Rearrangements In Acute Myeloid Leukemia Research Articles

BRAF Mutations in Patients with Myeloid Neoplasms: A Cancer Center Multigene Next-Generation Sequencing Analysis Experience.

BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.

Age-related dynamics in acute myeloid leukemia: Implications for prognosis, risk stratification, and treatment response

Abstract BACKGROUND: Acute myeloid leukemia (AML) is a complex, heterogeneous disease driven by acquired somatic mutations. The presence of specific mutations advances stratification, treatment, and prognosis. Linear accumulation of mutations over time is a crucial factor in cancer development, particularly among elderly patients. Our recent study on gene rearrangement in AML revealed a significant association between age and adverse risk cases. AIM: The aim of this study was to examine the distribution of age, molecular characteristics, risk stratification, and treatment response based on age among patients with de novo AML in Iraq. PATIENTS AND METHODS: A prospective cohort study enrolled 115 Iraqi adult patients diagnosed with de novo AML using morphology and flow cytometry from December 2020 to May 2022. The Leukemia Q-Fusion Screening Kit, employing multiplex reverse transcription–real-time quantitative polymerase chain reaction with 30 gene rearrangements, was employed for the identification of gene rearrangement. The patients received care and follow-up at the Hematology Unit of Baghdad Teaching Hospital in Medical City. Ethical approval from the College of Medicine’s Ethical Committee at the University of Baghdad was secured before commencing the research, ensuring adherence to ethical standards throughdout the study. RESULTS: The age distribution exhibited a bimodal pattern, with a mean of 45.1 ± 17.5 years, ranging from 18 to 84 years, and a median of 46 years. A total of 39.1% of patients were diagnosed with AML before the age of 35 years, while 43% were diagnosed after the age of 51 years. AML patients with RARA mutations, RUNX1:: RUNX1T1 alterations, and NPM1 mutations were predominantly observed in younger individuals, as well as those diagnosed with AML defined by differentiation. Conversely, KMT2A rearrangements were more prevalent among older age groups, with a statistically significant difference in the distribution of AML classifications according to the World Health Organization (WHO) by age categories (P = 0.001). The risk stratification based on age and response assessment showed a notable higher risk profile observed among elderly patients that was associated with adverse risk and poorer response and mortality (P < 0.05). The prediction of treatment response accuracy rate was improved by adding age to the WHO classification and ELN 2022 risk stratification (73.5%–87.9%). CONCLUSION: Age significantly influences AML prognosis and treatment response. Incorporating age into risk stratification improves accuracy. Tailored approaches considering age are vital for optimizing AML management and outcomes.

An Integrated Approach Including CRISPR/Cas9-Mediated Nanopore Sequencing, Mate Pair Sequencing, and Cytogenomic Methods to Characterize Complex Structural Rearrangements in Acute Myeloid Leukemia.

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach's value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.

Detection of Cryptic Gene Fusion and Chimeric RNA Variants in Relapsed/Refractory Acute Myeloid Leukemia Patients Diagnosed with KMT2A/Afdn Chromosomal Translocation

