NPM1 Mutation Analysis in Acute Myeloid Leukemia: Comparison of Three Techniques - Sanger Sequencing, Pyrosequencing, and Real-Time Polymerase Chain Reaction
Journal Title: Turkish Journal of Hematology - Year 2018, Vol 35, Issue 1
Abstract
Objective: Nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis. Approximately 30% of newly diagnosed cytogenetically normal AML (CN-AML) patients harbor the NPM1 mutation in India. In this study we compared the efficiency of three molecular techniques in detecting NPM1 mutation in peripheral blood and bone marrow samples. Materials and Methods: In a single-center cohort we analyzed 165 CN-AML bone marrow/peripheral blood samples for NPM1 mutation analysis. About 30% of the CN-AML samples revealed NPM1 mutations. For the detection, three methods were compared: Sanger sequencing, pyrosequencing, and real-time polymerase chain reaction (PCR). Results: NPM1 exon 12 mutations were observed in 52 (31.51%) of all CN-AML cases. The sensitivity of Sanger sequencing, pyrosequencing, and real-time PCR was 80%, 90%, and 95%, whereas specificity was 95%, 100%, and 100%, respectively. The minimum limit of mutation detection was 20%-30% for Sanger sequencing, 1%-5% for pyrosequencing, and 0.1%-1% for real-time PCR. Conclusion: The sequencing method, which is the reference method, has the lowest sensitivity and is sometimes difficult to interpret. Real-time PCR is a highly sensitive method for mutation detection but is limited for specific mutation types. In our study, pyrosequencing emerged as the most suitable technique for the detection of NPM1 mutations on the basis of its easy interpretation and less time-consuming processes than Sanger sequencing.
Authors and Affiliations
Dushyant Kumar, Anurag Mehta, Manoj Kumar Panigrahi, Sukanta Nath, Kandarpa Kumar Saikia
Keywords
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- EP ID EP268166
- DOI 10.4274/tjh.2017.0095
- Views 73
- Downloads 0
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