Detection of CD34, TdT, CD56, CD2, CD4, and CD14 by Flow Cytometry Is Associated With NPM1 and FLT3 Mutation Status in Cytogenetically Normal Acute Myeloid Leukemia

Abstract

Methods

We retrospectively correlated NPM1 and FLT3 mutation status with flow cytometric profile of leukemic blasts in 83 adult patients with cytogenetically normal acute myeloid leukemia (CN-AML).

Results

Mutation of the NPM1 gene (NPM1.mt) was found in 39 (47%) of 83 patients, and internal tandem duplication (ITD) of the FLT3 gene (FLT3-ITD) was seen in 38 (46%) of 83 patients. Patients with CN-AML and with NPM1.mt were less likely to express CD34 (33% vs. 93%; 2P = .0001), CD2 (0% vs. 14%; 2P = .0187), and CD14 (6% vs. 22%, 2P = .0476), and were more likely to express CD4 (65.5% vs. 37%; 2P = .0367) and CD19 (49% vs. 27%; 2P = .0506). The patients with CN-AML and with FLT3-ITD were more likely to express CD56 (47% vs. 23%; 2P = .0393). Moreover, patients with favorable prognostic combination of NPM1.mt and wild-type (wt) FLT3 (n = 18) were less likely to express CD34 (33% vs. 74% all others; 2P = .0021) and CD56 (6% vs. 37% all others; 2P = .0072). The group with an unfavorable prognostic combination of NPM1-wt and FLT3-ITD (n = 17) were more likely to express CD34 (88% vs. 45% all others; 2P = .0011) and TdT (40% vs. 2% all others; 2P = .0054).

Conclusions

In patients with CN-AML, characteristic flow cytometric profile is associated with NPM1 and FLT3 mutation status.

Introduction

Prognosis in acute myeloid leukemia (AML) is based on age, comorbidities, white blood cell count, the presence of a myelodysplastic state, cytotoxic therapy, and cytogenetic abnormalities.1, 2 Approximately half of the patients with AML can be cytogenetically stratified into high or low risk and treated with chemotherapy plus stem cell transplantation or chemotherapy alone, respectively.³ The remaining patients are placed into an intermediate (or indeterminate) risk category and that consists mostly of patients with cytogenetically normal AML (CN-AML). CN-AML is a heterogeneous group with a 5-year survival ranging from 24 to 42 months,⁴ and the place of stem cell transplantation in this group of patients is uncertain.⁵

Recent molecular techniques have identified several submicroscopic genetic abnormalities in patients with CN-AML4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 such as nucleophosmin 1 (NPM1), FMS-like tyrosine kinase gene 3 (FLT3), CCAAT/enhancer-binding protein-alpha (CEBPA), mixed-lineage leukemia, brain and acute leukemia cytoplasmic, meningioma 1, E-twenty six related gene 1, Wilms' tumor 1, P14 alternate reading frame, CD74, neuroblastoma RAS, and ecotropic viral integration site 1. Of these, the mutations of NPM1, FLT3 and CEBPA have confirmed prognostic significance6, 21, 22, 23 and are being used for treatment stratification of patients with CN-AML.2, 4, 24 NPM1 gene codes for a nuclear protein, nucleophosmin. Nucleophosmin assists in ribosome biogenesis, regulates centrosome duplication, modulates several tumor suppressor proteins, and stabilizes nuclear proteins. It constantly shuttles between nucleus and cytoplasm; mutations of NPM1 that involve C-terminal tryptophans 288 and 290 prevent nucleophosmin binding to the nucleolus and contribute to NPM1 accumulation in the cytoplasm.⁶ NPM1 mutation (NPM1.mt) is seen in 35% of all patients with AML and 50% to 60% of patients with CN-AML. NPM1.mt has a favorable impact on the remission rate,21, 25 event-free survival,26, 27 and overall survival (OS)16, 25 in patients with CN-AML. The FLT3 gene codes for a receptor tyrosine kinase that is normally expressed by hematopoietic progenitor cells and rapidly downregulated as they differentiate.⁹ The most common FLT3 mutation involves an internal tandem duplication (FLT3-ITD) in the juxtamembrane region of the gene. The mutated gene encodes for an abnormal protein that activates diverse signaling pathways, such as JAK2, STAT5, and MAPK, all involved in cell proliferation, differentiation, and survival. The FLT3-ITD is seen in 15% to 25% of all patients with AML and in 20% to 40% of patients with CN-AML.²⁴ The FLT3-ITD mutation has an unfavorable impact on remission rate,²⁸ event-free survival,⁹ and OS.²⁹

Recently, the combination of NPM1.mt and FLT3-wt has been shown to identify a favorable prognosis subgroup of patients with CN-AML,26, 30 and these patients are offered chemotherapy alone; by contrast, the subgroup of patients with CN-AML and with NPM1-wt and FLT3-ITD make up the unfavorable prognosis group³¹ and are unlikely to be cured by chemotherapy alone. Their prognosis may be improved after stem cell transplantation.¹⁶

Mutations of NPM1 and FLT3 are conventionally detected by polymerase chain reaction (PCR) amplification or direct sequencing technics. Some studies report that patients with AML whose blasts had “cuplike” nuclei were more likely to have NPM1.mt,³² FLT3-ITD,³³ or both together.³⁴ However, NPM1.mt is a favorable feature, whereas the FLT3-ITD is an unfavorable feature, both sharing a single morphologic finding. The cytoplasmic localization of nucleophosmin in patients with NPM1.mt, detected by immunohistochemistry,³⁵ has been confirmed by some researchers25, 36 but not by others.³⁷ Recently, flow cytometric correlation of NPM1 and FLT3 status has been reported in all patients with AML38, 39, 40, 41, 42 as well as the CN-AML subgroup. According to these reports, the patients with CN-AML and with NPM1.mt are less likely to express CD3425, 35, 43 and CD7,²⁵ whereas those with FLT3-ITD are more likely to express CD56 and CD7.44, 45 We report here further correlation of flow cytometric data with NPM1 and FLT3 status in our patients with CN-AML.