A New Gene Expression Profile Signature CRLF2 Overexpression Based Identifies Novel Adult "Triple Negative" Acute Lymphoblastic Leukemia Subgroups

Abstract Background: The heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL patients (pts) that doesn't have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as "triple negative" (Ph-/-/-) ALL. CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Clustering and biological characterization of Ph-/-/- ALL (that represents 61% of adult B-ALL; Roberts KG, J Clin Oncol. 2016), considering CRLF2 overexpression event, in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) were performed on 55 Ph-/-/- ALL, 29 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized with the Expression Console Software. Successively we cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in the top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (TruSight Pancancer-Illumina; MLPA and/or dMLPA-MRC-Holland; SNP Array-Affymetrix; 454 Junior-Roche and PCR). Results: Clustering our Ph-/-/- gene expression data using the impact of the 10 single genes in our cohort, we could identify a defined 2-clusters-subdivision (Gr1 and Gr2; Fig 1A). The Gr2 is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) overexpression and it represents 14.1% of all B-ALL. The Gr2 GEP is similar to Ph+ one. Fusion copy number alteration and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mutations or deletion), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. Molecular characterization shed light on a very heterogeneous scenario especially in the group 1, suggesting the need of a more discerning clustering for this group. In spite of the small number of cases is required, preliminary Gr1 subclustering discerns MLLr and ZNF384 gene expression subgroups. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upregulated in the group1 and CDK6 in the Gr2. CRLF2 and CD200 immunoblotting and CD200 immunohistochemistry preliminary analyses suggest that protein expression of CRFL2 and CD200 are higher in Gr2 in comparison to Gr1. Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. 3C-up represents 14.1% of all B-ALL and it is characterized by a) high co-expression of three main genes: CRLF2, CTGF and CD200; b) IKZF1 deletion; c) JAK-STAT pathway mutations/fusions/deletions. Gr1 represents 46.9% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identify new potential therapeutic targets with available drug (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; tyrosine kinase inhibitors already effective on Ph+ and Ph-like) to test. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Figure. Figure. Disclosures Cavo: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Novartis: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad/Incyte: Consultancy; Amgen: Consultancy.

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Inotuzumab Ozogamicin (Ino) May Overcome the Impact of Philadelphia Chromosome (Ph)-like Phenotype in Adult Patients (pts) with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood-2019-126940 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1641-1641 ◽

Cited By ~ 1

Author(s):

Elias Jabbour ◽

Kathryn G. Roberts ◽

Koji Sasaki ◽

Yaqi Zhao ◽

Chunxu Qu ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Advisory Committees ◽

The Impact

Background: Ino showed significant activity in phase II trials in pts with R/R ALL, that was subsequently confirmed in Phase III trial where Ino demonstrated higher response rates and superior overall survival vs standard of care chemotherapy (SOC) in adults with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (R/R ALL).Ph-like or BCR-ABL1-like ALL possesses a gene expression profile similar to that of BCR-ABL1 ALL but lacks the BCR-ABL1 fusion protein. It is characterized by increased expression of hematopoietic stem-cell genes, deletion of B-cell lineage genes and kinase-activating alterations. Ph-like ALL is associated with refractoriness to standard induction/consolidation chemotherapy and poor prognosis. Aim: To evaluate the outcomes of pts with R/R Ph-like ALL treated in phase II trial with Ino monotherapy. Methods: We performed an integrated analysis of whole genome sequencing (to identify sequence mutations, structural variations and DNA copy number alterations), and transcriptome sequencing (RNAseq; to quantify gene expression, determine Ph-like gene expression profile and identify fusions) on 53 patients' samples treated with Ino between June 2010 and September 2012. Results: Fifty-three evaluable pts with R/R ALL with stored baseline samples were analyzed. Pts characteristics are summarized in Table 1. Median age was 50 years. Ino was given as Salvage 1, Salvage 2, and Salvage 3 and beyond in 20 (38%), 18 (34%), and 15 (28%) pts, respectively. Figure 1 reflects the different genomic subgroups identified among 53 evaluable pts. Ph-like gene signature was found in 12 pts (22.6%). Among these 12 pts, 6 had IGH-CRLF2, 2 IGH-EPOR, 1 SNX2-ABL1, and 3 had no fusions identified. The overall response rates (ORR) were 54% [complete remission (CR) 20%, CR with partial hematologic recovery (CRh) 32%, and marrow CR (CRi) 2%]. Among pts with morphologic remission, 46% and 82% achieved minimal residual disease (MRD) negativity at CR and at any time, respectively. The ORR for pts with Ph-like ALL, Ph-positive ALL, ALL with KMT2A, and others were 58% (CR=25%; CRh=33%), 42% (CR=8%; CRh=33%), 57% (CR=14%; CRh=29%; CRi=14%), and 56% (CR=26%; CRh=30%), respectively. The respective overall MRD negativity rates were 71%, 100%, 75%, and 83% (Table 1). The median follow-up was 60 months. The median event-free (EFS) and overall survival (OS) were 3.3 and 5.4 months, respectively. There was no difference in EFS and OS between the subgroups analyzed (P=0.464; P=0.824). The median EFS and OS were 4.5 and 4.5 months for pts with Ph-like, 3.1 and 7.2 months for those with Ph-positive ALL, 2.8 and 4.4 months for those with KMT2A, and 2.2 and 4.6 months for others (Table 1). 21 (40%) pts had subsequent allogeneic stem cell transplant; 6 (50%), 3 (25%), 4 (57%), and 8 (36%) in each subgroup, respectively. The rate of VOD was 3 (6%) with no difference among different subgroups. Conclusion: The current analysis suggest that Ino therapy may overcome the impact of Ph-like phenotype in pts with ALL. Confirmation of these findings in a larger cohort and in frontline ALL patients is needed. Disclosures 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. Sasaki:Pfizer: Consultancy; Otsuka: Honoraria. Jain:Precision Biosciences: 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; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; 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; Incyte: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: 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. Ravandi:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Consultancy, Research Funding; Macrogenix: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding; Cyclacel LTD: Research Funding. Short:AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Research Funding; Amgen: Honoraria. 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. Konopleva:Cellectis: Research Funding; Agios: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Ascentage: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Kisoji: Consultancy, Honoraria; Ablynx: Research Funding; Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Research Funding. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Kantarjian:Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Research Funding; Immunogen: Research Funding; AbbVie: Honoraria, Research Funding; Astex: Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria.

