EVALUATION OF DNA AMPLIFICATION ASSAYS FOR THE DETECTION OF THE FLT3-ITD MUTATION
- 1Universidade Nilton Lins, Manaus, AM, Brazil
Abstract
Background: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the clonal proliferation of myeloid precursor cells and marked genetic heterogeneity, both of which are directly associated with prognosis and therapeutic response (WHO, 2022). Among the molecular alterations of greatest clinical relevance, the FLT3-ITD (Internal Tandem Duplication) mutation is found in 20–30% of AML patients and is associated with constitutive activation of the FLT3 tyrosine kinase receptor, increased cellular proliferation, a higher risk of relapse, and reduced overall survival (Bhansali, Pratz, & Lai, 2023). Early identification of this mutation is essential for prognostic stratification, molecular monitoring, and the selection of targeted therapies. However, the limitations of conventional diagnostic methods highlight the need for rapid, sensitive, specific, and accessible molecular methodologies (Hellwig, 2023). Aim: To evaluate DNA amplification methodologies for the detection of the FLT3-ITD mutation by comparing their sensitivity, specificity, reproducibility, and limit of detection, as well as their potential application in the molecular diagnosis of acute myeloid leukemia. Materials and methods: This experimental laboratory-based study was conducted at the Molecular Genetics Laboratory of Nilton Lins University. Synthetic plasmids containing FLT3 gene sequences in both the wild-type and mutant (FLT3-ITD) forms were used as molecular controls for analytical standardization and validation. Plasmid sequences and primer oligonucleotide sequences are not available. Conventional PCR and real-time PCR (qPCR) assays were standardized. The performance of the methodologies is being evaluated based on sensitivity, specificity, limit of detection, and reproducibility using serial dilutions of the synthetic controls. Results: Preliminary results demonstrated high specificity of the developed oligonucleotide primers, indicating accurate recognition of the region corresponding to the FLT3-ITD mutation. Standardization of conventional PCR demonstrated consistent amplification of the expected fragment using synthetic controls, enabling the establishment of reproducible experimental conditions. A comparative evaluation of conventional PCR, qPCR, and MIRA is currently underway to identify the methodology with the best analytical performance and the greatest potential for application in biological samples. Discussion: The use of synthetic controls represents a robust strategy for the initial validation of molecular assays, promoting greater experimental standardization while minimizing interference inherent to clinical samples. Furthermore, highly accurate molecular tools may expand access to specialized diagnostics and strengthen precision medicine in AML. Conclusion: The results obtained demonstrate the feasibility of standardizing molecular methodologies for the detection of the FLT3-ITD mutation using synthetic controls. The next stages of the study aim to develop a sensitive, specific, reproducible, and potentially applicable diagnostic method for routine laboratory use.
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BibTeX
@article{souza2026evaluation,
title = {{EVALUATION OF DNA AMPLIFICATION ASSAYS FOR THE DETECTION OF THE FLT3-ITD MUTATION}},
author = {Amanda Caroline Queiroz SOUZA and George Allan Villarouco SILVA},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.107588},
url = {https://doi.org/10.1016/j.htct.2026.107588},
note = {CC BY},
}
RIS
TY - JOUR
TI - EVALUATION OF DNA AMPLIFICATION ASSAYS FOR THE DETECTION OF THE FLT3-ITD MUTATION
AU - Amanda Caroline Queiroz SOUZA
AU - George Allan Villarouco SILVA
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.107588
UR - https://doi.org/10.1016/j.htct.2026.107588
AB - Background: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the clonal proliferation of myeloid precursor cells and marked genetic heterogeneity, both of which are directly associated with prognosis and therapeutic response (WHO, 2022). Among the molecular alterations of greatest clinical relevance, the FLT3-ITD (Internal Tandem Duplication) mutation is found in 20–30% of AML patients and is associated with constitutive activation of the FLT3 tyrosine kinase receptor, increased cellular proliferation, a higher risk of relapse, and reduced overall survival (Bhansali, Pratz, & Lai, 2023). Early identification of this mutation is essential for prognostic stratification, molecular monitoring, and the selection of targeted therapies. However, the limitations of conventional diagnostic methods highlight the need for rapid, sensitive, specific, and accessible molecular methodologies (Hellwig, 2023). Aim: To evaluate DNA amplification methodologies for the detection of the FLT3-ITD mutation by comparing their sensitivity, specificity, reproducibility, and limit of detection, as well as their potential application in the molecular diagnosis of acute myeloid leukemia. Materials and methods: This experimental laboratory-based study was conducted at the Molecular Genetics Laboratory of Nilton Lins University. Synthetic plasmids containing FLT3 gene sequences in both the wild-type and mutant (FLT3-ITD) forms were used as molecular controls for analytical standardization and validation. Plasmid sequences and primer oligonucleotide sequences are not available. Conventional PCR and real-time PCR (qPCR) assays were standardized. The performance of the methodologies is being evaluated based on sensitivity, specificity, limit of detection, and reproducibility using serial dilutions of the synthetic controls. Results: Preliminary results demonstrated high specificity of the developed oligonucleotide primers, indicating accurate recognition of the region corresponding to the FLT3-ITD mutation. Standardization of conventional PCR demonstrated consistent amplification of the expected fragment using synthetic controls, enabling the establishment of reproducible experimental conditions. A comparative evaluation of conventional PCR, qPCR, and MIRA is currently underway to identify the methodology with the best analytical performance and the greatest potential for application in biological samples. Discussion: The use of synthetic controls represents a robust strategy for the initial validation of molecular assays, promoting greater experimental standardization while minimizing interference inherent to clinical samples. Furthermore, highly accurate molecular tools may expand access to specialized diagnostics and strengthen precision medicine in AML. Conclusion: The results obtained demonstrate the feasibility of standardizing molecular methodologies for the detection of the FLT3-ITD mutation using synthetic controls. The next stages of the study aim to develop a sensitive, specific, reproducible, and potentially applicable diagnostic method for routine laboratory use.
SN - 2531-1379
SN - 2531-1387
ER -