients at risk for CD19Neg relapse together with the mechanism behind it. Future studies will be conducted to confirm unique tumorigenic pathways in CD19Neg B cells and determine their therapeutic potential. Disclosures Mullighan: Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Amgen: Honoraria, Other: speaker, sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Loxo Oncology: Research Funding. Grupp:Humanigen: Consultancy; CBMG: Consultancy; Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy.

Description: Minimal residual disease (MRD) at the end of remission-induction therapy predicts relapse in acute lymphoblastic leukemia (ALL). We examined the clinical significance of levels below the usual threshold value for MRD positivity (0.01%) in 455 children with B-lineage ALL, using polymerase chain reaction amplification of antigen-receptor genes capable of detecting at least 1 leukemic cell per 100 000 normal mononucleated cells (0.001%). Of the 455 clinical samples studied on day 46 of therapy, 139 (30.5%) had MRD 0.001% or more with 63 of these (45.3%) showing levels of 0.001% to less than 0.01%, whereas 316 (69.5%) had levels that were either less than 0.001% or undetectable. MRD measurements of 0.001% to less than 0.01% were not significantly related to presenting characteristics but were associated with a poorer leukemia cell clearance on day 19 of remission induction therapy. Patients with this low level of MRD had a 12.7% (± 5.1%; SE) cumulative risk of relapse at 5 years, compared with 5.0% (± 1.5%) for those with lower or undetectable MRD (P 〈 .047). Thus, low levels of MRD (0.001%-〈 0.01%) at the end of remission induction therapy have prognostic significance in childhood ALL, suggesting that patients with this finding should be monitored closely for adverse events.

Description: Abstract 182 Chromosomal alterations are a hallmark of acute lymphoblastic leukemia (ALL), but many cases lack a recurring cytogenetic abnormality. To identify novel alterations contributing to leukemogenesis, we previously performed genome-wide profiling of genetic alterations in pediatric ALL using single nucleotide polymorphism (SNP) microarrays. This identified a novel focal deletion involving the pseudoautosomal region (PAR1) of Xp/Yp in 15 B-progenitor ALL cases lacking sentinel chromosomal abnormalities, including six of eight cases of ALL associated with Down syndrome (DS-ALL). The deletion involved hematopoietic cytokine receptor genes, including IL3RA and CSF2RA, but due to poor array coverage, it was not possible to define the limits of deletion using SNP array data alone. To characterize this abnormality, we examined an expanded cohort of 329 B-ALL cases, including 22 B-progenitor DS-ALL cases. Strikingly, 12 (55%) DS-ALL cases harbored the PAR1 deletion. Mapping using high density CGH arrays showed the deletion to be identical in each case, and involved a 320kb region extending from intron 1 of the purinergic receptor gene P2RY8 to the promoter of CRLF2 (encoding cytokine receptor like factor 2, or thymic stromal lymphopoietin receptor). The deletion resulted in a novel fusion of the first, non-coding exon of P2RY8 to the entire coding region of CRLF2 in each case. The P2RY8-CRLF2 fusion resulted in elevated expression of CRLF2 detectable by quantitative RT-PCR, and flow cytometric analysis of leukemic cells. One DS-ALL case with elevated CRLF2 expression lacked the PAR1 deletion, but had an IGH@-CRLF2 translocation detected by fluorescence in situ hybridization (FISH). CRLF2 alteration was associated with gain of chromosome X (which was shown by FISH to result in duplication of the PAR1 deletion), deletion of 9p, and the presence of Janus kinase (JAK1 and JAK2) mutations. Ten (53%) of patients with CRLF2 alteration had JAK mutations, compared with two patients lacking CRLF2 abnormalities (P

Description: Abstract 289 Children with Down syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL), and consistently demonstrate poorer outcomes due to higher rates of both relapse and treatment-related mortality compared to other children with ALL. The biology of ALL in DS is unique, with lower frequency of the classic cytogenetic lesions generally observed in childhood ALL, and increased frequency of JAK2 mutations and CRLF2 rearrangements, which are not clearly associated with adverse prognosis in DS-ALL. In order to improve risk stratification and identify potential novel therapeutic targets in this vulnerable population, we analyzed 90 DS-ALL cases for prognostically significant copy number abnormalities (CNAs) in a collaborative cohort from the Children's Oncology Group (n=30), St. Jude Children's Research Hospital (n=22), AIEOP (n=16), Texas Children's Cancer Center (n=10), UKALL 2003 (n=6) and an archival UK sample (n=1), and Utah's Primary Children's Medical Center (n=5). Copy number profiling was performed using 500K, 6.0, CytoScan HD, and OncoScan FFPE Express arrays (Affymetrix), and Human CNV370-Duo arrays (Illumina). Gene expression profiling was performed using U133 Plus2.0 arrays (Affymetrix). Copy number was analyzed with Nexus Copy Number (BioDiscovery, Inc.) and gene expression was analyzed with Partek Genomics Suite (Partek, Inc.) and Gene Set Enrichment Analysis (Broad Institute). Deletions of a focal region on 22q11.22 (present in 28.9% of cases, similar to the incidence previously reported in a non-DS cohort [Mangum et al, ASH 2011:741]), and deletions of IKZF1 (present in 20.0% of cases), were significantly associated with poor event-free survival (EFS) (5-year EFS was 88.6 ± 6.3% in wild-type cases [n=31], 68.1 ± 13.3% in cases with deletion of 22q11.22 only [n=15], and 60.0 ± 21.9% in those with deletion of IKZF1 only [n=5]; combined deletion [n=6] was associated with an even poorer EFS (33.3 ± 19.3%, p

Description: Although the cure rate for acute lymphoblastic leukemia (ALL), a frequent pediatric leukemia, has improved dramatically, the overall prognosis remains dismal due to frequent disease relapse and the absence of non-cytotoxic targeted therapy options. Up to 25% of children fail frontline therapy and in these cases prognosis is dismal and the cure rate is approximate 20%. Main current therapies are based on intensive induction chemotherapy that is most frequently coupled to intrathecal chemotherapy alone or with cranial irradiation for central nervous system prophylaxis, which has severe short and long-term side effects. Thus, the ultimate and most critical aim for developing new treatments in different types of leukemia is to block the effects of specific cancer-inducing oncogenes. Others and we have previously shown that T cell ALL (T-ALL) is characterized by activating mutations in the NOTCH signaling pathway. It is currently unclear how key transcription factors in T-ALL such as NOTCH1 recruit the epigenetic machinery and bring together different chromosomal domain, in order to carry out the oncogenic transformation program. We generated evidence that NOTCH1 oncogenic action leads to important epigenetic changes through antagonizing the polycomb repressive complex 2 (PRC2) and leads to loss of the repressive mark histone 3 lysine 27 di/tri-methylation (H3K27me2/3). Moreover, we identified inactivating mutations of the polycomb repressive complex 2 (PRC2), the “writer” of Histone 3 lysine 27 methylation, in primary samples from human patients revealing a tumor suppressor role for the complex in T-ALL. Further extending our work on the H3K27me3 mark, we showed the oncogenic role for the Jumonji d3 (JMJD3) demethylase. Functionally, genomic ablation of the JMJD3 modulator as well as targeting with a specific chemical inhibitor, GSKJ4, generated by GlaxoSmithKline, leads to apoptosis and cell cycle arrest of T-ALL lines and primary cells. Genetic ablation of JMJD3 leads to slower initiation of the disease with significantly improved survival rates of the mice. Surprisingly, UTX acts as a tumor suppressor in the context of the same disease, as part of different transcriptional complexes, and we found that it is genetically inactivated in T-ALL patients. In light of recent developments on novel epigenetic inhibitors against JMJD3, these findings pave the way to specific pharmacological targeting of T cell leukemia. Based on this activity of Notch1 oncogene on epigenetic marks we further hypothesized that the switch from physiological to oncogenic activity might be mediated by changes in enhancer-promoter interaction networks forming chromosomal domains. A substantial percentage of these interactions are likely to be specific for the malignant state, and their disruption with epigenetic pharmacological inhibitors would not potentially affect healthy tissues. Studies in our laboratory show for the first time in leukemia that NOTCH1 chromatin binding sites are associated with enhancer-promoter interactions at oncogenic loci, using up-to-date chromosome conformation capture technology. We hereby show the importance of these interactions for oncogenic gene expression and pharmacological targeting of leukemic cells. These findings lend further rationale to the use of epigenetic drugs for targeted treatment of T cell leukemia. Disclosures Kruidenier: GlaxoSmithKline: Employment. Prinjha:GlaxoSmithKline: Employment.

