High-resolution melting analysis is a sensitive diagnostic tool to detect imatinib-resistant and imatinib-sensitive PDGFRA exon 18 mutations in gastrointestinal stromal tumors.
The mutational status of KIT and PDGFRA is highly relevant for prognosis and therapy prediction in gastrointestinal stromal tumors (GIST). PDGFRA exon 18 mutations have direct therapeutic implications since it is crucial to distinguish mutations associated with sensitivity to tyrosine kinase inhibitors from those causing primary resistance, eg, the most common exon 18 mutation p.D842V. In response to a growing demand for reliable, faster and more sensitive methods we established and validated a high-resolution melting (HRM) assay for PDGFRA exon 18. A total of 159 GIST samples were comparatively analyzed by HRM and direct Sanger sequencing. We demonstrate that HRM provides highly reliable mutational results with higher sensitivity and shorter time to diagnosis compared to Sanger sequencing. We determined the sensitivity threshold of HRM at 6% of mutated alleles. PDGFRA exon 18 wild-type status and the most common p.D842V resistance mutation (together representing >90% of the cases) can be detected specifically by HRM. Other rare mutations can be pre-screened by HRM and afterwards determined precisely by DNA sequencing. In this way we detected four novel mutations in PDGFRA exon 18, two of which were associated with an aggressive clinical course. Including these new mutations, we provide a comprehensive overview of all 60 currently known subtypes of PDGFRA exon 18 mutations in GIST. Seven of them (accounting for about 75% of all exon 18-mutated GISTs) are reported to be resistant to imatinib. However, there are at least 10 other mutations which are regarded as sensitive to tyrosine kinase inhibitors.
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany.
Helen
H
Künstlinger
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany.
Elke
E
Binot
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany.
Sabine
S
Merkelbach-Bruse
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany; Gerhard-Domagk-Institute of Pathology, University of Münster Medical Center, D-48149 Münster, Germany.
Sebastian
S
Huss
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany; Gerhard-Domagk-Institute of Pathology, University of Münster Medical Center, D-48149 Münster, Germany.
Eva
E
Wardelmann
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany.
Reinhard
R
Buettner
Institute of Pathology, University Hospital Cologne, D-50942 Cologne, Germany; Institute of Pathology, University Hospital Göttingen, D-37075 Göttingen, Germany. Electronic address: hans-ulrich.schildhaus@med.uni-goettingen.de.
Hans-Ulrich
HU
Schildhaus
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001549:Benzamides / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005091:Exons; D005260:Female; D046152:Gastrointestinal Stromal Tumors / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D005838:Genotype; D006801:Humans; D000068877:Imatinib Mesylate; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010879:Piperazines / Q000627:therapeutic use*; D011379:Prognosis; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D011743:Pyrimidines / Q000627:therapeutic use*; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics*
D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; D020796:Receptor, Platelet-Derived Growth Factor alpha
GIST; Gastrointestinal stromal tumor; High-resolution melting; Imatinib; PDGFRA