MicroRNAs in Intermediate Risk Cytogenetic Acute Myeloid Leukemia Add Relevant Prognostic Information to Molecular Categorization

Abstract Abstract 235 The prognosis of AML patients within the intermediate cytogenetics category is mainly determined by the mutational status of some relevant genes, such as NPM1 mutations (NPMmut), or biallelic CEBPA mutations (CEBPAmut), associated with a favorable outcome, and with the presence of FLT3 internal tandem duplication (FLT3-ITD), which correlates with an adverse prognosis. Nonetheless, additional biological features such as microRNA (miRNA) expression pattern might contribute to refine prognosis and guide therapy in this setting. The aim of the present study is to investigate whether miRNA expression is associated with molecular characteristics and clinical outcome in intermediate-risk AML patients (IR-AML). We have analyzed samples from 85 IR-AML patients (median age, 52 [range, 18–71]; 52% males) who received intensive therapy from 1994 to 2009. Forty-three patients (51%) harbored NPMmut, 37 (44%) harbored FLT3-ITD (including 23 with NPMmut), and 11 (13%) harbored CEBPAmut, including 7 with biallelic mutations. The expression of 670 mature miRNAs was analyzed by multiplex Real Time PCR using TaqMan Human MicroRNA Arrays (Applied Biosystems). All PCR reactions were performed using an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2−DDCt method, using RNU48 as endogenous control. Statistical analysis was performed with BRB Array Tools, SPSS version 15.0.1 and R software version 2.9.0. Supervised analysis by means of t-test based on multiplex permutations (class comparisons analysis, p<0.001) revealed a distinctive miRNA signature in patients with NPMmut, with overexpression of miR-10a, miR-10a*, miR-10b and miR-196b, and downregulation of miR-126, miR126*, miR-424, miR-424* and miR-335, as well as patients with biallelic CEBPAmut, characterized by downregulation of miR-196b and upregulation of miR-181a. Response rate in this series of patients was 84%, with 5-year survival of 43±11% and relapse incidence (RI) of 55±14%. Multivariate analysis for overall survival(OS) including NPM status, FLT3-ITD status, age, WBC, and Log Rank OS significant miRNAs (miR-632, miR-23b, miR-409-3p, let-7a*, miR-565 and miR-196b) identified age, absence of NPMmut, and FLT3-ITD as unfavorable variables together with low expression of miR-409-3p (p<0.001; HR=3.3, 95% CI: 1.7–6.4), and increased level of let-7a* (p=0.026; HR=5.1, 95% CI: 1.21–21.5) and miR-196b (p=0.056; HR=7.27, CI: 0.95–55.6). Concerning risk of relapse (RR), multivariate analysis including NPM status, age, FLT3-ITD, WBC, and Log Rank RR significant miRNAs (miR-632, miR-155*, miR-135a, miR-409-3p, miR-150, miR-23a* and miR-363) the absence of NPMmut, FLT3-ITD and increasing leukocyte count were associated with a higher RI. Remarkably, decreased miR-409-3p expression (p=0.011; HR=3.3, 95% CI: 1.3–8.2) and miR-135a (p=0.02; HR=4.2, 95% CI: 1.2–14.2), together with higher levels of miR-23a* (p<0.001; HR=6.2, 95% CI: 2.61–14.7) were independently associated with a higher relapse risk. Of note, a decreased miR-409-3p level retained its adverse prognosis value in the subgroup of patients without favorable molecular markers (i.e., wild-type NPM1 and CEBPA and/or FLT3-ITD;p=0.001) together with low miR-361-3p (p=0.013, HR= 2.4, CI: 1.2–5.1). On the contrary, let-7a* levels segregated subgroups of patients in the category of favorable genotype (i.e., mutated NPM1 without FLT3-ITD p=0.027). In this series of patients of intermediate-risk cytogenetic AML, measurement of expression levels of several miRNAs such as miR-409-3p, miR-135a, let-7a* or miR-23a* showed independent prognostic value, and contribute to predict the outcome within specific molecular subgroups. Nonetheless, confirmation of the prognostic impact of these miRNAs and investigation of possible underlying mechanisms account for this effect require future studies. Disclosures: No relevant conflicts of interest to declare.

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Prognostic Impact of Genetic Characterization in the GIMEMA LAM99P Study for Newly Diagnosed Adult AML. Relevance of Combined Analysis of Conventional Karyotyping, FLT3 and NPM Mutational Status.

Blood ◽

10.1182/blood.v106.11.226.226 ◽

2005 ◽

Vol 106 (11) ◽

pp. 226-226

Author(s):

G. Saglio ◽

Francesco Lo Coco ◽

A. Cuneo ◽

F. Pane ◽

G. Rege Cambrin ◽

...

Keyword(s):

Multivariate Analysis ◽

High Risk ◽

Genetic Characterization ◽

High Risk Group ◽

Low Risk ◽

Prognostic Impact ◽

Intermediate Risk ◽

Mutational Status ◽

Molecular Studies ◽

The Impact

Abstract Between 1998 and 2002, 509 patients with AML (median age 46 yrs, range 15–60) were enrolled in the multicenter LAM99P study of the Italian GIMEMA group. To better evaluate the clinical impact of genetic characterization, all patients received a uniform protocol and diagnostic samples were centralised for cytogenetic and molecular studies. Therapy consisted of HU pre-treatment (2g/m2 for 5 days) followed by induction with DNR (50 mg/m2 d 1, 3, 5), cytarabine (100 mg/m2 d 1–10) and etoposide (100 mg/m2 d 1–5) and consolidation with cytarabine (500 mg/m2/q12 hrs d 1–6) and DNR (50 mg/m2 d 4–6). After consolidation, eligible patients with an identical HLA donor were to receive allogeneic SCT and the remaining peripheral blood autologous SCT. Cytogenetic and molecular genetic characterization (including analysis of major fusion genes, FLT3 and NPM status) was available in 397 (78%) patients. Compared to previous GIMEMA studies, the possibility to collect samples during the 5d of HU pretreatment considerably improved genetic characterization and in particular centralised karyotyping by overcoming the problem of sampling and shipment over the w-end. After induction, 269/397 (68%) patients achieved CR. For induction response, conventional K identified 3 distinct risk groups as follows: low risk (inv. 16 and t8;21), intermediate (normal K and other anomalies not comprised in the high risk group) and high risk (t3;3, inv.3, t9;22, 11q23, 5/7 abnormalities complex K,) with CR rates of 92%, 67% and 39%, respectively (P<.0001). NPM mutations were significantly associated with older age, higher WBC, normal K and FLT3-ITD. CR rates in NPM+ (mutated) vs. NPM- (wildtype) groups were 76% vs. 60% for the whole population and 81% vs, 61% for patients in the normal K group (P<.001 for both comparisons). Multivariate analysis for CR indicated that low risk K and NPM+ were independent factors favorably affecting CR achievement while FLT3 status had no significant impact on CR. The analysis of prognostic factors for DFS and OS was carried out in 269 patients in CR (median follow-up of 39 mos.) and multivariate analysis performed after adjusting for unfavorable factors (WBC count). Multivariate analysis of variables influencing OS showed the following: low vs intermediate K, P=.0005; high vs intermediate K, P<.0001; FLT3+ vs. FLT3−, P=.06. Multivariate analysis for DFS showed: low risk vs. intermediate risk K, p=.01; high risk vs. intermediate risk K, p= .03; FLT3+ vs. FLT3-, p=.0002. NPM status did not significantly influence DFS in either the whole population or in the normal K group. In particular, there was no difference in the DFS rates among patients NPM+ and NPM- in the normal K/FLT3- group while in the normal K/FLT3+ group there was a trend (p=.06) for lower relapse rate for NPM+ patients as compared to NPM- ones. These results highlight the relevance of combining cytogenetic and molecular studies in the diagnostic work up of AML and confirm the impact of karyotype on all outcome estimates as well as of FLT3 status on DFS. As to NPM mutations, these appear a favorable predictor of CR achievement. Further investigations in large clinical trials are needed to assess the prognostic value of NPM mutations on outcome in AML with normal karyotype.