Description: Abstract 47 Lmo2 overexpression and Arf loss induce myeloid differentiation in primitive thymocytes LMO2 is a hematopoietic transcription factor that is deregulated as a consequence of chromosomal translocations in T-cell leukemia. Recently we reported that Lmo2 overexpression collaborates with loss of the p19Arf tumor suppressor to induce central T cell leukemias in mice, in part by conferring an increase in self-renewal and engraftment potential in thymic repopulating cells (Treanor LM et al, Blood 2011). Primary recipients were transplanted with Lmo2-transduced, Arf−/− DN2 (CD4−CD8−CD44+CD25+) thymocytes that were cultured on OP9-DL1 cells for 20 days before transplant. Primary recipients engrafted and secondary transplants were later performed. Several secondary recipients developed acute myelogenous leukemia originating from the thymocyte graft. These mice had elevated white blood counts between 200–500×103/μl and their spleen, thymus, bone marrow and peripheral blood contained 90% mCherry+, Gr1+, Mac1+ cells. Both the spleen and liver were infiltrated with myeloperoxidase positive blast cells and pathological review confirmed acute myeloid leukemia. Tertiary irradiated hosts transplanted with these cells developed a Gr1+ tumor with the same phenotype as the secondary animal. All of the tumor cells observed had a high expression level of the mCherry vector indicating that Lmo2 was expressed in the blast cells. Vector integration site clonality analyses confirmed that the vector was present in the blast cells and was from the same clone in the primary, secondary and tertiary recipient. This data led to the hypothesis that enforced Lmo2 expression and Arf loss may reprogram DN2 thymocytes to obtain myeloid differentiation potential. Transduced DN2 thymocytes were then assayed for myeloid colony formation in semisolid cultures containing cytokines that are specific for myeloid differentiation. Initially CD4−CD8− thymocytes were selected from Arf+/+ and Arf−/− thymi, transduced with either control vector or Lmo2 and cultured on OP9-DL1 stromal cells for 20 days. At day 20 the thymocytes were sorted for the vector positive DN2 population and 5×104 of these DN2 thymocytes were plated into these semisolid cultures. Only thymocytes that contained the Lmo2 vector were able to form myeloid colonies and this colony forming ability was greatly enhanced by the absence of Arf (n=3) as shown in figure 1. Moving to an in vivo assay, sublethally irradiated Rag2−/−γc−/−were transplanted with 2×105 transduced DN2 thymocytes. Three weeks after transplant the spleen, bone marrow and peripheral blood contained greater than 50% mCherry+ cells and of these cells between 3%-10% were Mac1+Gr1+ double positive. Vector+ (mCherry+) cells were sorted from the bone marrow and plated in semisolid culture with myeloid cytokines. After seven days the cultures were positive for myeloid colonies that were mCherry+, Gr1+, Mac1+. These in vivo and in vitro assays demonstrate that Lmo2 induces myeloid potential in DN2 thymocytes. These data indicate that Lmo2 expression combined with loss of the Arf locus may recapitulate a hematopoietic stem cell (HSC) “state” in the DN2 thymocytes as HSCs express relatively high levels of Lmo2 and do not express p19Arf due to Bmi1-mediated epigenetic suppression. The novel reprogramming events that we now report could have relevance to early thymic precursor leukemia, in which various degrees of myeloid conversion are noted. We recently documented high amounts of Lmo2 mRNA expression in pediatric early thymic precursor leukemia by expression array analysis in 11/12 cases. Disclosures: No relevant conflicts of interest to declare.