Cytogenetically Normal Acute Myeloid Leukemia Pts Research Articles

Recurrent Genetic Variants of Long Non-Coding RNAs (lncRNAs) Independently Associate with Clinical Outcome of Younger Adults with Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUNDAberrant expression of lncRNAs has been shown to independently associate with clinical outcome of younger adult patients (pts) [aged <60 years (y); Papaioannou et al. Haematologica 2017;102:1391-1400] and older pts (aged ≥60 y; Garzon et al. PNAS 2014;111:18679-84) with cytogenetically normal acute myeloid leukemia (CN-AML). However, the prognostic significance of genetic variants (single nucleotide polymorphisms or somatic mutations) in lncRNAs and their association with established prognostic gene mutations have not been studied. METHODSWe performed whole transcriptome profiling (RNA-seq) in 377 younger adults with de novo CN-AML and investigated the associations of recurrent genetic variants located within expressed lncRNAs with clinical outcome and gene mutations. All pts were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology protocols. Analysis of 11 gene mutations, reported to be prognostic in AML, was performed by targeted amplicon sequencing (ASXL1, DNMT3A, FLT3 -TKD, IDH2, IDH1, NPM1, RUNX1, TET2, WT1), Sanger sequencing (double CEBPA) and fragment analysis (FLT3 -ITD). RESULTSWe first generated a comprehensive list of the genetic variants that are located in lncRNAs in transcriptomes of younger CN-AML pts. To analyze unequivocally non-coding genetic variants and to avoid ambiguity in their genomic location, all variants that overlapped exons of protein coding genes and those that mapped to segmental duplications or repeat masked regions of the genome were excluded from further analyses. We focused on 207 variants whose frequency differed between our AML cohort and the healthy population, and on 88 variants that were novel. Of the variants tested, 8 showed significant association with at least 1 outcome endpoint [disease-free (DFS), overall (OS) or event-free (EFS) survival], and 3 of them significantly correlated with DFS, OS and EFS. There was no association between complete remission rates and lncRNA variants. The 3 variants which associated with all survival endpoints were: a thymidine (T)-to-cytosine (C) variant in lncRNA RP5-1074L1.4, an adenosine (A)-to-C variant in lncRNA HCG4, and a C-to-T variant in lncRNA HLA-V . The C variant of RP5-1074L1.4 was detected in 182 (64%) of the 284 pts who expressed the lncRNA and associated with longer DFS (P <.001; 5-y DFS, 41% v 27%), OS (P=.04; 5-y OS, 40% v 35%) and EFS (P <.001; 5-y EFS, 36% v 22%). The C variant of HCG4 was found in 41 (18%) of the 234 pts with detectable expression of the lncRNA. Pts harboring the C variant had longer DFS (P=.01; 5-y DFS, 61% v 35%), OS (P=.02; 5-y OS, 58% v 39%) and EFS (P=.04; 5-year EFS, 49% v 29%). Finally, the T variant of HLA-V, which was detected in 58 (26%) of the 227 pts who expressed the transcript, associated with longer DFS (P=.005; 5-y DFS, 53% v 30%), OS (P=.01; 5-y OS, 55% v 33%) and EFS (P=.007; 5-y EFS, 45% v 24%).With regard to associations with prognostic gene mutations, pts with the C variant of RP5-1074L1.4 were more likely to harbor FLT3 -TKD than pts with the T variant (P=.02). Pts with the T variant in HLA-V were less likely to harbor RUNX1 mutations than pts with the C variant (P=.02). The C variant of HCG4 did not associate with differences in the frequencies of prognostic mutations.In multivariable analyses, the variables considered in the models (other than the prognostic lncRNA variants) were: white blood cell and platelet counts, hemoglobin, extramedullary involvement, the presence of FLT3 -TKD, FLT3 -ITD and CEBPA, DNMT3A, NPM1, RUNX1 and WT1 mutations, and expression levels of MN1 and miR-155. The C variant of RP5-1074L1.4 was an independent marker for longer DFS (HR: 0.53, P <.001), OS (HR: 0.64, P=.007) and EFS (HR: 0.55, P <.001). The C variant of HCG4 independently associated with longer DFS (HR: 0.52, P=.03) and OS (HR: 0.61, P=.05), whereas the T variant of HLA-V independently associated with longer DFS (HR: 0.46, P=.003), OS (HR: 0.38, P <.001) and EFS (HR: 0.48, P=.001).PCR assays and targeted Sanger sequencing experiments in leukemic blasts and paired remission bone marrow samples are currently being conducted to examine whether the prognostic variants are acquired genetic events or germline variants. CONCLUSIONSWe conclude that a small subset of genetic variants that are located within lncRNAs are clinically relevant and provide independent prognostic information in younger adults with CN-AML. DisclosuresBlum:Astellas: Consultancy; Pfizer: Consultancy; Boerhinger Ingelheim: Research Funding. Uy:Boehringer Ingelheim: Consultancy; Novartis: Consultancy, Other: Travel Suppport; GlycoMimetics: Consultancy. Stone:Celgene: Consultancy; Novartis: Consultancy; Fuji Film: Consultancy; Ono: Consultancy; Agios: Consultancy; Astellas: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Jazz: Consultancy; Sumitomo: Consultancy; Pfizer: Consultancy; Arog: Consultancy. Byrd:Acerta Pharma: Research Funding; Janssen: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Pharmacyclics: Research Funding.

Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Distinct Gene Expression Profiles and Mutations Associate with Outcome in Younger Adults with De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) (Alliance)

Distinct Gene Expression Profiles and Mutations Associate with Outcome in Younger Adults with De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) (Alliance)

Prognostic and Biologic Significance of Transfer RNA-Derived Small RNAs (tsRNAs) Expression in Younger Adult Patients (Pts) with Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Introduction: tsRNAs constitute a novel class of small non-coding RNAs (~18-40 nucleotides), which are generated during stress-induced cleavage or the maturation processes of transfer RNAs (tRNAs). This class of RNA has been recently shown (Balatti et al. PNAS 2017;114:8071; Kim et al. Nature 2017;552:57) to be dysregulated in some forms of cancer (i.e., chronic lymphocytic leukemia, lung, colon, breast and ovarian cancer). However, to our knowledge, the prognostic value and biologic implications of tsRNA expression in AML pts have not been previously studied.Aims: The aims of our study were to determine whether tsRNA expression associates with pretreatment characteristics and clinical outcome of younger [aged <60 years (y)] adults with de novo CN-AML, and to gain biological insights into the functional role of tsRNA expression in AML.Methods: We conducted small RNAseq in a training set (n=208; median age, 48 y; range, 18-59 y) and a validation set (n=90; median age, 45 y; range 17-59 y) of younger adults with de novo CN-AML. None of the clinical or molecular parameters analyzed in this study were significantly different between the two sets. All pts were treated on frontline Cancer and Leukemia Group B/Alliance protocols. To obtain the expression of tsRNAs, we applied a bioinformatics workflow to small RNAseq data of a cohort of 20 CN-AML pts, in association with data, which were collected via custom array assay (Pekarsky et al. PNAS 2016;113:5071).Results: We obtained the expression data of 136 tsRNAs in both sets. Next, we derived a signature (3-tsRNA) composed of 3 tsRNAs (tsRNA20, tsRNA64, and tsRNA66), which were associated with event-free survival at the significance level of P<.001. We used the 3rd quartile as a cutoff in each set to discriminate between pts with a 3-tsRNAlow (1st to 3rd quartile) and those with 3-tsRNAhigh (4th quartile) score. With respect to clinical outcome, pts with 3-tsRNAhigh score had a worse disease-free survival (DFS; 5-y rates, 23% v 43%; P=.01) and overall survival (OS; 5-y rates, 25% v 45%; P=.003) in the training set (Figure 1). These results were confirmed in the validation set (5-y DFS rates, 12% v 50%; P=.003; 5-y OS rates, 18% v 58%; P=.002). In multivariable analyses, 3-tsRNAhigh score independently associated with shorter DFS (P=.02; HR: 2.34) and OS (P=.03; HR: 1.98) after adjusting for other co-variates [i.e., internal tandem duplication of the FLT3 gene (FLT3-ITD) and MN1 expression].Regarding pretreatment clinical and molecular features in the training set, pts with a 3-tsRNAhigh score had a higher percent of bone marrow blasts (P=.03) and tended to have a higher percent of blood blasts (P=.06). 3-tsRNAhigh scorers had a higher frequency of FLT3-ITD (P=.01) and DNMT3A non-R882 mutations (P=.03), and were marginally more likely to have an ASXL1 (P=.06) or RUNX1 mutation (P=.06). Pts with 3-tsRNAhigh score were less likely to harbor NPM1 mutations (P=.03).To gain initial insights into the biological significance of tsRNA expression in AML, we performed loss of function experiments and knocked-down (KD) all 3 tsRNAs using customized single strand RNA-inhibitors. Of the tsRNAs tested, only KD of tsRNA20 and tsRNA66 decreased the proliferative capacity of THP-1 and OCI-AML3 cells, as measured by MTS reagent degradation (tsRNA20: P=.002 and P=.04, respectively; tsRNA66: P=.009 and P=.07, respectively). Additionally, these cell lines showed higher frequency of apoptotic cells 48 hours after KD (tsRNA20: P=.007 and P=.008, respectively; tsRNA66: P<.001 and P=.009, respectively).Conclusion: We conclude that tsRNAs represent a novel prognostic and biologically important class of non-coding RNAs in CN-AML. DisclosuresPowell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Uy:Curis: Consultancy; GlycoMimetics: Consultancy. Kolitz:Magellan Health: Consultancy, Honoraria. Wang:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Jazz: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau.

Differential Clinical Impact Of Gene Mutations and Their Combinations In Primary Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

In CN-AML, mutations of specific genes have prognostic significance and are used for treatment guidance. However, these mutations are in general not mutually exclusive and CN-AML patients (pts) frequently harbor mutational combinations with unclear prognostic significance because of the concurrent presence of mutations with favorable (CEBPA, NPM1) and unfavorable (ASXL1, DNMT3A, IDH1, IDH2, FLT3-ITD, MLL-PTD, RUNX1, TET2, WT1) impact on outcome. Here, we report the frequency and clinical significance of isolated mutations and their combinations in 364 CN-AML pts. Younger [<60 years (y); n=163] and older (≥60 y; n=201) pts were intensively treated on frontline CALGB/Alliance protocols and, per protocol, did not undergo allogeneic stem cell transplantation in first complete remission (CR). Median follow-up for pts alive was 8.7 y. Pts were analyzed centrally for mutations in the ASXL1, CEBPA, DNMT3A, IDH1, IDH2, NPM1, RUNX1, TET2, and WT1 genes and the presence of FLT3-ITD, FLT3-TKD and MLL-PTD. We found that 99% of pts had ≥1 and 88% ≥2 of the 12 mutations. Specifically, 11% of pts had 1 gene mutated (n1), 40% had 2 (n2), 31% 3 (n3), and 18% harbored ≥4 mutated genes (n4). The distribution of the different n groups in younger and older pts was similar (Fig 1). The expected frequency of each mutated gene in every numerical (n) group was calculated as follows: [no. of pts with the mutated gene] x [total no. of mutations in the n group] / [total no. of mutations in all pts], and compared with the observed frequency. CEBPA, IDH2, NPM1, and RUNX1 mutations were overrepresented in the n1 and n2 groups (P<.001, P<.001, P=.002, P=.02, respectively) and thus likely represent “early” events in leukemogenesis. In contrast, DNMT3A, FLT3-ITD, and TET2 mutations were more frequent in the n3 and n4 groups (P=.008, P<.001, P=.02, respectively) suggesting that they are likely “late” events. The distribution among age groups and clinical impact of the “early” mutations (i.e., CEBPA, NPM1, IDH2) presenting as isolated mutations or in combination with other mutations were then investigated. Isolated RUNX1 mutations could not be evaluated due to small sample size. Younger pts were more likely to harbor an isolated “early” mutated gene than older pts (21% v 12%, P=.03). Specifically, isolated CEBPA and NPM1 mutations were more frequent in younger pts compared with the older (7% v 2%, P=.007; 7% v 3%, P=.07, respectively). In pts with isolated CEBPA mutations, these were always bi-allelic. Single “early” mutated genes varied in their impact on CR achievement (CEBPA: 100%, NPM1: 94%, IDH2: 67%), and overall survival (OS; 3-y OS: CEBPA: 87%, NPM1: 69%, IDH2: 22%). Acquisition of additional mutations adversely modified the prognostic significance of favorable “early” mutations in the CEBPA (Fig 2A) and NPM1 (Fig 2B) genes, but left unchanged the unfavorable effect of IDH2 mutations (Fig 2C). Two mutational combinations were exceptions to this: younger pts with both NPM1 and DNMT3A mutations (8 pts) had an excellent prognosis with 3-y OS of 100%; and older pts with concurrent NPM1 and IDH2 mutations (9 pts; all IDH2 mutations were in codon R140) had a high 3-y OS rate of 67%. We conclude that CEBPA and NPM1 mutations mainly define pts with favorable prognosis when they present as single markers, whereas mutated IDH2 predominantly associates with adverse outcome. The NPM1+DNMT3A and NPM1+IDH2 mutational patterns may define novel favorable molecular subtypes in younger and older CN-AML pts, respectively, but these results require corroboration in larger pt cohorts. [Display omitted] [Display omitted] Disclosures:Mrózek:AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 611 category Other. Eisfeld:AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 608 category Other.

