NPM1-Mutated Acute Myeloid Leukemia PDF

NPM mutation in acute myeloid leukemia (The NPM1 mutant defines AML irrespective of blast count) BY Sahar Jasim M. Supervisor Dr. Ihsan Mardan Nucleophosmin (NPM1) gene ► The nucleophosmin (NPM1) gene encodes for a multifunctional chaperone protein that...

NPM mutation in acute myeloid leukemia (The NPM1 mutant defines AML irrespective of blast count) BY Sahar Jasim M. Supervisor Dr. Ihsan Mardan Nucleophosmin (NPM1) gene ► The nucleophosmin (NPM1) gene encodes for a multifunctional chaperone protein that is localized in the nucleolus but continuously shuttles between the nucleus and cytoplasm.  NPM1 Structure and Functions Structurally, NPM1 wild type (NPMwt) is a 37 kDa protein with a hydrophobic N-terminus, a central acidic core and basic C terminus. The central acidic core The C-terminus contains a The N-terminus contains basic region involved in (contains aspartic and two leucine rich nuclear binding to nucleic acid. glutamic acid) export signals (NES). ✔ It is responsible for ✔ The region also contains ✔It is responsible for two tryptophan residues histone binding and formation of NPM1 (W288 and W290) ribonuclease activity. pentamers. responsible for the ✔It has a bipartite nuclear ✔It acts as a chaperone by nucleolar localization localization signal (NLS), preventing protein signal (NoLS) and responsible for nuclear misfolding in the interaction with localization of the nucleolus ribosomal DNA. NPM1wt. ► In normal physiological conditions, C-terminus NoLs dominate and direct the nucleolar location of the NPM1wt. Functions of NPM1wt. ► The NPM1wt participates in ribosomal biosynthesis and cell growth ► NPM1wt also binds to the centrosome and prevents uncontrolled duplication during the resting phase of the cell cycle. ► In case of oxidative injury, NPM1 via its interaction with p53 increases cellular stability and promotes growth arrest thereby preventing mutagenesis. Mechanism of leukemogenesis ► Despite its high prevalence, the mechanism(s) of action of NPM1c in AML is still poorly understood. ► Mutations in NPM1 are always heterozygous and result from 4-bp insertions causing a frameshift mutation at the C-terminus resulting in ✔ loss of tryptophan (W288 and W290 or W290 alone) ✔ Gain of new NES leading to disruption of the folded helix structure with loss of NoLS, which shifts the balance towards nuclear export and cytoplasmic localization of NPM1mut. ✔ All NPM1 mutations are “born to be exported” ✔ This extra-nuclear transfer is mediated by interaction between mutated NPM1 and exportin 1 (XPO1). ► Mutant NPM1 promotes leukemogenesis by interacting with chromatin-bound XPO1 at HOX loci to facilitate their expression and binding resulting in: 1.Cytoplasmic delocalization of several nuclear proteins involved in apoptosis, DNA repair and differentiation, including ARF, HEXIM1 FBW7, MIZ1,APE1, and caspases 6 and 8. 2-Recently, altered localization of the transcription factors CTCF65 and PU. 3-Export of tumor suppressor gene ► Physiologically, NPM1wt binds to a small nuclear pool of the transcriptional regulator BRD4, exerting an inhibitory effect on its transcriptional activity. ► NPM1 mutants alter this equilibrium, because a significant proportion of NPM1 is delocalized into the cytoplasm without BRD4, which is then free to drive the transcription of its target genes, especially BCL2 and MYC. NPM1-mutated acute myeloid leukemia (AML) ► It accounts for about 30%–35% of newly diagnosed cases in adults (50%–60% of all AML with normal karyotype ). ► It is characterized by distinctive clinicopathological and molecular features. For this reason, NPM1-mutated AML is now recognized as a distinct entity in both the International Consensus Classification (ICC) and the fifth revision of the (WHO) classification of myeloid neoplasms ✔ According to the 2016 WHO classification of myeloid neoplasms ≥20% bone marrow (BM) blast cells were required to diagnose NPM1-mutated AML. ✔ This blast threshold has been abolished in the fifth revision of WHO while the ICC still requires at least 10% blasts for the diagnosis of NPM1-mutated AML, similar to other AMLs with recurrent genetic abnormalities. ✔ They usually occur at exon-12. ✔ Frequently associate with FLT3-ITD, DNMT3A, TET2, and IDH1/25 mutations.  