TY - JOUR
T1 - Performance and clinical evaluation of a sensitive multiplex assay for the rapid detection of common NPM1 mutations
AU - Hafez, Michael
AU - Ye, Fei
AU - Jackson, Keith
AU - Yang, Zhe
AU - Karp, Judith E.
AU - Labourier, Emmanuel
AU - Gocke, Christopher D.
N1 - Funding Information:
Supported in part by the National Institutes of Health/National Cancer Institute (Small Business Innovation Research grant R43CA130501 ).
PY - 2010/9
Y1 - 2010/9
N2 - Determination of NPM1 mutation status has become essential for the molecular classification of acute myeloid leukemias (AML). Methods with high clinical sensitivity and specificity adapted to the molecular laboratory workflow are required for the diagnosis, prognosis, and monitoring of AML with normal karyotype. We report here the development and evaluation of a novel, streamlined, RNA-based assay for the rapid multiplex detection of common NPM1 mutations in a 96-well assay format. Using synthetic transcripts and total RNA from leukemic cell lines, we show that the assay can specifically detect NPM1 wildtype and mutants A, B, D, or J transcripts in the same reaction. Dilution experiments indicate an assay dynamic range >4 log units with an analytical sensitivity of approximately 0.01%. Evaluation of 69 clinical specimens at initial diagnosis resulted in 100% agreement with reference methods. Of patients with AML with normal karyotype, 53% carried one of four different mutations. The assay was also combined with other laboratory-developed tests to simultaneously detect NPM1 mutant transcripts and fusion transcripts resulting from t(8;21) or inv(16) in a single reaction well. Overall, these results show that the assay is a versatile and specific tool for the screening of NPM1 mutations in patients with AML. Its high analytical sensitivity further suggests potential utility for the monitoring of residual disease in AML with normal karyotype.
AB - Determination of NPM1 mutation status has become essential for the molecular classification of acute myeloid leukemias (AML). Methods with high clinical sensitivity and specificity adapted to the molecular laboratory workflow are required for the diagnosis, prognosis, and monitoring of AML with normal karyotype. We report here the development and evaluation of a novel, streamlined, RNA-based assay for the rapid multiplex detection of common NPM1 mutations in a 96-well assay format. Using synthetic transcripts and total RNA from leukemic cell lines, we show that the assay can specifically detect NPM1 wildtype and mutants A, B, D, or J transcripts in the same reaction. Dilution experiments indicate an assay dynamic range >4 log units with an analytical sensitivity of approximately 0.01%. Evaluation of 69 clinical specimens at initial diagnosis resulted in 100% agreement with reference methods. Of patients with AML with normal karyotype, 53% carried one of four different mutations. The assay was also combined with other laboratory-developed tests to simultaneously detect NPM1 mutant transcripts and fusion transcripts resulting from t(8;21) or inv(16) in a single reaction well. Overall, these results show that the assay is a versatile and specific tool for the screening of NPM1 mutations in patients with AML. Its high analytical sensitivity further suggests potential utility for the monitoring of residual disease in AML with normal karyotype.
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U2 - 10.2353/jmoldx.2010.090219
DO - 10.2353/jmoldx.2010.090219
M3 - Article
C2 - 20616361
AN - SCOPUS:77956794315
SN - 1525-1578
VL - 12
SP - 629
EP - 635
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 5
ER -