Chromosomal rearrangements, such as translocations, play a significant role in creating chimeric genes and oncofusion proteins initiating leukemogenesis in 52% of acute myeloid leukemia (AML) patients, and are associated with an adverse prognosis. Currently, there are limited targeted therapies available for AML patients with chromosome rearrangements primarily due to a lack of accurate molecular and genetic characterization of the mechanism of leukemic progression along the treatment course. In our study, we investigated the pathological landscape of treatment resistance and relapse in AML patients with KMT2A-rearranged gene fusions (previously known as mixed lineage leukemia, MLL gene, 11q23), which is one of the most common gene rearrangements in AML with a dismal prognosis. Our approach included chromosome analysis, fluorescence in situ hybridization (FISH), transcriptomic analyses (RNA-seq), polymerase chain reaction (PCR), DNA-sequencing and quantitative PCR (qPCR) were applied to monitor RNA- and genome-wide changes among newly diagnosed, refractory and relapsed AML patients. Bone marrow samples of seven AML patients with different chromosome were obtained and two samples were found to possess the chromosomal translocation t(6;11)(q27;q23) which leads to KMT2A/AFDN gene fusion. In these patients, RNA-seq identified a cryptic CCDC32 (15q15.1) /CBX3 (7p15.2) gene fusion in both newly diagnosed and relapsed samples, which is previously unreported in KMT2A/AFDN-rearranged AML patients. Quantitative gene expression measurement by qPCR demonstrated that CCDC32/CBX3 gene fusion significantly affected the expression of wild-type genes of CCDC32 (essential for embryonic development) and CBX3 (an oncogene for solid tumors) during the relapse, suggesting the potential oncogenic role for the CCDC32/CBX3 chimera in AML development. Our result is consistent with a previous study showing multiple breakpoints in AML patients with KMT2A/AFDN translocations. KMT2A rearrangements are known to have an unfavorable prognosis in AML and the specific t(6;11)(q27;23) translocation in AML patients leads to a median overall survival of only 12 months. Our novel findings also suggest that one reason for the adverse outcomes of KMT2A chromosomal rearrangements is their complex ability to generate chimeric variants and more aggressive subclones to elude the treatment. Our study implies that standard cytogenetic analyses and emerging NGS are not sufficient to fully diagnose a patient with chromosome rearrangement. Particularly, short-read RNA-seq presents problems because it cannot detect gene fusions with low expression level, like the KMT2A/AFDN gene fusion that was undetected in patient 7 by our short-read RNA-seq. Instead, long-read RNA-seq, combined with cytogenetics and molecular profiling through PCR/Sanger DNA-sequencing, should be utilized to identify complex fusion transcripts and different chimeric variants of multiple gene fusions.

Prevalence of Gene Rearrangement by Multiplex PCR in De Novo Acute Myeloid Leukemia in Adult Iraqi Patients.

Gene rearrangements of acute myeloid leukemia (AML) play a significant role in categorizing patients and provide valuable information about prognosis and treatment choices. However, in Iraq, the prevalence and prognostic significance of gene rearrangements in AML have not been previously examined. This study utilized a multiplex reverse transcription real-time PCR (RT-qPCR) system to identify gene rearrangements in a group of 115 adult patients from Iraq who had been diagnosed with De Novo AML. The diagnosis of AML was confirmed through blood film and flow cytometry. The ethical committee of the College of Medicine at the University of Baghdad provided approval for this research study. In this study, 66.1% of the patients diagnosed with acute myeloid leukemia (AML) exhibited distinct genetic abnormalities. Among these abnormalities, the most frequent was the rearrangement involving the KMT2A gene, observed in 19.9% of the patients. The risk stratification analysis revealed that 40% of the patients were classified as having a favorable risk, 4.3% as intermediate risk, and 25.2% as adverse risk. A subtype of AML known as core-binding factor (CBF) AML was identified in 21.7% of the cases, with 84% of these patients achieving complete remission. The NPM-RARA gene rearrangement, found in 43% of acute promyelocytic leukemia (APL) cases, was associated with a 71% complete remission rate. Among patients with KMT2A rearrangement, which accounted for 19.9% of all AML cases, the MLL-AF10 rearrangement was the most common, although only one patient with KMT2A rearrangement achieved complete remission. Furthermore, the analysis of demographic data revealed a significant association between increased risk and advanced age, presence of comorbidities, and FAB classification (M0 subtype). The prevalence of genetic rearrangements in Iraqi De Novo AML patients is higher than the global trend, highlighting the importance of genetic characterization in risk assessment and treatment decisions.