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ID1 Expression Correlates with CEBPA Mutational Status and Is Not An Independent Risk Factor in Cytogenetically Normal AML,

Blood ◽

10.1182/blood.v118.21.3554.3554 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3554-3554

Author(s):

Katharina Wagner ◽

Frederik Damm ◽

Michael A Morgan ◽

Felicitas Thol ◽

Haiyang Yun ◽

...

Keyword(s):

Multivariate Analysis ◽

Prognostic Factors ◽

Prognostic Factor ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

High Expression ◽

Prognostic Impact ◽

Mutational Status ◽

Negative Prognostic Factor ◽

Cebpa Mutations

Abstract Abstract 3554 Background: Acute myeloid leukemia with normal karyotype (CN-AML) is a heterogenous disease. During the last years, mutations in several genes (e.g. NPM1, FLT3, CEBPA, WT1, IDH1, IDH2) have been identified which are involved in the pathogenesis of AML and affect the prognosis of these patients. Moreover, deregulated expression of genes such as MN1, BAALC, ERG and WT1 was demonstrated to be predictive of outcome in CN-AML. Recently, high expression of the ID1 gene was described as a negative prognostic factor in AML (Tang et al. Blood 2009, 114:2993–3000). Aims: We have shown that C/EBPα, a transcription factor encoded by the CEBPA gene, binds to a regulatory element in the promoter region of the ID1 gene and regulates ID1 expression in leukemic cells (Wagner et al. Proc Natl Acad Sci USA 2006, 103:6338–6343). Therefore, we wanted to analyze the prognostic impact of ID1 expression in CN-AML in the context of other molecular markers, in particular CEBPA mutations. Methods: ID1 expression was quantified normalized to ABL by real time RT-PCR in 269 patients (age 16–60 years) with CN-AML treated with intensive double induction and consolidation therapy within the AMLSG 295 and 0199 trials (NCT00209833). The patients were also analyzed for mutations in the genes NPM1, FLT3, CEBPA, WT1, IDH1 and IDH2. Median follow up was 79 months. Results: Expression of ID1 varied over a 3-log range. High expression of ID1 (ID1high, defined as > median expression level) was significantly associated with the presence of a FLT3 -ITD or an IDH2 mutation and WT1 wildtype. Moreover, ID1 expression was closely associated with CEBPA mutational status. Altogether, 41 patients (15%) harboured a CEBPA mutation (24 monoallelic and 17 biallelic mutations). ID1 expression in the CEBPA wildtype patients was significantly higher than in patients with monoallelic CEBPA mutations and these patients had a significantly higher ID1 expression compared to patients with biallelic CEBPA mutations (p = 0.001). ID1high patients had a trend to a lower complete remission (CR) rate (74% vs. 84%; p = 0.07), but in multivariate analysis only blast clearance on day 15 after induction 1, age and WT1 SNP rs16754 were independent predictors for the achievement of CR. In univariate analysis, ID1high patients had an inferior overall survival (OS) compared to patients with low expression (median OS 29 vs. 78 months, 5 year OS 39% vs. 53%, p = 0.026). ID1high status was an independent negative prognostic factor in multivariate analysis when analyzed together with NPM1, FLT3 -ITD, WT1, IDH1, IDH2, extramedullary disease and platelet counts (HR 1.51; 95% CI 1.06–2.19). However, when also CEBPA mutational status was entered into the model, ID1 expression lost its prognostic impact and the only independent prognostic factors were age, platelets, CEBPA mutations, NPM1 /FLT3 -ITD risk group and WT1 SNP rs16754. Likewise, ID1high patients had a trend to an inferior relapse-free survival (RFS; HR 1.36, 95% CI 0.96–1.93, p = 0.086) in univariate analysis. However, in multivariate analysis including CEBPA mutational status, ID1 expression had no impact on RFS and the only prognostic factors for RFS were NPM1 and CEBPA mutations and WT1 SNP rs16754. In CEBPA wildtype patients, ID1 expression had no impact on CR-rate, OS or RFS in univariate or multivariate analysis. Conclusions: CEBPA mutations seem to deregulate ID1 expression in CN-AML. Therefore, ID1 expression is not an independent prognostic factor in CN-AML. Disclosures: No relevant conflicts of interest to declare.

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Comprehensive Genetic Screening of Chronic Myelomonocytic Leukemias (CMML)

Blood ◽

10.1182/blood.v120.21.3811.3811 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3811-3811

Author(s):

Raphael Itzykson ◽

Olivier Kosmider ◽

Aline Renneville ◽

Margot Morabito ◽

Céline Berthon ◽

...

