Fms-related Tyrosine Kinase 3 Mutations Research Articles

Real world molecular characterisation and clonal evolution of acute myeloid leukaemia reveals therapeutic opportunities and challenges

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with poor prognosis. Increasing understanding of the molecular mechanisms driving clonal proliferation has resulted in advancements in classification and available therapeutic targets. Fms-related tyrosine kinase 3 (FLT3) mutations are prognostically important and offer options for targeted inhibition, however they are not stable and can emerge or disappear at relapse. Our aim was to review diagnostic testing of consecutive cases of newly diagnosed and relapsed AML reported across Queensland in comparison to available literature. We conducted a retrospective review of 1531 samples from 1231 patients to identify patterns of molecular testing and AML subtypes in our cohort. Outcomes included World Health Organization (WHO) classification, European LeukaemiaNet (ELN) risk category and rates of missed FLT3 mutation testing. Patients aged <60 years had significantly more favourable risk AML (48% vs 25%, p<0.01), with favourable risk chromosomal translocations [t(8;21) and inv(16)] being more common. Thirteen patients (1%) did not have FLT3 mutation testing at diagnosis, with 103 relapse samples (39%) not being tested. Eighteen patients (10%) had FLT3 mutations lost at relapse, with five patients (3%) developing new FLT3 mutations at relapse. This study identifies the subtypes and risk stratification of a large cohort of AML patients over an extended period. The relatively high rate of absent FLT3 mutation testing at relapse as well as FLT3 loss or gain highlights the potential missed opportunities for salvage treatment strategies.

Safety and Efficacy of Midostaurin in Combination with High-Dose Daunorubicin in 7+3 Induction for Acute Myeloid Leukemia with FLT3 Mutation

Safety and Efficacy of Midostaurin in Combination with High-Dose Daunorubicin in 7+3 Induction for Acute Myeloid Leukemia with FLT3 Mutation

PS1011 MEASURABLE RESIDUAL DISEASE BY MULTIPARAMETRIC FLOW‐CYTOMETRY IS A RELIABLE TOOL FOR RISK‐STRATIFICATION OF FLT3‐MUTATED AML PATIENTS

Background:Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), both at the tyrosine kinase domain (TKD) and the juxta‐membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML),identifying a subgroup of patients with mainly unfavorable prognosis.FLT3 mutations are considered an unreliable tool for measurable (previously minimal) residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent a reliable tool to monitor MRD in this molecular subset. In fact,through the recognition and monitoring of leukemia associated immunophenotypes,MFC is applicable in up to 90% of AML patients with a sensitivity up to 10–4.Aims:The aim of our study was to investigate the feasibility of MRD assessment by MFC in FLT3 mutated AML. Furthermore, we investigated if MRD persistence in FLT3 mutated AML affects prognosis in terms of overall (OS) and disease‐free survival (DFS), as compared with FLT3 wildtype ones.Methods:We retrospectively analyzed a series of 39 patients with de novo AML carrying FLT3 mutations (34/39,87% ITD, 2/39, 5% TKD, 3/39, 8% ITD/TKD). We compared MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 128). Both groups were treated according to intensive EORTC/GIMEMA protocols. MRD was measured by MFC at the post‐consolidation timepoint. Patients were defined as MRD‐negative, when obtaining a residual leukemic cells (RLCs) count below the threshold of 3.5x10–4 (0.035%).Results:Overall median age was 47 (range 17–72). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3 mutated group, 22 out of 37 (59%) cases carried a concomitant NPM1 mutation vs 32 out of 124 (26%) of FLT3 wildtype ones (p &lt;0.001). Furthermore, FLT3 mutation was significantly associated with a WBC count &gt;50x109/L (20/39, 51%) vs 30/128 (23%) of those FLT3 wildtype (p &lt; 0.001). Post remission treatment allocation was different between FLT3 mutated patients as compared to. FLT3 wildtype ones; 16 out of 39 (41%) FLT3 mutated patients were submitted to allogenic stem cells transplantation (ASCT) as compared to 34 out of 128 (27%) FLT3 wildtype ones (p = 001). At the post‐consolidation time‐point, MRD negativity rate was significantly lower in FTL3 mutated patients (4/39, 10%) as compared to those FLT3 wildtype (40/128, 31%), (p = 0.009). In particular, 32 out of 35 (91%) FLT3 mutated patients were above the ELN recommended threshold of positivity (0.1%) and 11/35 (31%) were above 1% of RLC.Among FLT3 mutated patients, OS was significantly longer for patients obtaining MRD‐negativity as compared to those who did not (67% vs. 31%, p = 0.9). As for as DFS, we observed the same difference for MRD negative patients vs. MRD positive ones (67% vs. 29%, p = 0.8). The negative impact of MRD was proportional to the amount of RLC at post‐consolidation time‐point (Figure 1) (p = 0.045). However, among FLT3 mutated patients, ASCT was the only post remission treatment allowing a prolonged OS to be achieved as compared to autologous SCT or standard chemotherapy (4‐years OS 63% vs. 0% vs. 15%, p = 0.019, respectively).Summary/Conclusion:In conclusion, MRD as measured by MFC is a biomarker of response also in FLT3 mutated patients. In these patients, ASCT delivery is associated with a satisfactory long term outcome, however a longer duration of response can be pursued by administering FLT3 inhibitors before or after transplant, in order to improve the quality of remission and prevent leukemic recurrence.image

Mutational spectrum and associations with clinical features in patients with acute myeloid leukaemia based on next‑generation sequencing.

