Single-Cell Genomic Analysis Identifies Prognostically Significant Gene Expression Programs in Infant Acute Lymphoblastic Leukemia

Abstract Introduction: Infant acute lymphoblastic leukemia (ALL) is an aggressive subtype of leukemia with low rates of survival. Rearrangements involving the KMT2A gene are associated with poor prognosis for infants with this cancer. Although many patients with KMT2A rearrangements (KMT2A-r) ultimately relapse, some do not. The molecular basis for this distinction has not yet been determined. There is evidence to suggest that cancers with distinct KMT2A fusion partners show distinct patterns of gene expression, though these differences have not previously been linked to prognostic outcomes. Here we utilize single-cell genomic analysis for infant ALL samples with and without KMT2A-r taken at diagnosis to explore transcriptional dynamics and identify gene expression programs with potential prognostic value. Methods: We performed 10x Chromium single-cell multiome sequencing to obtain both gene expression (scRNA-seq) and chromatin accessibility (scATAC-seq) data for blood and/or bone marrow samples obtained from a total of 34 infant ALL patients at time of diagnosis. Of these, 19 KMT2A-r patients later relapsed, 6 KMT2A-r patients did not relapse, and 9 patients did not show KMT2A rearrangement. Sequencing data were processed and aggregated with cellranger-arc, with normalization and differential gene expression performed using the Seurat package for gene expression analysis. Differential gene expression was performed between the three groups described above, (KMT2A-r + relapse, KMT2A-r no relapse, no KMT2A-r), then further subdivided for KMT2A-r cases based on the KMT2A partner gene (MLLT1 n=13 or AFF1 n=12). Results: In preliminary scRNA- and scATAC-seq in infant ALL patients, we had observed that cancer cells from individual patients tend to cluster separately, while non-blast cell populations showed similar interindividual patterns, suggesting a high degree of divergence among blast cell transcriptional programs. We replicated this finding here in larger patient samples, demonstrating no overt similarity based on KMT2A-r status or whether patients later relapsed. Strikingly, samples did broadly cluster according to KMT2A rearrangement partner gene, indicating that this appears to be a major driver of the transcriptional program in infant ALL patients. Differential expression analysis within the KMT2A-MLLT1 and KMT2A-AFF1 samples separately revealed expression of other genes associated with particular KMT2A fusion genes. Specifically, we observed that expression of HOXA genes was mostly restricted to KMT2A-MLLT1 samples. Consistent with previous results, we observed that within KMT2A-AFF1 samples there were two distinct groups of cells with mutually exclusive expression of HOXA9 or IRX1. Furthermore, the limited HOX gene expression observed in KMT2A-AFF1 samples was enriched in patients who did not relapse, supporting previous observations that the KMT2A-AFF1 samples expressing IRX1 comprise a more aggressive leukemia. Conclusions: Utilizing a single-cell transcriptomic approach enabled us to identify gene expression programs associated with good and poor prognosis in KMT2A-r infant ALL cases. We found that the KMT2A fusion partner gene appears to drive a large portion of the transcriptional heterogeneity observed across KMT2A-r samples. By treating the KMT2A-MLLT1 and KMT2A-AFF1 samples separately and exploring transcriptional dynamics within these groups, we identified transcriptional differences between patients who did and did not relapse in the presence of a KMT2A-AFF1 fusion. We are continuing to explore these data to identify prognostic markers in KMT2A-MLLT1 patients. Figure 1. Mutually exclusive gene expression programs distinguish subsets of infant ALL samples. A) Uniform manifold approximation and projection (UMAP) visualization of gene expression in individual cells across all patient samples at diagnosis, each color corresponding to a single patient (n=34). B) Cells colored according to KMT2A-r status and relapse or lack thereof. C) Cells colored according to KMT2A fusion partner gene. D) HOXA9 expression among KMT2A-AFF1 patients is strongly biased to patients that do not relapse. E) In KMT2A-AFF1 samples, HOXA9 and IRX1 show mutually exclusive expression patterns. Figure 1 Figure 1. Disclosures Brown: Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Kura: Membership on an entity's Board of Directors or advisory committees; KIte: Membership on an entity's Board of Directors or advisory committees.

