NPM1 Mutation Detection: A Comparative Study of Quantitative Real-Time PCR and Capillary Electrophoresis in 157 Clinical Samples
FH Barakat, R Luthra, LJ Medieros, BA Barkoh, J Li, S Hai, W Jamil, YI Bhakta, S Herrmann, SS Chen, Z Zuo. MD Anderson Cancer Center, Houston, TX
Background: Nucleophosmin (NPM1) is one of the most commonly mutated genes in acute myeloid leukemia (AML) and has prognostic significance. The presence of NPM1 mutation predicts a better prognosis in AML patients, particularly in the subgroup with diploid cytogenetics and no evidence of FLT3 gene mutation. NPM1 mutations involve exon 12 and cause a frame shift in the translation and aberrant cytoplasmic dislocation of NPM1 protein. Most of these mutations involve a 4-bp insertion in a limited region, which can be detected by capillary electrophoresis (CE) or by real-time quantitative PCR (RQ-PCR) methods. besides its prognostic importance, NPM1 mutation can be helpful in monitoring minimal residual disease (MRD).
Design: This study compared CE and RQ-PCR, for assessing for NPM1 mutation in peripheral blood or marrow samples of 157 patients sent for NPM1 study. Our laboratory currently uses CE assay as routine test. For this study, we developed a multiplex RQ-PCR assay using single TaqMan probe and 6 mutation-specific primers to detect the most common NPM1 mutations, accounting for >95% of cases. This RQ-PCR method was run parallel with our CE assay. Analytical sensitivities of both methods were determined by serial dilution studies. Different mutation types and discordant results between the two methods were further validated by Sanger sequencing.
Results: Serial dilution studies showed that the RQ-PCR method has mutation detection sensitivity of higher than 1:100 cells, compared with approximately 1:40 cells for the CE method. The RQ-PCR assay involves only one step, unlike the two-step CE method, and therefore turn-around time was also cut in half using RQ-PCR. Among the 157 clinical samples tested, 155 (98.7%) showed concordant results. Two samples were discordant: 1 RQ-PCR+ and CE- and 1 RQ-PCR- and CE+. Sanger sequencing analysis in both cases was negative. Given the fact that the sensitivities of both assays are well above that of Sanger sequencing, we are unable to use Sanger sequencing to determine whether these two cases were true or false positives. Within this study cohort, frequency of NPM1 and FLT3 mutation was 19% and 23% in AML, respectively, and 7% and 0% in MDS, respectively.
Conclusions: The RQ-PCR method developed here generated highly concordant results compared with our CE assay for NPM1 mutation detection. In addition, the RQ-PCR assay is more sensitive, rapid and cost effective, making it ideal for monitoring MRD.
Monday, March 22, 2010 8:00 AM
Platform Session: Section G 1, Monday Morning