The aim of the present study was to examine the associations between 112 acute myeloid leukaemia (AML)-associated genes and the prognosis and clinical features of AML using bioinformatics analysis in 62 patients with AML. A total of 61 gene mutations were identified, and ≥1 mutations were detected in 96.77% of the patients. A total of 11 frequent mutations were identified, including nucleophosmin 1 (NPM1), Fms related tyrosine kinase 3 (FLT3), DNA methyltransferase 3α (DNMT3A) and Notch 2 (NOTCH2), with a mutation rate of ≥10%. The FLT3 mutation was significantly associated with the white blood cell count at the time of diagnosis, and DNMT3A was significantly associated with the French-American-British subtype and cytogenetics of patients with AML. The FLT3, NPM1 and DNMT3A mutations were significantly associated with a poor overall survival (OS) in patients with AML. In addition, the co-mutation of DNMT3A-CCAAT enhancer binding protein α (CEBPA) was observed to be significantly associated with a poor OS in patients with AML. Furthermore, the functional enrichment analysis revealed that the co-mutations of FLT3-NOTCH2, SETBP1-CREBBP and DNMT3A-CEBPA were significantly enriched in processes of ‘negative regulation of cell differentiation’ and ‘immune system development’, indicating that these mutations may serve crucial roles in the diagnosis and treatment of AML.

Midostaurin for FLT3-mutated Acute Myeloid Leukaemia – Considerations for Optimal Use

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Midostaurin for the treatment of adult patients with newly diagnosed acute myeloid leukemia that is FLT3 mutation-positive.

Midostaurin is a multitargeted tyrosine kinase inhibitor (TKI) that potently inhibits activated fms-related tyrosine kinase 3 (FLT3) in the nanomolar range and other kinases including platelet-derived growth factor receptors α (PDGFR- α) and β (PDGFR- β), cyclin-dependent kinase, proto-oncogene tyrosine-protein kinase Src, tyrosine-protein kinase Fgr, spleen tyrosine kinase (Syk), KIT proto-oncogene receptor tyrosine kinase and the major vascular endothelial growth factor receptor (VEGFR). Activating mutations in FLT3, which is one of the more common acute myeloid leukemia (AML) mutations, particularly those that result in an FLT3-ITD (internal tandem duplication) mutation, confer poor prognosis and represent a therapeutic target. Small molecule TKIs that vary in potency and selectivity for FLT3 are under investigation. Here, we provide a comprehensive review of the preclinical and clinical activity of midostaurin, a recently approved drug indicated to be used in combination with cytarabine and daunorubicin induction and cytarabine consolidation chemotherapy for the treatment of AML featuring an FLT3 mutation.

NT1721, a novel epidithiodiketopiperazine, exhibits potent in vitro and in vivo efficacy against acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by heterogeneous genetic and epigenetic changes in hematopoietic progenitors that lead to abnormal self-renewal and proliferation. Despite high initial remission rates, prognosis remains poor for most AML patients, especially for those harboring internal tandem duplication (ITD) mutations in the fms-related tyrosine kinase-3 (FLT3). Here, we report that a novel epidithiodiketopiperazine, NT1721, potently decreased the cell viability of FLT3-ITD+ AML cell lines, displaying IC50 values in the low nanomolar range, while leaving normal CD34+ bone marrow cells largely unaffected. The IC50 values for NT1721 were significantly lower than those for clinically used AML drugs (i.e. cytarabine, sorafenib) in all tested AML cell lines regardless of their FLT3 mutation status. Moreover, combinations of NT1721 with sorafenib or cytarabine showed better antileukemic effects than the single agents in vitro. Combining cytarabine with NT1721 also attenuated the cytarabine-induced FLT3 ligand surge that has been linked to resistance to tyrosine kinase inhibitors. Mechanistically, NT1721 depleted DNA methyltransferase 1 (DNMT1) protein levels, leading to the re-expression of silenced tumor suppressor genes and apoptosis induction. NT1721 concomitantly decreased the expression of EZH2 and BMI1, two genes that are associated with the maintenance of leukemic stem/progenitor cells. In a systemic FLT3-ITD+ AML mouse model, treatment with NT1721 reduced tumor burdens by > 95% compared to the control and significantly increased survival times. Taken together, our results suggest that NT1721 may represent a promising novel agent for the treatment of AML.