Abstract 1307: Heritable polymorphism predisposes to high expression of BAALC in cytogenetically normal acute myeloid leukemia (CN-AML)

Abstract Introduction: Risk-adapted therapy of AML patients (pts) is presently based on cytogenetic and molecular findings. However, the outcome remains poor for the majority of pts. Overexpression of BAALC is implicated in leukemogenesis and poor survival of CN-AML pts, but the underlying mechanisms are unknown. We hypothesized that the elevated expression of BAALC might result from a genetic variant in or close to the gene (in cis). Methods and Results: BAALC and RUNX1 expression were measured in pre-therapy samples from a test set of 253 CN-AML pts (Cancer And Leukemia Group B) using Affymetrix U133 plus 2.0 arrays. Pts were grouped as high or low BAALC and high or low RUNX1 expressers using the median expression value as the cut-off and screened for single nucleotide polymorphisms (SNPs) by direct sequencing of the BAALC gene. Nine informative SNPs were identified. By genotyping, marker rs62527607, a non-coding SNP in the promoter region correlated with high BAALC expression (rs62527607G&amp;gt;T: genotypes TT/GT vs. GG: P=2.01E-04). Luciferase reporter constructs (pGL4.11) spanning the respective alleles demonstrated that the T allele (“risk allele”) of rs62527607 increased luciferase activity compared to the G allele (P=1.0E-02). The T allele of rs62527607 is predicted to create a binding site for the transcription factor RUNX1. Indeed, RUNX1-cotransfection showed an increase in luciferase activity for the T allele of rs62527607 (P=2.0E-02) but not for the G allele (P=2.7E-01). An Electrophoretic Mobility Shift Assay comparing the binding activity of the two alleles showed highly increased binding activity of RUNX1 to the T allele vs. the G allele. Testing of the pt samples for a potential correlation of RUNX1 and BAALC expression levels revealed a positive correlation in pts with genotypes TT/GT but not GG when restricting the analysis to the high RUNX1 expressing group (P=1.18E-04). For validation of our findings, a set of 105 CN-AML pts comprising 52 high and 53 low BAALC expressers (AML Study Group [AMLSG], quantitative Real-Time PCR [qPCR]) was genotyped for rs62527607, determined for RUNX1 expresser status (qPCR) and analyzed alone and in combination with the test set. A non-significant association of TT/GT vs. GG with high BAALC expression (31% vs. 19%; P=1.6E-01) was seen. Analyzing both sets combined resulted in a P-value of 9.3E-05 (Odds Ratio [OR]=2.59 [1.62, 4.22; 95%CI]). Combining the results of the CALGB &amp; AMLSG series showed a highly significant association between RUNX1 and BAALC expression in pts with genotypes TT/GT belonging to the high RUNX1 expressing group (P=7.97E-05, OR=4.02 [2.05, 8.21; 95%CI]). Conclusion: We show that the risk allele of rs62527607 creates a RUNX1 binding site, thereby leading to increased transcription of BAALC. The observation that a heritable trait is involved in the leukemogenesis process might initiate a new era of personalized medicine in AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1307. doi:1538-7445.AM2012-1307