Keyword(s):

Multivariate Analysis ◽

Conflicts Of Interest ◽

Gene Dosage ◽

Univariate Analysis ◽

Continuous Variable ◽

Prognostic Impact ◽

Gene Dosage Effect ◽

Disease Evolution ◽

Mutational Status

Abstract Abstract 3811 Background: A large number of genes have been found mutated in CMML including 18 encoding signaling molecules (CBL, N/KRAS, JAK2, FLT3, KIT), epigenetic regulators (TET2, IDH1/2, DNMT3A, ASXL1, EZH2) transcription (RUNX1, NPM1) and splicing (SRSF2, SF3B1, U2AF1, ZRSR2) factors. We report the genotypic patterns, clinical correlates and prognostic impact of mutations in those 18 genes in a large cohort of CMML patients (pts). Methods: Bone marrow or peripheral CD14+ cells from 224 CMML pts from a non interventional study (n=186) or a phase II decitabine trial (n=38; Braun Blood 2011) were genotyped by mutation specific techniques and Sanger sequencing for up to 18 genes (depending on available material): TET2, IDH1, IDH2, DNMT3A, CBL, NRAS, KRAS, JAK2V617F, FLT3, KIT, NPM1, RUNX1, ASXL1, EZH2, SF3B1, SRSF2, U2AF1 and ZRSR2. The number of TET2 alleles with a functional Cystein Rich (CysR) domain (Delhommeau NEJM 2009) was predicted based on mutation type and zygosity, assuming that double mutations affect independent alleles. Overall (OS) and AML-free (AMLFS) survival were analyzed from the date of genotyping. Results: 224 CMML pts (152M/72F, median age 75y) were genotyped at diagnosis (37%) or after a median of 7.2 months of evolution (none had received hypomethylating agents [HMA] before genotyping); WHO diagnosis was CMML-1/2 in 78%/22%, 70% pts had normal karyotype, 22% had extramedullary disease (EMD); 13 pts had autoimmune manifestations (AIM). The most frequently mutated genes were TET2 (58%), SRSF2 (47%) and ASXL1 (38%). Mutations in RUNX1, CBL and NRAS were found in 14%, 11% and 10% of pts, respectively (resp). All other genes were mutated in <10% of pts. Only 5% pts lacked any mutation, and 70% had ≥2 mutated genes; TET2, IDH1 and IDH2 mutations, present in 64% of pts, were mutually exclusive. Mutations in splice and signaling genes were present in 63% and 35% of pts, with 2 mutated genes within each group in 3% and 2% pts, resp. ASXL1 mutations were less frequent in the presence of TET2 mutations (P<.0001). Significant mutual associations included ASXL1/RUNX1, ASXL1/NRAS, TET2/SRSF2, RUNX1/SRSF2 and U2AF1/IDH2. In multivariate analysis accounting for those interactions, TET2 status was the only independent predictor of hemoglobin values (median 10.2 vs 11.9 g/dL in wildtype [wt] vs mutated pts, P<.0001) with a gene dosage effect (P=.0003). Platelet counts were higher in JAK2V617F pts, and lower in pts with RUNX1, TET2 or SRSF2 mutations. WBC and monocyte counts were higher in pts with ASXL1 and NRAS mutations. EMD was associated to ASXL1, CBL, KRAS and JAK2 mutations. EMD, CMML-2 and abnormal karyotype were less frequent in TET2 mutated pts. All 13 pts with AIM had at least one mutated gene, with no specific genotype spectrum. With a median follow-up of 25.4 months, median OS and AMLFS were 32.2 and 28.0 months resp. In univariate analysis, OS was decreased in pts with IDH2 mutations (P=.04), and AMLFS was shorter in pts with NRAS (P=.04), RUNX1 (P=.03) and SRSF2 (P=.04) mutations. ASXL1 mutations markedly reduced OS (median 18.5 vs 35.7 months in wt pts) and AMLFS (median 12.5 vs 34.7 months, both P<.0001), with similar results in the 74 pts who received HMA during follow-up. In the 224 pts, there was no significant effect of overall TET2 status (wt vs mutated) on OS or AMLFS (both P=.09) but median OS was 29.3, 35.7 months and not reached in pts with 2 (57%), 1 (30%) and 0 (13%) TET2 alleles with a putatively functional CysR domain (P=.01). Similar differences were noted for AMLFS (P=.008). In multivariate analysis including peripheral blood counts, WHO classification, cytogenetics, disease evolution and therapy, ASXL1 was the only gene whose mutations independently predicted inferior OS (HR: 2.44, 95% CI: 1.36–4.37, P=.003)and AMLFS (HR: 2.54, 95% CI: 1.46–4.42, P=.001); SRSF2 mutations only predicted inferior AMLFS (HR: 2.05, 95% CI: 1.15–3.65, P=.02). The total number of mutated genes as a continuous variable, which was higher in ASXL1 mutated pts (mean 3.0 vs 1.8, P<.0001), was the only genetic variable to retain prognostic value when added to those models (OS and AMLFS both P<.0001). Conclusion: TET2, SRSF2 and ASXL1 are the most frequent mutated genes in CMML. The number and location of TET2 mutations may impact CMML presentation and outcome. The total number of mutated genes has the strongest prognostic relevance, but ASXL1 mutational status provides a robust surrogate prognostic marker for daily practice. Disclosures: No relevant conflicts of interest to declare.

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New TET2, ASXL1 and CBL Mutations Have Poor Prognostic Impact In Systemic Mastocytosis and Related Disorders

Blood ◽

10.1182/blood.v116.21.3076.3076 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3076-3076

Author(s):

Fabiola Traina ◽

Ania Jankowska ◽

Hideki Makishima ◽

Fred H. Hsieh ◽

Yingchun Han ◽

...

Keyword(s):