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DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-123475 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2742-2742

Author(s):

Christian Hurtz ◽

Gerald Wertheim ◽

Rahul S. Bhansali ◽

Anne Lehman ◽

Grace Jeschke ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Negative Regulator ◽

Advisory Committees ◽

Normal Tissues ◽

Pharmacologic Inhibition

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

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Multiomic Mapping of Copy Number and Structural Variation on Chromosome 1 (Chr1) Highlights Multiple Recurrent Disease Drivers

Blood ◽

10.1182/blood-2021-148439 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 721-721

Author(s):

Patrick Blaney ◽

Eileen M Boyle ◽

Yubao Wang ◽

Hussein Ghamlouch ◽

Jinyoung Choi ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Chromatin Structure ◽

Copy Number ◽

Research Funding ◽

Chromosome 1 ◽

Structural Variants ◽

Advisory Committees

Abstract Introduction Copy number abnormalities (CNA) and structural variants (SV) are crucial to driving cancer progression and in multiple myeloma (MM). Chr1 CNA are seen in up to 40% of cases and associate with poor prognosis. Variants include deletions, gains, translocations and complex SV events such as chromothripsis (CT), chromoplexy (CP) and templated insertions (TI) which result in aberrant transcriptional patterns. Abnormal expression of genes on chr1 lead to the adverse clinical outcome and studies focussed on 1p12, 1p32.3 and 1q12-21 identified potential causal genes including TENT5C, CDKN2C, CKS1B, PDZK1, BCL9, ANP32E, ILF2, ADAR, MDM2 and MCL1 but none fully explain the clinical behavior. To address this deficiency and to relate chromatin structure to gene deregulation we present a multiomic bioinformatic analysis of SV, CNA, mutation and expression changes in relation to the chromatin structure of chr1. Methods We analysed data derived from 1,154 CoMMpass trial patients. We analyzed 972 NDMM patients with whole exome for mutations, and 752 whole genomes for copy number, translocations, complex rearrangements such as CP, CT and TI as previously described. Using GISTIC 2.0, we identified hotspots of CNA. This information was then analyzed in conjunction to the RNA-seq data derived from 643 patients to determine the aberrant transcriptional landscape of chr1. Using HiC data derived from U266 MM cell line, we associated these changes with TAD structures, A/B compartments, and histone marks along chr1, to gene expression changes, and recurrent SV. Using the cell line dependency map for CRISPR knockdown of the gene set on chr1 derived from 20 MM cell lines we related cell viability to chr1 copy number status. Results We identified 7 hotspots of deletion, 9 of gain, 3 of CT and 2 of templated-insertion across chr1. We mapped these regions to epigenetic plots and show that gained regions are hypomethylated compared to the rest of chr1 (Wilcoxon, p=0.0002). Overall 69% of gain(1q) and 45% of the non-gained hotspots were in A compartments (χ 2=11, p=0.0009) and had an overall higher compartment score (p=0.01).The recurrent regions of loss on 1p confirm the clinical relevance of this region. The critical importance of TENT5C, CDKN2C and RPL5 is identified by the impact of deletion, mutation and the rearrangement of superenhancers. Further this convergence of multiple oncogeneic mechanisms to a single locus points to a number of novel candidate drivers including FUB1 and NTRK1.We provide important new information on 1q21.1-1q25.2 encompassing 145-180Mb a transcriptionally dense region containing 6 GISTIC 2.0 hotspots of gain (G2-G7). The hotspots occur within TAD structures that correlate upregulation of known drivers listed above and also identified novel potential upregulated drivers including POU2F1, a transcription factor, CREG1, an adenovirus E1A protein that both activates and represses gene expression promoting proliferation and inhibiting differentiation (G6) and BTG2 a G1/S transition regulator (G8). These data for copy number gain provides strong evidence for the prognostic relevance of of multiple drivers within deregulated TADs rather than single candidate genes. It also highlights the importance of the chromatin structure of Chr1 in the generation of these events.Using dependency map CRISPR data we identified 320 essential genes for at least one cell line (>1). A common set of 31 genes were identified including 3 proteasome subunits (PSMA5, PSMB2, PSMB4), three regulators of ubiquitin-protein transferase activity (RPL5, RPL11, CDC20), splicing (SF3B4, SF3A3, SFPQ, RNPC3, SRNPE, PRPF38A, PRPF38B) and DTL. A common dependency for 1q+ or 1p- was not identified but a number of dependencies were identified in more than one cell line including UQCRH, SLCA1, CLSPN in 1p- cell lines and IPO9, PPIAL4G, and MRPS2 in 1q+. Conclusion We present an elegant anatomic map of chr1 at the genetic and epigenetic levels providing an unprecedented level of resolution for the relationships of structural variants to epigenetic, expression and mutation status. The analysis highlights the importance of active chromatin in gene deregulation by SV and CNA where the importance of multiple gene deregulation within TAD structures is critical to MM pathogenesis. The implications are that we could improve prognostic assignment and identify new targets for therapy by further characterizing these relationships. Figure 1 Figure 1. Disclosures Braunstein: Jansen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Epizyme: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Davies: Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Constellation: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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The Genomic Landscape of Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-124881 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 649-649