Fusion Gene-Based Classification of Variant Cytogenetic Rearrangements in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) represents a heterogeneous disease entity that is continuously moving to a more genetically defined classification. The classification of AML with recurrent chromosomal translocations, including those involving core binding factor subunits, plays a critical role in diagnosis, prognosis, treatment stratification, and residual disease evaluation. Accurate classification of variant cytogenetic rearrangements in AML contributes to effective clinical management. We report here the identification of four variant t(8;V;21) translocations in newly diagnosed AML patients. Two patients showed a t(8;14) and a t(8;10) variation, respectively, with a morphologically normal-appearing chromosome 21 in each initial karyotype. Subsequent fluorescence in situ hybridization (FISH) on metaphase cells revealed cryptic three-way translocations t(8;14;21) and t(8;10;21). Each resulted in RUNX1::RUNX1T1 fusion. The other two patients showed karyotypically visible three-way translocations t(8;16;21) and t(8;20;21), respectively. Each resulted in RUNX1::RUNX1T1 fusion. Our findings demonstrate the importance of recognizing variant forms of t(8;21) translocations and emphasize the value of applying RUNX1::RUNX1T1 FISH for the detection of cryptic and complex rearrangements when abnormalities involving chromosome band 8q22 are observed in patients with AML.

Molecular cytogenetics in acute myeloid leukemia in adult patients: practical implications.

Throughout the last 50 years, cytogenetic analyses of pretreatment bone marrow and / or blood samples from patients diagnosed with acute myeloid leukemia (AML) revealed a large number of recurring chromosome aberrations, both structural and numerical. Using standard banding methods and, more recently, molecular cytogenetic techniques, such as fluorescence in situ hybridization, spectral karyotyping, multiplex fluorescence in situ hybridization and comparative genomic hybridization, cytogenetic investigations detect acquired abnormalities that, together with submicroscopic gene mutations and changes in gene expression, strongly influence the clinical features and prognosis of patients with AML. Selected reciprocal translocations and inversions and their molecular counterparts, as well as a number of unbalanced chromosome abnormalities are used, together with bone marrow morphology, immunophenotype, and clinical characteristics, to define separate AML entities in the World Health Organization Classification of Haematolymphoid Tumours. Moreover, cytogenetic findings (and specific gene mutations) are being used in geneticrisk classifications, such as the 2022 European LeukemiaNet classification. Such classifications divide patients into broad prognostic categories: favorable, intermediate, and adverse, which are useful in the management of adults with AML. In this article, I review the present data on recurrent chromosome rearrangements in AML and on correlations between cytogenetic findings and clinical features and treatment outcomes of adult patients diagnosed with AML.

TXNIP induces growth arrest and enhances ABT263-induced apoptosis in mixed-lineage leukemia-rearranged acute myeloid leukemia cells.

Thioredoxin‐interacting protein (TXNIP) has been widely recognized as a tumor suppressor in various cancers, including liver, breast, and thyroid cancers. Although TXNIP is epigenetically silenced in acute myeloid leukemia (AML) cells, as in many cancer cells, its role in leukemogenesis remains elusive. Mixed‐lineage leukemia (MLL) gene rearrangements in AML are associated with poor prognosis, and the development of a new treatment method is eagerly anticipated. In this study, we first reveal that lower expression of TXNIP is correlated with shortened overall survival periods in AML patients. Moreover, we demonstrated that TXNIP overexpression significantly suppresses proliferation in AML cells harboring MLL fusion genes. TXNIP promotes autophagy by increasing expression of the autophagy protein, Beclin 1, and lipidation of LC3B. We also show that TXNIP overexpression combined with ABT263, a potent inhibitor of Bcl‐2 and Bcl‐xL, is highly effective at inducing cell death in MLL‐rearranged (MLL‐r) AML cells. In summary, this study provides insights into the molecular mechanism of TXNIP‐mediated tumor suppression and furthermore underscores the potential of TXNIP as a promising therapeutic target for MLL‐r AML.