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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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A Comprehensive Analysis of Single-Cell Chromatin Accessibility and Gene Expression Identifies Intra-Tumor Heterogeneity and Molecular Treatment Responses in Relapsed/Refractory Multiple Myeloma

Blood ◽

10.1182/blood-2019-130051 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 575-575

Author(s):

Alexandra M Poos ◽

Jan-Philipp Mallm ◽

Stephan M Tirier ◽

Nicola Casiraghi ◽

Hana Susak ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Tumor Heterogeneity ◽

Mek Inhibitor ◽

Chromatin Accessibility ◽

University Research ◽

Sample Processing ◽

Advisory Committees

Introduction: Multiple myeloma (MM) is a heterogeneous malignancy of clonal plasma cells that accumulate in the bone marrow (BM). Despite new treatment approaches, in most patients resistant subclones are selected by therapy, resulting in the development of refractory disease. While the subclonal architecture in newly diagnosed patients has been investigated in great detail, intra-tumor heterogeneity in relapsed/refractory (RR) MM is poorly characterized. Recent technological and computational advances provide the opportunity to systematically analyze tumor samples at single-cell (sc) level with high accuracy and througput. Here, we present a pilot study for an integrative analysis of sc Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) and scRNA-seq with the aim to comprehensively study the regulatory landscape, gene expression, and evolution of individual subclones in RRMM patients. Methods: We have included 20 RRMM patients with longitudinally collected paired BM samples. scATAC- and scRNA-seq data were generated using the 10X Genomics platform. Pre-processing of the sc-seq data was performed with the CellRanger software (reference genome GRCh38). For downstream analyses the R-packages Seurat and Signac (Satija Lab) as well as Cicero (Trapnell Lab) were used. For all patients bulk whole genome sequencing (WGS) data was available, which we used for confirmatory studies of intra-tumor heterogeneity. Results: A comprehensive study at the sc level requires extensive quality controls (QC). All scATAC-seq files passed the QC, including the detected number of cells, number of fragments in peaks or the ratio of mononucleosomal to nucleosome-free fragments. Yet, unsupervised clustering of the differentially accessible regions resulted in two main clusters, strongly associated with sample processing time. Delay of sample processing by 1-2 days, e.g. due to shipment from participating centers, resulted in global change of chromatin accessibility with more than 10,000 regions showing differences compared to directly processed samples. The corresponding scRNA-seq files also consistently failed QC, including detectable genes per cell and the percentage of mitochondrial RNA. We excluded these samples from the study. Analysing scATAC-seq data, we observed distinct clusters before and after treatment of RRMM, indicating clonal adaptation or selection in all samples. Treatment with carfilzomib resulted in highly increased co-accessibility and >100 genes were differentially accessible upon treatment. These genes are related to the activation of immune cells (including T-, and B-cells), cell-cell adhesion, apoptosis and signaling pathways (e.g. NFκB) and include several chaperone proteins (e.g. HSPH1) which were upregulated in the scRNA-seq data upon proteasome inhibition. The power of our comprehensive approach for detection of individual subclones and their evolution is exemplarily illustrated in a patient who was treated with a MEK inhibitor and achieved complete remission. This patient showed two main clusters in the scATAC-seq data before treatment, suggesting presence of two subclones. Using copy number profiles based on WGS and scRNA-seq data and performing a trajectory analysis based on scATAC-seq data, we could confirm two different subclones. At relapse, a seemingly independent dominant clone emerged. Upon comprehensive integration of the datasets, one of the initial subclones could be identified as the precursor of this dominant clone. We observed increased accessibility for 108 regions (e.g. JUND, HSPA5, EGR1, FOSB, ETS1, FOXP2) upon MEK inhibition. The most significant differentially accessible region in this clone and its precursor included the gene coding for krüppel-like factor 2 (KLF2). scRNA-seq data showed overexpression of KLF2 in the MEK-inhibitor resistant clone, confirming KLF2 scATAC-seq data. KLF2 has been reported to play an essential role together with KDM3A and IRF1 for MM cell survival and adhesion to stromal cells in the BM. Conclusions: Our data strongly suggest to use only immediately processed samples for single cell technologies. Integrating scATAC- and scRNA-seq together with bulk WGS data showed that detection of individual clones and longitudinal changes in the activity of cis-regulatory regions and gene expression is feasible and informative in RRMM. Disclosures Goldschmidt: John-Hopkins University: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees.

