Introduction

The presence of FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in patients with AML is associated with a poor prognosis, characterized by an increased risk of relapse and shorter overall survival. Recent studies have indicated that specific factors significantly influence the prognosis of patients with FLT3-ITD mutations. However, there is currently no consensus on the impact of these factors on prognosis, and further validation with a large number of clinical samples is needed.

Methods

A novel Next Generation Sequencing panel encompassing 70 genes related to the diagnostic classification of AML, prognosis evaluation, and treatment guidance has been developed. This panel includes all exons of the FLT3gene, as well as the regions of intron 13 and intron 14 for the detection of FLT3-ITD mutations. For ITD analysis, we employed the software pindel (version 0.2.5b9) and utilized the default parameters for analyzing FLT3-ITD mutations. All patients underwent intensified induction chemotherapy and consolidation treatment tailored to the ELN-2017 recommendations.

Results

To validate the detection capacity of the AML panel, 20 samples confirmed positive for FLT3-ITD through capillary electrophoresis analysis were collected (5 cases had insertion lengths of less than 50 bp, 8 cases were between 50 bp and 100 bp, 4 cases were between 100 bp and 150 bp, and 3 cases were >150 bp). All FLT3-ITD mutations were successfully detected in these samples using the AML panel. The number of detected inserted bases precisely matched the results obtained from the capillary electrophoresis analysis. Moreover, the mutation frequency identified by the AML panel exhibited a correlation coefficient (R²) of 0.88 with the mutation rates obtained from capillary electrophoresis analysis.

From November 2022 to February 2023, the AML panel identified a total of 342 non-M3 AML Chinese patients who were positive for FLT3-ITD, included 180 males and 162 females, with a median age of 53 years.

A total of 428 ITD mutations were identified among 342 patients. Of these, 276 patients (80.7%) exhibited only one ITD mutation, 51 patients (14.9%) had two ITD mutations, and 15 patients (4.4%) displayed three or more ITD mutations, with the highest number recorded being five ITD mutations in a single patient. The median insertion length of ITD mutations was 42 bp (3 bp to 210 bp). The most common insertions measured were 24 bp (9.8%), 21 bp (8.6%), and 30 bp (8.4%). The median VAF was 25.1% (1% to 91.8%).

The FLT3-ITD mutation was significant positively related to NPM1mutation (p<0.0001). Conversely, the FLT3-ITD mutation showed significant negative correlations with the FLT3-TKD (p<0.0001), NRAS(p<0.0001) and TP53(p=0.0001) mutations.

Kaplan-Meier analysis revealed that, FLT3-ITD inserted >42 bp (p=0.008), FLT3-ITD mutations located in the TKD1 region (p=0.012), and the presence of FLT3-TKD mutations (p=0.01) were associated with significantly poorer relapse-free survival (RFS). The factors of sex, age, the number of blast cells, the VAF of FLT3-ITD, the number of FLT3-ITD, the accompanied by NPM1or KRAS/NRASmutations, do not significantly impact RFS.

Univariate Cox regression analysis revealed that patients exhibited significantly poorer RFS when the FLT3-ITD inserted >42 bp [hazard ratio (HR): 2.42; 95% CI: 1.22-4.79; p=0.012], when the FLT3-ITD mutation was located in the TKD1 region (HR: 2.39; 95% CI: 1.18-4.83; p=0.016), and when accompanied by FLT3-TKD mutations (HR: 2.59; 95% CI: 1.21-5.59; p=0.015). Importantly, after adjusting for other confounding variables, the multivariate Cox regression analysis indicated that the larger ITD size (>42 bp) was an independent prognostic factor for poorer RFS, with a HR of 2.45 (95% CI: 1.17-5.11, p=0.017).

Conclusions

Following verification, the AML panel demonstrates the capability to accurately detect FLT3-ITD mutations with varying insertion lengths. AML patients who possess FLT3-ITD mutations featuring insertions >42 bp, where the FLT3-ITD mutation occurs within the TKD1 region, and when accompanied by FLT3-TKD mutations, experience significantly shorter RFS times.

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2025
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