Bioinformatics Analysis of Differentially Expressed Genes and Their Functional Enrichment in Acute Myeloid Leukemia Bearing MLL Translocation

Background: Chromosomal translocations of the mixed-lineage leukemia gene (MLL; tMLL) correlate with resistance to therapy and an extremely poor prognosis for individuals with Acute Myeloid Leukemia (AML). The underlying mechanisms are still unknown. This study aims to identify the key genes and the potential molecular mechanisms involved in MLL rearrangement in AML using a bioinformatics approach. Methods: The gene expression profiles from 15 individuals with partial tandem duplication of the MLL gene (MLL-PTD)-AML and 10 tMLL-AML samples were downloaded from the Gene Expression Omnibus (GEO) database. The Differentially Expressed Genes (DEGs) were selected and functional enrichment analyses were performed. The Protein-Protein Interaction (PPI) network was established and visualized in Cytoscape. The hub genes were identified by CytoHubba and significant modules were screened out by Molecular Complex Detection (MCODE). Results: We categorized a total of 885 DEGs comprising 330 upregulated and 555 downregulated genes. The majority of DEGs were significantly enriched for calcium ion transmembrane transport, embryonic skeletal system morphogenesis and cell proliferation processes. Several pathways were enriched, including those associated with PI3K-Akt signaling and insulin resistance. We identified 32 hub genes and screened out 2 modules. Conclusion: The genes we have identified in this study may represent potential biomarkers for MLL-rearranged AML and contribute to the development of novel therapeutic strategies.

Unique RUNX1 Gene Rearrangements in Acute Myeloid Leukemia Identified (AML)

Unique RUNX1 Gene Rearrangements in Acute Myeloid Leukemia Identified (AML)

Next-generation sequencing and molecular cytogenetic characterization of ETV6-LYN fusion due to chromosomes 1, 8 and 12 rearrangement in acute myeloid leukemia

In a newly diagnosed patient with acute myeloid leukemia (AML) and complex cytogenetics and negative for gene mutations associated with myeloid neoplasms, RNA sequencing by next-generation sequencing (NGS) through a large cancer-related gene panel showed ETV6-LYN leukemic fusion transcript. Breakpoint analysis of the NGS reads showed fusion of exon 5 of the ETV6 gene to exon 8 of the LYN gene. Metaphase fluorescence in situ hybridization (FISH) inferred a four-break rearrangement of three chromosomes, namely 1, 8 and 12. First, there was a balanced translocation t(1;12)(p13;p13.2) in which the ETV6 was split between der(1) and der(12). Second, an inverted insertion of 8q12.1~q24.21 into 1p13 occurred, thus bringing ETV6 and LYN into juxtaposition in the correct 5' to 3' orientation to produce an in-frame chimeric fusion gene on der(1). Notwithstanding two previous reports of ETV6-LYN fusion in myeloproliferative neoplasms (MPN), we report the first case of this fusion in AML and hence broaden its disease association. We also illustrate the clinical utility of NGS based detection of gene fusion in the setting of complex karyotype or cryptic aberration, since this method does not require a priori knowledge of the translocation partner and exact breakpoints to guide the application of appropriate primers or probes.

Rare CBFB-MYH11 cryptic rearrangement in acute myeloid leukemia with ins(16)(p13.1q22q22)

<p>Acute myeloid leukemia (AML) with <em>CBFB-MYH11</em> fusion gene is almost always associated with either inv(16)(p13.1q22) or t(16;16)(p13.1;q22). Only a few variants leading to formation of this chimeric gene have been reported. In this report, we describe a novel type of <em>CBFB-MYH11</em> fusion variant generated by an insertion of <em>CBFB</em> into the <em>MYH11</em> gene, which is identifiable by fluorescence <em>in situ</em> hybridization (FISH).</p>

Aberrant expression of ecotropic viral integration site-1 in acute myeloid leukemia and acute lymphoblastic leukemia

Ecotropic viral integration site-1 (EVI1) proto-oncogene expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) requires further investigation. Here, EVI1 expression levels were measured in 216 Chinese patients with AML and 67 with ALL via quantitative real-time polymerase chain reaction. We found that EVI1 expressed at a high level (H-EVI1) was present in 11.1% of patients with AML versus 20.9% with ALL. Low levels of EVI1 expression occurred in 23.1% with AML versus 43.3% with ALL. This suggested that alteration of EVI1 expression was more profound in ALL than in AML. H-EVI1 was significantly enriched in 30–60-year-old patients. French–American–British (FAB) M3 subtype was significantly correlated with H-EVI1. Interestingly, we found that EVI1 expression was negatively associated with presence of the Philadelphia chromosome (Ph+) and MLL rearrangements in AML. However, Ph+, but not MLL rearrangements, was inversely correlated with EVI1 expression in B-ALL. These results for the first time suggest a mutually exclusive relationship between EVI1 expression and Ph+ karyotype.

