Targeted informatics for optimal FLT3-ITD detection, characterization, and quantification across multiple NGS platforms.

Abstract Assessment of internal tandem duplications in FLT3 (FLT3-ITDs) and their allelic ratio is recommended by clinical guidelines for diagnostic workup of AML and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary NGS panels, estimation of ITD allelic ratio is not routinely part of clinical NGS practice due to inherent biases and challenges. In this study, data from multiple NGS platforms -- anchored multiplex PCR (AMP), amplicon (TSCA), and hybrid-capture (HC) – was analyzed through a custom algorithm including platform-specific measures of allelic ratio. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE was 100% (42/42) and 99.4% (1076/1083) by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48) by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, which was estimated to occur in ∼9% of unselected ITDs. Allelic fractions under AMP or TSCA were highly correlated to CE with linear regression slopes near 1 for ITDs not duplicating primers and were systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. In summary, we provide an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms and find AMP capable of near perfect sensitivity and specificity with relatively accurate estimates of allelic ratios.