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.
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.
Taira
Chiaki
C
Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto 390-8621, Japan.
Matsuda
Kazuyuki
K
Kamijyo
Yuka
Y
Sakashita
Kazuo
K
Ishida
Fumihiro
F
Kumagai
Toshiko
T
Yamauchi
Kazuyoshi
K
Okumura
Nobuo
N
Honda
Takayuki
T
D000328:Adult; D000483:Alleles; D002648:Child; D002675:Child, Preschool; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D019337:Hematologic Neoplasms / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D025721:WT1 Proteins / Q000235:genetics; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics
D050676:Core Binding Factor Alpha 2 Subunit; D009841:Oligonucleotides; D025721:WT1 Proteins; C477371:locked nucleic acid; C497970:FLT3 protein, human; D019009:Proto-Oncogene Proteins c-kit; D051941:fms-Like Tyrosine Kinase 3