Mutant Garrett-Bakelman, Francine E; Melnick, Ari M
Epigenomics,
2016-July-01, Letnik:
8, Številka:
7
Journal Article
Recenzirano
Aberrant epigenomic programming is a hallmark of acute myeloid leukemia. This is partially due to somatic mutations that perturb cytosine methylation, histone post-translational modifications and ...transcription factors. Remarkably, mutations in the
and
genes perturb the epigenome through all three of these mechanisms. Mutant IDH enzymes produce high levels of the oncometabolite (R)-2-hydroxyglutarate that competitively inhibits dioxygenase enzymes that modify methylcytosine to hydroxymethylcytosine and histone tail methylation. The development of IDH mutant specific inhibitors may now enable the therapeutic reprogramming of both layers of the epigenome spontaneously to revert the malignant phenotype of these leukemias and improve clinical outcome for acute myeloid leukemia patients with IDH mutations.
Recently discovered recurrent so- matic mutations in the key metabolic enzymes IDH1 and IDH2 produce the aberrant oncometabolite 2-HG and contribute to malignant trans- formation of hematopoietic and ...glial cells. Two recent reports in Science describe the first IDH1 and IDH2 mutant-specific small-molecule inhib- itors, which induce cell differentiation of myeloid leukemias and malignant gliomas.
The majority of pediatric acute myeloid leukemia (AML) patients achieve complete remission. However, after this initial response to therapy, relapse occur in around forty per cent. The failure of ...therapy has been thought to be due to drug resistance resulting either from accumulating mutations, or already a characteristic of leukemic stem cells present at diagnosis. These mechanisms are shown to be true in part of the patients, however in quite a number of these relapses other mechanisms must play a role. In adult leukemias high epigenetic heterogeneity is associated with worse clinical outcomes. Remarkably, the DNMT3, TET and IDH mutations often found in adults are almost non-existent in children, despite the fact the leukemias are phenotypically very alike. We tested the hypothesis that, apart from genetic heterogeneity, epigenetic heterogeneity contributes to therapy resistance and thereby initiation of relapse.
Methods: We performed flow cytometric immunophenotypic analyses, whole exome sequencing and epigenome sequencing (by enhanced reduced representation bisulfite sequencing) on bone marrow (BM) samples taken at time of diagnoses, within two weeks after achievement of CR and at time of relapse of three selected patients (diagnosed between 2007-2014, treated according to the Dutch-Belgian DB-AML-01 protocol). Of each patient, apart from whole diagnosis sample and whole relapse sample (all with a minimum of 90% blast cells), the MRD sample was FACS sorted to select for blast cells.
Results: Leukemia associated immunophenotypes phenotype found at time of diagnosis remained the same over time with no emerging new immunophenotypes coinciding with emergence of relapses. We further evaluated whether changes in variant allele frequency (VAF) of mutations between diagnosis and subsequent time points could be linked to the development of relapse. Generally, VAF numbers decreased or remained stable over time, with no new mutations arising that characterized relapse. None of the patients had a mutation in DNTM3A, TET or IDH genes, accounting for any epigenetic dysregulation.
In contrast to the findings for immunophenotypes and genetics, extensive epigenetic changes were found at the different time points. In samples taken within two weeks after reaching complete remission, a median of 23,665 (range 19,303-28,808) differentially methylated regions (DMRs) was found compared with the diagnosis samples. When comparing the corresponding relapse sample with this CR/MRD sample, another set of DMRs was found: median 19,625 (range 14,680-30,410). In all three patients, the majority of exons, intergenic, intron and promoter regions are uniquely hyper or hypo methylated at time of relapse (median of 88%, range 81%-95%), with a small proportion already differentially methylated at time of remission (median 11%, range 4%-18%). We hypothesize that these changes in epigenetic status occurring after CR, possibly contributes to relapse. Pathway analysis will probably give clues for understanding the pathophysiology and hopefully new targets for treatment.
Conclusion: Our preliminary data suggest that epigenetic regulation may play a vital role in development of relapse. Ultimately, a better understanding of the role of epigenetics could open new therapeutic approaches for pediatric AML.