MiR-3151, a Novel MicroRNA Embedded in BAALC, Is Only Weakly Co-Expressed with Its Host Gene and Independently Impacts on the Clinical Outcome of Older Patients (Pts) with De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Abstract 1462Despite progress made in understanding the biology and risk-adapted treatment of AML, prognosis of older pts [age ≥60 years (y)] remains poor. We have shown that high expression of the BAALC gene bestows worse outcome in older CN-AML pts. Yet, the mechanism(s) by which BAALC affects response to therapy remain unknown. MicroRNAs (miRs) are small noncoding RNAs that hybridize to target mRNAs and inhibit their translation or promote degradation, resulting in downregulation of the corresponding proteins. Recently, miR-3151 was discovered embedded in intron 1 of BAALC. We thus hypothesized that miR-3151 might be co-expressed with BAALC and contributes alone or in concert with its host to poor prognostic impact in older CN-AML. MiR-3151 and BAALC expression were measured by quantitative real time (RT) PCR in pretreatment blood of 179 older CN-AML pts enrolled on Cancer and Leukemia Group B (CALGB) cytarabine/daunorubicin-based protocols. The expression values of miR-3151 and BAALC were normalized to the internal controls SNORD44 and RN18S1, respectively. MiR-3151 expression correlated only weakly with the expression of BAALC (Spearman correlation r= 0.3). For clinical and prognostic analyses pts were dichotomized into high and low miR-3151 and BAALC expressers at the median miR and gene transcript expression values and analyzed for other molecular prognosticators (FLT3 -ITD, FLT3 -TKD, CEBPA, IDH1, IDH2, NPM1, TET2, WT1 mutations and ERG expression). Gene (GEP) and microRNA (MEP) expression profiles associated with miR-3151 expression were derived, using Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays, respectively. At diagnosis, higher miR-3151 expression was associated with lower % blood blasts (P =.02), FAB types M4 and M5 (P =.05), and wild-type NPM1 (P<. 001). High miR-3151 expressers had a lower complete remission (CR) rate (62% v 81%, P=. 005), and, with a median follow-up of 5.1 y for pts alive, shorter disease-free (DFS; hazard ratio [HR]=1.76, P=. 003; 3y rates, 7% v 26%; Figure 1A) and overall survival (OS; HR=1.86, P <.001; 3y rates, 10% v 32%; Figure 1B) than low expressers. In multivariable analyses (MVA), higher miR-3151 expression no longer remained in the model for CR after adjusting for BAALC expression and white blood count (WBC), but higher miR-3151 expression remained associated with shorter DFS (HR=2.32, P <.001), after adjusting for FLT3 -TKD, ERG expression status and WBC, and shorter OS (HR=1.77, P=. 003), after adjusting for ERG and BAALC expression status. To gain biological insights of miR-3151 -associated AML, we derived a GEP comprising 377 annotated genes and a MEP comprising 14 miRs. High miR-3151 expressers exhibited upregulation of genes associated with immature differentiation stage and adverse outcome (eg, MN1, ID1), downregulation of genes involved in hematopoietic differentiation (eg, MEIS1, PDCD4, PBX3) and transcriptional regulators (ZNF s, E2F3), including predicted targets of miR-3151 (eg, MEIS1). In the MEP, high miR-3151 expressers showed downregulation of let-7a/b/c that play crucial roles in cell cycle and proliferation regulation. While miR-3151- associated GEP/MEP shared genes with BAALC expression-associated GEP/MEP (eg, upregulated MN1 and DNTT and downregulated MEIS1, let-7b, miR-10a/b), other genes were unique to miR-3151- associated GEP/MEP (eg, upregulated ID1 and downregulated ZNFs, miR-206 and let-7a/c), suggesting biologic mechanisms distinct from those directly connected with BAALC upregulation operating in miR-3151- associated leukemia. In conclusion, high miR-3151 expression is independently associated with worse survival in older CN-AML pts. The weak correlation between expression of miR-3151 and its host gene BAALC, the independent impact of each on outcome endpoints (BAALC was a strong predictor of CR but did not predict DFS while miR-3151 was not independently associated with CR, but remained in the DFS MVA) and differences in GEP/MEP suggest that miR-3151 and BAALC are deregulated and contribute to disease outcome differently. We surmise that determining the expression levels of miR-3151 at diagnosis might help to improve the risk-stratification of older CN-AML. Development of therapies targeting miR-3151 upregulation with synthetic inhibitors may provide novel, effective strategies for personalized treatment of older CN-AML pts. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Poor Outcome of RUNX1-Mutated (RUNX1-mut) Patients (Pts) with Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) and Associated Gene- and MicroRNA (miR) Expression Signatures,

Abstract 3454RUNX1 encodes the α subunit of core binding factor, a heterodimeric transcription factor required for normal hematopoiesis. Acquired RUNX1 mutations (muts) have been associated with poor clinical outcome in AML; however, prior studies analyzed pts heterogeneous for cytogenetics, age, AML type (primary or secondary), and treatment received [including allogeneic stem cell transplant (alloSCT) in 1st complete remission (CR1)] and contained limited data regarding the potential molecular drivers of the worse outcome. We report a relatively large study testing the prognostic impact of RUNX1 muts in primary CN-AML pts (n=392) treated similarly with intensive cytarabine/anthracycline-based 1st-line therapy and without alloSCT in CR1. This cohort comprised both younger [<60 years (y); n=173] and older (≥60 y; n=219) pts. Pretreatment marrow (n=303) and blood (n=89) were analyzed centrally for RUNX1 muts by PCR and direct sequencing, and for FLT3-ITD, FLT3-TKD, MLL-PTD and NPM1, CEBPA, WT1, IDH1, IDH2 and TET2 muts. Gene and miR expression profiles were derived using microarrays. RUNX1 muts were found in 12.5% of pts (8% younger, 16% older), and were associated with lower hemoglobin (P=.01), white blood cells (WBC; P=.04), and blood blasts (P=.006). RUNX1-mut pts harbored NPM1 (P<.001) and CEBPA muts (P=.06) less frequently than RUNX1-wild-type (RUNX1-wt) pts. RUNX1-mut pts had lower CR rates (P=.005 in younger; P=.006 in older), and shorter disease-free (DFS; P=.058 in younger; P<.001 in older), overall (OS; P=.003 in younger; P<.001 in older) and event-free (EFS; P<.001 for younger and older; Figures 1 and 2) survival than RUNX1-wt pts. In multivariable models, RUNX1 muts remained associated with lower CR rate (P<.001) and shorter DFS (P<.001), OS (P<.001), and EFS (P<.001; Table) after adjustment for clinical and molecular variables. [Display omitted] [Display omitted] Table 1Multivariable analysis for EFS according to RUNX1-mut status in all CN-AML ptsHREFSPRUNX1, mut v wt2.271.65–3.12<.001FLT3-ITD, ITD v no ITD1.571.27–1.95<.001WT1, mut v wt1.441.02–2.01.04WBC, continuous 50 unit increase1.131.04–1.23.006Age group, ≥60y v <60y1.801.46–2.22<.001Note: A hazard ratio (HR) >1 corresponds to a higher risk for higher values of continuous variables and the 1st level listed of a dichotomous variable.To gain biological insight, RUNX1 mut-associated gene and miR expression signatures were derived in CN-AML for the first time. Older, NPM1-wt pts were analyzed since RUNX1 muts are more common in this age group and are nearly exclusive from NPM1 muts, which have their own characteristic gene-expression signature. This yielded 484 probe sets representing 278 named genes differentially expressed between RUNX1-mut (n=31) and RUNX1-wt (n=45) pts (P<.001). Genes normally expressed in hematopoietic stem (HSC) and early progenitor cells, including DNTT, BAALC, MN-1, CD109, P2RY14, FOXO1 and FLT-3 were upregulated in RUNX1-mut pts, as were components of the Wnt-signaling pathway, LRP6 and TCF4, that promote self-renewal and proliferation of HSCs. Genes upregulated (SETBP1, RBPMS, and SLC37A3) and downregulated (CCNA1 and RNASE3) in AML stem cells relative to AML progenitors were similarly deregulated in the RUNX1-mut signature. B cell lineage genes BLNK, IGHM, IRF8 and several class II MHC molecules were upregulated in RUNX1-mut pts while CEBPA, a key promoter of granulopoiesis, was downregulated. Genes implicated in chemoresistance, GAS6, PRKCE, and PTK2, were upregulated and MYCN, a promoter of both proliferation and apoptosis of myeloid cells, was downregulated in RUNX1-mut pts. Seven miRs were differentially expressed between RUNX1-mut and RUNX1-wt pts. Two members of the let-7 tumor suppressor family, which represses self-renewal and promotes differentiation of stem cells, were downregulated, as was miR-223, a positive regulator of granulopoiesis. MiRs -99a and -100 were also downregulated and miRs -211 and -595 upregulated in association with RUNX1 muts.In summary, RUNX1 muts are twice as common in older CN-AML pts than younger. They negatively impact on outcome in both younger and older pts not receiving alloSCT in CR1. RUNX1-mut blasts have molecular features of normal/malignant stem cells and B cells, which may explain their chemoresistance and guide novel therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Mutations In the Tet Oncogene Family Member 2 (TET2) Gene Refine the New European LeukemiaNet Risk Classification of Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) In Adults: A Cancer and Leukemia Group B (CALGB) Study

