Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course

Abstract

Background: Monitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients' therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. Methods:We developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G > T, KIT 2446G > T, and KIT 2447A > T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1-RUNX1T1 fusion gene and WT1 by conventional quantitative PCR. Results:The AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1-RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations. Conclusions:The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored. AS-qPCR for monitoring MRD 3

Spliceosome-related gene mutations in myelodysplastic syndrome can be used as stable markers for monitoring minimal residual disease during follow-up

“…The mutation-specific primers designed according to the previously reports [12,13,17] and TaqMan probes for the U2AF1, SRSF2, or SF3B1 genes were exploited to quantify the mutant DNA by AS-qPCR (Table 1). Primers and the TaqMan probe for the albumin (ALB) gene were used to calculate the relative quantity of the target genes with respect to the level of the ALB gene [18] (Table 1).…”

“…Genetic alterations, including single nucleotide mutations, can be considered applicable targets for monitoring MRD. We have previously described the comparable accuracy of allele-specific quantitative PCR (AS-qPCR) for single nucleotide mutations by using genomic DNA or mRNA with conventional short tandem repeat-PCR for chimerism analyses and with qPCR for the RUNX1/RUNX1T1 fusion gene and the WT1 gene [12][13][14][15].…”

Genome-wide analysis of 1-amino-cyclopropane-1-carboxylate synthase gene family in Arabidopsis, rice, grapevine and poplar

“…The robustness of the trees was assessed by bootstrapping (500 pseudo-replicates) in Phyml. Bayesian analysis was conducted using the same model with MrBayes v3.1.2 (Huelsenbeck and Ronquist.. 2001;Taira et al, 2011).…”

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR

“…We previously reported the use of AS-qPCR to quantify single nucleotide mutations in hematological malignancies [27][28][29] and demonstrated that the method was able to detect mutant DNAs when the recipient DNA was less than 5% or negative [29]. The present AS-qPCR method for SNP detection showed 1.0% sensitivity and provided a chimerism assay with high specificity and sensitivity that was better or equal to that of STR-PCR [21].…”