Ossenkoppele:Roche: Honoraria; Novartis: Research Funding; Karyopharm: Consultancy, Research Funding; J&J: Consultancy, Honoraria; Celgene: Honoraria, Research Funding. Kaspers:Janssen-Cilag: Research Funding. Cloos:Takeda: Honoraria.
Despite recent advances in our understanding of the genetic origins of acute myeloid leukemia (AML), clinical outcomes remain poor. While standard induction chemotherapy induces remission in most ...patients, the majority of patients eventually relapses and dies from progressive disease. A number of cell surface proteins have been shown to be expressed at high levels on AML stem cells compared to normal HSCs including CD47, CD44, CD96, TIM3, CD123, CD25 and IL1RAP. Despite the attention these antigens have received, data supporting their roles as cell-intrinsic regulators of LSCs are more limited. We have identified CD97, a member of the adhesion class of GPCR, as a frequently upregulated antigen expressed on AML blasts that is a critical regulator of LSC function.
In order to determine whether CD97 is expressed on LSCs, we assessed CD97 expression in human AML primary samples by flow cytometry. This study revealed that CD97 was 10 fold increased on LSC-enriched (CD34+CD38-) blasts compared to cord blood HSCs in all 30 patients tested. In contrast, the expression of other previously reported LSC was more variable and less frequently increased over HSCs. To test if CD97 may regulate leukemic cell function, we knocked-down (KD) CD97 in human AML cell lines. CD97-KD in HL60 and MOLM-13 AML cell lines increased apoptosis, reduced cell growth 4-fold, and delayed mortality once injected into NSG mice (20 vs. 28 days, p=0.0012). As CD97 expression increases during normal myelomonocytic differentiation, we cultured control and CD97-KD HL60 or U937 cells (which can be induced to differentiate along the myelomonocytic lineage following all-trans-retinoic acid (ATRA) treatment) for 4 days in the presence of 10nM ATRA or vehicle control. Flow cytometric analysis revealed that CD97-KD cells expressed higher levels of the myeloid differentiation antigens CD11b and CD15 both before and after ATRA treatment. In addition, mRNA expression of CD11b and PU.1, key genes associated with myeloid differentiation, were increased in CD97-KD cells compared to controls.
To investigate whether CD97 regulates leukemic initiation, purified HSPCs from WT and CD97-/- mice were transduced with the MLL-AF9 oncogene (MA9). CD97-/--MA9 cells (GFP+) showed a 3-fold reduction in colony formation in serial replating assays in methylcellulose, consistent with a reduction in self-renewal. Mice transplanted with 5,000 CD97-/--MA9 blasts survived significantly longer than those receiving WT-MA9 blasts (70 vs 100 days, p=0.0039). Consistent with reduced disease aggressiveness, at the time of sacrifice mice injected with WT-MA9 cells contained a higher percentage of total GFP+ cells, L-GMPs and CD11b+Gr1+ cells in the BM than those receiving CD97-/--MA9 grafts. Secondary transplant experiments revealed that CD97-/--MA9 blasts exhibit a 25-fold lower LIC frequency than WT-MA9 blasts. Similar results were obtained using the AML1-ETO9a mice model.
To better understand the molecular pathways underlying CD97 regulation of AML cells, we performed a combination of RNA-sequencing and protein phospho-array analyses in CD97-KD and scramble control cells. Gene Ontology and GSEA analyses revealed enrichment of transcripts encoding genes that regulate apoptosis as well as downregulation of cell cycle regulators in CD97-KD cells. Consistent with our flow cytometry data, CD97-KD induced differentiation, as evidenced by increased expression of myeloid differentiation genes and global increases in granulocyte and monocyte fingerprint signatures. Phosphoproteome analysis revealed downregulation of several mediators of the MAPK cascade and MTOR signaling pathways in CD97-KD cells. These signaling changes were confirmed by western blot, as activating phosphorylation events of ERK 1/2, c-Jun, and STAT3 were decreased upon CD97-KD. Together, these data indicate that functional alterations in CD97-KD AML cells are due to the modulation of signaling cascades including MAPK and mTOR.
Our studies indicate that CD97 is expressed at a higher frequency in AML patient samples than previously identified LSC antigens and that its expression is elevated, irrespective of genetic subtype. Studies using human cell lines and mouse models of AML demonstrate that CD97 regulates the proliferation and survival of leukemic blasts as well as disease initiation and self-renewal. Collectively, these data credential CD97 as a promising therapeutic target in AML.
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No relevant conflicts of interest to declare.
Hematopoietic differentiation is characterized by the organized epigenetic reprogramming of hematopoietic stem cells to terminally differentiated cells. Acute leukemias represent a disruption of this ...process and are categorized into myeloid and lymphoid subtypes on the basis of surface marker patterns identified by flow cytometry. Biphenotypic and other poorly specified leukemias such as early T-cell precursor acute lymphoblastic leukemias (ETP T-ALLs) highlight the limitations of this technology. Here we describe a novel approach to characterize leukemia lineage on the basis of epigenetic characteristics. Using model-based deconvolution, we show that DNA methylation profiling and ATAC-seq chromatin accessibility maps can be used to specify leukemia lineage characteristics more precisely. On in-vitro mixtures of purified cells, it can quantitatively determine sample composition with R2 = 0.89. Extending this method to evaluate primary leukemia samples, we show that ETP T-ALLs exhibit a mixture of lineage characteristics that varies by patient. Typical T-ALL patient samples exhibit predominantly lymphoid characteristics most similar to the common lymphoid progenitor (CLP) stage. In contrast, ETP T-ALL cases exhibit a mixture of myeloid and lymphoid features, with some showing dominant CLP features, some a mix of both CLP and granulocytic macrophage progenitor (GMP) characteristics, and one with dominant GMP features. Biphenotypic leukemias also contained CLP dominant, GMP dominant, and mixed cases. CEBPa-silenced AMLs, which had previously been described as exhibiting a T-cell like phenotype, exhibited dominant CLP characteristics in contrast to other AMLs that exhibit dominant GMP features. In the future, we hope to adapt this method to assist in determining whether myeloid or lymphoid directed therapy should be used for leukemias with ambiguous lineage characteristics.
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Levine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.
Acute Myeloid Leukemia (AML) in patients over the age of 60 (elderly AML) remains a clinical challenge. Despite advances in the field, outcomes for elderly AML patients remains poor regardless of ...treatment administered. We hypothesize that elderly AML is characterized by the acquisition of somatic mutations that enhance leukemogenic potential and associate with worse clinical outcomes. To test this hypothesis, we determined the landscape of somatic mutations in de novo AML patient samples from the phase III prospective ECOG-ACRIN Cancer Research Group clinical trial 3999 (E3999; NCT00046930; ages 60-93). DNA was isolated from patient specimens processed to isolate blast-enriched cells (CD3 and CD19 depletion using Miltenyi magnetic beads) and patient-matched CD3-positive cells (flow cytometry) as germline controls. Exome capture was performed in paired disease and germline samples from 210 patients. Samples were sequenced on a HiSeq 2500 obtaining a median coverage of 150x bases per position. Low-coverage whole genome sequencing was used to identify copy number alterations (CNAs) in 202 of the samples. Indexed Illumina libraries were sequenced to a coverage of approximately 3 million reads per sample.
The Cancer Genome Project pipeline guidelines were utilized to determine somatic mutation events in the whole exome data. We aligned the reads with BWA mem, determined somatic copy number aberrations using CNVKit, short insertions and deletions (including FLT3 Internal Tandem Duplication) using Pindel, and substitutions using CaVEMan. We then used classical heuristics including strand bias, mapping quality and high rate of presence in the ExAC database to remove artifacts and non-somatic variants. Finally, we used COSMIC and published data in the literature to triage events into known oncogenic events, unknown but likely oncogenic events and variants of unknown significance. CNAs were determined from the whole genome sequencing data by partitioning the human genome into 60 thousands bins (i.e. resolution of 50kb) and counting uniquely mapped reads within the genomic bins/intervals. Read counts within bins were subsequently processed and transformed into integer copy number states.
