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Clinical Chemistry 49: 1642-1650, 2003; 10.1373/49.10.1642
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(Clinical Chemistry. 2003;49:1642-1650.)
© 2003 American Association for Clinical Chemistry, Inc.

Hematology

Rapid Detection of Flt3 Mutations in Acute Myeloid Leukemia Patients by Denaturing HPLC

Michele Bianchini1, Emanuela Ottaviani1, Tiziana Grafone1, Barbara Giannini1, Simona Soverini1, Carolina Terragna1, Marilina Amabile1, Pier Paolo Piccaluga1, Michele Malagola1, Michela Rondoni1, Costanza Bosi1, Michele Baccarani1 and Giovanni Martinellia,1

1 Institute of Hematology and Medical Oncology "L. e A. Seràgnoli", University of Bologna, Via Massarenti No. 9, 40138 Bologna, Italy.

aAuthor for correspondence. Fax 39-051-6364037; e-mail gmartino{at}kaiser.alma.unibo.it.

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.




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