title: Improved minimal residual disease detection by targeted quantitative polymerase chain reaction in Nucleophosmin 1 type a mutated acute myeloid leukemia.
abstract: Multicolor flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR) are important independent techniques to determine minimal residual disease (MRD) in acute myeloid leukemia (AML). MFC is the standard method, but may be unreliable. Therefore, MFC-based determination of MRD with an RQ-PCR-based approach targeting the nucleophosmin 1 (NPM1) type A mutation was set out to compare. Since most current NPM1 RQ-PCR MRD protocols suffer from clear definitions of quantifiability, we sought to define quantifiability in a reproducible and standardized manner. The limit of quantifiability of our RQ-PCR protocol for the NPM1 type A mutation varied between 0.002% and 0.04% residual leukemic cells depending on the features of the standard curve for each PCR experiment. The limit of detection was close to 0.001% leukemic cells. The limit of detection by MFC ranged from 0.01% to 1% depending on the phenotype of the leukemic cells as compared with non-leukemic bone marrow cells. Forty-five MRD samples from 15 patients using both NPM1 mutation specific RQ-PCR and MFC were analyzed. In 32 of the 45 samples (71%), an MRD-signal could be detected with RQ-PCR. A quantifiable NPM1 mutation signal was found in 15 samples (33%) (range 0.003%-2.6% leukemic cells). By contrast, only two follow-up samples (4%) showed residual leukemic cells (0.04% and 0.3%, respectively) by MFC. Thus, RQ-PCR of the NPM1 type A mutation was more sensitive and reliable than MFC for determination of MRD, which might have clinical implications. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Pathology, Halland Hospital Halmstad, Halmstad, Sweden.
authors.forename: Louise
authors.initials: L
authors.lastname: Pettersson
authors.affiliation: Department of Pathology, University and Regional Laboratories, Skåne University Hospital, Lund, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Levéen
authors.affiliation: Department of Pathology, University and Regional Laboratories, Skåne University Hospital, Lund, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Axler
authors.affiliation: Department of Internal Medicine-Hematology and Oncology, Center of Molecular Biology and Gene Therapy, Masaryk University and University Hospital Brno, Brno, Czech Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Dvorakova
authors.affiliation: Department of Hematology, Skåne University Hospital, Lund, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Juliusson
authors.affiliation: Department of Pathology, University and Regional Laboratories, Skåne University Hospital, Lund, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Ehinger
mesh_terms: D000328:Adult; D000368:Aged; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification; D000090243:Nucleophosmin; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin