Dataset Information

Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data.

ABSTRACT: FLT3 internal tandem duplications (ITDs) are found in approximately one-third of patients with acute myeloid leukemia and have important prognostic and therapeutic implications that have supported their assessment in routine clinical practice. Conventional methods for assessing FLT3-ITD status and allele burden have been primarily limited to PCR fragment size analysis because of the inherent difficulty in detecting large ITD variants by next-generation sequencing (NGS). In this study, we assess the performance of publicly available bioinformatic tools for the detection and quantification of FLT3-ITDs in clinical hybridization-capture NGS data. We found that FLT3_ITD_ext had the highest overall accuracy for detecting FLT3-ITDs and was able to accurately quantify allele burden. Although all other tools evaluated were able to detect FLT3-ITDs reasonably well, allele burden was consistently underestimated. We were able to significantly improve quantification of FLT3-ITD allelic burden independent of the detection method by utilizing soft-clipped reads and/or ITD junctional sequences. In addition, we show that identifying mutant reads by previously identified junctional sequences further improves the sensitivity of detecting FLT3-ITDs in post-treatment samples. Our results demonstrate that FLT3-ITDs can be reliably detected in clinical NGS data using available bioinformatic tools. We further describe how accurate quantification of FLT3-ITD allele burden can be added on to existing clinical NGS pipelines for routine assessment of FLT3-ITD status in patients with acute myeloid leukemia.

SUBMITTER: Tung JK

PROVIDER: S-EPMC8527870 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data.

Tung Jack K JK Suarez Carlos J CJ Chiang Tsoyu T Zehnder James L JL Stehr Henning H

The Journal of molecular diagnostics : JMD 20210805 10

FLT3 internal tandem duplications (ITDs) are found in approximately one-third of patients with acute myeloid leukemia and have important prognostic and therapeutic implications that have supported their assessment in routine clinical practice. Conventional methods for assessing FLT3-ITD status and allele burden have been primarily limited to PCR fragment size analysis because of the inherent difficulty in detecting large ITD variants by next-generation sequencing (NGS). In this study, we assess ...[more]

PMID: 34363960

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Project description:FLT3-internal tandem duplication occurs in 20-30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3-231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.

Project description:Activating internal tandem duplications (ITD) in the juxtamembrane domain of receptor tyrosine kinase FLT3 occur frequently in patients with acute myeloid leukemia (AML). Constitutive active FLT3-ITD mutations induce aberrant signaling and promote leukemic cell transformation. Inactivation of the attenuating receptor protein tyrosine phosphatase CD45 (PTPRC) in FLT3-ITD mice resulted in the development of a severe hematopoietic phenotype with characteristics of AML. In addition, abnormal bone structures and ectopic bone formation were observed in these mice, suggesting a previously unknown role of FLT3 to control bone development and remodeling. While Ptprc knockout and Flt3-ITD mutant mice showed a largely normal bone microarchitecture, micro-CT analysis of femurs from Flt3-ITD Ptprc knockout mice revealed trabecularization of the cortical bone. This resulted in increased trabecular bone volume at the metaphysis, while the cortical bone at the diaphysis was thinner and less dense. In the metaphysis, severely reduced osteoclast and osteoblast numbers were observed. Reduced capacity of ex vivo differentiation of CD11b-positive bone marrow stem cells to mature osteoclast was accompanied by their abnormal morphology and reduced size. Transcriptome analysis revealed reduced expression of osteoclastogenic genes. Unexpectedly, cumulative resorption activity of osteoclasts was increased. Size and structure of resorption pits of differentiated osteoclasts remained similar to those observed in osteoclast cultures derived from control animals. Enhanced proliferation of cells in osteoclast cultures derived from FLT3-ITD-expressing mice was mediated by increased expression of STAT5 target genes. Transcriptome analysis of differentiated osteoclasts showed dysregulated signaling pathways influencing their differentiation as well as the coupling of bone resorption and formation. Taken together, inactivation of attenuating CD45 in mice expressing oncogenic FLT3-ITD resulted in marked abnormalities of the osteo-hematopoietic niche, which can be explained by aberrant STAT5 activation.

Project description:We conducted a single-arm, phase 2 trial (German-Austrian Acute Myeloid Leukemia Study Group [AMLSG] 16-10) to evaluate midostaurin with intensive chemotherapy followed by allogeneic hematopoietic-cell transplantation (HCT) and a 1-year midosta urin maintenance therapy in adult patients with acute myeloid leukemia (AML) and fms-related tyrosine kinase 3 (FLT3) internal tandem duplication (ITD). Patients 18 to 70 years of age with newly diagnosed FLT3-ITD-positive AML were eligible. Primary and key secondary endpoints were event-free survival (EFS) and overall survival (OS). Results were compared with a historical cohort of 415 patients treated on 5 prior AMLSG trials; statistical analysis was performed using a double-robust adjustment with propensity score weighting and covariate adjustment. Results were also compared with patients (18-59 years) treated on the placebo arm of the Cancer and Leukemia Group B (CALGB) 10603/RATIFY trial. The trial accrued 440 patients (18-60 years, n = 312; 61-70 years, n = 128). In multivariate analysis, EFS was significantly in favor of patients treated within the AMLSG 16-10 trial compared with the AMLSG control (hazard ratio [HR], 0.55; P < .001); both in younger (HR, 0.59; P < .001) and older patients (HR, 0.42; P < .001). Multivariate analysis also showed a significant beneficial effect on OS compared with the AMLSG control (HR, 0.57; P < .001) as well as to the CALGB 10603/RATIFY trial (HR, 0.71; P = .005). The treatment effect of midostaurin remained significant in sensitivity analysis including allogeneic HCT as a time-dependent covariate. Addition of midostaurin to chemotherapy was safe in younger and older patients. In comparison with historical controls, the addition of midostaurin to intensive therapy led to a significant improvement in outcome in younger and older patients with AML and FLT3-ITD. This trial is registered at clinicaltrialsregistry.eu as Eudra-CT number 2011-003168-63 and at clinicaltrials.gov as NCT01477606.

Dataset Information

Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data.

Publications

Accurate Detection and Quantification of FLT3 Internal Tandem Duplications in Clinical Hybrid Capture Next-Generation Sequencing Data.

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