Minimal resdiual disease getITD for FLT 3-ITD-based MRD monitoring in AML

The clinical relevance of measurable residual disease (MRD) monitoring has been well recognized in acute myeloid leukemia (AML) [1] and respective assays have been established for several recurrent leukemic markers [2, 3]. However, although internal tandem duplications in the FLT3 gene (FLT3-ITDs) are the most common poor prognosis AML drivers [4], they have remained a challenging target: Their heterogeneity makes conventional PCRbased methods either insensitive or laborious [5]. Yet with recently approved FLT3-kinase inhibitors available [6], a specific monitoring of FLT3-mutation loads, and thus response to targeted therapy, is of particular interest. Nextgeneration sequencing (NGS) workflows for FLT3-ITD monitoring have been described, but were previously either proprietary or undisclosed [7, 8], unable to detect and correctly annotate all of the tested ITDs [9, 10], or used in conjunction with manual analysis with inherently subjective results [10]. We have therefore developed a new method based on targeted high-coverage NGS and our novel, opensource analysis program getITD. For assay assessment, we sequenced 3 human AML cell lines (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), 2 healthy volunteers, and 57 samples from 28 AML patients who were all included in the AMLSG BiO Registry study (NCT01252485) and gave their informed consent according to the Declaration of Helsinki. We show that our workflow detects ITDs of a broad range of lengths, insertion sites and variant allele frequencies (VAFs) with high accuracy and precision, is fully objective without any requirement for manual analysis, and thus applicable to routine clinical monitoring. Sample and method details are provided as supplementary information. getITD is freely available at https://github.com/tjblaette/getitd. To demonstrate our assay’s specificity, we analyzed three FLT3-ITD negative control samples (peripheral blood of healthy volunteers, n= 2; AML cell line HL-60). No ITDs were reported, indicating an assay specificity of 100% (coverage: 1.1–4.2 million, mean 2.6 million paired-end reads). To assess sensitivity, we analyzed two serial dilutions of FLT3-ITD positive in FLT3-ITD-negative DNA from AML cell lines MOLM-14 (21 bp ITD, 67% VAF [11]), PL-21 (126 bp ITD, 33% VAF [11]), and HL-60 (FLT3-ITD negative [11]). For each of the ITD-positive cell lines, we sequenced undiluted DNA and 3–4 serial 1:10 dilutions in HL-60 (1.1–2.9 million, mean 2.0 million paired-end reads). The expected ITDs were detected in all samples and VAF estimates were accurate and decreased linearly as expected (MOLM-14: R > 0.999; PL-21: R > 0.998; Fig. 1a). The most diluted sample, the 1:10,000 MOLM-14:HL-60 dilution, harbored the ITD at 0.0067% VAF (6.7 × 10), demonstrating that our limit of detection is at least this low. Note that while MOLM-14’s 21 bp ITD was correctly identified as such, PL-21’s 126 bp ITD was estimated to be 125 bp long. PL-21’s ITD is too long for the insert and its wild-type (WT) tandem to be spanned by the same read (2 × 126 bp > 250 bp) and, for these trailing ITDs, length estimates are non-exact but should deviate by ≤3 bp (Fig. 1b). To determine assay accuracy, we sequenced the diagnostic FLT3-ITD positive bone marrow samples of 28 AML patients (0.04–2.6 million, mean 1.4 million paired-end These authors contributed equally: Konstanze Döhner, Lars Bullinger