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 1:1-1:10³. 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. Conclusion: 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.