AbstractAbstract 98Mutations in the TET2 gene were recently identified in a variety of myeloid neoplasms including AML. However, the frequency and clinical relevance of TET2 mutations in CN-AML, the largest cytogenetic subgroup of adult AML, have not been well defined. We report here the frequency and spectrum of TET2 mutations, and their associations with clinical and molecular characteristics, treatment outcomes and genome-wide gene- and microRNA (miR)-expression signatures in a relatively large cohort of 427 patients (pts) with primary CN-AML. The pts, aged 18–83 years, were intensively treated on CALGB frontline protocols, and were analyzed centrally for TET2 mutations by PCR and direct sequencing, and for other prognostic gene mutations (FLT3 internal tandem duplications [ITD] and tyrosine kinase domain mutations, MLL partial tandem duplications, and mutations in NPM1, CEBPA, WT1 and IDH1&IDH2). If available, buccal swabs or remission marrow samples were used to determine TET2 germline status. Gene- and miR-expression profiles were derived using microarrays (Affymetrix HG-U133 plus 2.0 and OSUCCC custom miR array v4.0). At least 1 sequence variation in TET2 was found in 104 pts. Frameshift (n=59) and nonsense (n=34) variations were distributed throughout all coding exons, while missense changes (n=37) clustered mainly (28/37) in 2 evolutionarily conserved domains of TET2. The remaining missense variations in 9 pts were located outside the conserved domains, and analysis of available buccal swabs or remission samples showed that these sequence changes were present in the germline. Since it is unclear whether they represent innocent polymorphisms or disease-relevant mutations, these 9 pts were excluded from further analyses. TET2-mutated (TET2-mut) pts were older (P<.001), had higher white blood counts (P=.04), a lower frequency of IDH1 and IDH2 mutations (P<.001), and showed a trend towards higher frequency of CEBPA mutations (P=.07) compared with TET2 wild-type (TET2-wt) pts. The European LeukemiaNet (ELN) recently proposed a standardized reporting system for AML, in which CN-AML pts are assigned to Favorable-risk (Fav; pts with mutated CEBPA and/or mutated NPM1 without FLT3-ITD) or Intermediate-I-risk (Int-I; all remaining CN-AML pts) categories. We assessed the prognostic relevance of TET2 mutations in the context of the Fav (n=199 pts) and Int-I (n=219) ELN categories. TET2 mutations tended to be more frequent in Fav than in Int-I CN-AML pts (27% v 19%, P=.08), even though types and location of mutations were similar in both groups. Within the Fav category, TET2-mut pts had shorter event-free survival (EFS; P<.001), a lower complete remission (CR) rate (P=.007) and shorter disease-free survival (DFS; P=.003; Fig 1), and shorter overall survival (OS; P=.001; Fig 2) compared with TET2-wt pts. In contrast, in the Int-I category, no difference in EFS (P=.45), CR rates (P=1.0), DFS (P=.36; Fig 1) or OS (P=.72; Fig 2) was found between TET2-mut and TET2-wt pts. In multivariable models, TET2 mutations associated with shorter EFS (P=.004; hazard ratio [HR], 1.71), lower CR rate (P=.03; odds ratio, 0.62) and shorter DFS (P=.049; HR, 1.54) only among Fav, but not among Int-I, CN-AML pts. A TET2 mutation-associated gene-expression signature consisting of 213 probe sets (136 named genes) was identified in ELN Fav CN-AML pts and included genes previously implicated in AML pathogenesis, e.g., upregulated CEBPA, APP, NCAM1 and IDH1, and downregulated MLL. In contrast, no signature of differentially expressed genes was identified in Int-I pts. miR profiling revealed distinct TET2 mutation-associated miR-expression signatures in the ELN Fav and Int-I risk groups. Among miRs upregulated in ELN Fav/TET2-mut pts were miR-148a (targeting DNA methyltransferases, highly expressed in refractory chronic lymphocytic leukemia) and miR-24 (stimulating myeloid cell proliferation, blocking granulocytic and erythroid differentiation). In Int-I/TET2-mut pts, one of the upregulated miRs was miR-204 (targeting HOXA10 and MEIS1, downregulated in NPM1-mut AML). We conclude that TET2 mutations are associated with lower remission rates and inferior survival in the ELN Fav category of CN-AML, and may be useful to refine the ELN molecular classification. TET2 mutation-associated gene- and miR-expression signatures, first identified here, may contribute to our understanding of the biology of TET2-mutated CN-AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 2717: Higher MN1 expression is an unfavorable prognosticator in older patients (Pts) with cytogenetically normal acute myeloid leukemia (CN-AML): A CALGB study