Author(s):

Kathryn G. Roberts ◽

Samuel W. Brady ◽

Zhaohui Gu ◽

Lei Shi ◽

Stanley Pounds ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Driver Genes ◽

Advisory Committees ◽

Single Nucleotide ◽

To Come ◽

Commercial Research ◽

Age Range

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.

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Mutational Type and Configuration of an Individual Gene May Differentially Impact the Clinical and Phenotypic Features

Blood ◽

10.1182/blood-2019-128893 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2992-2992

Author(s):

Yasunobu Nagata ◽

Hideki Makishima ◽

Cassandra M Kerr ◽

Bhumika J. Patel ◽

Hassan Awada ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Somatic Mutations ◽

The Other ◽

Missense Mutations ◽

Advisory Committees ◽

Different Types ◽

Equity Ownership ◽

The Impact

Myelodysplastic syndromes (MDS) arise in older adults through the stepwise acquisition of multiple somatic mutations. The genetic heterogeneity that results includes mutations of diverse genes and their combinations, clonal hierarchy, genetic configuration (e.g., bi-allelic or compound heterozygous, hemizygous lesions), specific positions within a gene including canonical hotspots vs. other positions, and types of mutation (truncations vs. missense), all of which could differentially affect pathogenesis. Given the binary status (e.g. mutated vs. wild-type) used in many clinical analyses, the true impact of specific types of mutations may be obscured and their specific roles underestimated. Deep targeted NGS was carried out for a panel of the 36 most frequently mutated genes in 1,809 MDS patients (low-risk MDS (n=839) vs. high-risk MDS (n=607), MDS/MPN (n=212), and sAML n=151). Copy number alterations (CNA) were also evaluated by combining karyotyping, microarray, and digital copy number analysis. With a mean coverage of 862x, after removing SNPs and errors, 3,971 somatic mutations were identified, the most common (>10% of cases) being TET2, SF3B1, ASXL1, del(5q), SRSF2, complex karyotype, and del(7q). For the purpose of this proof of concept analysis we focused on illustrative genes (TP53, RUNX1, TET2, and EZH2) affected by 2 recurrent hits. Bi-allelic TET2 or TP53 mutations were found in 15% (271/1,809) and 4% (72/1,809) of patients, respectively. TET2 and RUNX1 were most likely biallelic, whereas TP53 and EZH2 were most often affected by mutations and somatic deletion. Comparing the distribution of canonical vs. other types of mutations in genes, DNMT3A mutations affected the canonical site (R882) in 17% (35/203) of patients, were truncating in 39% (79/203) and missense in 44% (89/203) have also been found; deletions affecting the DNMT3A locus are rare. Within U2AF1, U2AF1Q157 are more frequent than U2AF1S34 (54% vs. 35%). Next, we checked correlation between these different types of mutations of one gene. 78 significant combinations were found. For instance, U2AF1Q157 mutations more commonly accompanied ASXL1 mutations and del(7q) and less frequently DNMT3A and BCOR mutations, trisomy8 and del(20) when compared to U2AF1S34 mutations [ASXL1 mutations 53% (42/80) in U2AF1Q157 vs. 16% (8/49) in U2AF1S34, P < .0001]. TET2 Bi-allelic mutations were more commonly associated with ZRSR2 and SRSF2 mutations, and less frequently del(5q) when compared to TET2 mono-allelic mutations [SRSF2 mutations 29% (80/276) in TET2-bi vs. 15% (34/227) in TET2-mono, P = .003]. In addition, patients with SRSF2 missense mutations were more likely to have RUNX1 bi-allelic mutations than those with SRSF2 in-frame mutations. We evaluated the impact of different types of mutations and combinations of them on disease phenotypes and survival. We then evaluated the impact of different types of mutations and their combinations on clinical phenotypes including dichotomous morphological (MDS vs. MDS/MPN) features, progressive (low- vs. high risk) subtypes. EZH2 bi-allelic alterations were more commonly associated with myleoproliferative features` compared to EZH2 mono-allelic alteration (q=.016). TET2 bi-allelic alterations and truncating mutations were found more frequently in higher-risk subtypes than TET2 mono-allelic and missense mutations (q<.001). In survival analyses, patients with DNMT3AR882 mutations had a poorer prognosis than those with truncating and the other missense mutations [P = .033, HR 1.86 (1.05-3.3)]. Next, using the PyClone bioanalytic pipeline, we recapitulated for each patient the clonal hierarchy and defined "dominant" vs. "secondary" mutations. DNMT3AR882 mutations were likely to be dominant/founder lesions compared to truncating or the other missense mutations: 77% (27/35) for R882 vs. 51% (40/79) for truncating vs. 45% (47/98) for the other missense, p=.0046. Specific dominant and secondary mutational pairs also differentially affected survival compared to the reverse configuration (q<.1) including EZH2 and RUNX1 or BCOR and U2AF1 or RUNX1 and BCOR. In conclusion, we report a comprehensive analysis of various types and configurations of lesions of individual commonly affected genes. Our results indicate that establishment of clinical or phenotypic correlations requires consideration of the type, rank and configuration of somatic mutations. Disclosures Mukherjee: McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Nazha:Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

