FLT3 internal tandem duplications (ITDs) are found in > 20% of pediatric and adult acute myeloid leukemia (AML) cases and are generally associated with a poor prognosis. Nevertheless, detection of FLT3-ITDs presents a challenge to NGS-based approaches, as many variant callers fail to identify the highly variable repeated sequences associated with FLT3-ITDs.

We have developed a novel targeted assay, the Archer™ VariantPlex™ Core AML Panel, that utilizes Anchored Multiplex PCR (AMP™) with probes in multiple locations proximal to exons 13, 14, and 15 of FLT3. These exons encompass the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. This panel yields high-complexity, dual-strand coverage of known FLT3-ITD locations. Because AMP probes, unlike traditional PCR probes, function independently of each other, we are able to produce multiple overlapping “snapshots” of the region of interest, thereby enhancing the ability to confidently identify complex mutation types. The sequenced reads are assembled using a novel de novo assembly algorithm in the Archer Analysis bioinformatics pipeline, in which the resulting consensus is annotated and events that involve FLT3 exons 13 14 or 15 are marked as FLT3 abnormalities.

In order to assess Archer Analysis and the VariantPlex Core AML panel, we tested it on >20 patient-derived samples with known FLT3-ITDs. In addition, we generated over two thousand in silico datasets, representing the spectrum of known ITDs, to further test our panel design and analysis algorithm. Each in silico dataset was constructed to simulate reads originating from probes in VariantPlex Core AML Panel. This methodology permitted rigorous optimization of both the VariantPlex Core AML Panel and the analysis algorithm.

The VariantPlex Core AML Panel, in conjunction with our novel detection algorithm, showed both exceptional sensitivity and specificity in the detection of FLT3-ITDs. FLT3-ITDs were successfully identified in all patient samples tested, and no false positives were detected. Our in silico datasets showed similarly high sensitivity and specificity.

These data indicate that AMP libraries targeting the FLT3-ITD region are ideal for the detection of this complex mutation type, largely because of the overlapping and anchored read structure. Therefore, the Archer VariantPlex Core AML panel, in conjunction with Archer Analysis represents a reliable platform for the detection of FLT3-ITDs, in addition to other mutations commonly found in AML.

Citation Format: Marc Bessette, Benjamin Van Deusen, Michael Banos, Laura Johnson, Aaron Berlin, Erik Reckase, Joshua Stahl, Abel Licon, Brian Kudlow. NGS-based detection of FLT3-ITDs with Anchored Multiplex PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3618.