Abstract In younger (&amp;lt;60 y) adult CN-AML pts, high MN1 expression associates with adverse outcome. The prognostic impact of MN1 expression in older pts (&amp;gt;60 y) requires further evaluation. Pre-therapy bone marrow samples from 140 older de novo CN-AML pts (median age 68 y; range 60 – 81 y) enrolled on cytarabine/daunorubicin-based protocols were studied centrally by RT-PCR. Using the median expression value for MN1, as a cutoff pts were grouped as high &amp; low MN1 expressers. Analysis of the mutation status for FLT3-ITD, FLT3-TKD, NPM1, CEBPA &amp; WT1 &amp; gene &amp; microRNA (miR) expression profiling using, respectively, Affymetrix U133 plus 2.0 &amp; OSU CCC v4.0 arrays, were performed centrally. At diagnosis, higher MN1 associated with wild-type NPM1 (P&amp;lt;.001). Median follow-up was 4.0 y for pts alive. High MN1 expressers had lower complete remission (CR; 53% v 80%, P=.001), shorter event-free (EFS; P=.004; 3y 6% v 14%) &amp; overall survival (OS; P=.03; 3y 10% v 23%; Table) rates. In multivariable models, higher MN1 expression independently associated with a lower CR rate &amp; shorter EFS. Comparison of age groups (60-69 y v ≥70 y) revealed that MN1 expression had a stronger prognostic impact on pts ≥70 y (n=61; Table). Gene (294 genes) &amp; miR (21 miRs) expression signatures associated with high MN1 expression were derived. High MN1 pts had higher expression of precursor markers (CD34, PROM1, CD44, KIT, HLA-DRB1, DNTT), known adverse outcome predictors (BAALC, ERG, HGF), anti-apoptotic BCL2, &amp; miR-101 &amp; miR-126, which are linked to hematologic differentiation. Underexpressed were HOX genes, their co-regulators (MEIS1) &amp; HOX-gene embedded miRs (miR-10a, miR-10b). In conclusion, higher MN1 associated with adverse outcome in older CN-AML pts independently of other established prognosticators &amp; had a stronger impact on ≥70 y pts. The expression signatures provide novel information that may elucidate how MN1 contributes to leukemia &amp; help design new therapeutic approaches.Table.Outcomes according to MN1 expression of all pts and, separately, those age 60-69 y and ≥70yOutcome End PointsMN1 expressionAll Pts (n=140)Pts age 60-69 y (n=79)Pts age ≥70 y (n=61)HighLowPHighLowPHighLowP% achieving CR5380.0015480.035181.03EFS, %event-free at 3 y614.00467.11627.007OS, %alive at 3 y1023.031118.44931.006 The clinical endpoints significantly associated with MN1 expression levels are denoted by bold type.Pts were enrolled on CALGB protocols [CALGB 8525, 8923, 9420, 9720 &amp; 10201]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2717.

Increased Expression of Macrophage Migration Inhibitory Factor (MIF) Receptor CD74 Is Associated with Inferior Outcome in Younger Patients (Pts) with Cytogenetically Normal Acute Myeloid Leukemia (CN-AML): a Cancer and Leukemia Group B (CALGB) Study.

Abstract 1616Poster Board I-642CD74 is a type II integral membrane protein receptor that binds its ligand MIF to induce phosphorylation of the extracellular signal-regulated kinase-1/2 (ERK-1/2) and drive cellular proliferation via nuclear factor-kappa B (NF-kB) activation. CD74 expression has been identified in human solid tumors, and its expression is associated with adverse prognosis in advanced pancreatic cancer. As CD74 is expressed and NF-kB constitutively activated in myeloblasts, we hypothesized that CD74 expression might also be associated with adverse outcome in AML. To investigate the prognostic impact of CD74 expression in the context of other predictive molecular markers in CN-AML, we assessed CD74 expression levels by Affymetrix HG-U133 Plus 2.0 microarray in 102 younger [<60 years (y)] adults with primary CN-AML, treated on the front-line CALGB 19808 trial with an induction regimen containing daunorubicin, cytarabine, etoposide and, in some cases, the inhibitor of multidrug resistance valspodar, and consolidation with autologous stem cell transplantation. Microarray data were analyzed using the Robust Multichip Average method, making use of a GeneAnnot chip definition file, which resulted in a single probe-set measurement for CD74. At diagnosis, CD74 expression, when assessed as a continuous variable, was significantly associated only with extramedullary disease involvement (P=.006) among clinical features, and with none of the molecular prognostic variables tested, including NPM1, WT1, CEBPA, FLT3 (FLT3-ITD and FLT3-TKD) mutations, MLL partial tandem duplication, or differential BAALC and ERG expression levels. Although CD74 expression levels were not associated with achievement of complete remission (CR; 83% vs 81%), higher levels of CD74 were associated with shorter disease-free survival [DFS; P=.046, hazard ratio (HR) 1.85, 95% confidence interval (CI) 1.12-3.08] and with shorter overall survival (OS; P=.02, HR 1.32, CI 1.04-1.67). In multivariable analyses, higher CD74 expression was independently associated with shorter DFS (P=.045, HR 1.98, CI 1.16-3.40), after adjusting for WT1 mutations (P<.001) and FLT3-TKD (P=.04), and shorter OS (P=.01, HR 1.58, CI 1.11-2.25) after adjusting for FLT3-TKD (P=.02), WT1 mutations (P=.007), BAALC expression levels (P=.02), white blood counts (P=.007), and extramedullary involvement (P=.04). As quartiles 2-4 had similar expression levels distinct from the lowest quartile, to display the impact of CD74 expression levels on clinical outcome only, pts were dichotomized into low (the lowest quartile) and high (the top three quartiles) CD74 expressers. The Kaplan-Meier curves for DFS and OS (Figures 1 and 2) are shown below. In conclusion, our study identifies elevated CD74 expression as associated with adverse prognosis in younger CN-AML pts. Since we previously reported that higher CD74 expression was favorably associated with achievement of CR in AML patients receiving chemotherapy plus bortezomib, an inhibitor of the proteasome and NF-kB (Attar et al., Clin Cancer Res, 2008;14:1446-54), it is possible that in future studies elevated CD74 levels can be used not only for prognostication, but also to stratify CN-AML pts to study of bortezomib-containing chemotherapy regimens. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aberrant Gene Expression of BAALC and ERG in Older [≥60 Years (y)] De Novo Cytogenetically Normal Acute Myeloid Leukemia (CN-AML): A Cancer and Leukemia Group B (CALGB) Study.