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Integration of Hi-C and Nanopore Sequencing for Structural Variant Analysis in AML with a Complex Karyotype: (Chromothripsis)²

Blood ◽

10.1182/blood-2020-133787 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Marius-Konstantin Klever ◽

Eric Sträng ◽

Julius Jungnitsch ◽

Uirá Souto Melo ◽

Sara Hetzel ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Research Funding ◽

Molecular Mechanisms ◽

Complex Karyotype ◽

Rna Seq ◽

Advisory Committees ◽

Functional Interpretation ◽

Hematopoietic Stem ◽

The Impact

Background. Acute myeloid leukemia (AML) with a complex karyotype (CK-AML) is an AML subtype with a still dismal outcome despite recent therapeutic advances. The prognosis is even worse when the underlying structural variants (SVs) lead to an extremely complex pattern of rearrangements, called chromothripsis, with a median overall survival of only 120 days. Except for the presence of inactivating TP53 aberrations in about 70% of all AML-CK cases, the pathogenesis is poorly understood. To gain novel insights into the molecular mechanisms underlying CK-AML reliable high precision SV delineation is needed, which so far has been a major limitation in cancer research. Aim. We developed a SV detection pipeline by integrating Oxford Nanopore Technology (ONT) based whole genome sequencing (WGS) and Hi-C sequencing. This pipeline generated precise characterization of SVs for which the impact on gene expression and the emergence of novel fusion genes was studied by RNA-seq and ONT transcriptome sequencing. Patients and Methods. We applied our WGS and Hi-C SV detection pipeline to a cohort of 11 AML-CK cases. Nanopore DNA Sequencing was performed until a genomic coverage >10x per patient was reached. The samples of 9 patients were also subjected to Nanopore cDNA sequencing for fusion gene analysis and Illumina based RNA-seq for transcript quantification. As controls for Hi-C and Illumina RNA sequencing, CD34+ hematopoietic stem cell enriched samples from five healthy donors were used. Results. Our SV detection pipeline enabled us to fully reconstruct the derivate chromosome structure even of very complex, chromothriptic rearrangements in CK-AML. This enabled us to identify features of chromothripsis, that could previously not be detected using conventual technologies. We found local clustering of breakpoints in three of the patients with up to 31 Inversions and Translocations located in a genomic region of just 2.7 kb. These breakpoints were present in the Hi-C as well as in our Nanopore SV dataset. Our SV pipeline also showed that in these highly clustered regions, the very small rejoined fragments (in many cases less than 1 kb in size) often showed an elevated copy number (CN) state, i.e. small amplifications. We termed this newly discovered phenomenon chromothripsis-in-chromothripsis or (chromothripsis)². The precise knowledge about these breakpoints, which were validated by two different technologies, enabled us to study the pathogenesis of CK-AML at a so far unprecedented resolution. Fusion transcripts could be very precisely mapped and the impact of the breakpoints and CN changes on gene expression levels could be validated, thereby indicating functional relevance of the respective aberrations. Conclusions. The combination of Hi-C and long-read sequencing for SV detection proved to be a powerful tool for precise SV detection. Our SV pipeline allowed us to discover a new level of complexity in chromothripsis. Application of this pipeline to leukemias as well as other types of cancer can improve the precision of SV detection, thereby raising new opportunities for functional interpretation of complex genomic aberrations of pathogenic relevance. Disclosures Döhner: Sunesis Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding; Roche: Consultancy; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Abbvie: Consultancy; Agios: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Celgene: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Bullinger:Amgen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees.