Molecular Features of Three Children Diagnosed With Early T-Cell Precursor Acute Lymphoblastic Leukemia.

Molecular Features of Three Children Diagnosed With Early T-Cell Precursor Acute Lymphoblastic Leukemia.

Constitutive Activation of FLT3 Is a Positive Prognostic Factor in Infants with MLL-Rearranged Acute Lymphoblastic Leukemia

Constitutive activation of the FMS-related tyrosine kinase-3 (FLT3) is a common feature of acute leukemias which can be caused by activating mutations or by high expression levels. Aberrant FLT3 downstream signaling is mediated via the RAS/MAPK, PI-3-Kinase/AKT, and STAT5 pathways leading to proliferation, survival, and therapy resistance. In MLL-rearranged acute lymphoblastic leukemia (ALL) activating FLT3 tyrosine kinase domain mutations (TKDs) affecting codons D835 and I836 are frequently identified (Armstrong et al., 2003; Taketani et al., 2004). In addition, MLL-rearranged ALLs are consistently characterized by an exceptionally high FLT3 expression (Armstrong et al., 2002) which has been shown to be associated with ligand independent signaling (Stam et al., 2005). However, the prognostic impact of a constitutive activation of FLT3 in this ALL subset remains controversial.Here, we report on the FLT3 mutational status and gene transcription levels in a large cohort of 95 infants and 72 children with MLL-rearranged ALL. Results obtained were further complemented by a high resolution melting (HRM) screen for common activating NRAS and KRAS mutations at codons 12, 13, and 61. All patients were enrolled in the multicenter trials ALL-BFM 86, 90, 95, 2000, and AIEOP-BFM ALL 2009 as well as Interfant-99 and Interfant-06.In infants, FLT3-TKD mutations were identified in 12/95 patients (12.6%) including one novel insertion/deletion involving codons D835 to S838. In only 2/95 patients (2.1%) an alteration in the juxtamembrane domain of FLT3 was detected. Of the 12 infants with mutation only 2 suffered from a relapse, 2-years cumulative incidence of relapse (CIR) 18%± 12%. In children, only one FLT3 aberration (FLT3-TKD D835 mutation) was detected (1/72, 1.4%). FLT3 transcript levels were analyzed by quantitative real-time PCR in 124 patients (69 infants and 55 children) with available RNA. When we separated the infant cohort into two groups according to the median RQ value, FLT3high and FLT3low, the CIR was significantly different (CIR 19%±7% vs. 66%± 9%, Gray p=0.0001). Of the 6 patients with low FLT3 transcription level, but with presence of a mutation, only one had a relapse. These results indicate that activating FLT3 mutations may compensate for the high relapse risk of patients with a low FLT3 expression. Accordingly, we could show that the CIR was significantly reduced for infants with a low FLT3activation (low transcription, no mutation 19%±7%) compared to those with a high FLT3 activation (high transcription or mutation 75%±9%, Gray p<0.0001, Figure 1). In multivariate analysis, the high prognostic impact of the FLT3 transcription level in infants was independent of age (< vs. ≥6 month), white blood cell count at diagnosis (WBC ≥ vs. <300000), or prednisone response (poor vs. good). This influence of the FLT3 expression could not be seen in children: CIR 13%±7% for low transcription vs. 12%±7% for high transcription.The only other known recurrent mutations in pediatric MLL-rearranged ALL are activating N- and KRAS mutations at codons 12, 13, and 61 (Liang et al., 2006; Andersson et al., 2015). As RAS signaling is considered as a putative downstream target of FLT3, we investigated the frequency of these mutations and their impact in the context of the prognostic value of FLT3 activation. We identified non-synonymous N/KRAS mutations in 21/95 (22.1%) infants and in 10/72 (13.9%) children. The presence of activating RAS mutations correlated with a higher rate of relapse and a lower probability of event-free survival (pEFS). For infants alone, constitutive activation of N/KRAS resulted in a lower pEFS (43%±6% wt vs. 11%±8%, p=0.01), but there were no significant differences in the CIR (40%±6% wt vs. 51±12%, p=0.40).In summary, we confirm that MLL-rearranged infant ALL represents a biologically distinctive entity with unique molecular genetic features. However, in contrast to published studies, we report that hyperactivation of FLT3 signaling is associated with a good prognosis in MLL-rearranged infant ALL. Our data has important implications for the design of rational therapies in MLL-rearranged ALL as the use of small molecule FLT3 inhibitors may be disadvantageous in some infants depending on FLT3 expression levels and FLT3 and RAS mutational status. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characteristics of Sweet Syndrome in patients with acute myeloid leukemia.