Abstract 214BAALC &ERG are aberrantly expressed in younger (<60 y) adult CN-AML patients (pts), where expression levels of these genes are also associated with clinical outcome. Whether aberrant BAALC &ERG expression also occurs in older (≥60 y) CN-AML pts is unknown. To assess the BAALC &ERG expression levels in older CN-AML & their impact on outcome we studied pts ≥60 y (median age, 68 y; range 60–83) enrolled on cytarabine/daunorubicin-based protocols [CALGB 10201, 9720, 9420, 8923, 8525], with diagnostic blood samples available for quantitative RT-PCR analysis (n=158), that were also characterized for other molecular prognosticators (FLT3-ITD, FLT3-TKD, NPM1 &WT1 mutations). BAALC &ERG expression values were normalized to an internal control (ABL1), & the median gene expression value was used to define high & low expressers for BAALC &ERG. Gene (GEP) & microRNA (MEP) expression profiling were done using, respectively, Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays. At diagnosis, lower BAALC expression was associated with mutated NPM1 (P<.001) & low ERG expression (P<.001). Lower BAALC expressers had a higher complete remission rate (CR; 86% v 54%, P<.001) & longer disease-free (DFS; P=.006; 3y rates 19% v 12%) & overall survival (OS; P<.001; 3y 29% v 10%) than higher expressers. Lower ERG expression was associated with lower WBC (P=.005), % marrow (BM; P=.001) & blood (P<.001) blasts & absent FLT3-ITD (P<.001) & low BAALC expression (P<.001). Lower ERG expression also associated with longer DFS (P=.001; 3y 18% v 14%) & OS (P<.001; 3y 24% v 15%). In multivariable models (Table 1), low BAALC expression independently associated with CR & longer DFS. BAALC &ERG expression were the only factors associated with OS. Comparison of age-groups (60-69 y v ≥70 y; Table 2) showed BAALC expression had a stronger prognostic impact in ≥70 y pts; lower expression was associated with higher CR rates & longer DFS & OS. ERG expression had instead stronger prognostic impact in 60-69 y pts (Table 2); lower expression was associated with longer DFS & OS. GEP (482 probes) & MEP (22 probes) differentiated low from high BAALC expressers. Low BAALC expressers had down-regulation of stem cell markers (CD34, CD133) & unfavorable outcome predictors (HGF, MN1, CD200), & up-regulation of HOX genes &miR-10a &miR-10b. GEP (1554 probes) & MEP (11 probes) differentiating low from high ERG expressers showed low ERG expressers had down-regulation of DNMT3B & up-regulation of topoisomerase 1 (TOP1), which is associated with enhanced chemotherapy sensitivity. Among up-regulated microRNAs in low ERG expressers was miR-208a, which is predicted to target ERG. In conclusion, lower expression of both BAALC &ERG associated with better outcome in older CN-AML pts even in the context of other established prognostic molecular markers, but have different impact on age-groups. GEP & MEP provided novel information that may elucidate how differential expression levels of these genes contribute to leukemogenesis & aid in developing novel risk-adapted therapies.Table 1:Multivariable analyses for CR, DFS & OSVariables in the final modelCRDFSOSOR*PHR†PHR†PBAALC, low v high (median cut)4.32<.0010.64.030.49<.001‡ERG, low v high (median cut)----0.69.03WBC, each 50 unit increase0.68.02----NPM1, mutated v wild-type2.34.04----FLT3-ITD, any ITD v no ITD--1.79<.001‡--Age, each 10 year increase--0.65.047--*OR, odd ratio, >1 (<1) mean higher (lower) CR rate for the 1st category listed for the dichotomous variables & for the higher values of the continuous variables.†HR, hazard ratio, <1 (>1) indicate lower (higher) risk for an event for the first category listed for the dichotomous variables & for the higher values of the continuous variables. Variables considered in the models were those significant at α=0.20 in univariable analyses.‡Variable did not meet the proportional hazards assumption, a covariate was used to account for time dependence.Table 2:Age-Group comparison for CR, DFS & OSClinical endpointBAALC expressionERG expression60-69 y Pts (n=86)≥70 y Pts (n=72)60-69 y Pts (n=86)≥70 y Pts (n=72)LowHighPLowHighPLowHighPLowHighP% achieving CR8471.198837<.0018076.807153.15DFS, % in CR at 3 y1610.102314.03196<.0011725.48OS, % alive at 3 y2412.01358<.0012412.0062318.03The age-group clinical endpoints significantly associated with BAALC or ERG expression levels are denoted by bold type. Disclosures:No relevant conflicts of interest to declare.