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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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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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Gene Expression Profiling of CD93-Selected CP-CML Stem Cells Confirms Their Quiescent Character and Biomarker Potential

Blood ◽

10.1182/blood.v128.22.4231.4231 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4231-4231

Author(s):

Gillian A. Horne ◽

Chinmay Rajiv Munje ◽

Ross Kinstrie ◽

Eduardo Gómez-Castañeda ◽

Helen Wheadon ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Single Cells ◽

Gene Clustering ◽

Single Cell Level ◽

Cell Level ◽

Advisory Committees ◽

Two Populations

Abstract The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". Disease persistence suggests, that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of a small population of cells, termed leukemic stem cells (LSC). We previously identified CD93 expression as a promising biomarker of LSC in chronic phase (CP)-CML. Our group has described the long term self-renewal potential of Lin-CD34+93+ CP-CML cells compared to their Lin-CD34+93- counterparts through LTCIC assays (n=3, p<0.0001) and NSG engraftment models (3.5-30-fold increased in engraftment with Lin-CD34+93+ cells, p<0.03). We hypothesized that CD93+-selected cells would represent a more immature functional phenotype compared to CD93- selected cells. The aim of this study was to characterize differences in the gene expression profile between CD93+ and CD93- CML LSC populations and determine heterogeneity of each population at a single cell level. To interrogate this, we initially identified CP-CML subpopulations with the greatest functional capability compared to normal. Normal and CP-CML samples were FACS-sorted into HSC/LSC, CMP, GMP, and MEP sub-populations. Results suggest a significant change in functional status between normal and CP-CML subpopulations within the HSC/LSC compartment (lin-CD34+CD38-CD45RA-CD90+), where CML LSC demonstrated significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture in physiological growth factors. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively (p<0.05)). Furthermore, fluorescence in situ hybridization demonstrated that >90% of lin-CD34+CD38-CD45RA-CD90+ CML LSC from all patient samples were BCR-ABL positive. Subsequent experiments were confined to the LSC population. We hypothesized that lin-CD34+CD38-CD90+CD93- CML cells would have a more mature gene expression profile compared to lin-CD34+CD38-CD90+CD93+ cells. CP-CML cells were sorted into (1) lin-CD34+, (2) lin-CD34+CD38-CD90+CD93- and (3) lin-CD34+CD38-CD90+CD93+ populations. RNA was harvested at baseline from bulk populations (1) to (3) and cDNA was generated from single cells using the Fluidigm C1 autoprep system. Using Fluidigm technology, quantitative PCR of 90 lineage-specific and cell survival genes was performed within all populations of cells (1) to (3) in 'bulk' samples (n=3), and at single cell level (n=123 CD93+, n=120 CD93-single cells; n=3 samples in total). Bulk sample analysis demonstrated a significant increase in expression of lineage commitment genes within the lin-CD34+CD38-CD90+CD93- population, as shown by increased expression of GATA1 (p=0.0007), and CBX8 (p=0.0002). The lin-CD34+CD38-CD90+CD93+ population displayed a less lineage-restricted profile with increased expression of CDK6 (p=0.05), HOXA6 (ns), CDKN1C (ns) and CKIT (p=0.0014), compared to the lin-CD34+CD38-CD90+CD93- population. Furthermore, the two populations could be segregated by differential gene expression through gene clustering. At a single cell level, differences were noted in the frequency of expression between lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations, particularly in GATA1, TPOR, and VWF. Although a statistically significant change was demonstrated in gene expression between the lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations in a number of genes, we were not able to segregate the populations by differential expression using gene clustering. This highlights the heterogeneous nature of the cell populations and the inability to distinctly characterize between the two populations at a single cell level. Our results validate CD93 as a potential biomarker to separate the primitive CP-CML LSC population and highlight key lineage and cell survival pathways that are altered in CML LSC. The results demonstrate the heterogeneity seen within gene expression at the single cell level, which may allow for further insight into the CML LSC compartment with further analyses. Disclosures Wheadon: GlaxoSmithKline: Research Funding. Copland:Shire: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

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Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽

10.1182/blood-2020-142157 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-34

Author(s):

Masahiro Marshall Nakagawa ◽

Ryosaku Inagaki ◽

Yutaka Kuroda ◽

Yasuhito Nannya ◽

Lanying Zhao ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Simultaneous Detection ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Single Cell Sequencing ◽

Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Dissecting Heterogeneity of Tumor Cells and Their Microenvironment in Refractory Multiple Myeloma

Blood ◽

10.1182/blood-2019-128625 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 571-571

Author(s):