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Characterization of the "Immune Evasion" Phenotype of Richter Syndrome and the Implications for Immune-Checkpoint Inhibitor Therapy

Blood ◽

10.1182/blood-2019-127800 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4290-4290

Author(s):

Clare Gould ◽

Jennifer Lickiss ◽

Yamuna Kankanige ◽

Satwica Yerneni ◽

John Markham ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Board Of Directors ◽

Immune Checkpoint ◽

Copy Number ◽

Research Funding ◽

De Novo ◽

Advisory Committees ◽

Immune Checkpoint Molecules ◽

Checkpoint Inhibitor Therapy

Richter syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) to a high-grade B-cell lymphoma and is associated with an aggressive clinical course and poor prognosis. Conventional treatment options for RS are generally associated with low response rates and limited durability making this entity an area of significant unmet therapeutic need. Immune checkpoint inhibitor therapy has shown promise in the treatment of some aggressive lymphoma subtypes. In RS, modest benefits have been reported in small phase two trials of anti-PD-1 monotherapy and in combination with ibrutinib, however larger scale studies are lacking (Ding et al Blood, 2017; Jain et al Blood, 2016). We sought to characterise the immune-evasion phenotype of RS focussing on potential genetic biomarkers which may inform the selection of patients who are most likely to benefit from immune-directed therapies. We first assessed the gene expression of immune-checkpoint molecules given their potential clinical relevance and ability to be targeted by available therapeutic agents. Given immunohistochemical (IHC) assessment of immune-checkpoint molecules is recognized to be associated with high inter-observer variability and there is a high correlation between gene expression of immune-checkpoint molecules and IHC, we performed gene expression quantification using the Nanostring nCounter Human Immunology V2 panel (Nanostring Technologies, USA). Nanostring analysis was performed on samples from 17 patients with histologically confirmed RS (DLBCL subtype) and compared to 73 cases of de novo (non-transformed) DLBCL. Significant differences in the gene expression of checkpoint molecules was observed between RS and DLBCL biopsies, including higher expression of LAG3, PD1 and TIGIT in RS (p=0.0001, logFC 1.9; p=0.0017, logFC 1.1 and p=0.0437, logFC 0.7 vs DLBCL, respectively). PD-L2 and TIM3 gene expression were both significantly lower in RS compared to DLBCL (p = 0.0059, logFC 0.8; p = 0.012, logFC 0.8). PDL1 and CTLA4 gene expression did not significantly differ between RS and DLBCL. We next assessed the gene expression of T- and NK- cell markers (including CD3, CD4, CD8, FOXP3 and CD56) and the ratios of these markers to malignant B-cells (CD19). We observed no significant difference between RS and DLBCL, consistent with a similar relative quantity of immune cell infiltration between the two entities. Significantly higher gene expression of CD39, a marker of CD8+ T-cell exhaustion, was observed in RS than DLBCL (p = 0.031; logFC 0.5). Additional immune-related genes were next assessed, including those involved in antigen presentation (e.g. B2M, HLA molecules, TAP), immunosuppressive cytokine generation (e.g. ARG1, IDO1) and apoptosis resistance (e.g. FAS) which showed no significant differences in expression between RS and DLBCL. To assess whether these findings were consistent across other transformed lymphoma subtypes, we compared RS to a cohort of transformed follicular lymphoma (tFL, n=16) and transformed marginal zone lymphoma (tMZL, n=25). LAG3 expression was significantly higher in RS compared to both tFL and tMZL (p=0.0002, logFC 2.7; p=0.019, logFC 1.7). PD1 expression was also significantly higher in RS than tFL but not tMZL (p=0.0045, logFC 1.7; p=0.39, logFC 0.4). Given the established association of copy number amplifications involving immune checkpoint molecules (e.g. PD-L1/PD-L2 on 9p24.1) representing a potential predictive biomarker of response in other lymphomas, we performed hybridization-based NGS with whole genome copy number assessment to evaluate immune checkpoint gene loci in the three cohorts. No significant focal amplifications were detected in RS samples with overexpressed immune-checkpoint molecules. In contrast, three patients in the DLBCL/transformed cohort had focal copy number amplifications involving PD-L1. No copy number amplification of LAG3 was observed in either RS or DLBCL. In summary, we have observed significantly increased gene expression of LAG3, PD1 and TIGIT in RS compared to de novo DLBCL. Combined with increased gene expression of the exhausted cytotoxic T-cell marker CD39, these data provide a strong biological rationale for pursuing LAG3 inhibition either alone or in combination with other immune checkpoint blockade to enhance anti-tumour T cell responses in this difficult-to-treat entity. CG/JL/YK co-first authors Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Neeson:Roche Genetech: Research Funding; Allergan: Research Funding; Juno/Celgene: Research Funding; Compugen: Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Seymour:Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding. Dickinson:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Blombery:Invivoscribe: Honoraria; Novartis: Consultancy; Janssen: Honoraria.