Types of NPM1 Mutations Type A Mutation Description: Characterized by a duplication of four nucleotides (TCTG) between nucleotides 860 and 863, leading to a frameshift. Prevalence: Accounts for approximately 70-80% of NPM1 mutations in AML cases. Type B Mutation Description: Involves an insertion of CATG at the same position as Type A. Prevalence: Found in about 5-11% of cases. Type D Mutation Description: Characterized by an insertion of CCTG. Prevalence: Comprises around 5-8% of NPM1 mutations. Other Types More than 50 different mutations have been identified, but they collectively account for a smaller percentage of cases compared to Types A, B, and D. Genetic Features of NPM1 mutations Heterozygous mutations detected in about one third of adult AML (50-60% of AML with normal cytogenetic). Less frequent in children (7%). Mostly restricted to exon 12 (1% other exons). Probably arising from replication errors primed by illegitimate terminal deoxynucleotidyl transferase activity. Found in the whole leukemic population (as proven by IHC) and stable over time (being detected at relapse). Not detected in individuals with clonal hematopoiesis Frequently co-occur with mutations of FLT3, DNMT3A, and IDH1/2 genes. Prognosis may vary according to the associated mutations. Mutually exclusive with AML entities defined by recurrent genetic abnormalities in the 2017 WHO classification of hematopoietic tumors Close association with normal karyotype (85% of cases). About 15% of cases carry chromosome aberrations, especially 18, del(9q), 14. Associated with distinct gene expression profile characterized by upregulation of HOX genes (HOXA, HOXB) and low expression of CD34 and CD133. DIAGNOSIS OF NPM1-MUTATED AML The diagnosis of NPM1-mutated AML requires the demonstration of NPM1 mutation and/or aberrant cytoplasmic expression of NPM1 mut. in a patient otherwise meeting conventional criteria for AML diagnosis.  NPM mutated AML should be suspected in ✔ A middle-age or older patient presenting with AML showing myelomonocytic or monocytic (M4–M5) features, cup-like nuclei. ✔ Approximately 23% of cases show multilineage dysplasia. ✔ CD34 negativity and a normal karyotype. ✔ High white blood cell (WBC) is frequent when concomitant FLT3 and RAS mutations are present and may associate with a starry sky pattern in the BM biopsy. ✔ A low WBC count does not exclude NPM1-mutated AML since cases with FLT3 wild-type may present in this way. ✔ The number of platelets is relatively preserved despite high WBC count. ✔ NPM1 mutations may associate with disseminated intravascular coagulation. ✔ The BM, is usually hypercellular without significant increase in the reticulin fibers. ✔ BM biopsy should be considered in all patients carrying an NPM1 mutation and showing a low percentage of blasts in the BM aspirate. ✔ Myeloid sarcomas expressing cytoplasmic NPM139 may occur, especially in the skin. PB film from a patient with AML with a normal karyotype who had both an NPM1 mutation and FLT3-ITD showing blast cells with cup-shaped nuclei PB film from a patient with acute monocytic leukemia with NPM1 and FLT3 mutation immunohistochemistry in NPM1-mutated AML? The most peculiar characteristic of NPM1-mutated AML is the ectopic localization of NPM1 mut. in the cytoplasm of leukemic cells The best control for the specificity of NPM1 cytoplasmic positivity is immunostaining for C23/nucleolin that, in NPM1-mutated AML, is expressed only in the nuclei of the leukemic cells ❖ Immunohistochemical detection of cytoplasmic NPM1 may help in Defining myeloid sarcoma Identify multilineage dysplasia To diagnose cases with aplastic or necrotic BM resulting in dry tap. Immunohistochemistry can predict NPM1 mutations occurring at exons other than 12 (e.- g., exons 9, 11, and 5) that may be missed by standard molecular assays Antibodies specific for the NPM1 mutant may also have a potential impact in assessing MRD post-therapy in the cytoplasm of leukemic cells. Challenges in the diagnosis of NPM1-mutated AML ► NPM1-mutated myeloid neoplasms showing