Quantitative Detection of Circulating Nucleophosmin Mutations DNA in the Plasma of Patients with Acute Myeloid Leukemia

Abstract

Objective: The aim of this study was to quantify the copies of circulating nucleophosmin (NPM) mutations DNA in the plasma of patients with acute myeloid leukemia (AML) and to explore the association of circulating NPM mutation levels with clinical characteristics.Design and Methods: The presence of NPM mutations in 100 Chinese patients newly diagnosed with AML were identified by RT-PCR and sequencing analysis. Copies of circulating NPM mutation A (NPM mut.A) DNA in the plasma of mutation-positive cases were quantified by real-time quantitative PCR (qRT-PCR). Furthermore, the association of circulating NPM mutation levels and clinical characteristics was analyzed.Results: NPM mutations were identified in 37 of the 100 patients and all cases were NPM mut.A. The circulating NPM mut.A levels ranged from 0.35×108 copies/ml to 6.0×108 copies/ml in the 37 mutation-positive cases. The medium and quartile M (P25, P75) of the circulating NPM mut.A levels in patients classified as M2, M4 and M5 morphological subtypes were 1.35×108 (0.76×108, 1.91×108) copies/ml, 1.81×108 (1.47×108, 2.2×108) copies/ml and 2.50×108 (2.42×108, 3.05×108) copies/ml, respectively. Circulating NPM mut.A levels were significantly higher in patients with the M5 subtype of AML compared to patients with the M2 and M4 subtypes (p=0.000, p=0.046). In addition, circulating NPM mut.A copies were significantly associated with a higher white blood cell count, platelet count and bone marrow blast percentage (p<0.05).Conclusion: Our results suggest that circulating NPM mutations DNA assay serves as a complementary to the routine investigative protocol of NPM-mutated leukemia.

Cell‐free DNA — Minimally invasive marker of hematological malignancies

“…Mutation of NPM1 gene was identified in 37 of 100 patients using plasma cfDNA, but only in 35 patients using PB cells. It was found that copies of NPM1 mutations in PB were significantly associated with different subtypes of AML and also with higher white blood cells count, platelets, and percentage of blast cells in BM …”

“…The first study dealing with analysis of cfDNA in hematological malignancies, especially in MDS and AML, was published in 1994 . Since then, a few studies on cfDNA in hematological malignancies showed that cfDNA derived from tumor cells is potentially useful for diagnosis, prognosis, monitoring of tumor burden, and response to treatment . Most of these studies focused on lymphoid malignancies, especially those derived from B cells.…”

“…Furthermore, specific genetic markers, for example, somatic mutations of FLT3 as well as cytogenetic abnormalities, were analyzed in cfDNA of AML patients. In one study, LOH and chromosome X inactivation were analyzed as the representatives of genetic abnormalities in AML and MDS patients by the comparison of cfDNA from PB and gDNA from BM samples.…”

“…In a more recent study, Quan et al . performed quantification of copies of NPM1 gene mutations in cfDNA from PB of AML patients by qPCR and NGS analysis.…”

Liquid biopsies for liquid tumors: emerging potential of circulating free nucleic acid evaluation for the management of hematologic malignancies

“…Cytogenetics and specific molecular profiles (NPM1, c-Kit, FLT3, CEBPA) in AML are used to define prognosis and guide treatment decisions following induction 23 . It has been shown in a recently published study that NPM mutations can be detected and quantified in the PB 24 at a rate commensurate with the rate of detection in historical BM samples 25 . However, this study did not report parallel assessment of NPM mutations in paired cfDNA with BM samples and consequently conclusions about the sensitivity of cfDNA detection could not be drawn.…”