We found a range of 1 to 37 mutational events per sample (median of 15), including 0 to 7 oncogenic or likely oncogenic events (median of 2). Most commonly mutated genes were NPM1 (25.7%); epigenetic modifier genes: TET2 (24.7%), DNMT3A (20.5%), ASXL1 (12.9%), and IDH2 (11.4%); FLT3 (20%); spliceosome complex genes: SRSF2 (19.5%), U2AF1 (10%) and STAG2 (10%); RUNX1 (15.7%); TP53 (12.4%), and NRAS (11.9%). Comparing our AML cohort to the mutation landscape reported by Papaemmanuil et. al., NEJM 2016 (age range: 18 to 84 years), we found that the elderly group was enriched in oncogenic mutations in the SRSF2, TET2, U2AF1 and ASXL1 and depleted for IDH1 and FLT3 (Fisher exact test adjusted p-value <0.05) genes. Furthermore, the elderly AML patient cohort was enriched in spliceosome/cohesin modifier and TP53-complex karyotype subtypes (Fisher exact test: p<2.8*10^-8 and p<10^-5 respectively).
Detailed analyses of the mutational spectra in the E3999 clinical trial suggests enrichment for mutations in epigenetic modifiers, spliceosome factors, and cytogenetic events associated with poor prognosis in AML. These may be underlying the aggressive phenotype in this age range and contribute to disease pathogenesis.
Authors contributed equally: Ari Melnick and Ross Levine
Co-corresponding authors: Maria Kleppe and Francine E. Garrett-Bakelman
Lowe:Mirimus Inc.: Consultancy. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.
Abstract
The purpose of this study is to identify key regulatory pathways that potentially drive abnormal gene expression program in relapsed Acute Myeloid Leukemia (AML) patients, by integrative ...computational analyses on multi-omics molecular profiles. Relapsed AML remains a clinical challenge. Epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML. However, what specific transcriptional programs and potential regulatory mechanisms contribute to disease relapse are not yet well understood. To characterize the global transcriptional landscapes in relapsed AML, we integrated genomics data from two cohorts of matched diagnosis and relapse patient specimens. We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in Cohort I. Unsupervised clustering yielded three distinct DEG groups: group A, B and C genes that were predominantly (88%) down-regulated, divergently regulated, or predominantly (65%) up-regulated, respectively, upon relapse. The expression pattern of all DEGs separated the patients into two clusters, most robustly by Group B genes. Interestingly, the majority of DEGs did not associate with changes in gene promoter methylation. Similar patterns were observed in Cohort II. We used Binding Analysis for Regulation of Transcription (BART) to identify transcriptional regulators (TRs) that potentially regulated the DEGs not associated with DNA methylation changes, and assessed the differential expression of identified TRs during disease progression. PU.1 was identified as a potential TR for Group A genes and was down-regulated upon relapse. GATA1 and TAL1 were identified as regulating Group B genes and were up-regulated in patient cluster1 and down-regulated in cluster2, consistent with the expression pattern of Group B genes. RBBP5 was a top predicted TR for Group C genes and was up-regulated upon relapse. We next validated the potential functionality of those predicted factors. In NSG mice transplanted with a human AML specimen, TAL1 and GATA1 were downregulated in AML cells collected four weeks after chemotherapy treatment, and were inferred as TRs for the down-regulated genes, similar to the patient data. PU.1 was inferred as regulating the up-regulated genes. Furthermore, we found that the level of differential expression of TAL1, GATA1, and PU.1 in each patient specimen associated with the correlation of DEG profiles between the patient specimen and TR perturbation in human-derived hematopoietic cell lines. Our results support the possibility that in some AML patients, TRs with roles in hematopoiesis and leukemia might contribute to disease relapse. Further mechanistic studies deciphering the molecular and phenotypic events facilitated by these TRs will yield significant insight into disease biology and possible therapeutic targeting approaches in relapsed AML.
