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 0.35×10(8) copies/ml to 6.0×10(8) 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 1.35×10(8) (0.76×10(8), 1.91×10(8)) copies/ml, 1.81×10(8) (1.47×10(8), 2.2×10(8)) copies/ml and 2.50×10(8) (2.42×10(8), 3.05×10(8)) 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.

Keywords: acute myeloid leukemia; circulating DNA; mutation; nucleophosmin; real-time quantitative polymerase chain reaction..

Figure 1

Establishment of the standard curve for qRT-PCR. (A) Representative amplification plots of serial plasmid dilutions ranging from 10⁵ to 10¹¹ copies/ml in each reaction. (B) Standard curve of the real-time amplification of NPM mut.A derived from plots in (A) with a correlation of 0.99812.

Figure 2

Circulating NPM mut.A copies in patients with AML. The circulating NPM mut.A copy numbers in 37 NPM mutation-positive cases were determined by qRT-PCR assay and summarized as median and quartile M (P25, P75).

Figure 3

Circulating NPM mut.A copies and laboratory parameters of NPM-mutated AML. Dot plots show the distribution of NPM mut.A copies in AML patients. Each laboratory parameter (WBC count, platelet count and BM blast percentage) was divided into two categories (low/high).