High Sensitivity Detection of FLT3-ITD Measurable Residual Disease By Deep Sequencing Prior to Hematopoietic Cell Transplant Is Highly Prognostic for Outcome in Acute Myeloid Leukemia
Authors: Loo Sun , Dillon Richard , Anstee Natasha S , Tiong Ing S , Ivey Adam , Potter Nicola , Othman Jad , Runglall Manohursingh , Jovanovic Jelena , Gilkes Amanda , Thomas Ian , Johnson Sean , Russell Nigel H. , Wei Andrew H. 
Published: November 2021

Abstract
Introduction
Allogeneic hematopoietic cell transplant (allo-HCT) in first or second complete remission (CR1 or CR2) plays a fundamental role in the management of FLT3-ITD mutant acute myeloid leukemia (AML). Measurable residual disease (MRD) detected prior to HCT by flow cytometry or NPM1 RT-qPCR indicates a high risk of post-HCT relapse (Dillon et al, 2020). FLT3-ITD has not routinely been used as an MRD marker due to concerns relating to clonal stability and assay sensitivity, however increasing use of FLT3 inhibitors post-HCT is likely to alter this recommendation, particularly in view of the routine use of midostaurin, which may increase clonal instability (Schmalbrock et al, 2021). To date evaluation of the prognostic utility of FLT3-ITD quantitation in the pre-HCT setting has mainly been limited to capillary electrophoresis (CE) (Gaballa et al, 2017, Helbig et al, 2020) which has low sensitivity (1-5%). Next-generation sequencing (NGS) enables FLT3-ITD MRD detection with higher sensitivity and specificity (lower limit of detection (LLD) 0.001%) (Blätte et al, 2019). Preliminary data indicate that pre-HCT detection of FLT3-ITD may provide powerful prognostic information (Hourigan et al, 2019). The current work evaluates the prognostic impact of FLT3-ITD MRD via deep NGS sequencing in the pre-HCT setting.
Methods
FLT3-ITD NGS was performed on stored DNA pre-HCT (69 bone marrow, 9 peripheral blood) from the UK NCRI AML17 trial (ISRCTN 55675535) and Alfred Hospital transplant database. Quantitation of FLT3-ITD was by amplicon-based sequencing of exons 14-15, detecting ITDs &gt;6 base pairs (bp) length. Prepared libraries were sequenced (Illumina) using 150 bp paired-end reads with minimum coverage of 400,000 reads and bioinformatics analysis using getITD (Blatte et al, 2019). Sensitivity ~0.01% and LLD 0.001%. Kaplan-Meier method was used to determine relapse-free survival (RFS; HCT date to relapse or death from any cause) and overall survival (OS; HCT date to death or last follow-up). Cumulative incidence of relapse was estimated considering competing risk. Cox proportional hazards model was used to assess factors impacting RFS and OS. Categorical data evaluated using Fisher's exact and continuous variables with Mann-Whitney-U tests. The study was approved by Alfred Health Ethics Committee (181/21).
Results
The retrospective cohort included 78 patients with FLT3-ITD AML who received first allo-HCT in morphologic remission between 2010-2020. At diagnosis, median age was 48, 83% had NPM1 mutation, 45% FLT3-ITD allelic ratio (AR) ≥0.5, 43% received myeloablative conditioning, 82% transplanted in CR1, unrelated donor in 53% and 14% had received upfront FLT3-inhibitor. Pre-HCT FLT3-ITD MRD by NGS was not detected in 62% (MRD-neg) and detected in 38% (MRD-pos). Among FLT3-ITD MRD-pos pre-HCT, 90% had 1 ITD, median bp 54 (21-210) and median VAF 0.0632% (0.001-9.3519). Overall, 73% with MRD had FLT3-ITD VAF &lt;1%, nominally below limit of detection by CE. CE (sensitivity ~1% and LLD 0.5%) performed on 20 FLT3-ITD NGS MRD-pos cases failed to detect any ITDs with VAF &lt;1%, with 4/4 cases positive by both CE and NGS (VAF ≥1%). With median follow-up of 58 months, patients FLT3-ITD MRD-pos by NGS had a median time to relapse of 4 months (range, 1-17) vs not reached (p= &lt;0.0001), with poorer 1-year RFS 27% vs 77% (p= &lt;0.0001) and OS 37% vs 77% (p= &lt;0.0001) compared to MRD-neg patients. Multivariable analysis considering factors in table 1, showed that RFS was independently influenced by pre-HCT FLT3-ITD MRD (hazard ratio (HR) 4.36; p=0.0004) and T-cell depletion (HR 3.72, p=0.0044). Among patients who were both FLT3-ITD and NPM1 mutant at diagnosis, best prognosis post-HCT was among patients who were both NPM1 and FLT3-ITD MRD negative pre-HCT (1-year OS 96%). The additional value of FLT3-ITD MRD was illustrated by the inferior prognosis for patients who were both NPM1 and FLT3-ITD MRD-pos (1-year OS 32%), vs NPM1 MRD-pos but FLT3-ITD MRD-neg (1-year OS 64%) (p=0.014) pre-HCT (Figure 4).
Conclusions
Pre-HCT FLT3-ITD MRD detection at any level by deep NGS is associated with a higher risk of post-HCT relapse and poorer long-term survival. Among patients who were also NPM1 MRD-pos, FLT3-ITD MRD was more informative for outcome. Our findings support the use of FLT3-ITD NGS to assess pre-HCT risk and allows rational application of FLT3 directed targeted therapy in the pre- or post- HCT setting in this high-risk AML population.
Figure 1 Figure 1.

Disclosures
Dillon: Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events; Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Jazz: Other: Education events; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees. Tiong: Amgen: Speakers Bureau; Servier: Consultancy, Speakers Bureau; Pfizer: Consultancy. Russell: Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Jazz: Research Funding, Speakers Bureau. Wei: Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Macrogenics: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.