Citation Format: Zhenjia Wang, Yaseswini Neelamraju, Cem Meydan, Nicholas Dunham, Jorge Gandara, Tak Lee, Subhash Prajapati, Franck Rapaport, Caroline Sheridan, Paul Zumbo, Michael Becker, Lars Bullinger, Martin Carroll, Richard D’Andrea, Richard Dillon, Ross Levine, Christopher E. Mason, Ari Melnick, Donna Neuberg, Stefan Bekiranov, Chongzhi Zang, Francine E. Garrett-Bakelman. Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3155.
We have previously reported that hypermethylationof the GADD45A promoter (GADD45AmeHI) occurs frequently in AML at a specific CpG residue (CpG1) and associates with poor overall survival for patients ...on standard chemotherapy (Perugini et al, Leukemia 2013). Sequenom multiplex analysis of 195 AML patients revealed a co-occurrence of GADD45AmeHI with recurrent mutations at conserved residues in IDH1 and IDH2 (p<0.0001, Fisher’s exact test). These mutations in IDH1 and IDH2 result in enzyme isoforms that produce high levels of the onco-metabolite 2-hydroxyglutarate with a wide-range of effects including inhibition of α-KG-dependent dioxygenases and association with a profound DNA hypermethylation phenotype in AML (Figueroa et al, Cancer Cell 2010). Furthermore these mutations are found in pre-leukemic AML clones (Shlush et al, Nature 2014) and lead to pre-leukaemic phenotypes in mouse models (Sasaki et al, Nature 2012, Kats et al, Cell Stem Cell 2014, Ogawara et al, Cancer Research 2015). Here we investigated the relationship between hypermethylation at GADD45A CpG1, IDH1/2 mutation status, global methylation patterns and patient survival.
We performed survival analysis to determine disease-free survival (DFS) and relapse-free survival (RFS) for AML patients with GADD45AmeHI or IDH1/2-mutations. This showed that GADD45AmeHI is a significant independent predictor of poor DFS and RFS, particularly in normal karyotype AML (Cox regression analysis, NK-AML DFS, P=0.009 HR=2.55, RFS, P=0.003 HR=2.75). Despite the co-association of GADD45AmeHI with mutations in IDH1 and IDH2, the mutation status of IDH1/2 did not predict DFS or RFS in these patients. To examine further the relationship between GADD45AmeHI and IDH1/2-mutation, and to investigate how this might influence tumour cell biology in AML, we determined global methylation patterns for a panel of AML diagnosis (Dx) samples (base-pair-resolution analysis using enhanced reduced representation bisulfite sequencing; ERRBS) in which both GADD45AmeHI and IDH mutation status has been determined. Unsupervised analyses of global methylation patterns grouped the AML Dx samples into three clusters including cluster 1 (n=12) which was associated with GADD45AmeHI samples with IDH- mutations, cluster 2 (n=13) which was enriched for GADD45AmeHI lacking IDH- mutations, and cluster 3 (n=9) which was associated with GADD45AmeLO (low CpG1 methylation) IDH-WT AML. We propose that this CpG in the GADD45A promoter may be subject to alternative events affecting DNA methylation in AML pathogenesis, including events distinct from IDH1/2 mutation. Finally, in GADD45AmeHI AML we detected hypermethylated regions compared to CD34+ normal bone marrow controls within 2016 gene promoters, 848 of which were unique to the GADD45AmeHI samples and not present in IDH1/2-mutant samples. We hypothesize that these differentially methylated genes may contribute mechanistically to the poor survival observed for this subtype.
To determine how GADD45AmeHI status might associate with disease progression, DNA methylation assessment was performed on the patient panel-matched relapse samples (Rx). While GADD45AmeHI occurs frequently in both cluster 1 and 2 there is a significant difference in level of GADD45A CpG1 methylation at Dx and Rx for samples in cluster 1 vs cluster 2 and 3 (Figure 1), consistent with mutant IDH1/2 activity influencing methylation levels at this CpG site. Given that GADD45A has an established basal role in the maintenance of genomic stability (Liebermann & Hoffman, Springer 2013), and is a determinant of HSC self-renewal and response to genotoxic insult (Wingert et al, Stem Cells 2016, Chen et al, Blood 2014) we are also investigating whether GADD45A methylation and silencing plays a direct role in determining aggressiveness and response to chemotherapy for GADD45AmeHIAML.