Conflicts Of Interest ◽

Systemic Mastocytosis ◽

Single Nucleotide Polymorphism Array ◽

Myeloproliferative Neoplasms ◽

Uniparental Disomy ◽

Prognostic Impact ◽

Biallelic Mutation ◽

Prognostic Information ◽

Kit Mutation ◽

Disease Spectrum

Abstract Abstract 3076 Mastocytosis is a heterogeneous hematopoietic neoplasm characterized by proliferation and organ infiltration by clonal mast cells (MC). The disease spectrum encompasses chronic indolent forms such as cutaneous mastocytosis (CM)/indolent systemic mastocytosis (ISM) to more aggressive types such as SM with associated clonal hematologic non-mast-cell disease (SM-AHNMD), the latter most closely related to myeloproliferative neoplasms (MPN) or MDS/MPN overlap syndromes. Molecular pathogenesis of mastocytosis involves acquisition of c-KIT mutations, particularly D816V, which is present in many cases and confers resistance to imatinib. TET2 mutations are often found in MPN and MDS/MPN and also in ∼20% of SM patients without noticeable impact on survival. We have hypothesized that analysis of molecular defects in mastocytosis may shed light on disease pathogenesis and possibly convey prognostic information that may help in diagnosis and selection of rational therapies. To investigate these molecular events, we have applied single nucleotide polymorphism array-based karyotyping (SNP-A) (Affymetrix 6.0) to identify recurrent areas of loss of heterozygosity and performed a broad screen for mutations which could be present in mastocytosis including c-KIT, TET2, CBL gene family (CBL, CBLB, CBLC), ASXL1, IDH1/IDH2, which have been found in hematologic disorders related to or associated with SM. Overall survival (OS) was analyzed using the Kaplan-Meier method (Log-Rank). We studied a total of 35 mastocytosis patients classified using WHO criteria (CM, N=9; ISM, N=14; SM-AHNMD, N=9; [CMML, N=6; AML, N=1; NHL, N=2], aggressive SM (ASM) N=2; MC sarcoma, N=1). Median age of the cohort was 51 yrs (13-71). SNP-A showed a total of 20 new lesions (13 gains, 3 losses and 4 uniparental disomy [UPD]) in 10 patients (CM=1, ISM=4, SM-AHNMD=4, ASM=1). The most frequently affected chromosomes were 2, 7, 12, 13, 14 and X. UPD was only found in SM-AHNMD and ASM and it involved chromosomes 2p, 4q, 7p and 13q. No OS difference were observed between patients with new SNP lesions compared to those without (47 mo vs. 38 mo; p=.84). c-KIT sequencing showed D816V in 29% of patients (ISM=29%; SM-AHNMD=44%, ASM=100%). A total of 15 additional mutations were found in 9/35 patients. TET2 mutations were found in 8/35 (23%), including 2 patients with biallelic mutations (3 frameshift, 2 nonsense and 5 missense). TET2 mutational frequencies for CM, ISM and SM-AHNMD (only CMML) were 22% (2/9), 7% (1/14) and 56% (5/9). Majority of TET2 mutations were heterozygous, except one that was homozygous. These mutations have not been previously described in mastocytosis. We have also detected ASXL1 mutations in 3/35 (9%) patients, with biallelic mutation seen in one patient (1 frameshift, 1 nonsense and 2 missense). ASXL1 mutations were seen in 1/14 ISM and 2/9 SM-AHNMD (with CMML). To our knowledge, ASXL1 mutations have not been described in mastocytosis. A heterozygous CBL mutation was found in 1/35 patients with SM-AHNMD (CMML). No mutations were found in CBLB, CBLC and IDH1/IDH2. Interestingly, 5 patients were found to have >1 mutation, c-KIT and TET2 in 2, c-KIT/TET2/ASXL1 in 2 and TET2/CBL in 1 patient. The median OS of the cohort was 18 mo (1-85). As expected, for patients with only SM (excluding CM cases), c-KIT mutants had a worse OS than wild type (WT) c-KIT patients (17 mo vs. 52 mo; p=.02). SM patients with TET2, ASXL1 or CBL mutations, independently of c-KIT, had a worse OS than those with WT genes (17 mo vs. 52; p=.01). SM patients with c-KIT mutation who carry additional mutations had a worse OS, c-KIT + any mutation [11 mo] vs. TET2/ASXL1/CBL mutant [32 mo] vs. c-KIT mutant alone [NR] vs. WT [NR]; p<.0001. Similarly, when TET2 and c-KIT mutations were analyzed independent of CBL and ASXL1, patients with mutant c-KIT and TET2 had the poorest OS in the group (c-KIT plus TET2 [10 mo] vs. TET2 alone [32 mo] vs. c-KIT alone [NR] vs. WT [NR]; p<.0001). All patients with CM were still alive at the time of analysis. In conclusion, SNP-A lesions including UPD are karyotypic changes also seen in mastocytosis. TET2 mutations are frequently found in mastocytosis, particularly in SM-AHNMD (CMML). Novel molecular mutations frequently found in MDS and MPN, as ASXL1 and CBL, are also found in mastocytosis but at lower frequencies. More importantly, these new mutations may affect prognosis, as demonstrated by poor OS in patients who carry these mutations independently of c-KIT. Disclosures: No relevant conflicts of interest to declare.

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DNMT3A mutations Predict for Inferior Outcome in NPM1-Wildtype and Molecular Unfavorable Cytogenetically-Normal Acute Myeloid Leukemia: A Study of the German-Austrian AMLSG

Blood ◽

10.1182/blood.v118.21.415.415 ◽

2011 ◽

Vol 118 (21) ◽

pp. 415-415 ◽

Cited By ~ 1

Author(s):

Verena I. Gaidzik ◽

Richard F. Schlenk ◽

Peter Paschka ◽

Anja Stölzle ◽

Andrea Corbacioglu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Negative Impact ◽