Stephan M Tirier ◽

Jan-Philipp Mallm ◽

Nicola Casiraghi ◽

Hana Susak ◽

Nicola Giesen ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Types ◽

Specific Gene ◽

Refractory Disease ◽

Advisory Committees ◽

Cell Trajectory

Introduction: Despite significant improvements in therapy during the last decade, most multiple myeloma (MM) patients develop refractory disease over time. Treatment of refractory MM is a major challenge, likely due to the still poorly characterized inter- and intratumor heterogeneity at this stage of the disease, and the complex interplay of MM cells with the microenvironment (ME). In particular, there is an urgent need to unravel how these features of MM are linked to molecular mechanisms of drug resistance. Methods: We resolved the cellular composition, underlying transcriptional inter- and intra-patient heterogeneity and molecular treatment response of relapsed/refractory MM by single cell RNA sequencing (scRNA-seq). Using droplet-based microfluidics, ~230,000 single cell gene expression profiles from bone marrow (BM) aspirates of 21 patients sorted into CD138+and CD138- fractions were acquired, allowing for a comprehensive analysis of both MM cells as well as their ME. Patients had a median of 4 prior lines of therapy including both a proteasome inhibitor and an immunomodulator and were refractory to their immediate prior line of therapy at time of sampling. In addition, paired samples before either pomalidomide- or carfilzomib-based therapies were analyzed for 16/21 patients. Genomic aberrations in individual patients were mapped by interphase fluorescence in situ hybridization. Cells were clustered and CD138+ MM subtypes as well as immune cell-types of the ME were identified from their single cell transcriptomes and a copy number variation (CNV) analysis. As a reference for non-malignant cells and to construct a developmental B-cell trajectory the Human Cell Atlas BM scRNA-seq reference dataset was used. To characterize interactions of MM cells with their ME, the correlated expression of ligand-receptor pairs was exploited. Results: The analysis of inter- and intra-tumor heterogeneity of molecular MM subgroups revealed distinct transcriptome signatures with contributions that could be assigned to differences in heavy and light chain immunoglobulin expression as well as known genomic alterations, including t(11;14), t(4;14) and hyperdiploidy. MM cells from individual patients largely maintained a plasma cell specific gene expression profile but a partial loss of plasma cell identity was detected based on mapping to a developmental B-cell trajectory. It was characterized by the upregulation of subgroup transcriptome signatures associated with earlier stages of B-cell development in almost 50% of patients, such as a pre-B or mature B cell-like phenotype. Within individual samples, subclonal MM cell populations with specific gene expression programs were resolved based on the CNV analysis and included those characterized by expression of the immune-activator CD27 and the modulator of WNT signaling FRZB. The analysis of longitudinally collected samples revealed both changes in the cell subtype cluster structure as well as drug-specific adaptation of gene expression programs in distinct subpopulations persisting or emerging at relapse. These profile changes were characterized by e.g. downregulation of Myc target genes upon pomalidomide treatment or induction of heat shock proteins under carfilzomib. Within the ME of refractory MM patients, we observed that the fraction of B cells and CD4+ T cells was strongly reduced while CD14+ and CD16+ monocytes as well as dendritic cells expanded. Notably, the immune checkpoint protein PD-1H (aka VISTA) that inhibits T cell activation was highly expressed in cell types from the myeloid compartment in contrast to healthy donors. Further, a ligand-receptor analysis revealed that MM cells displayed the strongest interactions with monocytes, which were mediated by MIF, BAFF and other cytokines. Conclusions: Our study demonstrates the value of scRNA-seq analysis for identifying crucial transcriptome features that classify refractory MM subtypes and their evolution in response to treatment including regulation of drug resistance associated signaling pathways. Our data suggest that refractory MM cells shape the myeloid compartment in the BM to generate an immune suppressive ME. Understanding the evolution of MM cell heterogeneity and the bone marrow milieu in refractory disease will lead to novel treatment approaches and eventually improve patient outcome. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:John-Hopkins University: Research Funding; Molecular Partners: Research Funding; Amgen: Consultancy, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding.

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Lack of Somatic Sequence Mutations In Protein Tyrosine Kinase Genes Other Than the JAK Kinase Family In High Risk B-Precursor Childhood Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG) High-Risk (HR) ALL TARGET Project

Blood ◽

10.1182/blood.v116.21.2752.2752 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2752-2752 ◽

Cited By ~ 1

Author(s):

Jinghui Zhang ◽

Charles Mullighan ◽

Richard Harvey ◽

William L. Carroll ◽

I-Ming L. Chen ◽

...

Keyword(s):

Gene Expression ◽

High Risk ◽

Tyrosine Kinase ◽

Board Of Directors ◽

Expression Profile ◽

Tyrosine Kinases ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Kinase Signaling ◽

Advisory Committees

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

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

Blood ◽

10.1182/blood-2018-99-118062 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5284-5284

Author(s):

Anna Ferrari ◽

Silvia Vitali ◽

Valentina Robustelli ◽

Andrea Ghelli Luserna Di Rora ◽

Simona Righi ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Triple Negative ◽

Lymphoblastic Leukemia ◽

Gene List ◽

Advisory Committees ◽

The Impact ◽

Stat Pathway

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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