Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells

Abstract

Abstract. Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61˚C and 68˚C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68˚C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

Detection of the MYD88 mutation by the combination of the allele‐specific PCR and quenching probe methods

“…Quenching probe analysis was performed using a LightCycler Nano as previously described . The QP was complimentary to the mutant allele and was labeled with BODIPY FL at the 3′ end.…”

“…Recently, we demonstrated the advantages of the quenching probe (QP) technique to detect mutations in the JAK2 and NPM1 genes. The QP is an oligonucleotide with a terminal fluorescent dye‐modified cytosine used for melting curve analysis following PCR amplification of the target region.…”

Nucleophosmin in Its Interaction with Ligands

“…In 2016, AML with mutated npm1 has been defined as a distinct entity in the World Health Organization (WHO) classification of hematopoietic malignancies [75]. Until today about 50 mutations in the exon-12 of npm1 have been reported, all between nucleotide positions 861 and 894 [3,5,39,76]. The most common NPM1 mutation is type-A, accounting for about 75-80% of the patients, and consisting of a "TCTG" tetranucleotide tandem duplication [65,77].…”

NPM1A in plasma is a potential prognostic biomarker in acute myeloid leukemia

“…In previous studies, overexpression of NPM has been reported to be in proliferating cells and involved in the tumorigenesis of various tumors, such as thyroid cancer [ 14 ], bladder cancer [ 15 ] and hepatocellular carcinoma [ 16 ]. As the most frequent mutation type, NPM1A with an insertion of TCTG at position 956-959, occurs in approximately 80% of patients with NPM1 mutation [ 17 ]. Moreover, Su et al [ 18 ] confirmed that patients in morphologic CR had achieved complete loss of NPM1-mutA after chemotherapy.…”