Fnih MRD in AML Biomarkers Consortium: Evaluation of Methods for Detection of FLT3 internal Tandem Duplication Measurable Residual Disease in Acute Myeloid Leukemia

Introduction FLT3 internal tandem duplication (ITD) is one of the most frequently observed mutations in acute myeloid leukemia (AML) and persistent detection in complete remission (CR) prior to allogeneic hematopoietic transplant (alloHCT) is highly predictive of increased incidence of relapse and death. A variant allele fraction (VAF) of ≥0.01% is associated with the greatest risk, but there is increasing evidence that some of this risk can be mitigated by intervention. Objective There is an immediate need for commercially available assays for FLT3 -ITD measurable residual disease (MRD) detection. The FNIH MRD in AML Biomarkers Consortium here benchmarked technologies for FLT3 -ITD MRD detection. Methods Assay characteristics from five next-generation sequencing (NGS)-based assays for sensitive FLT3 -ITD detection, available commercially as a service or kit, were collected from the providers. In phase 1, 48 contrived standards were generated by serially diluting DNA from FLT3 -ITD cell lines into control DNA in isolation or multiplexed (VAF 0.01-1%). The top four performing assays were carried forward to phase 2, where testing of DNA from peripheral blood or bone marrow of 16 AML patients with FLT3 -ITD(s) (12-192bp) detected by capillary gel electrophoresis at a low allelic ratio (0.01-0.18) and four controls was performed. All testing was performed blinded. Results The five NGS-based technologies evaluated utilized a variety of enrichment strategies, error-correction methods, and sequencing platforms ( Figure 1 ). In phase 1 testing of standards, four technologies reliably detected all ITDs down to 0.05-0.01% VAF, while 2 ITDs were missed by one technology due to primer design and bioinformatic filtering. Significant correlation between expected and observed VAFs was seen for three technologies ( Figure 2) , while one technology exhibited conflicting results related to ITD length. In all cases, the presence of multiple ITDs did not impact performance. In phase 2 testing of primary samples, all technologies correctly assigned patients as FLT3 -ITD positive or negative. While 18 of 20 ITDs were identified by all four assays, one technology missed 2 complex ITDs due to bioinformatic filtering. A significant correlation between expected and observed VAF was observed for all but one method. Additional analyses including further assay optimization and comparison to reported assay performance will be presented. Conclusion The evaluation of NGS-based technologies for FLT3 -ITD MRD detection showed the ability of multiple strategies to detect and quantify ITDs of different lengths down to a clinically relevant level. However, multiple factors, both technical and bioinformatic, can impact assay performance. Given test heterogeneity, careful consideration of the performance characteristics of any assay proposed for clinical decision making prior to alloHCT is recommended.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.