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Published on: September 20, 2016
Rapid detection of Flt3 mutations in acute myeloid leukemia patients by denaturing HPLC
Michele Bianchini1, Emanuela Ottaviani, Tiziana Grafone
1Institute of Hematology and Medical Oncology L. e A. Seràgnoli, University of Bologna, Via Massarenti No. 9, 40138 Bologna, Italy.
Background:
fms-related tyrosine kinase 3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has been implicated in its pathogenesis. Because screening of Flt3 in AML patients by PCR followed by gel electrophoresis is time-consuming and fails to detect some very small internal tandem duplications (ITDs), we developed a method for screening of FLT3 receptor mutations with PCR plus denaturing HPLC (D-HPLC).
Methods:
Total mRNAs extracted from 34 AML patients were first analyzed for the presence of juxtamembrane length mutations and tyrosine kinase domain point mutations by a conventional method involving PCR amplification, restriction enzyme digestion, and agarose gel electrophoresis (PCR-RED-AGE). Subsequently, the same patient panel was analyzed by D-HPLC, using specifically designed primers and optimized running temperatures for the length and point mutation analysis.
Results:
Thirty-four patients were analyzed by PCR-RED-AGE; 9 were positive for known Flt3 mutations: 6 of 34 (18%) for ITDs in exon 14 and 3 of 34 (9%) for point mutations in exon 20. The same patient panel was analyzed by D-HPLC, and additional nucleotide changes were discovered; in total, 14 sequence variations were identified: 7 of 34 (21%) for ITDs in exon 14; 2 of 34 (6%) for point mutations in exon 20; 1 of 34 (3%) for a new point mutation in exon 16; and 4 of 34 (12%) for polymorphisms in exons 13 and 14. Direct sequencing analysis identified nucleotide alterations in each of the "D-HPLC positives" but in none of the "D-HPLC negatives", yielding a specificity and sensitivity of 100% for D-HPLC-based screening.
Conclusions:
This novel D-HPLC-based procedure, which is optimized for identification of new point mutations in the catalytic and regulatory domains of FLT3 receptor, could potentially be useful for studies involving precise genotype determination, which could be critical for selection of innovative AML therapies targeting the FLT3 protein.
Insights
A new denaturing HPLC (D-HPLC) method effectively screens for fms-related tyrosine kinase 3 (Flt3) mutations in acute myeloid leukemia (AML) patients. This D-HPLC approach offers 100% sensitivity and specificity, improving upon traditional PCR methods for FLT3 mutation detection.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Fms-related tyrosine kinase 3 (Flt3) is frequently mutated in acute myeloid leukemia (AML).
- Conventional PCR methods for Flt3 mutation screening are time-consuming and may miss certain mutations like internal tandem duplications (ITDs).
- A more efficient and sensitive screening method for Flt3 mutations in AML is needed.
Purpose of the Study:
- To develop and evaluate a novel method for screening Flt3 receptor mutations in AML patients.
- To compare the efficacy of PCR plus denaturing HPLC (D-HPLC) against conventional PCR-RED-AGE for Flt3 mutation detection.
Main Methods:
- Total mRNAs from 34 AML patients were analyzed.
- Conventional PCR amplification, restriction enzyme digestion, and agarose gel electrophoresis (PCR-RED-AGE) were used for initial analysis.
- The same patient samples were subsequently analyzed using D-HPLC with specifically designed primers and optimized temperatures.
Main Results:
- PCR-RED-AGE identified 9 Flt3 mutations (6 ITDs, 3 point mutations).
- D-HPLC identified a total of 14 sequence variations, including 7 ITDs, 2 point mutations, 1 new point mutation, and 4 polymorphisms.
- D-HPLC demonstrated 100% sensitivity and specificity compared to direct sequencing, detecting all nucleotide alterations identified by sequencing.
Conclusions:
- The novel D-HPLC-based procedure is highly effective for identifying Flt3 mutations, including new point mutations in critical domains.
- This method offers improved precision for genotype determination in AML patients.
- The D-HPLC approach could be valuable for selecting targeted therapies for AML patients with FLT3 mutations.