Sweet syndrome (SS) is associated with hematologic malignancies including acute myeloid leukemia (AML). Records of patients with AML treated at our institution were reviewed to identify those with SS. Patient characteristics, laboratory values, and cytogenetic and molecular abnormalities were retrospectively reviewed. We identified 21 of 2178 (1%) AML patients who demonstrated clinical signs and symptoms, and histological features consistent with SS. Eleven patients (52%) were classified as AML with myelodysplasia-related features and 3 patients had therapy-related AML. Three patients had received treatment with granulocyte colony stimulation factor, 1 patient liposomal all-trans-retinoic acid, and 2 patients received hypomethylating agents before development of SS. Cytogenetic analysis revealed diploid karyotype in 7 patients (33%); -5/del(5q) in 8 patients (38%): 3 patients had -5/del(5q) as the sole abnormality and 5 patients had -5/del(5q) as part of complex cytogenetics; and complex cytogenetics in 5 patients (24%). Gene mutations in FMS-related tyrosine kinase-3 (FLT3) gene were identified in 7 of 18 evaluable patients (39%), including FLT3-internal tandem duplication in 4 patients and FLT3-D835 tyrosine kinase domain mutation in 3 patients. SS occurs in 1% of AML patients; -5/del(5q) karyotype, FLT3 mutations, and AML with myelodysplasia-related features were more frequent among patients with SS.

Identification of Mutations Coexisting with FLT3 and NPM1 Mutations in AML Patient Samples Using the MyAML™ Targeting Sequencing Strategy

Introduction: Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults, with roughly 19,000 new diagnoses expected yearly in the United States. Mutations in fms-related tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) are observed in over one third of all AML patients. These mutations include internal tandem duplications within the juxtamembrane domain (FLT3-ITD; 15-30% of patients) and substitutions within the tyrosine kinase domain (FLT3-TKD; 5-10% of patients) of FLT3, along with 4bp insertions (15-30% of patients) within the C-terminal domain of NPM1. These mutations have significant impacts on prognosis; patients with FLT3-ITDs have poor prognosis while patients with NPM1 mutations without an associated FLT3-ITD mutation have better long-term outcomes. Since characterization of these mutations is critical for accurate therapeutic decisions, assays have been developed to accurately identify these mutations in AML patients. However, these assays lack greater context because they do not identify coexisting mutations in other AML associated genes. As such, they fail to characterize additional prognostic markers that may more fully predict and stratify AML patients’ disease progression. To investigate the limitations of AML individual mutations assays, we identified coexisting mutations in 22 AML patients with known FLT3 and NPM1 mutations using the MyAML™ targeted sequencing panel.Methods: Isolated DNA from 22 AML samples with known FLT3-ITD, FLT3-TKD and NPM1 mutation status was sheared then hybridized to MyAML oligonucleotide baits comprised of exons (coding and non-coding) and breakpoint hotspots from 194 genes known or predicted to be involved in AML pathogenesis. Targeted loci were sequenced on an Illumina MiSeq utilizing v3 chemistry with the 600-cycle kit. By indexing two samples per flowcell, we were able to sequence 12.6 to 32.9 M unique reads per sample, providing an average depth of 985x across the ~3.5Mb target. Using a custom bioinformatics pipeline, we performed mutation detection analyses to identify single nucleotide variants, indels, and structural variant breakpoints. We also calculated variant allelic frequencies to investigate potential aneuploidy, loss of heterozygosity and clonality.Results: Using individual PCR with capillary electrophoresis (PCR/CE) assays, 14 FLT3-ITD, 7 FLT-TKD and 10 NPM1 mutations were initially detected in the 22 AML patient samples. These mutations were 100% concordant with results from the MyAML sequencing data. However, while the individual PCR/CE assays were limited to detecting specific mutations in two genes, the MyAML panel detected 4,172 protein altering variants in 155 of the 192 additionally targeted genes. These include 35 potential mutations in five key AML genes: 13 in DNMT3A, 5 in IDH1, 8 in IDH2, 4 in KIT and 5 in CEBPA. Eight of the 22 patients contained at least two potentially pathogenic mutations in these five genes, with one patient containing mutations in four of the genes. Interestingly, while some co-existing mutations appear to have the same mutant to wild-type allelic ratio as the main FLT3 or NPM1 mutations, others have distinct ratios that may suggest the presence of subclonal cellular populations. For example, we identified an NPM1 Mutation A (c.859_860insTCTG; p.W288fs*>9; COSM158604) in 40.0% of a patient’s sequencing reads, while a co-existing CEPBA missense mutation (c.961A>G; p.N321D; COSM96570) was only present in 4.3% of the sequencing reads.Conclusions: While individual assays for mutations in FLT3, NPM1 and other common AML genes are useful for patient stratification and prognosis, it is crucial to understand these mutations in a greater genomic context. As more AML-related mutations are detected, such as resistance mutations to treatments with novel tyrosine kinase inhibitors, it becomes increasing important to fully characterize a patient’s tumor genome in order to successfully classify and treat their disease. In addition, as more AML patient samples are sequenced, relationships between mutations and their clonal populations can be elucidated, potentially leading to more effective combination therapies. MyAML targeted gene sequencing is the most comprehensive AML specific assay for the identification of somatic and germline driver mutations in their clonal context for the prediction of recurrence and response to various treatment regimens. DisclosuresPatay:Genection, Inc.: Consultancy. Cubbon:LabPMM LLC: Employment. Stenzel:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership.