Epigenetic Modification ◽

Methylation Status ◽

Prognostic Impact ◽

Sequencing Analysis ◽

Mutational Status ◽

Acute Myeloid

Abstract Abstract 415 Background: Alteration of DNA methylation, a hallmark of epigenetic modification, is currently discussed as one important pathomechanism in leukemogenesis. Using a next-generation sequencing approach, a frameshift mutation of the gene encoding the DNA methyltransferase (DNMT3A) in an acute myeloid leukemia (AML) case was identified. DNMT3A catalyses the addition of a methyl group to the cytosine residue of CpG dinucleotides, thereby affecting promoter methylation status and gene expression. Subsequent sequencing analysis in an independent cohort of 288 AML patients (pts) revealed DNMT3A mutations (DNMT3Amut) in 22% of the pts; mutations were associated with intermediate-risk cytogenetics and poor outcome. Aims: To evaluate frequency and clinical impact of DNMT3Amut in pts with AML aged 18 to 61 years who were treated within AMLSG treatment trials AML HD98A (Schlenk et al., J Clin Oncol 2010;28:4642–8) and AMLSG 07–04 (NCT00151242). Methods: DNMT3A mutation analysis was performed in 1218 AML (HD98A, n=685; AMLSG 07–04, n=533; de novo AML, n=1102; s-AML, n=45; t-AML, n=69) using a DNA-based PCR assay for all coding exons (1 to 23) followed by direct sequencing. The median follow-up was 5.06 years. Results: DNMT3A mut were found with an overall frequency of 19.6% (239/1218); 189 mutations were located in the MTase domain clustering at amino acid R882 (79%). All but one mutation were heterozygous; only 4 cases had two mutations. DNMT3A sequence alterations included 17 frameshift, 4 nonsense, and 222 missense mutations. DNMT3A mut pts were significantly older (P=.01), more frequently females (P=.001), had higher white blood cell and platelet counts (both P<.0001), and higher bone marrow blasts percentage (P=.001). DNMT3Amut were associated with cytogenetically-normal AML (CN-AML, P<.0001), while DNMT3Amut were rare in favorable and adverse-risk karyotypes (P<.0001). Correlations with other molecular markers (NPM1, CEBPA, FLT3, IDH1/2, TET2, ASXL1) revealed a significant association with NPM1 (P<.0001), FLT3-ITD (P<.0001), and IDH1/2 (IDH1R132, P<.0001; IDH2R140, P=.0003; IDH2R172, P=.03) mutations, while co-occurrence of CEBPA (P=.02) and ASXL1 (P=.02) mutations was less frequent. DNMT3A mutational status did not impact complete remission (CR) rate, event-free (EFS) and relapse-free survival (RFS), neither in the whole cohort (P=.09, P=.98, P=.11; respectively) nor in the subgroup of CN-AML (P=.39, P=.79, P=.19, respectively). DNMT3Amut had a negative impact on overall survival (OS) in trend in the whole cohort (P=.07) and significantly in CN-AML (P=.02). In multivariable analyses, DNMT3Amut were in trend associated with a negative prognostic impact on OS (hazard ratio, 1.24; P=.06). In addition, we performed subgroup analyses according to (1) the NPM1 mutational status, and (2) the molecular risk groups of CN-AML (as defined by the European LeukemiaNet classification). DNMT3Amut did not impact OS in NPM1-mutated patients in the whole cohort as well as in CN-AML (P=.34; P=.22; respectively), while in NPM1-wildtype patients DNMT3Amut were associated with inferior OS in both, the whole cohort and in CN-AML (P=.001; P=.005; respectively). In molecular unfavorable CN-AML (NPM1-wildtype with or without FLT3-ITD, NPM1-mutated with FLT3-ITD, CEBPA-wildtype), DNMT3Amut were significantly associated with worse OS (P=.002) compared with DNMT3A-wildtype pts, even outweighing FLT3-ITD as an unfavorable prognostic marker. There was no effect of DNMT3Amut in molecular favorable-risk CN-AML. Conclusions: DNMT3A mutations are confirmed as frequent genetic aberrations in AML, associated with normal karyotype, NPM1, FLT3-ITD, and IDH1/2 mutations. DNMT3Amut predicts for inferior outcome in molecularly-defined subsets of AML, that is, NPM1-wildtype AML and molecular unfavorable CN-AML. As a single marker, DNMT3Amut only had a moderate effect on outcome. Disclosures: No relevant conflicts of interest to declare.

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Clinical Monoclonal B Lymphocytosis (cMBL), Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL): Diagnostic Criteria, Features At Diagnosis and Natural History

Blood ◽

10.1182/blood.v122.21.5273.5273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5273-5273

Author(s):

Rodrigo Santacruz ◽

Julio Delgado ◽

Tycho Baumann ◽

Maria Rozman ◽

Martha Aymerich ◽

...

Keyword(s):

Overall Survival ◽

Multivariate Analysis ◽

B Cells ◽

B Cell ◽

Cell Count ◽

Diagnostic Criteria ◽

Conflicts Of Interest ◽

Consistent Difference ◽

Transformation Rate ◽

Mutational Status

Abstract Introduction CLL, SLL and cMBL are considered to be part of the same spectrum of clonal expansions of CD5+ B cells. Clinically, the transition from one to another of these forms over time is a well recognized event. Diagnostic criteria to separate these disorders have been proposed (IWCLL, 2008; WHO, 2008). Aim To compare presenting and evolving features of three groups of patients with cMBL, Rai 0 CLL or SLL and to ascertain the usefulness of current diagnostic criteria for these disorders. Patients and Methods Retrospective study of clinical, biologic and evolving characteristics of patients diagnosed with cMBL, Rai 0 CLL or SLL according to current criteria (CLL: ≥5x109 clonal B cells/L in peripheral blood; SLL <5x109/L clonal B cells with lymphadenopathy, organomegaly, cytopenia or disease-related symptoms; cMBL <5x109 clonal B cells with no signs or symptoms). Results Baseline characteristics of the patients are shown in the Table. Median age was 68 years (range, 24-94) and 57% of patients were males. Out of 1,093 patients, 79 had cMBL (7.2%), 522 Rai 0 CLL (48%) and 94 SLL (8.6%). Overall, adverse biomarkers such as high LDH (p<0.001), high B2M (p<0.001), increased ZAP70 (p<0.001), high CD38 (p<0.001), unmutated IGHV status (p=0.002), +12 (p=0.02) and 11q- (p=0.01) were significantly more frequent in SLL. In subgroup analyses, the only difference between cMBL and Rai 0 CLL was a higher proportion of cases with mutated IGHV in cMBL (p=0.008). Furthermore, when SLL was compared to Rai I to IV CLL no differences were observed (data not shown). The actuarial risk for transformation from cMBL or SLL to CLL was 4.2% and 4.4 % per year (p=0.5), respectively. Median TTFT was significantly shorter in the SLL group (12 m.) than in Rai 0 CLL (174 m.) or cMBL (244 m.) (p<0.001). Median overall survival was also significantly shorter for SLL (94 m.) compared with Rai 0 CLL and cMBL (153 and 157 m., respectively) (p=0.028). Multivariate analysis of 695 patients (cMBL/Rai 0-CLL/SLL) revealed four variables independently correlated with shorter TTFT: diagnosis of SLL vs. Rai 0 CLL vs. cMBL (HR 2.28; p=0.008), high ZAP70 (HR 4.08; p<0.001), high CD38 (HR 4.68; p=0.001) and increased serum B2M levels (HR 1.54; p = 0.031). Importantly, however, when the multivariate analysis was restricted to patients with cMBL and Rai 0 CLL, variables correlated with TTFT were the clonal B-cell count (HR 3.76; p=0.01), ZAP70 (HR 3.31; p<0.001), and CD38 (HR 4.61; p=0.02). FISH cytogenetics, IGHV mutational status,NOTCH1 or SF3B1 mutations were not included in the analysis because of missing data. Conclusions Disparities in biologic and clinical features of cMBL, CLL and SLL mainly reflect the different tumor burden in this spectrum of CD5+ monoclonal B cell disorders. In this study, the transformation rate from either cMBL or SLL to CLL was around 4% per year. As a result of differences in tumor mass, the need for therapy was shorter in SLL than in Rai 0 CLL and cMBL, and the overall survival poorer. Biologically, the only consistent difference between cMBL and Rai O CLL was a higher proportion of mutated IGHV in cMBL. Clinically, however, no differences in median survival were observed. Moreover, the clonal B cell count was the most reliable predictor of disease outcome in both cMBL and Rai 0 CLL. Therefore, the distinction between cMBL and Rai 0 CLL seems hardly justified. Disclosures: No relevant conflicts of interest to declare.

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ASXL1 Mutations in AML: Molecular Biomarker for Secondary AML?

