Targeted Informatics for Optimal Detection, Characterization, and Quantification of FLT3 Internal Tandem Duplications Across Multiple Next-Generation Sequencing Platforms

Abstract

Assessment of internal tandem duplications in FLT3 (FLT3-ITDs) and their allelic ratio (AR) is recommended by clinical guidelines for diagnostic workup of acute myeloid leukemia and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary next-generation sequencing (NGS) panels, AR estimation is not routinely part of clinical NGS practice because of inherent biases and challenges. In this study, data from multiple NGS platformsdanchored multiplex PCR (AMP), amplicon [TruSeq Custom Amplicon (TSCA)], and hybrid-capturedwere analyzed through a custom algorithm, including platform-specific measures of AR. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE were 100% (42/ 42) and 99.4% (1076/1083), respectively, by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48), respectively, by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, estimated to occur in approximately 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 systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. This article provides an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms; AMP was found capable of near perfect sensitivity and specificity with relatively accurate estimates of ARs.

A comparative study of next-generation sequencing and fragment analysis for the detection and allelic ratio determination of FLT3 internal tandem duplication

“…Recently, FLT3 inhibitors were introduced, leading FLT3 ITD to become an even more important indicator for determining subsequent treatment modalities [ 18 , 19 ]. Minimal residual disease (MRD) studies conducted using quantitative reverse-transcription PCR have made it clear that FLT3 ITD MRD levels following induction chemotherapy are a predictive marker of the duration of complete remission.…”

Role of Biomarkers in FLT3 AML

“…FLT3-ITD mut, are in-frame mutations consisting of duplications of 3-400 base pairs which lead to an elongated JM. This results in constitutive activation of the FLT3 receptor and the downstream signaling (Figure 3) [68,69]. The prognostic value of FLT3-ITD is determined by various factors, including the allele ratio (AR), ITD length, karyotype, insertion site, and co-mutations (NPM1) [11,70].…”

“…FLT3-ITD mut, are in-frame mutations consisting of duplications of 3–400 base pairs which lead to an elongated JM. This results in constitutive activation of the FLT3 receptor and the downstream signaling ( Figure 3 ) [ 68 , 69 ].…”

Revealing molecular architecture of FLT3 internal tandem duplication: Development and clinical validation of a web‐based application to generate accurate nomenclature

“…Novel bioinformatic analysis is required to bridge this clinical gap, improve the sensitivity and accuracy of ITD characterization, [23][24][25][26] as well as accurately calculate the allele ratio. 21,22,27,28 In the published studies, 13,21,22 multiple steps and tools are usually needed to create a final report of FLT3-ITD. An NGS assay using the Archer ® VariantPlex ® Myeloid panel, coupled with the proprietary Archer ® Analysis pipeline (ArcherDX, Inc., Boulder, CO), is a highly sensitive assay for detecting FLT3-ITD, especially for intermediate to long ITDs, compared to traditional PCR-based fragment length analysis (Ding and Zhang, unpublished clinical validation data).…”

“…However, it is a well‐known challenge that insertion mutations over 25 bp length are difficult to analyze and many times being missed by most bioinformatic pipelines. Novel bioinformatic analysis is required to bridge this clinical gap, improve the sensitivity and accuracy of ITD characterization, 23–26 as well as accurately calculate the allele ratio 21,22,27,28 . In the published studies, 13,21,22 multiple steps and tools are usually needed to create a final report of FLT3 ‐ITD.…”