title: Development of a flow cytometric method to detect the presence of mutated nucleophosmin 1 in acute myeloid leukemia.
abstract: OBJECTIVE
  Nucleophosmin 1 (NPM1) plays multiple roles in cell growth and proliferation. Deletion/insertion mutations in exon 12 of NPM1 (NPM1-DIM), commonly found in patients with acute myeloid leukemia (AML), alter the C-terminal amino acids and disrupt the normal nucleocytoplasmic shuttling function of the protein, which in turn leads to disease pathogenesis. However, this altered function as a result of NPM1-DIM positivity is actually associated with a significantly better response to therapy and overall survival, and thus it is of clinical relevance to investigate the mutation status at diagnosis. Our objective was to design a reliable flow cytometry assay to detect mutated NPM1 in peripheral blood (PB) samples from AML patients, using a polyclonal mutation-specific antibody.
  
  
  METHODS
  A commercially available NPM1 mutation-specific polyclonal antibody in combination with a secondary goat antirabbit antibody was used to detect the C-terminal-mutated NPM1 by flow cytometry. OCI/AML3 (+) cell line and clinical PB controls were used to optimize the assay and determine sensitivity, reliability, and reproducibility parameters. The assay was then tested on a small cohort of 12 AML patients at diagnosis and compared with NPM1-DIM testing on a standard polymerase chain reaction (PCR) platform.
  
  
  RESULTS
  Flow cytometry using the polyclonal antibody was able to reliably detect mutated NPM1 populations of at least 10%. Using an objective analysis of the mean fluorescent intensity, clear positive and negative mutated cell populations could be distinguished using the clinical AML samples. From the analysis of 12 patients, 2 were found to be positive using this assay, which corresponded with conventional PCR methodology.
  
  
  CONCLUSIONS
  Flow cytometry may be used to detect NPM1 C-terminal mutations in AML patients using a polyclonal anti-NPM1 antibody, allowing rapid mutation status determination at diagnosis.
authors.affiliation: Division of Haematology, Department of Clinical laboratory Sciences, University of Cape Town, South Africa; National Health Laboratory Services/Groote Schuur Hospital, Haematology, Cape Town, South Africa.
authors.forename: Louis Almero
authors.initials: LA
authors.lastname: Du Pisani
authors.affiliation: Division of Haematology, Department of Clinical laboratory Sciences, University of Cape Town, South Africa; National Health Laboratory Services/Groote Schuur Hospital, Haematology, Cape Town, South Africa. Electronic address: karen.shires@uct.ac.za.
authors.forename: Karen
authors.initials: K
authors.lastname: Shires
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000906:Antibodies / Q000737:chemistry; D015331:Cohort Studies; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000097:blood* / Q000235:genetics*; D017493:Leukocyte Common Antigens / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D000906:Antibodies; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
keywords: Acute myeloid leukemia; Flow cytometry; Nucleophosmin 1