Blood ◽

10.1182/blood.v124.21.2343.2343 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2343-2343

Author(s):

Jingmei Hsu ◽

Anita J. Kumar ◽

Martin P. Carroll ◽

Noelle V. Frey ◽

Nirav N. Shah ◽

...

Keyword(s):

Overall Survival ◽

De Novo ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Myeloproliferative Neoplasm ◽

Molecular Characteristics ◽

Intermediate Risk ◽

Kaplan Meier ◽

The Impact

Abstract Background: Additional sex combs like transcription factor 1 (ASXL1) is a member of the polycomb group protein. ASXL1 mutation has been implicated in myeloid malignancy transformation. It is hypothesized that mutated ASXL1 leads to the loss of polycomb repressive complex 2 (PRC2) mediated gene repression and subsequent transforming events. Recent studies identify ASXL1 mutation as a poor prognostic marker in patients (pts) with de novo acute myeloid leukemia (AML) who present with intermediate–risk cytogenetic lesions (Patel, NEJM 2012; Schnittger, Leukemia2013). To study the impact of ASXL1 mutations in an unselected AML population, we analyzed clinical and molecular characteristics of patients with untreated AML who express ASXL1 mutation at presentation. Methods: Using next generation sequencing, 254 adult patients with AML seen at the Hospital of the University of Pennsylvania were analyzed for mutations, including ASXL1, using a 33-gene hematologic malignancy panel. Clinical characteristics were obtained from retrospective chart review. Kaplan-Meier estimates were used to calculate overall survival (OS) from time of diagnosis. Living patients were censored at date last seen. Results: ASXL1 mutations were detected in 36/254 (14%) AML pts. There were 29 known pathologic mutations, 1 benign, 1 probable pathologic, and 9 variants of unknown clinical significance (VUS). In 6/36 (16.7%) pts, ASXL1 was the sole mutation identified. Of the 30 pts with additional mutations (Figure 1), 6/30 (20%) pts harbored 2 independent ASXL1 mutations. When the 27 patients with pathologic ASCL mutations were analyzed for co-mutations, TET2 (13/27, 48%) was the most frequent ASXL1 co-mutation. FLT3 (0/27, 0%) and NPM1 (1/27, 3.7%) were notable for their absence. Median age of pts at diagnosis was 69 years (range 23-80). Prior myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN) was noted in 9/36 (25%) and 11/36 (30.6%) pts, respectively. Four pts (11.1%) had received chemotherapy and/or radiation therapy for a prior non-myeloid neoplasm. Karyotype was normal in 18/36 (50%) pts, and 7 additional pts had intermediate cytogenetic lesions. There were 7 pts (19.4%) with unfavorable cytogenetics (complex karyotype (3 pts), 7q- (3 pts), and 5q- (1 pt)). Four pts (11.1%) had a favorable karyotype, with t(8;21) in 3 pts and t(15;17) in 1 pt. At presentation, median white blood cell count (WBC) was 6.4x103/uL (1.0 x -103). In pts whose AML transformed from prior MPN, median WBC was 50 X103/uL (3.3-140). Standard induction chemotherapy with an anthracycline and cytarabine was given to 17/36 (47%) pts. An additional 3/36 (8.3%) pts underwent induction therapy with clofarabine. Complete remission (CR) was documented in 14/20 (70%) evaluable pts. Of the remaining pts, 11 received a hypomethylating agent, and 5 received other therapies. Thirty-day treatment mortality for all 36 pts and for 27 pts with known ASXL1 pathologic mutation was 13.4% and 18.5% respectively. Kaplan-Meier estimate showed a median overall survival of 349 days (median follow up of 107 days (range 15-1570)). For the 27 pts with a pathologic ASXL1 mutation, the OS was 276 days (Figure 2, median follow up of 145 days (range 18-1570)). Conclusion: ASXL1 mutations in de novo AML with intermediate-risk cytogenetics is associated with poor clinical outcome in cooperative group trials. Strikingly we demonstrate in a single institution, retrospective analysis that 66.7% of pts who present with ASXL1 mutations in the setting of previously untreated AML had documented MDS, MPN and/or prior chemotherapy/radiation. Further studies are necessary to evaluate if ASXL1 mutation has independent prognostic significance in AML or if it is primarily a marker for secondary leukemia. Figure 1: ASXL1 and co-mutations Figure 1:. ASXL1 and co-mutations Figure 2: Overall survival for AML patients with ASXL1 pathologic mutation Figure 2:. Overall survival for AML patients with ASXL1 pathologic mutation Disclosures No relevant conflicts of interest to declare.

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Clinical Impact of Clonal and Subclonal TP53, SF3B1, BIRC3, and ATM Mutations in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.4138.4138 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4138-4138

Author(s):

Ferran Nadeu ◽

Julio Delgado ◽

Cristina Royo ◽

Tycho Bauman ◽

Tatjana Stankovic ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Allele Frequency ◽