FLT3 Variant Detection In Cell Lines

BackgroundMassive parallel sequencing (MPS) reveals many common drivers of cancer and spurs the broad development of targeted diagnostics and therapeutics. Precise characterization of immortalized fms-related tyrosine kinase 3 (FLT3) mutant cell lines that arise spontaneously and cell lines engineered to incorporate recurrent driver mutations will be needed to assist in clinical diagnostic and therapeutic translation. Two major classes of variants in the FLT3 gene drive cytogenetically normal acute myeloid leukemia (AML): nonsynonymous somatic mutations, predominantly in the tyrosine kinase domain (TKD), and somatic internal tandem duplications (ITD) in and immediately adjacent to the juxtamembrane domain (JMD). While clinical laboratories utilizes PCR and electrophoresis-based product sizing to detect these mutations, this approach does not fully characterize their sequence content or the context of FLT3 mutations. Specifically, ITD mutations are intractable due to their high degree of variability in length (3 to over 300 basepairs), sequence, copy number, and the insertion position. Next-generation sequencing (NGS) offers the potential for deriving a fuller understanding of these mutations and their context; however, the performance of most NGS analysis tools for identifying, quantifying the mutant to wildtype allelic ratios, and assigning specific intragenic locations to these heterogeneous FLT3 ITD mutations is largely unknown. MethodsWe coupled targeted baiting assays from Nimblegen and Agilent with the Illumina MiSeq sequencing platform to deep sequence FLT3 in five mutation containing cell lines (MV4-11, MOLM-13, PL-21, IVS-0059, IVS-0062) and a FLT3 wild-type cell line (NALM-6). We used this strategy to examine the performance of commonly used NGS analysis tools in identifying FLT3 mutations, including ITDs of varying sizes. An optimized bioinformatic pipeline was developed that used BWA to align raw reads, followed by Picard and GATK to process the resulting aligned reads. These aligned reads were then analyzed by Pindel and GATK to detect FLT3 mutations in each cell line. Moreover, we developed a novel strategy that used an additional eight targeted short tandem repeats (STRs) across seven chromosomes to calibrate allele frequencies. To validate the copy numbers for this calibration, we also performed cytogenetics and fluorescence in situ hybridization (FISH) on each cell line. ResultsUsing our custom target enrichment design, we were able to generate sequence reads covering all coding bases of FLT3 to a minimum read depth of 80x (average=2793x; median=1120x). This coverage and read depth was sufficient to detect all known FLT3 mutations in the five mutation containing cell lines while confirming the absence of these FLT3 mutations in the NALM-6 wild-type cell line. Also, we were able to confidently determine that no novel FLT3 mutations were present in any of these cell lines. In addition to identifying the known mutations, the employed sequencing strategy was also able to accurately characterize the size and sequence content of each mutation. These mutations include large ITD mutations of 126bp (PL-21) and 279bp (IVS-0062), as well as shorter insertions of 30bp (MV4-11) and 21bp (MOLM-13). As expected, all ITDs were identified as in-frame insertions within the JMD. Along with the more difficult ITD mutations, we were able to detect a somatic single nucleotide missense TKD mutation (c.2503G>A, p.D835N, COSMIC Mutation Id:789.) in the IVS-0059 cell line. For each mutation, the allelic frequency was calculated using read depths calibrated against known STRs across the genome. These STRs were confirmed to be biallelic in each cell line using cytogenetic and FISH analyses. Although these cytogenetic tests revealed complex karyotypes, such as additional copies of chromosome 13 in PL-21, FLT3 mutations could still be detected and their allele frequencies effectively calculated. ConclusionsThese data demonstrate FLT3 variants, including ITDs, can be reliably detected, characterized and quantified with next-generation sequencing assays using targeted, deep sequencing with an appropriate bioinformatic strategy. Disclosures:Carson:Genection: Consultancy. Patay:Genection: Consultancy, Equity Ownership. Graham:Genection: Employment. Osgood:Invivoscribe: Employment. Miller:Invivoscribe: Employment, Equity Ownership.

