NPM Mutation in Acute Myeloid Leukemia

NPM Mutation in Acute Myeloid Leukemia

• NPM1 mutations are a frequent finding in acute myeloid leukemia (AML), representing 30-35% of newly diagnosed cases in adults (50-60% of AML with normal karyotype).
• NPM1, the nucleophosmin gene, encodes a multifunctional chaperone protein localized in the nucleolus but shuttling between the nucleus and cytoplasm.
• Structurally, wild-type NPM1 (NPMwt) is a 37 kDa protein with a hydrophobic N-terminus, a central acidic core, and a basic C-terminus.
• The N-terminus contains two leucine-rich nuclear export signals (NES), responsible for NPM1 pentamer formation.
• The central acidic core of NPMwt is responsible for histone binding and ribonuclease activity. Also, it has a bipartite nuclear localization signal (NLS).
• The basic C-terminus of NPMwt binds to nucleic acids and contains tryptophan residues (W288 and W290) involved in nucleolar localization signal (NoLS) and interaction with ribosomal DNA.
• In normal conditions, the C-terminus NoLS dominates the nucleolar localization of NPMwt.
• NPMwt participates in ribosomal biosynthesis and cell growth, binds to the centrosome to prevent uncontrolled cell cycle duplication, and increases cellular stability in case of oxidative injury by interacting with p53.
• NPM1 mutations are always heterozygous and arise from 4-bp insertions causing a frameshift mutation at the C-terminus.
• Common results of these mutations include loss of tryptophan residues (W288 and/or W290) and gain of new NES.
• Loss of the tryptophan residues disrupts the folded helix structure, leading to nuclear export and cytoplasmic localization of NPM1 mutant (NPM1mut).
• All NPM1 mutations are "born to be exported" via interaction with exportin 1 (XPO1).
• Mutant NPM1 promotes leukemogenesis by interacting with chromatin-bound XPO1 at HOX loci, facilitating their expression and binding, leading to cytoplasmic delocalization of several nuclear proteins involved in apoptosis, DNA repair, and differentiation.
• Examples include ARF, HEXIM1, FBW7, MIZ1, APE1, and caspases 6 and 8 and also altered localization of the transcription factors CTCF65 and PU.
• Physiologically, NPMwt binds to a small nuclear pool of the transcriptional regulator BRD4, suppressing its activity.
• NPM1 mutants disrupt this equilibrium, resulting in cytoplasmic delocalization and free BRD4 to drive the transcription of target genes, such as BCL2 and MYC.
• NPM1-mutated AML has distinctive clinicopathological features and is recognized as a distinct entity in the International Consensus Classification (ICC) and the WHO classification of myeloid neoplasms.
• The diagnosis requires demonstrating the NPM1 mutation and/or aberrant cytoplasmic expression of NPM1 mutant in a patient meeting conventional AML criteria.
• Suspicion of NPM1-mutated AML often arises in middle-aged to older patients with AML, exhibiting myelomonocytic or monocytic features (M4-M5), CD34 negativity, and a normal karyotype.
• High white blood cell count is more frequent in cases with concomitant FLT3 and RAS mutations, potentially presenting as a starry sky pattern in bone marrow biopsy.
• Platelet count is typically preserved despite high WBC count.
• Bone marrow biopsies show hypercellularity without significant reticulin fiber increase.
• Myeloid sarcomas expressing cytoplasmic NPM139 can occur, especially in the skin.
• The most distinctive characteristic of NPM1-mutated AML is the ectopic localization of the NPM1 mutant in the cytoplasm of leukemic cells.

Types of NPM1 Mutations

• Type A: Duplication of four nucleotides (TCTG) between nucleotides 860 and 863, leading to a frameshift; accounts for 70-80% of cases.
• Type B: Insertion of CATG at the same position as Type A; prevalent in 5-11% of cases.
• Type D: Insertion of CCTG; comprises 5-8% of cases.
• Other types: Over 50 different mutations exist but account for a smaller portion of cases compared to the three main types.

Genetic Features of NPM1 Mutations

• Heterozygous mutations are more common in adults with normal cytogenetics (50-60% of AML with normal karyotypes).
• Primarily restricted to exon 12.
• Possibly arising from replication errors.
• Found throughout cells with leukemia, including at relapse.
• Frequently associated with mutations in FLT3, DNMT3A, and IDH1/2 genes.
• Mutually exclusive with AMLs defined by recurrent genetic abnormalities.
• Most cases (85%) have a normal karyotype.
• 15% display chromosomal aberrations (e.g., 18, del(9q), 14).
• Associated with distinct gene expression profiles, including upregulation of HOX genes (HOXA, HOXB) and low expression of CD34 and CD133.

Challenges in Diagnosing NPM1-Mutated AML

• NPM1-mutated myeloid neoplasms (<20% bone marrow blasts) are rare (1.6%) and often misdiagnosed as MDS or MDS/MPN, particularly chronic myelomonocytic leukemia (CMML).
• Diagnosis is often supported by multilineage dysplasia (usually ~25% of NPM1-mutated AML).
• NPM1-mutated MDS or CMML patients generally resemble typical NPM1-mutated AML rather than MDS or CMML with wild-type NPM1.

Immunohistochemistry in NPM1-Mutated AML

• The peculiar characteristic is ectopic NPM1 localization in leukemic cell cytoplasm.
• Immunohistochemistry for C23/nucleolin helps with specificity, as it's nucleus-localized in NPM1-mutated AML.
• Immunohistochemical detection of cytoplasmic NPM1 helps define myeloid sarcoma, identify multilineage dysplasia, and diagnose cases with aplastic or necrotic bone marrow.

Next-Generation Sequencing (NGS) in NPM1-Mutated Cases

• NGS reveals that NPM1-mutated MDS or CMML frequently resembles NPM1-mutant AML.
• Typically lacking typical MDS/AML mutations (e.g., ASXL1).
• Additional mutations (e.g., DNMT3A, TET2, NRAS) are frequently present for those cases.

Prognostic Impact of NPM1 Mutations

• NPM1 mutations, absent of FLT3-ITD, are associated with a favorable risk group.
• FLT3-ITD, regardless of allelic ratio, is associated with an intermediate risk group.
• Mutations in NRAS, KRAS, PTPN11, and RAD21 have a favorable prognosis.
• Triple mutations (NPM1, FLT3-ITD, and DNMT3A) show a poor prognosis.

Molecular MRD Assessment in NPM1-Mutated AML

• NPM1 mutations show high frequency, stability at relapse and absent from clonal hematopoiesis.
• The frequent and stable nature of the NPM1 mutation makes it an ideal target for MRD monitoring.

Monitoring during and after Treatment

• In NPM1-mutated AML, MRD assessment is performed after two chemotherapy cycles and the end of treatment, and before allogeneic stem cell transplantation.
• Persistent MRD positivity is associated with poor outcomes, with increased relapse and lower survival independent of cytogenetics.
• Limitations of MRD monitoring: Sampling problems, and the possible presence of relapse despite negative MRD status.
• RQ-PCR is the standard MRD method, helping predict relapse and long-term survival. Digital droplet PCR provides high sensitivity.

Explore the significance of NPM mutations in acute myeloid leukemia (AML). This quiz highlights the structure and functionality of the NPM1 gene and its role in tumorigenesis. Test your knowledge on this critical aspect of hematological malignancies.