Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography

M. W N Deininger, L. McGreevey, S. Willis, T. M. Bainbridge, Brian Druker, Michael Heinrich

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

Mutations of the ABL kinase domain (KD) are common in patients with chronic myelogenous leukemia (CML) who develop resistance to imatinib. We developed an RT-PCR-based denaturing high-performance liquid chromatography (D-HPLC) assay to detect mutations of the ABL KD. Validation experiments using mixtures of wild type and mutant amplicons showed that the D-HPLC assay could detect mutant transcripts when they represented at least 15% of the total, and was thus twice as sensitive as automated sequencing. When D-HPLC was applied to 30 cDNAs from patients with imatinib resistance that had previously been characterized for KD mutations by direct sequencing of BCR-ABL RT-PCR products, there was concordance in 97% of samples. Resequencing confirmed the original mutations in all cases. In addition, sequencing of individual clones detected a mutation in one sample that had been mutation-positive by D-HPLC but wild type by conventional sequencing. In serial samples from the same individuals, D-HPLC detected mutations as early as 260 days before hematological relapse. D-HPLC is suitable for routine clinical monitoring of CML patients for emergence of KD mutations and may be useful for optimizing therapy. Early detection of emerging mutant clones may aid in guiding decisions regarding alternative treatment options.

Original languageEnglish (US)
Pages (from-to)864-871
Number of pages8
JournalLeukemia
Volume18
Issue number4
DOIs
StatePublished - Apr 2004

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Phosphotransferases
High Pressure Liquid Chromatography
Mutation
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Clone Cells
Polymerase Chain Reaction
Complementary DNA
Recurrence
Therapeutics

Keywords

  • BCR-ABL
  • CML
  • Imatinib
  • Mutation
  • Resistance

ASJC Scopus subject areas

  • Hematology
  • Cancer Research

Cite this

Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography. / Deininger, M. W N; McGreevey, L.; Willis, S.; Bainbridge, T. M.; Druker, Brian; Heinrich, Michael.

In: Leukemia, Vol. 18, No. 4, 04.2004, p. 864-871.

Research output: Contribution to journalArticle

Deininger, M. W N ; McGreevey, L. ; Willis, S. ; Bainbridge, T. M. ; Druker, Brian ; Heinrich, Michael. / Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography. In: Leukemia. 2004 ; Vol. 18, No. 4. pp. 864-871.
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