Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

Abstract

Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities. ' 2012 International Society for Advancement of Cytometry Key termsimaging flow cytometry; acute leukemia; nucleophosmin; hematology NUCLEOPHOSMIN (NPM) is both a nuclear (predominantly nucleolar) and cytoplasmic protein that functions as a molecular chaperone (1). It has many functions including the prevention of protein aggregation in the nucleolus and regulation of p53 levels (2). Mutations within the C-terminal region (exon 12) of the NPM1 gene occur in approximately one-third of cases of de novo acute myeloid leukemia (AML) (3,4) leading to increased nuclear export and aberrant cytoplasmic accumulation of the mutant NPM protein (5). A number of methods have been assessed to detect cytoplasmic NPM (cNPM) in clinical specimens. Immunohistochemistry has been reported to be reliable, especially on B5-fixed bone marrow trephines, with close correlation between cNPM and the presence of mutated NPM1 (6). Immunocytochemistry of air-dried blood and bone marrow smears can also detect cNPM in myeloblasts but is insufficiently reliable for diagnostic use (7). Fluorescent confocal microscopy and standard flow cytometry have both been evaluated for cNPM detection (8,9). Standard flow cytometry cannot be used to assess the localization of molecules within specific cellular compartments, but two alternative approaches have been assessed. The first, using antibodies to mutant NPM1, was able to identify NPM1-mutated AML cases exclusively with or without the addition of a de novo NPM ''control'' antibody (8,9). The second method specifically addressed mean fluorescence intensity (MFI) and showed higher MFI in NPM1 mutated than wild-type AML cases (10). However, discrepancie...

Should evaluation for minimal residual disease be routine in acute myeloid leukemia?

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Development of a robust immuno‐S‐FISH protocol using imaging flow cytometry

“…For example, in acute promyelocytic leukemia abnormal small punctate PML bodies can be distinguished from the normal nuclear pattern using the modulation feature. In other types of acute myeloid leukemia, cytoplasmic nucleophosmin can be discriminated from that in the nucleus in cases with NPM1 mutations . We now show that the genetic component could also be incorporated.…”