Effect of NPM1 and FLT3 Mutations on the Outcomes of Elderly Patients With Acute Myeloid Leukemia Receiving Standard Chemotherapy

The effect of prognostically important gene mutations (MUTs), nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1) and fms-related tyrosine kinase 3 (FLT3), in elderly patients with acute myeloid leukemia (AML) is not well defined. We analyzed 557 patients, 65 years of age or older with newly diagnosed AML, treated at our institution between 2000 and 2010 with cytotoxic chemotherapy. NPM1 and FLT3 analysis were available in 146 patients (26%) and 388 patients (70%), respectively. NPM1 and FLT3 MUTs occurred in 16% and 12% of patients, respectively. No difference in median overall survival was observed between FLT3-MUT and NPM1-MUT patients who received cytotoxic chemotherapy. Outcome was significantly better among patients with NPM1-MUT/FLT3-wild type (WT) genotype (n= 14) compared with patients carrying FLT3/NPM1 genotypes other than NPM1-MUT/FLT3-WT (n= 125). The complete remission rates were 71% and 49%, respectively (P= .11). The median survival was 21.5 months vs. 9.0 months and estimated 2-year survival rates were 51% vs. 38%, respectively (P= .003). NPM1 and FLT3 MUTs appear to occur less frequently in elderly AML patients. The prognostic effect of isolated NPM1- or isolated FLT3-MUT is minimal. Elderly AML patients with NPM1-MUT/FLT3-WT genotype have significantly improved outcomes compared with patients with other NPM1/FLT3 genotypes when treated with cytotoxic chemotherapy.

Exome Sequencing Identifies Recurring FLT3 N676K Mutations in Core Binding Factor Leukemia

AbstractAbstract 404Inversion inv(16) or translocation t(16;16) and translocation t(8;21) are recurring rearrangements in acute myeloid leukemia (AML), which result in the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1, respectively. These rearrangements are found in 15–20% of adult de novo AML cases and are associated with favourable prognosis. CBFB and RUNX1 form the core binding factor (CBF), which is a transcription factor essential for normal hematopoiesis and myeloid development. By disrupting the physiological transcription factor activity of CBF the fusion proteins causes repression of the CBF-target genes resulting in a block of differentiation. Since expression of CBFB/MYH11 or RUNX1/RUNX1T1 on their own is not sufficient to cause leukemia it is likely that additional mutations are required for malignant transformation.To systematically identify mutations which may collaborate with CBFB/MYH11 during leukemogenesis we performed exome sequencing of an AML sample with an inv(16). The sample was selected based on availability and absence of known additional genetic alterations. By comparing the AML exome sequence with the exome sequence of a remission sample from the same patient we were able to identify leukemia-specific sequence variants as described previously (Greif et al., 2012, Blood). Using this approach we found an N676K mutation in the ATP-binding domain (TKD1) of the fms-related tyrosine kinase 3 (FLT3) gene. Mutations affecting N676 resulting in variable amino acid changes (N676D or N676S) were initially discovered in a screen for resistance to tyrosine kinase inhibitors (TKI) in FLT3 internal tandem duplication (ITD) expressing Ba/F3 cells (Cools et al., 2004, Cancer Res). An N676K point mutation has been reported in a cytogenetically normal (CN) AML patient with FLT3-ITD and TKI-resistance (Heidel et al., 2006, Blood). In contrast, our patient with inv(16) and the FLT3 N676K did not carry an additional FLT3-ITD. In a cohort of 69 patients with inv(16) we found a total of 4 patients with FLT3 N676K mutations (4/69, 6%). In 14 patients with t(16;16) and in 36 patients with t(8;21) we identified one patient each with FLT3 N676K (1/14, 7% and 1/36, 3%). Thus, the overall mutation frequency for patients with CBFB/MYH11 rearrangement was 6% (5/83). None of the CBF AML patients with FLT3 N676K mutation had an additional FLT3-ITD. In 90 CN-AML patients we detected only a single FLT3 N676K mutation and the affected patient had a concurrent FLT3-ITD. We are currently testing the frequency of FLT3 N676K mutations in independent CBF-AML cohorts.To test the transforming potential we expressed the FLT3 N676K mutant in Ba/F3 cells. As controls we expressed FLT3 wild type (WT), FLT3 mutants D835Y or ITD in parallel. Cell surface expression of N676K was similar to WT, but increased compared to D835Y and ITD. Cell proliferation assays were done in presence and absence of IL-3 or FLT3 ligand (FL). FLT3 N676K leads to IL-3 and FL independent cell growth reaching 25% of IL-3 mediated growth. FLT3 inhibition by AC220 or PKC412 abrogates this proliferation, but N676K is slightly more resistant to inhibition than ITD. In contrast to ITD expression that results in STAT5 phosphorylation, N676K expression leads to phosphorylation of MAPK and AKT.Gene expression profiling of patients with inv(16) revealed that patients with additional FLT3 N676K mutation show a significant enrichment of gene sets including ubiquitin mediated proteolysis, adherens junction and JAK/STAT signaling pathway.According to a structural model, the N676K mutation stabilizes the fold of the kinase domain between the juxtamembrane domain (JMD) and a hydrophobic pocket that is the target of FLT3 inhibitors. N676K mutations could therefore reduce the autoinhibition of FLT3 by the JMD and also negatively affect the binding affinity of FLT3 inhibitors.Ours is the first report of recurring FLT3 N676 mutations in the absence of FLT3-ITD. Our findings point towards a specific association of FLT3 N676K mutations with CBF leukemia. Based on our functional assays N676K acts as a gain-of-function mutation. Although FLT3 has been known for more than a decade to be mutated in one third of AML patients, it appears that the spectrum of FLT3 mutations is still not fully understood, in particular, in defined cytogenetic subgroups of AML. Unbiased mutation screening by exome sequencing allows the detection of novel sequence variations even in extensively studied genes. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Mutations in the Fms-Related Tyrosine Kinase 3 (FLT3) Gene Independently Predict Poor Outcome in Acute Myeloid Leukemia (AML) with t(8;21): A Study of the German-Austrian AML Study Group (AMLSG).

