Mutant Enrichment with 3'-Modified Oligonucleotides (MEMO)-Quantitative PCR for Detection of NPM1 Mutations in Acute Myeloid Leukemia.
BACKGROUND
Detection of NPM1 mutations in acute myeloid leukemia (AML) is important for risk stratification, treatment decision, and therapeutic monitoring. We have designed a real-time PCR method implementing the Mutant enrichment with 3'-modified oligonucleotides (MEMO) technique to detect NPM1 mutations and validated its utility in clinical samples.


METHODS
Sensitivity and linearity were evaluated using serially diluted NPM1-positive samples. Clinical usefulness was assessed by measuring the levels of mutant alleles in 29 patients at diagnosis and in ten patients after induction chemotherapy.


RESULTS
Excellent linear relationships between the mutant allele proportion and the threshold cycle (Ct) values (r = 0.999) were observed in a range of 1:1-1:10(3) . MEMO-PCR was able to detect NPM1 mutations regardless of mutant type and also detected novel mutants (964_967delTGGAinsATGATGTC, 957_959delCTGinsATGCATG, 960insTAAG, and 960insTCAG). The concentrations of NPM1 mutant alleles decreased after induction chemotherapy in accordance with the reduction of tumor cells, and in one case, NPM1 mutant alleles were detectable about 7 months before morphological relapse.


CONCLUSIONS
MEMO-quantitative PCR was shown to detect virtually all types of NPM1 mutants with high sensitivity and specificity. This novel method may be useful in the diagnosis of AML with an NPM1 mutation, the detection of minimal residual disease, and the monitoring of treatment response.
Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Sang-Yong
SY
Shin
Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Chang-Seok
CS
Ki
Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Hee-Jin
HJ
Kim
Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Jong-Won
JW
Kim
Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Sun-Hee
SH
Kim
Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Seung-Tae
ST
Lee
D000328:Adult; D000368:Aged; D002675:Child, Preschool; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin; D009841:Oligonucleotides / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D055815:Young Adult
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D009841:Oligonucleotides; D000090243:Nucleophosmin
AML; NPM1; real-time PCR