getITD for FLT3-ITD-based MRD monitoring in AML

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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 PCR-based 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. Next-generation 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, open-source 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 2 > 0.999; PL-21: R 2 > 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 −5 ), 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 ). Fig. 1 Assessment of assay sensitivity, accuracy, and reproducibility. a DNA of FLT3 internal tandem duplication ( FLT3 -ITD) positive acute myeloid leukemia (AML) cell lines MOLM-14 and PL-21 was each serially diluted in DNA of the FLT3 -ITD negative AML cell line HL-60; replicates were diluted independently. The expected ITDs were detected in all samples, down to a variant allele frequency (VAF) of 6.7 × 10 −5 . Exact numbers and statistics are presented in the supplementary table S1 . b Exemplary alignments created by getITD , showing the two types of ITDs detected by our assay: a non-trailing 21 bp ITD (left) and a trailing 198 bp ITD (right). ITD inserts are highlighted in red; the respective wild-type (WT) tandems are highlighted in green. For the trailing ITD, a lighter green mark presumably duplicated WT sequence that was not actually sequenced. Symbols “|” and “.” connect matching and mismatching bases, respectively; gaps indicating insertions and deletions are annotated with “-”. c Comparison of VAF estimates from PCR- and capillary electrophoresis-based fragment analysis (FA) and our next-generation sequencing (NGS)-based assay for 28 FLT3 -ITD positive diagnostic AML samples (left) and two independent analyses of 14/28 FLT3 -ITD positive diagnostic AML patient samples (right). Allelic ratios (ARs) obtained by FA were converted to VAFs for this comparison as described in the supplement Full size image 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 reads) and compared results to those of previously obtained fragment and Sanger sequencing analyses. In these 28 samples, PCR- and capillary electrophoresis-based fragment analysis (FA) had identified a total of 34 ITDs (1–2 per sample, mean 1.2). All 34 ITDs were also detected by our NGS-based assay. Insertion sites identified by our assay were all identical to those identified by Sanger sequencing and VAF estimates were highly correlated with those from FA ( R 2 = 0.978; Fig. 1c ). Estimated ITD lengths were identical to those previously determined for 33/34 ITDs; the 198 bp ITD was reported as 197 bp and trailing . In 19/28 samples, our assay identified one or more additional ITDs not detected by FA. In total, our assay detected 105 ITDs in these 28 samples (1–16 per sample, mean 3.8). The 71/105 ITDs, detected by our assay but not conventional FA, were similar in length to those detected by both assays (both: 12–198 bp, mean 50.4 bp; getITD only: 9–194 bp, mean 51.6 bp) but present at lower VAFs and thus mostly below the detection limit of conventional methods (both: 9.3–94.5%, mean 38.2%; getITD only: 0.006–2.7%, mean 0.2%). To ensure assay reproducibility, we independently re-sequenced 14/28 samples at low coverage (0.2–1.3 million, mean 0.5 million paired-end reads). Results were entirely reproducible between these low- and the above-mentioned high-coverage replicates, with identical insertion sites and lengths and again highly correlated VAFs ( R 2 = 0.986; Fig. 1c ). To study MRD in FLT3 -ITD positive AML patients, we analyzed serial samples of 10/28 patients. A total of 18 follow-up samples was sequenced for five patients that relapsed during therapy (0.7–3.0 million, mean 1.8 million paired-end reads) and 11 follow-up samples were sequenced for five patients who remained in continuous complete remission (1.4–3.0 million, mean 2.1 million paired-end reads). Serial samples of patients in continuous complete remission were all FLT3 -ITD negative by our assay, at all of the sampled time points (Fig. 2a , left). The relapsed patients all tested ITD positive with our assay at the time of relapse (Fig. 2a , right), whereas FA had identified only three as FLT3 -ITD positive at relapse and two patients were considered to have lost the FLT3 -ITD. In these two patients, our assay detected the ITDs at very low VAFs (0.06% and 0.2%, patient IDs 13 and 28, respectively; Fig. 2a ), whereas no ITD could be detected in any of their preceding follow-up samples. For the other three relapsing patients, who tested FLT3 -ITD positive at relapse also by FA, our assay detected ITDs at high VAFs at relapse (18.0–37.2%, mean 30.3%, patient IDs 21, 25, and 19; Fig. 2a ) and at lower levels in at least one of the preceding follow-up samples from complete remission. Notably, in all five of the relapsed patients, there was at least one diagnostic FLT3 -ITD clone that was detected also at relapse (Fig. 2b , blue circles). These ITD clones must have persisted during remission, at a VAF at times even below our limit of detection. Finally, getITD also identified, in all 10 patients, additional ITD cl

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