Abstract 825▪▪This icon denotes an abstract that is clinically relevant. Background:Acute myeloid leukemia (AML) with t(8;21)(q22;q22) is considered as a prognostically favorable subgroup of AML. However, outcome is heterogeneous and almost half of adult patients (pts) cannot be cured by current treatment. Candidate molecular markers have been assessed in an effort to predict outcome in AML with t(8;21) at the time of diagnosis and to potentially guide the development of genotype-specific approaches. In most, but not all studies, KIT mutations were associated with adverse prognosis in AML with t(8;21). However, no larger study has elucidated the independent prognostic impact of various gene mutations in a comprehensive molecular analysis. Methods:Bone marrow and/or blood specimens from 146 adult pts diagnosed with de novo (n=137), therapy-related (n=5) or unknown history (n=4) t(8;21) AML were studied for the presence of additional chromosome abnormalities and for mutations in FLT3 [internal tandem duplications (ITD) and tyrosine kinase domain mutations (TKD)], N-/K-RAS, KIT and JAK2 (V617F) genes. All pts were treated on one of 7 prospective protocols of the German-Austrian AML Study Group (AMLSG). For induction pts received anthracycline-and cytarabine-based therapy regimens; pts achieving a complete remission (CR) were assigned to postremission therapy incorporating higher doses of cytarabine in various settings or to autologous stem cell transplantation. Multivariable analyses were performed to assess the prognostic value of gene mutations on relapse-free (RFS) and overall survival (OS) and were stratified for treatment protocols. Results:Mutations were identified in 56% of the pts with the highest frequency observed in KIT (30%), followed by mutations in RAS (21%), FLT3 (13%) [ITD (9.5%) and TKD (3.5%)] and JAK2 (3.5%) genes. When correlating gene mutations with clinical features, pts with RAS mutations had a higher WBC (P=0.003) and a lower frequency of the most common secondary chromosome abnormality represented by the loss of a sex chromosome (LOS; P=0.03) when compared to pts with wild-type RAS; for the other genes studied no differences in pretreatment characteristics were observed. The median age of the study cohort at diagnosis was 46 years (yrs; range, 17-73 yrs), and the median white blood count (WBC) was 8.7 × 109/l (range, 0.9-152 × 109/l). Median follow-up for survival according to Korn was 3.4 yrs [95%-confidence interval (CI), 2.6.-5.2 yrs]. The CR rate in the entire study cohort was 89% and none of the gene mutations impacted as single marker on the CR rate. In univariable and multivariable analyses, only FLT3 mutations significantly affected relapse-free survival (RFS) and overall survival (OS). No significant difference in RFS and OS was observed with respect to the mutational status of KIT, RAS and JAK2 genes. In univariable analyses, pts with FLT3 mutations relapsed more frequently (P=0.03; 3-yr RFS rates, 22% vs 58%) and had a shorter survival time (P=0.006; 3-yr OS rates, 26% vs 63%) than those without FLT3 mutations. Multivariable analyses revealed the mutational status of FLT3 as independent prognostic variable for RFS and OS. Age was a significant risk factor for OS. Additional variables that were also included in multivariable models were mutational status of KIT and RAS, log10(WBC), and presence of LOS. Pts harboring FLT3 mutations relapsed more frequently (HR, 3.20, P=0.01) than pts with FLT3 wild-type. In addition, the risk of death in pts with FLT3 mutations was more than four times higher (HR, 4.24, P=0.004) than in pts lacking these mutations. Conclusions:In conclusion, we show here in a large group of adult AML pts with t(8;21) that the presence of activating FLT3 mutations independently predicts for poor outcome within this favorable subset of AML. Thus, adults with t(8;21)-positive AML and FLT3 mutations require alternative treatment strategies. Our data support the rationale of evaluating FLT3 tyrosine kinase inhibitors in these pts. Disclosures:No relevant conflicts of interest to declare.

Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis

FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients.

Trisomy 13 Defines a Subgroup of Myeloid Malignancies with an Extremely High Frequency of AML1 Gene Mutations and Newly Defined VEGFR-1 Mutations.

