Validation of digital droplet PCR in detection and quantification of FLT3-TKD mutations in acute myeloid leukaemia

Abstract

Background: Oncogenic mutation in the tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is a known recurrent mutation in AML.1 Com mon techniques used in its detection and/or characterisation include Sanger sequencing and nextgeneration sequencing, with recognised sensitivity limitations.2 Aim/Objective: To validate the use of digital droplet PCR (ddPCR) in the detection and quantification of common FLT3-TKD mutations (D835Y, D835H, D835V). Patients/Method: Samples from patients (seven D835Y, four D835H, two D835V), one external QAP and a commercial D835Y positive control were tested using a commercial Bio-rad ddPCR assay. Results: All samples with known FLT3-TKD mutations were correctly detected (15/15) with no false positives in the normal control. The assay showed cross-reactivity against FLT3-TKD mutations they were not-specific for, which persisted despite annealing temperature and digestion enzyme augmentation. The cross reactive events were reliably distinguishable from true positive events due to lower fluorescence amplitude. For the D835Y assay, repeatability studies showed a percentage relative standard deviation of 0.78% and a limit of detection of 0.01%. Conclusion: ddPCR is suitable for the detection and quantification of FLT3-TKD mutation in the diagnostic setting. Methodology optimisation may further improve upon on the sensitivity allowing for its use in minimal residual disease detection3.