Pontin is a critical regulator for AML1-ETO-induced leukemia.

The oncogenic fusion protein AML1-ETO, also known as RUNX1-RUNX1T1 is generated by the t(8;21)(q22;q22) translocation, one of the most frequent chromosomal rearrangements in acute myeloid leukemia (AML). Identifying the genes that cooperate with or are required for the oncogenic activity of this chimeric transcription factor remains a major challenge. Our previous studies showed that Drosophila provides a genuine model to study how AML1-ETO promotes leukemia. Here, using an in vivo RNA interference screen for suppressors of AML1-ETO activity, we identified pontin/RUVBL1 as a gene required for AML1-ETO-induced lethality and blood cell proliferation in Drosophila. We further show that PONTIN inhibition strongly impaired the growth of human t(8;21)(+) or AML1-ETO-expressing leukemic blood cells. Interestingly, AML1-ETO promoted the transcription of PONTIN. Moreover, transcriptome analysis in Kasumi-1 cells revealed a strong correlation between PONTIN and AML1-ETO gene signatures and demonstrated that PONTIN chiefly regulated the expression of genes implicated in cell cycle progression. Concordantly, PONTIN depletion inhibited leukemic self-renewal and caused cell cycle arrest. All together our data suggest that the upregulation of PONTIN by AML1-ETO participate in the oncogenic growth of t(8;21) cells.

A novel t(4;16)(q25;q23.1) associated with EGF and ELOVL6 deregulation in acute myeloid leukemia

About 50% of acute myeloid leukemia (AML) patients show the occurrence of non-random chromosome rearrangements. Most of the recurrent karyotypic rearrangements in AML have been defined as distinct disease entities in the 2008 World Health Organization (WHO) classification. In this paper we report an AML case showing a novel t(4;16)(q25;q23.1) rearrangement causing the activation of epidermal growth factor (EGF) and elongation of long-chain fatty acids family member 6 (ELOVL6) genes, rather than the generation of a novel fusion gene.

Novel Real-Time Polymerase Chain Reaction Assay for Simultaneous Detection of Recurrent Fusion Genes in Acute Myeloid Leukemia

The recent World Health Organization classification recognizes different subtypes of acute myeloid leukemia (AML) according to the presence of several recurrent genetic abnormalities. Detection of these abnormalities and other molecular changes is of increasing interest because it contributes to a refined diagnosis and prognostic assessment in AML and enables monitoring of minimal residual disease. These genetic abnormalities can be detected using single RT-PCR, although the screening is still labor intensive and costly. We have developed a novel real-time RT-PCR assay to simultaneously detect 15 AML-associated rearrangements that is a simple and easily applicable method for use in clinical diagnostic laboratories. This method showed 100% specificity and sensitivity (95% confidence interval, 91% to 100% and 92% to 100%, respectively). The procedure was validated in a series of 105 patients with AML. The method confirmed all translocations detected using standard cytogenetics and fluorescence in situ hybridization and some additional undetected rearrangements. Two patients demonstrated two molecular rearrangements simultaneously, with BCR-ABL1 implicated in both, in addition to RUNX1-MECOM in one patient and PML-RARA in another. In conclusion, this novel real-time RT-PCR assay for simultaneous detection of multiple AML-associated fusion genes is a versatile and sensitive method for reliable screening of recurrent rearrangements in AML.

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.