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Favorable Outcomes for Older Adolescents and Young Adults (AYA) with Acute Lymphoblastic Leukemia (ALL): Early Results of U.S. Intergroup Trial C10403

Blood ◽

10.1182/blood.v124.21.796.796 ◽

2014 ◽

Vol 124 (21) ◽

pp. 796-796 ◽

Cited By ~ 72

Author(s):

Wendy Stock ◽

Selina M. Luger ◽

Anjali S. Advani ◽

Susan Geyer ◽

Richard C. Harvey ◽

...

Keyword(s):

Gene Expression ◽

Young Adults ◽

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Multivariable Analysis ◽

Gene Expression Signature ◽

Remission Induction ◽

Advisory Committees ◽

Intergroup Trial

Abstract Background: Retrospective analyses have demonstrated significantly improved survival for AYA ALL patients (pts) aged 16-21 years (yrs) when treated on pediatric versus adult U.S. NCI Cooperative group regimens where 2-yr event-free survivals (EFS) have been only 35-40%. The purpose of C10403, a large prospective US intergroup trial, was to evaluate the feasibility and effectiveness [with EFS as a primary endpoint), of treating AYA ALL pts (ages 16-39 yrs) using the standard arm of the successful Children's Oncology Group regimen (COG AALL0232) . Methods: Newly diagnosed AYA patients with B-precursor (B-ALL) or T-precursor (T-ALL) ALL were eligible to enroll on C10403. Burkitt type and Ph+ ALL were excluded. The regimen was identical to the Capizzi methotrexate arm of COG AALL0232 (Larsen E. JCO 2011; 29 suppl:3) and consisted of four intensive courses: remission induction, remission consolidation, interim maintenance, delayed intensification, and prolonged maintenance therapy. Pts with M2 marrow response (>5% but < 25% lymphoblasts) after remission induction received an extended remission induction course of therapy. Events were defined as induction failure (M3 [³25% blasts] day 29 of induction or M2 day 43 of extended induction), death, relapse, or second malignancy. Key correlative science studies in a subset of the total accrued population included assessment of Minimal Residual Disease (MRD) using quantitative real-time PCR of clonal IgH or TCR gene rearrangements as well as Low Density Microarray (LDA) assays designed to detect a previously validated gene expression profile (Harvey, R; ASH 2013, abstract 826) which can prospectively identify ALL pts with Ph-like (BCR-ABL1-like) ALL. Results: 318 pts were enrolled on C10403 from 11/2007 to 8/2012; 22 withdrew prior to therapy. Of 296 evaluable pts, the median age at diagnosis was 24 yrs (range: 17 to 39): 25% were 17-20 yrs, 53% were 21-29 yrs, and 22% were 30-39 yrs. The majority had B-ALL (76%) and were male ( 61%). Approximately 25% were non-Caucasian and 15% were Hispanic or Latino. 32% of pts were obese (BMI³30). There were 5 (2%) treatment-related deaths during protocol therapy: liver failure (n=2, both during induction), infection (1 in induction, 1 in consolidation), and ventricular arrhythmia (1 in induction). Overall, treatment toxicities were similar to those reported in the standard arm of COG AALL0232, with an increased thrombosis and early hyperbilirubinemia for C10403 pts, as reported previously (Advani A, ASH 2013, abstract 303). To date, 70 deaths have been reported and 87 pts remain on protocol therapy. With a median follow-up of 28 months for surviving pts, 105 events have been observed. The median EFS overall is 59.4 months (95% CI: 38.4 to NR) and the 2-yr EFS rate overall is 66% (95% CI: 60 – 72%) [Fig 1a] with similar 2-yr EFS rates for B and T- ALL pts (65%, and 68%). The 2-yr OS rate is 78% (95% CI: 72 – 83%)[Fig 1b], which is similar for B (78%, 95% CI: 72 – 84%) and T-ALL, (80%, 95% CI: 70 – 91%). These results allow rejection of the null hypothesis of this Phase II trial that the true median EFS is, at most, 32 months. In multivariable analysis of presenting clinical features, age > 20 years and initial WBC count ³ 30k/microliter were significantly associated with worse EFS and OS. Presence of MRD at day 28 following initiation of induction therapy and presence of a Ph-like gene expression signature were significantly associated with both worse EFS and OS. Notably, absence of detectable MRD noted in 22/58 [38%] evaluable pts at day 28 of induction was associated with 100% EFS (p=0.0006). The Ph-like like signature was detected in 28% of pts tested on C10403 and the 2 yr EFS for these patients was only 52%, compared to 81% for those without Ph-like disease (p = 0.04). Conclusions: This large prospective US adult intergroup trial (C10403) for pts 16-39 years old employing an intensive pediatric regimen demonstrates a significant improvement (compared to historical controls) in AYA EFS and OS and validates this approach for treatment of AYA with ALL by adult hematologists. The improved clinical outcomes and the predictive value of the correlative studies in this trial lay the foundation for the design of future trials, where incorporation of novel agents to eradicate MRD, and/or use of tyrosine kinase inhibitors to target the frequently detected Ph-like ALL in AYA pts may further improve survival for young adults with ALL. Figure 1a Figure 1a. Figure 1b Figure 1b. Disclosures Stock: Sigma-Tau: Membership on an entity's Board of Directors or advisory committees, Research Funding. Advani:Sigma Tau: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees. Liedtke:Onyx: Membership on an entity's Board of Directors or advisory committees. Larson:Novartis: Consultancy, Research Funding.