In conclusion this data suggests that methylation at this specific CpGof the GADD45A promoter, in combination with IDH1/2 mutation status, associate with varying global methylation phenotypes. Importantly, we demonstrate that GADD45AmeHI better predicts poorer prognosis than IDH1/2 mutation status, despite the significant co-association of these characteristics in AML.
SES and FEGB contributed equally to this work.
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Guzman:Cellectis: Research Funding. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Melnick:Janssen: Research Funding.
Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Unfortunately, a significant proportion of patients relapse after responding to initial treatment reflecting our poor ...understanding of the mechanisms mediating therapy resistance and relapse. We hypothesized that understanding the evolution of the mutational landscape between diagnosis and relapse is essential in order to identify mutational markers associated with sensitivity or resistance to treatment. To address this hypothesis we assembled a cohort of 53 clinically annotated, paired AML patient samples (diagnosis, relapse and patient-matched germline samples; mean age = 52 years). All patients achieved clinical remission after treatment with combination chemotherapy (cytarabine arabinoside and an anthracycline) during induction phase followed by consolidation chemotherapy treatment with or without a stem cell transplantation in first remission. Serial samples were collected at the time of initial diagnosis and within three months of relapse (mean time to relapse 455 days).
We performed whole-exome and targeted capture followed by high-throughput sequencing. We aligned samples with BWA, recalibrated them with The Genome Analysis Toolkit (GATK) and then compiled integrated calls from substitution and indel callers (Mutect, Scalpel, Strelka, Varscan and Somatic Sniper). We performed several layers of post-processing filtering on these calls, including removing non-oncogenic mutations and previously documented non-somatic variants, and correcting for the variant allele fraction of indel calls. We filtered out the variants that were found to occur in non-copy number neutral re-arrangements using the clinically determined cytogenetic data. Furthermore, we assessed for copy number events, including loss of heterozygosity events, and for the presence and the variant allele frequency of the FLT3-ITD in our samples.
We observed a median of 4.5 and 5 mutations per patient at diagnosis and relapse, respectively, with 3.5 mutations being shared by paired diagnosis and relapse samples. When limiting our analysis to genes previously shown to contribute to leukemogenesis, we found a median of 1.5 and 2 mutations per patient at diagnosis and relapse, with 1 mutation being shared. FLT3, DNMT3A, IDH2, NRAS, RUNX1 and TET2 were among the most commonly mutated genes, with a detected presence rate of 28%, 25%, 19%, 19%, 11% and 11%, respectively, in the diagnosis samples and 39%, 23%, 19%, 4%, 13% and 11% in the relapse samples. We identified significant variation in the variant allele frequency (VAF) for several of the mutations related to these genes and others, denoting variations in the cellular prevalence of the related clones after adjustment for tumor content using the mutations with the highest VAF to delineate clonal architecture. Specifically, we observed that DNMT3A, IDH2, TET2 variants are most commonly present in the bulk AML clone, and persist after treatment. WT1, GATA2 and FLT3mutations are predicted to confer relative resistance to standard combination chemotherapy treatment based on their increased VAF at relapse, whereas KRAS and NRAS subclone(s) are more sensitive to chemotherapy since their VAFs decrease following multiagent chemotherapy. Fifteen patients presented new events in leukemogenesis-related genes at relapse.
Overall, our results support a model of AML as a disease with a complex mutational hierarchy and clonal architecture and provide further insight into how these change in response to standard induction therapy. Our data suggests that future efforts to develop targeted therapies with maximal clinical benefit in combination with standard induction treatments should be placed on mutated genes identified to be more strongly associated with disease relapse.
Authors contributed equally: F. Rapaport and M.R. De Massy
Authors contributed equally: A. al Hinai and M.A. Sanders
Guzman:Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Melnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.
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