Clinically relevant variation in FLT3-ITD quantitation as a result of PCR cycle number and ITD insertion size

FLT3 internal tandem duplication (ITD) quantitation is key to prognostication in acute myeloid leukaemia (AML). One potential source of variability in the allelic ratio (AR) is the number of polymerase chain reaction (PCR) cycles used. Using 30 archived samples of varying ITD lengths and AR, we compared two FLT3-ITD assays (Huang and RATIFY), evaluated the effect of PCR cycle number on each assay, and examined the potential clinical consequences. Huang and RATIFY assays at 35 and 27 PCR cycles, respectively, were highly concordant. A progressive decrease in AR (median 47%) was observed with the RATIFY assay when the PCR cycles were increased from 27 to 35 cycles, potentially impacting risk categorisation in 29% of patients. In contrast, minimal change in AR was observed with the Huang assay. Hence, both FLT3-ITD assays were almost identical using respective standard conditions, but the effect of PCR cycle number is assay-dependent, which may impact risk stratification in AML.

Automated summary

FLT3 internal tandem duplication (ITD) quantitation is crucial for predicting prognosis in acute myeloid leukaemia (AML). The number of polymerase chain reaction (PCR) cycles used can influence the allelic ratio (AR) and potentially impact risk categorisation in AML. Two FLT3-ITD assays, Huang and RATIFY, were compared and found to be highly concordant under standard conditions. However, the RATIFY assay showed a progressive decrease in AR when PCR cycles were increased, while the Huang assay showed minimal change in AR. Therefore, the effect of PCR cycle number on FLT3-ITD quantitation is assay-dependent and may have implications for risk stratification in AML.