Multiplex high-throughput gene mutation analysis in acute myeloid leukemia.
Classification of acute myeloid leukemia increasingly depends on genetic analysis. However, the number of known mutations in acute myeloid leukemia is expanding rapidly. Therefore, we tested a high-throughput screening method for acute myeloid leukemia mutation analysis using a multiplex mass spectrometry-based approach. To our knowledge, this is the first reported application of this approach to genotype leukemias in a clinical setting. One hundred seven acute myeloid leukemia cases were screened for mutations using a panel that covers 344 point mutations across 31 genes known to be associated with leukemia. The analysis was performed by multiplex polymerase chain reaction for mutations in genes of interest followed by primer extension reactions. Products were analyzed on a Sequenom MassARRAY system (San Diego, CA). The multiplex panel yielded mutations in 58% of acute myeloid leukemia cases with normal cytogenetics and 21% of cases with abnormal cytogenetics. Cytogenetics and routine polymerase chain reaction-based screening of NPM1, CEBPA, FLT3-ITD, and KIT was also performed on a subset of cases. When combined with the results of these standard polymerase chain reaction-based tests, the mutation frequency reached 78% in cases with normal cytogenetics. Of these, 42% harbored multiple mutations primarily involving NPM1 with NRAS, KRAS, CEBPA, PTPN11, IDH1, or FLT3. In contrast, cases with abnormal cytogenetics rarely harbored more than 1 mutation (1.5%), suggesting different underlying biology. This study demonstrates the feasibility and utility of broad-based mutation profiling of acute myeloid leukemia in a clinical setting. This approach will be helpful in defining prognostic subgroups of acute myeloid leukemia and contribute to the selection of patients for enrollment into trials with novel inhibitors.
Department of Pathology, Oregon Health & Science University, Portland, OR 97239, USA.
Jennifer
J
Dunlap
Carol
C
Beadling
Andrea
A
Warrick
Tanaya
T
Neff
William H
WH
Fleming
Marc
M
Loriaux
Michael C
MC
Heinrich
Tibor
T
Kovacsovics
Katalin
K
Kelemen
Nicky
N
Leeborg
Ken
K
Gatter
Rita M
RM
Braziel
Richard
R
Press
Christopher L
CL
Corless
Guang
G
Fan
D014408:Biomarkers, Tumor / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009687:Nuclear Proteins / Q000235:genetics; D000090243:Nucleophosmin; D011379:Prognosis; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics
D014408:Biomarkers, Tumor; D022762:CCAAT-Enhancer-Binding Proteins; C515848:CEBPA protein, human; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; C497970:FLT3 protein, human; D019009:Proto-Oncogene Proteins c-kit; D051941:fms-Like Tyrosine Kinase 3