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Integrative Analysis of Copy Number and Gene Expression Data Suggests Novel Pathogenetic Mechanisms in Primary Myelofibrosis

Blood ◽

10.1182/blood.v126.23.2830.2830 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2830-2830

Author(s):

Simona Salati ◽

Roberta Zini ◽

Simona Nuzzo ◽

Paola Guglielmelli ◽

Valentina Pennucci ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Primary Myelofibrosis ◽

Advisory Committees ◽

Treated Sample ◽

Cd34 Cells ◽

Megakaryocyte Differentiation

Abstract Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, we integrated gene expression and copy number (CN) signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is accompanied by a coordinated transcriptional up-regulation in PMF patients. To assess the impact of PAOX deregulation on the survival of PMF and normal primary hematopoietic cells, PMF and normal donors cells were incubated with increasing doses of the PAOX inhibitor MDL-72,572. PAOX inhibition resulted in rapid cell death of PMF progenitor cells (5,7±0,9% of late apoptotic cells in NT sample vs 20,1±3,2% in 150μM treated-sample, p<0.05), while survival of normal CD34+ cells was not affected (0,97±0,09 of late apoptotic cells in NT sample vs 2,7±0,4 in 150μM treated-sample, p<0.05), as monitored by Annexin V/PI staining. These data suggest that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, our integrative analysis of gene expression and CN data pointed out the concomitant copy number loss and transcriptional down-regulation in PMF patients of the chromatin modifier HMGXB4. Interestingly, silencing of HMGXB4 in CD34+ stem/progenitor cells induced megakaryocyte differentiation, as demonstrated by increased expression of CD41 (36±1,9% vs 26,1±2,9% at day 12, 52,5±4,9% vs 33±1% at day 14 of serum free liquid culture, p<0.05) and increased number of CFU-MK colonies (27,7±2,5 vs 18,1±1,3% in small CFU-MK colonies, p<0.05). On the other hand, HMGXB4 silencing repressed the erythroid differentiation, as indicated by a decrease in the percentage of cells positive for the erythroid marker GPA (10.8%±5.6 vs 24.8%±5, p<0.05) and significant decrease in Burst Forming Unit-Erythroid (BFU-E) and Colony-Forming Unit-Erythroid (CFU-E) (47,9±6 vs 60,5±7,6, p<0.05). Taken together, these data suggest that HMGXB4 silencing in human HSPCs favors megakaryocyte differentiation while restraining the erythroid lineage. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterize PMF patients. In conclusion, our work sheds light on the influence of genomic abnormalities on gene expression regulation in PMF CD34+ cells and on their impact to features typical of PMF, such as a hyperplastic megakaryopoiesis and resistance to apoptosis, and therefore potentially contributing to the development of the myeloproliferative disease. Disclosures Vannucchi: Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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The BMP/SMAD Pathway Is a Key Mediator of Leukemic Transformation of TP53-Mutant Post-MPN AML

Blood ◽

10.1182/blood-2021-152036 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 626-626

Author(s):

Bing Li ◽

Wenbin An ◽

Hua Wang ◽

Aishwarya Krishnan ◽

Shoron Mowla ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Sequencing Analysis ◽