Lymphocytic Leukemia ◽

Molecular Characteristics ◽

Prognostic Impact ◽

Driver Genes ◽

Germline Variants ◽

Tp53 Mutations ◽

Mutational Status ◽

The Impact

Abstract Genomic studies have provided a complete profile of somatic mutations in chronic lymphocytic leukemia (CLL). These comprehensive approaches have revealed a relatively large number of mutated genes, the adverse prognostic value of some of which has been demonstrated in a number of reports. Recent studies have shown the clinical relevance of TP53 mutations at very low allele frequency. The presence and prognostic impact of minor mutated clones of other CLL driver genes and their clonal dynamics in the evolution of the disease is not well known. The goal of this study was to explore the presence of clonal and subclonal mutations of TP53, SF3B1, BIRC3, and ATM using an ultra-deep next-generation sequencing (NGS) strategy, to define the evolution of these subclones in different time-points of the disease, and to determine their influence in the outcome of the patients. Samples from 363 untreated CLL cases were included in this study. Copy number alterations were investigated by high density SNP-arrays or by quantitative PCR in 341 and 16 cases, respectively. Targeted ultra-deep NGS of TP53 (exons 4-10), ATM (exons 2-63), BIRC3 (exons 2-9), and SF3B1 (exons 14-16 and 18), including splicing sites, was performed using the Access-Array system (Fluidigm) and sequenced in a MiSeq equipment (Illumina). This methodology combined with a robust bioinformatic analysis based on well-known available tools allowed the identification of mutations down to 0.3% of variant allele frequency (VAF). Results obtained were fully verified by orthogonal techniques. Twelve per cent of VAF was used as threshold for the classification of clonal or subclonal mutations since 12% was the cut-off for detection of mutations by Sanger sequencing. Deletions of 11q comprising ATM or BIRC3 were found in 7% of the cases and were associated with mutations of the other ATM allele in 19/26 (73%) cases and BIRC3 in 3/23 (13%). Deletions of 17p were found in 19 (5%) cases and co-existed with TP53 mutations in 15 (79%) of them. Regarding the mutational status of the studied genes, TP53 mutations were present in 11.6% of patients (7.2% clonal, 4.4% subclonal), ATM mutations in 10% (7% clonal, 1% subclonal, 2% germline mutations considered pathogenic), SF3B1 mutations in 12% (7% clonal, 5% subclonal), and BIRC3 mutations in 4% (2% clonal, 2% subclonal). These subclonal mutations had similar molecular characteristics to their respective high-allele frequency mutations supporting a comparable pathogenic effect. In this regard, clonal and subclonal SF3B1 mutations were associated with shorter time to first treatment (TTT) independently of IGHV mutations. Clonal and subclonal TP53 mutations predicted for shorter overall survival (OS) together with the IGHV mutational status, although the impact of isolated TP53 mutations (i.e. without 17p deletion) on OS was not so evident, as has been the case in other studies. In addition, the outcome of patients with clonal and subclonal BIRC3 mutations showed a similar significant shorter OS. Regarding ATM, the effect of isolated subclonal ATM mutations could not be evaluated because of their low number, but ATM mutations as a whole had a significant impact on TTT even in the absence of 11q deletions. This study also reinforces the need to study the germline of the patients to fully characterize the ATM mutations observed in the tumors. Of note, germline variants previously described as pathogenic were associated with 11q deletions, confirming the hypothesis already suggested that these germline variants may influence disease progression through loss of the otherallele. Clonal dynamics was examined in longitudinal samples of 45 CLL patients. We confirmed the expansion of most TP53 mutated clones after therapy. However, both TP53 and SF3B1 mutations expanded also before any therapy in some patients, indicating that progressive dynamics of these clones is not only dependent on therapy selection. On the contrary, small ATM mutated clones seemed to be more stable. Although the number of cases is limited, we observed that clonal evolution in longitudinal samples had an unfavorable impact on OS. In conclusion, this study shows the presence of a high number of subclonal mutations of different driver genes in CLL and provides insights on the impact of these mutations on the outcome of the patients. These findings suggest that the characterization of the subclonal architecture may be relevant for a better management of CLL patients. Disclosures No relevant conflicts of interest to declare.

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Prognostic Impact of the Interaction between DNMT3A mutation and Internal Tandem Duplication of the FLT3 Gene (FLT3-ITD) in Patients with De Novo Mutated NPM1 (NPM1mut) acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-115337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1492-1492

Author(s):

Guadalupe Oñate ◽

Ana Garrido ◽

Jordi Esteve ◽

Rosa Coll ◽

Montserrat Arnan Sangerman ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Complete Remission ◽

Myeloid Leukemia ◽

De Novo ◽

Prognostic Impact ◽

Intermediate Risk ◽

Mutational Status ◽

De Novo Aml ◽

Acute Myeloid

Abstract Introduction The association of NPM1mut and FLT3-ITD in de novo acute myeloid leukemia (AML) with intermediate-risk cytogenetics has different prognostic impact depending on the FLT3 allelic burden. Previous studies published by our cooperative group showed that patients with de novo AML of intermediate-risk cytogenetics with NPM1mut and FLT3-ITD low ratio (<0.5, FLT3low) at diagnosis presented an overall survival and relapse rate similar to those with NPM1mut and FLT3wt. Therefore, in the CETLAM-2012 protocol, patients with FLT3low NPM1mut AML are not considered for allogenic hematopoietic stem cell transplant (allo-HSCT) in first complete remission (CR1). Recent studies suggest that the co-occurrence of DNMT3A mutation in FLT3-ITD NPM1mut AML patients confers a worse prognosis regardless of FLT3-ITD ratio. We analysed our data to determine whether these findings were confirmed in our cohort, specifically in the low FLT3-ITD ratio patients, since this could have therapeutic implications. Methods and patients A total of 163 patients with de novo AML, intermediate-risk cytogenetics and NPM1mut were analysed (median age 53 years (18-72); male:female 72:91 (0.79)). Eighty patients (49%) harboured an FLT3-ITD, with a high allelic ratio in 42 of 76 patients with available ITD/wt ratio (55%). They were included in the AML-2003 (n=49) and AML-2012 (n=114) CETLAM protocols. Proportion of patients undergoing alloHSCT in CR1 is detailed in table 1. Bone marrow samples from diagnosis were studied for DNMT3A mutations as previously described. The definition of complete remission (CR), overall survival (OS), leukemia-free survival (LFS) and risk of relapse (RR) followed recommended ELN criteria. The Kaplan-Meier method was used to estimate the distribution of LFS and OS, for RR cumulative incidence was used. Results Out of the 163 patients with AML of intermediate risk cytogenetics and NPM1mut, 78 presented DNMT3A mutations (48%). Of these, 62 (79%) presented mutations in codon R882 or corresponded to DNA insertions/deletions while 16 (21%) harboured missense mutations. Presence of DNMT3A mutation did not associate with FLT3-ITD (ITD/85 DNMT3Awt vs ITD/78 DNMT3Amut, p=0.394). In the entire cohort, 5-year OS, LFS and RR were 58±4.5%, 59±4.6% and 27±13.9%. FLT3-ITD ratio confirmed its prognostic impact when analysing FLT3wt (n=83) vs FLT3low (n=34) vs FLT3high (n=42) patients (5-year OS of 68±6% vs 62±8.7% vs 37±8.6%; p=0.002; and 5-year RR of 18±9.4% vs 27±16.1% vs 41±23.2%; p=0.023). On the contrary, DNMT3Amut did not exert any effect on overall outcome (5-yr OS DNMT3Awt vs DNMT3Amut 61±6.2% vs 55±6.2%; p=0.234) When DNTM3A mutational status was considered, the impact of FLT3-ITD on outcome was mitigated in wild-type DNMT3A population. Thus, we found that DNMT3Awt patients presented no statistical differences in OS according to FLT3 mutational status or ratio: FLT3wt (n=46) vs FLT3-ITD (n=39) was 67±8.5% vs 57±8.2%; p=0.122, whereas FLT3wt (n=46) vs FLT3low (n=18) vs. FLT3high (n=19) was 67±8.5% vs. 66±11.5% vs 46±11.8%; p=0.088 (image 1A).This was also seen in relation to LFS and RR according to FLT3 ratio: 5-yr LFS of FLT3wt vs FLT3low vs FLT3high was 72±7.9% vs 61±12.6% vs 51±13.4%; p=0.244 and 5-year RR of the same groups: 19±8.8% vs 26±12.5% vs 27±21.9%; p=0.724 (image 2A). In the DNMT3Amut group, patients with FLT3-ITD (n=41) presented shorter OS than those with FLT3wt (n=37) with an OS of 37±10.7% vs 69±7.8%; p=0.028. When FLT3 ratio was considered, FLT3wt (n=37) vs FLT3low (n=16) vs FLT3high (n=23) showed an OS of 69±7.8% vs. 58±13.2% vs 27±13.1%; p=0.038 (image 1B). Similar results were seen in LFS according to FLT3 ratio (FLT3wt (n=29) vs FLT3low (n=16) vs FLT3high (n=20) 71±8.6% vs 53±12.9% vs 18±13.8%; p=0.012). Finally, we observed significant differences in the 5-year RR when considering DNMT3Amut patients in relation to FLT3 ratio (FLT3wt vs FLT3low vs FLT3high 18±10.6% vs 27±20% vs 54±28.8%; p=0.021)(image 2B). Conclusions In this study, patients with NPM1mut and FLT3-ITDlow presented a similar outcome to patients with NPM1mut and FLT3wt regardless of DNMT3A mutational status. These results support the modification of alloHCST policy in CR1 in CETLAM-2012, which do not consider alloHSCT for patients with FLT3low. On the other hand, concurrence of DNMT3A mutation may have an added negative effect in patients with NPM1mut and FLT3-ITDhigh, which should be further confirmed in larger studies. Disclosures No relevant conflicts of interest to declare.

