NPM1-Mutated Acute Myeloid Leukemia: Recent Developments and Open Questions

Abstract

Somatic mutations in the nucleophosmin (NPM1) gene occur in approximately 30% of de novo acute myeloid leukemias (AMLs) and are relatively enriched in normal karyotype AMLs. Earlier World Health Organization (WHO) classification schema recognized NPM1-mutated AMLs as a unique subtype of AML, while the latest WHO and International Consensus Classification (ICC) now consider NPM1 mutations as AML-defining, albeit at different blast count thresholds. NPM1 mutational load correlates closely with disease status, particularly in the post-therapy setting, and therefore high sensitivity-based methods for detection of the mutant allele have proven useful for minimal/measurable residual disease (MRD) monitoring. MRD status has been conventionally measured by either multiparameter flow cytometry (MFC) and/or molecular diagnostic techniques, although recent data suggest that MFC data may be potentially more challenging to interpret in this AML subtype. Of note, MRD status does not predict patient outcome in all cases, and therefore a deeper understanding of the biological significance of MRD may be required. Recent studies have confirmed that NPM1-mutated cells rely on overexpression of HOX/MEIS1, which is dependent on the presence of the aberrant cytoplasmic localization of mutant NPM1 protein (NPM1c); this biology may explain the promising response to novel agents, including menin inhibitors and second-generation XPO1 inhibitors. In this review, these and other recent developments around NPM1-mutated AML, in addition to open questions warranting further investigation, will be discussed.

Keywords: Acute myeloid leukemia; Minimal residual disease; NPM1; Nucleophosmin.

Fig. 1.

A representative case of NPM1-mutated AML. a Peripheral blood smear preparation showing frequent abnormal large blasts (∼93% of nucleated cells by manual differential) with high N:C, irregular to occasionally folded nuclei, open chromatin pattern, and occasional “cup-like” nuclear invagination (inset) [Wright-Giemsa, ×1,000]. b Multiparameter flow cytometry (MFC) performed on the peripheral blood detected a predominant population in the conventional CD45+ (dim)/low side scatter (SSC) blast gate (∼88% of viable cells), which is predominantly positive for CD117 with only dim/partial expression of CD34. Color scheme for gated populations: mature lymphocytes (green), monocytes (orange), granulocytes (aqua), “blast” gate (red). Axes are on log scale, and major hashmarks denote 10²–10⁵. c Histomorphologic evaluation of the corresponding Bouin-fixed paraffin-embedded trephine biopsy shows a nearly ∼100% cellular marrow, markedly hypercellular for the patient’s age (H&E, ×40). d Higher magnification reveals cellular composition by a predominant population of medium to large sized atypical blasts (H&E, ×600). e The abnormal blasts exhibit cytoplasmic positivity for mutant NPM1 protein by chromogenic immunohistochemistry (Fast red, antibody clone: PA1-46356 [Thermo Fisher], ×600).

Fig. 2.

Longitudinal assessment pre- and post-induction therapy in a representative case of NPM1-mutated AML with monocytic differentiation. a Multiparameter flow cytometry (MFC) was performed on a peripheral blood specimen characterized by an exuberant monocytosis (∼86%) including immature forms and ∼8% circulating blasts by manual differential count. MFC shows a predominant abnormal population in the conventional CD45+ (moderate to bright)/intermediate side scatter (SSC) monocyte gate (∼91.6% of viable cells), which expresses several monocytic antigens (not shown) and aberrant partial CD56 positivity. Color scheme for gated populations: mature lymphocytes (green), monocytes (orange), granulocytes (aqua), “blast” gate (red). Axes are on log scale, and major hashmarks denote 10²–10⁵. b Multiparameter flow cytometry (MFC) was performed on a post-induction therapy bone marrow aspirate specimen with ∼13% monocytosis and ∼3% blasts by manual differential count. MFC shows a small but discrete abnormal population in the conventional CD45+ (moderate to bright)/intermediate side scatter (SSC) monocyte gate (∼17% of viable cells), which expresses several monocytic antigens (not shown) and aberrant CD56 positivity, suspicious for minimal/measurable residual disease. Color scheme for gated populations: mature lymphocytes (green), monocytes (orange), granulocytes (aqua), “blast” gate (red). Axes are on log scale, and major hashmarks denote 10²–10⁵. c, d Mutant NPM1 protein immunohistochemistry performed on the accompanying Bouin-fixed paraffin-embedded trephine biopsy (fast red, antibody clone: PA1-46356 [Thermo Fisher], ×1,000) highlights foci of clustered immature-appearing mononuclear cells (c) as well as conspicuous terminally differentiated megakaryocytes (d).

Fig. 3.

Two representative examples of mutant NPM1 protein-specific immunohistochemistry in bone marrow trephine biopsy specimens from NPM1-mutated AML patients obtained following induction chemotherapy. a Case 1: NPM1-mutated cells in clusters (left) and singly dispersed (right). The aspirated marrow material was insufficient for reliable flow cytometry- or PCR-based MRD assessment in this case. b Case 2: NPM1-mutated cells in clusters (left) and singly dispersed (right). Using a conventional NGS-based assay (non-deep sequencing), the type A mutant NPM1 allele was detected at a variant allele frequency of 2.1% (data not shown). Chromogenic immunohistochemistry performed on Bouin-fixed paraffin-embedded tissues, using anti-mutant NPM1 protein-specific antibody (fast red, antibody clone: PA1-46356 [Thermo Fisher], ×1,000).