Low Meningioma 1 (MN1) gene expression to predict outcome in cytogenetically normal acute myeloid leukemia (CN-AML): A Cancer and Leukemia Group B (CALGB) study

7011 Background: Expression of the MN1 gene, which encodes a transcription co-activator implicated in myeloid leukemogenesis, has been shown to predict outcome in CN-AML patients (pts) in 1 study (Heuser et al. Blood 2006;108:3898). Here we present the first validation of the prognostic impact of MN1 expression in CN-AML in the context of not only mutated (mut) FLT3 and NPM1 and MLL-partial tandem duplication but also other established prognostic markers (i.e., CEBPAmut, WT1mut, ERG and BAALC expression levels). Methods: MN1 expression was measured by quantitative real-time RT-PCR in pretreatment bone marrow samples from 119 de novo CN-AML adult pts <60 years with material available for MN1 expression analyses who were treated on CALGB protocols 9621 and 19808. MN1 copy numbers were measured and normalized to ABL (internal control) copy numbers. Pts were dichotomized at the MN1 median expression and analyzed for outcome. Results: At diagnosis, low MN1 expression was associated with NPM1mut (P<0.0001) and the molecular low risk group (FLT3-internal tandem duplication (ITD)negative and NPM1mut; P<0.001). Complete remission (CR) rate was higher in low MN1 pts (93% v 73%); low MN1 pts were 6 times more likely to achieve a CR than high MN1 pts (P=0.005), once adjusting for hemoglobin (P=0.01) (Table). With a median follow-up of 4.6 years, low MN1 pts also had longer disease-free survival (DFS) (P<0.005; 3-year DFS: 60% v 32%) and overall survival (OS) (P<0.001; 3-year OS: 68% v 40%) than high MN1 pts. In multivariable analyses, MN1 expression independently predicted for DFS (P=0.01) and OS (P=0.03). For low MN1 pts, the risk for an event was reduced by approximately 50% compared with high MN1 pts (Table). Conclusions: MN1 expression predicts outcome independent of other established molecular markers in CN-AML pts. Particularly, low MN1 expression defines a group of CN-AML pts with favorable clinical outcome. Multivariable analyses for achievement of CR, DFS and OS Variables in Final Models CR DFS OS OR * P HR ** P HR ** P MN1 expression, low v high 6.4 0.005 0.46 0.01 0.54 0.03 WT1, mutated v wild-type – – 5.6 <0.0001 7.0 <0.0001 FLT3-ITD/NPM1 status, high v low risk – – – – 3.0 0.005 WBC, each 50 unit increase – – 2.1 0.002 1.7 <0.0001 Hemoglobin 1.7 0.01 – – – – * OR: odds ratio; ** HR: hazard ratio. Variables considered in the models were those significant at α=0.20 in univariable analyses as follows: for CR, MN1 expression, FLT3-ITD/NPM1 status (molecular high risk is FLT3-ITDpositive and/or NPM1wt; molecular low risk is FLT3-ITDnegative and NPM1mut), WBC, hemoglobin, age, race; for DFS, MN1 expression, FLT3-ITD/NPM1 status, ERG expression, WT1 and CEBPA mutations, WBC, race; for OS, MN1 expression, FLT3-ITD/NPM1 status, ERG and BAALC expression, WT1 mutations, WBC, age, race, hemoglobin, % blood blasts, extramedullary involvement. No significant financial relationships to disclose.

Prognostic Impact of BAALC Expression in the Context of Other Molecular Markers in Cytogenetically Normal Acute Myeloid Leukemia.

Background: In previous studies, high blood expression values of BAALC (brain and acute leukemia cytoplasmic) have been shown to be an adverse risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). So far, the prognostic value of BAALC expression has not been evaluated in the context of other relevant molecular markers such as mutations (mut) in the NPM1 or CEBPA genes.Aims: To evaluate the prognostic impact of BAALC expression in the context of NPM1, FLT3-ITD (internal tandem duplication)/TKD (tyrosine kinase domain mutations at codons 835/836), CEBPA, and MLL-PTD (partial tandem duplication) mutation status on response to induction therapy, relapse-free (RFS) and overall survival (OS).Methods: Patients (pts) 16 to 60 years of age were entered on three treatment trials conducted by the German-Austrian AML Study Gorup (AML HD93, AML HD98A, AML04–07). In all three trials, a genetic randomization was performed assigning all pts with an HLA-matched family donor to allogeneic stem cell transplantation (SCT) in first complete remission (CR). BAALC expression was determined in peripheral blood from 339 CN-AML pts by real-time RT-PCR as recently described; gene mutation screening was performed as previously reported.Results: BAALC expression levels were heavily scattered in the 339 pts, with a median of 4,716 (range 11–1,120,212). High and low BAALC expression was determined by dichotomizing BAALC at the median. Correlation of BAALC expression levels with FLT3-ITD/TKD, NPM1, CEBPA and MLL-PTD mutation status revealed high BAALC expression to be significantly associated with FLT3-ITDpos (p=0.001), NPM1WT (p<0.0001) and CEBPAmut (p=0.006). In addition, when correlating BAALC expression levels with the combined FLT3-ITD/NPM1 genotypes that recently have been shown to be of prognostic relevance in CN-AML, a significant correlation of low BAALC expression and the favorable FLT3-ITDneg/NPM1mut (p<0.0001) genotype was present. In contrast, high expression levels were significantly correlated with the other unfavorable genotypes. There was a trend towards a lower CR rate in high BAALC expressers (p=0.07). However, in a multivariable logistic regression model, BAALC was not significant (p=0.75); the only significant variables for induction success were the FLT3-ITDneg/NPM1mut (p=0.03) and CEBPAmut genotypes (p=0.04), as well as logarithm of white blood cell count (WBC, p=0.03). In univariable analyses for OS and RFS, high BAALC expressers had significantly inferior clinical outcome (p=0.05 and p=0.02, respectively). Again, multivariable Cox proportional hazard models for RFS and OS revealed no significant impact of BAALC expression (p=0.15 and p=0.96, respectively); significant variables for OS and RFS were the FLT3-ITDneg/NPM1mut (p<0.0001 and p<0.0001) genotype, availability of an HLA-matched family donor (p=0.02 and p=0.001), and WBC (p=0.0002 and p=0.0001); in addition, the CEBPAmut genotype was a significant variable for OS (p=0.002).Conclusions: In our study on a large series of molecularly well characterized CN-AML, BAALC expression levels were significantly correlated with FLT3-ITD/NPM1 genotypes. In the context of these genotypes, BAALC expression lost its independent prognostic impact.