Erythroid Progenitors ◽

Leukemic Transformation ◽

Advisory Committees ◽

Smad Pathway ◽

The Impact

Abstract Leukemic transformation (LT) after an antecedent myeloproliferative neoplasm (MPN) carries a dismal prognosis. As such, there is a pressing need for new mechanistic insights into LT as well as novel therapeutic approaches. Mutational inactivation of TP53 is the most common somatic mutation in LT. However, the impact of TP53 allelic state on the ability to potentiate LT, as well as the pathways involved in this process, have largely remained unresolved. To investigate the role of Tp53 alterations in LT, we generated an allelic series of mouse models with Jak2V617F/+ combined with conditional Tp53 knockout and point mutant alleles (all crossed to Rosa-CreERT2); Jak2V617F/+(J VF) , Jak2V617F/+-Tp53fl/+(J VFP +/-), Jak2V617F/+-Tp53fl/fl (J VFP -/-), Jak2V617F/+-Tp53R172H/+(J VFP R172H/+), Jak2V617F/+-Tp53R172H/fl (J VFP R172H/-). After tamoxifen-induced recombination, mice transplanted with J VF, J VFP +/- and J VFP R172H/+ cells developed an MPN phenotype, whereas all the recipients of J VFP -/- and J VFP R172H/- bone marrow initially developed an MPN phenotype followed by transformation to acute leukemia with significantly impaired survival, and changes in blood counts and organ weights, compared to other genotypes (Fig 1A/B). Histopathology of J VFP -/- and J VFP R172H/- mice was consistent with pure erythroleukemia (PEL; Fig 1C). Analysis of stem and progenitor compartments demonstrated that the MEP (Megakaryocyte Erythroid Progenitors) compartment was significantly expanded in the bone marrow and spleen of both J VFP -/- and J VFP R172H/- mice, compared to other genotypes, at both the MPN and PEL stages of disease, consistent with erythroid-biased hematopoiesis (Fig 1D). Given we observed sequential MPN->AML progression, we hypothesized that additional genetic/biological events were required to promote LT. Sparse whole genome sequencing analysis revealed that transformation to PEL was associated with the development of recurrent copy number alterations (CNA) . Importantly, CNAs were restricted to the MEP compartment and not identified in the GMP compartment (Fig 1E), suggesting that MEPs might represent the leukemia initiating population with capability of acquiring additional genomic instability. Consistent with this hypothesis, mice transplanted with MEPs, but not GMPs from J VFP -/- and J VFP R172H/- mice at the MPN stage developed PEL. Further, single-cell RNA sequencing of J VF and J VFP -/- (at both MPN and PEL stage) demonstrated that the gene-expression signature of the leukemic population was most similar to that of erythroid progenitors and erythroblasts, and that by copy number inference analysis, CNAs were restricted to the leukemic population. We identified 617 genes up-regulated in both J VFP -/- and J VFP R172H/- leukemic MEPs when compared to J VF MEPs using RNA-seq. Pathway analysis demonstrated increased expression of Bone morphogenetic protein (BMP) pathway genes in both J VFP -/- and J VFP R172H/- leukemic mice (Fig 1F). Importantly, similar observations were made in human PEL samples as well. To investigate the function of this pathway, leukemic MEPs from J VFP -/- and J VFP R172H/- mice were transduced with an shRNA-targeting Bmp2 or a control and injected into lethally irradiated recipient mice. Mice injected with Bmp2-shRNA MEPs demonstrated leukemic regression and restoration of normal hematopoiesis as evidenced by significant reductions in leukocytosis (p<0.05) and increased HGB (p<0.05) and an increase in PLT count (p<0.05/p<0.01) (Fig 1G). Finally, as compared to mice injected with leukemic MEPs with control shRNA, mice injected with Bmp2-shRNA had significantly longer survival (p<0.05) (Fig 1H). Thus, downregulation of Bmp2 results in attenuation of the leukemic phenotype. Using novel models, we have identified that bi-allelic, but not mono-allelic Tp53 alteration is required for LT of MPN. The leukemia initiating population arises within the MEP compartment and is characterized by recurrent CNAs acquired in a specific hematopoietic compartment. Moreover, the BMP/SMAD pathway is upregulated in leukemic MEPs and plays a functional role in LT. Collectively, our data yields novel biological insights into the process of leukemic transformation mediated by Tp53 alterations. Data on selective therapeutic targeting of p53-mutant PEL will be presented at the meeting. Figure 1 Figure 1. Disclosures Xiao: Stemline Therapeutics: Research Funding. Lowe: Oric Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Mirimus, Inc: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Faeth Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; PMV Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Gilead: Honoraria; Amgen: Honoraria; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Janssen: Consultancy; Astellas: Consultancy. Rampal: Pharmaessentia: Consultancy; Abbvie: Consultancy; Kartos: Consultancy; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Novartis: Consultancy; CTI: Consultancy; Sierra Oncology: Consultancy; Stemline: Consultancy, Research Funding; Blueprint: Consultancy; Memorial Sloan Kettering: Current Employment.

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