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Detection of c-kit–Activating Mutations in Gastrointestinal Stromal Tumors by High-Resolution Amplicon Melting Analysis

Carlynn Willmore MT(ASCP), Joseph A. Holden MD, PhD, Luming Zhou PhD, Sheryl Tripp MT(ASCP), QIHC(ASCP), Carl T. Wittwer MD, PhD, Lester J. Layfield MD
DOI: http://dx.doi.org/10.1309/4E6UYBY62N2FCA6N 206-216 First published online: 1 August 2004


High-resolution amplicon melting analysis was used to scan for c-kit–activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD117 positivity and might represent true smooth muscle tumors or c-kit–negative GISTs. No c-kit–activating mutations were detected in the 7 CD117– cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit–activating mutations and can aid in the diagnosis of GISTs.

Key Words:
  • Gastrointestinal stromal tumor
  • c-kit–activating mutation
  • High-resolution amplicon melting analysis
  • Polymerase chain reaction