Clinical Laboratory Validation and Implementation of Quantitative, Real-Time PCR-based Monitoring of NPM1 Type A Mutation

Abstract

NPM1 is frequently mutated in acute myeloid leukemia (AML) and the mutations consist of a variety of small insertions; the most common NPM1 mutation (ie, Type A) accounts for approximately 80% of all NPM1 mutations. NPM1 mutations have been reported to have prognostic significance in AML and, more recently, monitoring mutant NPM1 levels during and after therapy has been described to predict relapse and survival. Despite the published relevance of this molecular biomarker, routine residual disease monitoring for mutant NPM1 levels has not been widely adopted in many academic clinical laboratories. In this manuscript, we present the validation and implementation of a quantitative, real-time PCR-based method for the monitoring of NPM1 Type A mutant transcripts, for use in routine practice in a clinical laboratory. We have found that this assay format is sensitive and reproducible. We describe the rigorous validation procedure and share observations that will help other clinical laboratories that may wish to implement this type of testing. We show comparison of the results from this assay to other assays and a representative case is provided which highlights the utility of the findings from this assay. Taken together, in the context of molecular testing for residual disease in AML, which is an area of practice that continues to expand, we have found that this method and protocol will serve as an appropriate option for monitoring NPM1 Type A mutation levels in clinical practice.