Somatic mutations in the DNA-binding domain, the socalled Runt domain, of the AML1 gene have been identified to occur in AML with the highest incidence in the AML M0 subgroup as well as in myeloid malignancies displaying acquired trisomy 21. To characterize our own cohort of AML M0 patients (n = 83) for AML1 mutations, we analyzed AML1 transcripts by a combination of denaturing high performance liquid chromatography (dHPLC) and direct sequencing. In this cohort 37 cases (45%) carried an AML1 mutation. The AML1 mutated cases showed a remarkable high incidence of trisomy 13 (14/37 pts; 38%). In contrast to previous data only one case had a trisomy 21 (3%). The group without AML1 mutations (n = 46) included only one case with trisomy 13 (2%) and no trisomy 21. To extent this observation beyond the AML M0 subtype, we analyzed 14 additional trisomy 13 cases from other FAB subtypes. 79% (11/14) of the non-AML M0 trisomy 13 cases displayed an AML1 mutation. To evaluate the role of trisomy 13 in the context of AML1 mutations we looked for tyrosine kinase receptors on chromosome 13, which might play a cooperative role in concert with mutated AML1. The fms-related tyrosine kinase 3 (FLT3) and the fms-related tyrosine kinase 1 (FLT1, VEGFR-1) were identified as candidates on chromosome 13. In the AML M0 group 70 of 83 cases were analyzed for activating FLT3 length mutations (FLT3-LM) and/or activating point mutations in the tyrosine kinase domain (FLT3-TKD). 12 cases (18%) were positive for such activating mutations (9 FLT3-LM, 2 FLT3-TKD, 1 FLT3-LM+TKD) with 4 of these cases having an AML1 mutation in parallel. However, none of the 14 analyzed M0 cases with trisomy 13 were affected. FLT3 overexpression has also been suspected as a mechanism for constitutive FLT3 activation. Therefore, we hypothesized that trisomy 13 by means of a gene dosage effect might contribute to an increase in FLT3 expression. When we compared FLT3 RNA expression levels as determined by Affymetrix U133 Plus 2.0 gene expression arrays between AML M0 trisomy 13 samples (n = 9) versus other AML M0 samples (n = 19), we observed a significantly higher FLT3 expression in the trisomy 13 cases (p = 0.048, t-test). In the group of non M0 cases with trisomy 13 (n = 14) 3 cases (21%) had activating FLT3 mutations (2 FLT3-LM, 1 FLT3-TKD). Only 1 of these cases carried an AML1 mutation in parallel. In the trisomy 13 group we detected novel mutations in VEGFR-1 in 3 of 21 cases (14.2%). All three cases had an M0 phenotype as well as an AML1 mutation. Thus, 3 of 5 M0/trisomy 13 cases were positive for this new kind of mutation. In conclusionwe confirm the high incidence of AML1 somatic mutations in the AML M0 subgroup (45%).We identified a new entity of AML characterized by trisomy 13 with an incidence of AML1 mutations of about 80%.We suggest that trisomy 13 might be a mechanism for the amplification of FLT3 transcript levels for the activation of FLT3 signaling which collaborates with AML1 somatic mutations in leukemogenesis.We identified a new kind of mutation in the tyrosine kinase domain of VEGFR-1 which might be a specific cooperating mutation for AML1 mutation.

Prognostic Gene-Expression Signatures in Adult Acute Myeloid Leukemia with Normal Karyotype.

Acute myeloid leukemia (AML) encompasses a large number of morphologically similar but molecularly distinct variants. Recurrent cytogenetic aberrations have been shown to constitute markers of diagnostic and prognostic value. However, despite recent successes in detecting novel molecular markers like FLT3 (fms-related tyrosine kinase 3) mutation, treatment stratification is still difficult, especially for the 40–45% of patients with intermediate-risk, normal karyotype disease. To better characterize AML at the molecular level, and to address the need for improved risk stratification, we recently profiled gene expression in a large series of adult AML patients (Bullinger et al., N Engl J Med 350:1605, 2004). By unsupervised analysis we identified new prognostically-relevant AML subgroups, and using a supervised learning algorithm we constructed a gene-expression based outcome predictor, which accurately predicted overall survival across all patients, including for the subset of AML cases normal karyotype. Having demonstrated the presence at diagnosis of normal karyotype signatures correlating with clinical outcome, we have now sought to refine a prognostic signature specific for normal karyotype disease. Towards this goal, we have now profiled 119 samples of adult AML patients with normal karyotype using 42k cDNA microarrays from the Stanford Functional Genomics Facility. By semi-supervised analysis using the supervised principal component method (Bair et al., PLoS Biology 2:511, 2004), we built a cross-validated gene-expression based outcome predictor in a randomly partitioned training set (n=60 samples). This outcome signature, comprising only 16 genes, significantly predicted outcome class for normal karyotype samples in the independent test set (n=59 samples; P=0.001). In multivariate analysis, the 16-gene signature was a strong [odds ratio=0.35 (0.13 to 0.91); P=0.01] factor in predicting overall survival, independent of known prognostic factors including FLT3 mutations and preceeding malignancy. Our findings support the utility of expression profiling for improved risk stratification and clinical management of the clinically important subclass of AML patients with normal karyotype disease.