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Fludarabine, High Dose Cytarabine and Idarubicin-Based Induction Is Highly Effective in Young AML Patients with Concomitant NPM1 and FLT3-ITD Mutation Irrespectively of FLT3-ITD Allelic Burden

Blood ◽

10.1182/blood-2019-131303 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3828-3828

Author(s):

Paola Minetto ◽

Anna Candoni ◽

Fabio Guolo ◽

Marino Clavio ◽

Maria Elena Zannier ◽

...

Keyword(s):

Multivariate Analysis ◽

Leukemic Cells ◽

High Dose ◽

Prognostic Impact ◽

Allogeneic Bone ◽

Wild Type ◽

Npm1 Mutation ◽

Mutational Status ◽

High Dose Cytarabine ◽

Allelic Burden

Background: The presence of FLT3 "internal tandem duplication" (FLT3-ITD) mutation is associated with poor prognosis in acute myeloid leukemia (AML). However, the concomitant presence of NPM1 mutation (NPM1-mut) partially overcomes the negative prognostic impact of FLT3-ITD, which is also modulated by FLT3-ITD/wild-type allelic ratio. NPM1 and FLT3 mutational status assessment is strongly recommended for risk stratificationat diagnosis by the last European Leukemia Net (ELN) guidelines. Aims: To investigate the efficacy of an intensive fludarabine-containing induction regimen (FLAI) for fit, de novo AML patients according to NPM1 and FLT3-ITD mutational status. Methods: One-hundred and sixteen consecutive AML patients, treated in 3 Hematology Italian centers from January 2008 to January 2018, were included in this analysis. Twenty five patients showed isolated FLT3-ITD, 39 concomitant FLT3-ITD and NPM1-mutation and 52 isolated NPM-1-mutation.Median age was 52 yrs (range: 18-65 years). All patients received fludarabine, high dose cytarabine-based induction (fludarabine 30 mg/sqm and ARA-C 2g/sqm ondays 1 to 5 plus idarubicin 10 mg/sqm on days 1-3-5). For patients achieving CR fludarabine was omitted on II induction course and idarubicin dose was increased to 12 mg/sqm. Before2017 patients with isolated FLT3-ITD mutation were scheduled to receive allogeneic bone marrow transplantation (BMT) in first complete remission regardless of allelic burden whereas after 2017 only patients with high allelic burden received BMT in 1st CR. Minimal residual disease was evaluated on marrow samples using multicolor flow-cytometry (MFC)or NPM1 expression levels. Negative MFC-MRD wasdefined by the presence of less than 25 clustered leukemic cells/105 total events (threshold of 0.025% residual leukemic cells, Minetto et. al, BJH 2019). NPM1mutation (NPM1-A, B and D) was measured using Muta Quant Kit Ipsogen from Qiagen. FLT3-ITD allelic burden was available in31/64 of FLT3-ITD patients. Results: Overall 60-days mortality was 4%. After two induction cycles, 101 patients achieved CR (87%). Thirty-three/101 (33%) CR patients underwent BMT in first CR. After a median follow up of 61 months, 3-year overall survival (OS) was 56.8% (median not reached). In univariate analysis OS duration was favorably affected by age <55 yrs (p<0.05), MRD-negative status after induction (both MFC and NPM1 based MRD) (p<0.001) and wild-type FLT3(p<0.05). However, in multivariate analysis only MRD status significantly affected OS (p<0.003). The probability of achieving a molecular MRD negative CR after first cycle among NPM1-mut patients was similar between patients with or without concomitant FLT3-ITD mutation (61% and 66%, respectively, p=n.s.). Interestingly, in younger patients (< 55yrs) OS was comparable in case of isolated NPM1 mutation or concomitant NPM1/FLT3-ITD mutation (3-year OS 68.4% and 62.3%, respectively, p= n.s, Figure 1), regardless of FLT3-ITD allelic burden.Patients with isolated FLT3-ITD mutation had a significantly worse prognosis (3-year OS 34.4%, p<0.05). Again, multivariate analysis showed that persistence of MRD (by any method, p <0.05) was the strongest predictor of outcome. Moreover, performing BMT infirst CR did not impact OS neither in univariate or multivariate analysis. Conclusion: Despite the potential bias due to the retrospective nature of the analysis, our data indicate that intensive fludarabine-high dose cytarabine-based induction exerts a strong anti-leukemic efficacy in younger AML patients carrying NPM1 mutation irrespectively of FLT3 mutational status.Thus, the synergism of fludarabine and cytarabine seems to affect the intrinsic chemosensitivity of NPM1-mut leukemic cells, overcoming the negative impact of FLT3-ITD. Moreover, our data suggest the strong prognostic impact on survival of MRD clearance and question the role of BMT in I CR in this setting of patients. Figure 1 Disclosures Candoni: Gilead: Honoraria, Speakers Bureau; Celgene: Honoraria; Pfizer: Honoraria; Janssen: Honoraria; Merck SD: Honoraria, Speakers Bureau. Bocchia:Incyte: Honoraria; Novartis: Honoraria; BMS: Honoraria.

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