Pericentric chromosome 8 inversion, inv(8)(p11.2q22), associated with RUNX1/RUNX1T1 rearrangement in acute myeloid leukemia

Pericentric chromosome 8 inversion, inv(8)(p11.2q22), associated with RUNX1/RUNX1T1 rearrangement in acute myeloid leukemia

ETV6 rearrangements are recurrent in myeloid malignancies and are frequently associated with other genetic events

ETV6 (TEL) rearrangements are favorable in pediatric acute lymphoblastic leukemia but are less well characterized in myeloid malignancies. We investigated 9,550 patients with myeloid disorders for ETV6 rearrangements by chromosome banding analysis and interphase fluorescence in situ hybridization. ETV6 rearrangements were identified in 51 of 9,550 (0.5%) patients (range, 19.2-85.3 years). Frequencies were in detail: acute myeloid leukemia (AML): 40 of 3,798, 1.1%; myelodysplastic syndromes (MDS): 6 of 3,375, 0.2%; myeloproliferative neoplasms (MPNs): 5 of 1,720, 0.3%; MDS/MPN: 0 of 210; and chronic myelomonocytic leukemia: 0 of 447. Thirty-three different partner bands of ETV6 were identified, and most were recurrent: 3q26 (n = 10), 5q33 (n = 4), 17q11 (n = 3), 22q12 (n = 3), 5q31 (n = 2), and 2q31 (n = 2). Additional chromosomal abnormalities were identified in 29 of 51 (57%) ETV6 rearranged cases. In AML, ETV6 rearrangements were frequently associated with NPM1 (9/39, 23%) and RUNX1 mutations (6/31, 19%). The FAB M0 subtype was more frequent in ETV6 rearranged de novo AML than other AML (P < 0.001); expression of CD7 and CD34 by immunophenotyping was higher in ETV6 rearranged AML compared with other subgroups. Survival of 29 ETV6 rearranged de novo AML was compared with 818 AML from other cytogenetic subgroups. Median overall and event-free survival of ETV6 rearranged cases was similar to the intermediate-risk cohort (26.3 vs. 62.2 months and 14.0 vs. 15.4 months) defined according to Medical Research Council criteria. Our study confirms the variety of ETV6 rearrangements in AML, MDS, and MPNs often in association with other genetic events. Prognosis of ETV6 rearranged de novo AML seems to be intermediate, which should be independently confirmed.

Fluorescence in situ hybridization study of acute myeloid leukemia with cryptic chromosome rearrangements

To detect specific chromosome rearrangements in acute myeloid leukemia (AML) using interphase-fluorescence in situ hybridization (FISH). All cases were studied by R-band karyotypic analysis using direct method and/or short-term culture for chromosomes preparation. Interphase-FISH was performed in 108 cases of AML with M5, M4, M2, M3 subtypes including 103 cases with normal karyotypes, 4 cases with chromosomal abnormalities other than specific chromosomal rearrangements using chromosome translocation probe such as AML1/ETO, PML/RARα, CBFβ/MYH11 and MLL. Of 38 cases of M2-AML without t(8;21) on conventional cytogenetics(CC) analysis, 4 cases showed positivity for AML1/ETO fusion transcript, which included 2 cases with typical signal model and 2 with insertion. Of 9 cases of M3-AML without t(15;17) on CC analysis, 6 showed positivity for PML/RARα fusion transcript including 2 with typical signal model, 3 with insertion, one without PML/RARα rearrangement on reverse transcription-PCR and FISH assay using PML/RARα probe. FISH assay using the RARα dual color, break-apart rearrangement probe indicated a partial deletion of RARα. Of 23 cases with M4 or M4EO-AML without inv(16) on CC analysis, 3 showed positivity for CBFβ/MYH11 fusion transcript. Of 38 cases without 11q23 translocation on CC analysis, all cases were negative for MLL rearrangement. Interphase-FISH can detect specific chromosome rearrangements such as AML1/ETO, PML/RARα or CBFβ/MYH11 in some AML cases with normal karyotype, though it seemed less useful for the detection of MLL rearrangement.