Detection of C-KIT and PDGFRA Mutations in Gastrointestinal Stromal Tumors
Detection of C-KIT and PDGFRA Mutations in Gastrointestinal Stromal Tumors
Mutational analysis of c-KIT or PDGFRA has become an important laboratory assay for patients with gastrointestinal stromal tumors (GISTs) because the results are useful in predicting the responsiveness to imatinib. To assess the diagnostic usefulness of denaturing high-pressure liquid chromatography (DHPLC) in this setting, we performed DHPLC and DNA sequencing to study exons 9, 11, 13, and 17 of c-KIT and exons 12 and 18 of PDGFRA in 54 consecutive cases of GIST collected from a single population. Most (40/54 [74%]) carried c-KIT mutations, and 7 (13%) carried PDGFRA mutations. These results were similar to those described in the literature. It is important to note that DHPLC was found to be highly sensitive, detecting all of the mutations in these 6 exons that were identified by DNA sequencing. Our data suggest that DHPLC is a cost-effective, rapid, and sensitive test for screening for mutations of c-KIT and PDGFRA in GISTs.
Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms typically arising in the stomach, small intestine, colon, and other anatomic sites in the abdominal cavity. These tumors are characterized by the strong expression of the receptor tyrosine kinase c-KIT (CD117), which is now accepted as the most specific immunohistochemical marker for GIST. Recent studies have shown that approximately 60% to 70% of GISTs carry mutations in c-KIT, which encodes a member of the type III tyrosine kinase gene family. The 4 involved exons, ie, 9, 11, 13, and 17, correspond to the extracellular, juxtamembranous, tyrosine kinase 1, and tyrosine kinase 2 domains of the c-KIT protein, respectively. These mutations result in constitutive activation of the tyrosine kinase activity of c-KIT, which is believed to be the major oncogenic event in these tumors. For cases showing no evidence of c-KIT mutations, most carry mutations of the PDGFRA gene. Similar to c-KIT, PDGFRA is also a member of the type III tyrosine kinase family, and PDGFRA gene mutations found in GISTs result in constitutive activation of this tyrosine kinase, which contributes to the tumorigenesis. It is interesting that mutations of these 6 exons (ie, 4 in c-KIT and 2 in PDGFRA) are mutually exclusive in the majority of GISTs; excluding single nucleotide polymorphisms (SNPs), cases carrying multiple mutations of these 6 exons are exceedingly rare.
In view of the importance of c-KIT and PDGFRA in GIST, tyrosine kinase inhibitors such as imatinib have been used to treat GIST. Imatinib resistance, found in a proportion of patients with GIST, correlates with mutations of specific exons in c-KIT. Specifically, patients with tumors carrying mutations of exon 9 are more likely to show resistance, whereas those with tumors carrying mutations of exon 11 are more likely to show a good response. Mutations of exons 12 and 18 of PDGFRA also correlate with good responsiveness to imatinib, except for a point mutation in amino acid 842 in PDGFRA exon 18, which confers absolute resistance. Thus, detection and analysis of gene mutations of c-KIT and PDGFRA have become a standard of care for patients diagnosed with GIST.
With regard to the detection method, most of the previously published studies used DNA sequencing, which is a relatively labor-intensive and time-consuming procedure. A more efficient method for detecting gene mutations is that of denaturing high-pressure liquid chromatography (DHPLC), which has been used extensively to rapidly screen for gene mutations in research settings. The other major advantage of DHPLC over DNA sequencing is that interpretation of the generated data is less subjective and labor-intensive. Because the vast majority of GISTs carry only 1 mutation in the 6 implicated exons, it would be more cost-effective if DNA sequencing were performed only to confirm and analyze mutations that are initially detected by DHPLC.
To our knowledge, there are only a small number of publications describing the use of DHPLC to detect c-KIT and PDGFRA mutations in GIST. In most of these studies, the focus was not related to the validation of the DHPLC assay, and therefore, these studies are usually restricted to specific c-KIT exons or a relatively small sample. Furthermore, only 1 of these studies used a well-defined single population cohort.
In this study, we aimed to validate the clinical diagnostic usefulness of DHPLC in detecting mutations of c-KIT and PDGFRA in 54 consecutive cases of GIST that were derived from a well-defined population.
Abstract and Introduction
Abstract
Mutational analysis of c-KIT or PDGFRA has become an important laboratory assay for patients with gastrointestinal stromal tumors (GISTs) because the results are useful in predicting the responsiveness to imatinib. To assess the diagnostic usefulness of denaturing high-pressure liquid chromatography (DHPLC) in this setting, we performed DHPLC and DNA sequencing to study exons 9, 11, 13, and 17 of c-KIT and exons 12 and 18 of PDGFRA in 54 consecutive cases of GIST collected from a single population. Most (40/54 [74%]) carried c-KIT mutations, and 7 (13%) carried PDGFRA mutations. These results were similar to those described in the literature. It is important to note that DHPLC was found to be highly sensitive, detecting all of the mutations in these 6 exons that were identified by DNA sequencing. Our data suggest that DHPLC is a cost-effective, rapid, and sensitive test for screening for mutations of c-KIT and PDGFRA in GISTs.
Introduction
Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms typically arising in the stomach, small intestine, colon, and other anatomic sites in the abdominal cavity. These tumors are characterized by the strong expression of the receptor tyrosine kinase c-KIT (CD117), which is now accepted as the most specific immunohistochemical marker for GIST. Recent studies have shown that approximately 60% to 70% of GISTs carry mutations in c-KIT, which encodes a member of the type III tyrosine kinase gene family. The 4 involved exons, ie, 9, 11, 13, and 17, correspond to the extracellular, juxtamembranous, tyrosine kinase 1, and tyrosine kinase 2 domains of the c-KIT protein, respectively. These mutations result in constitutive activation of the tyrosine kinase activity of c-KIT, which is believed to be the major oncogenic event in these tumors. For cases showing no evidence of c-KIT mutations, most carry mutations of the PDGFRA gene. Similar to c-KIT, PDGFRA is also a member of the type III tyrosine kinase family, and PDGFRA gene mutations found in GISTs result in constitutive activation of this tyrosine kinase, which contributes to the tumorigenesis. It is interesting that mutations of these 6 exons (ie, 4 in c-KIT and 2 in PDGFRA) are mutually exclusive in the majority of GISTs; excluding single nucleotide polymorphisms (SNPs), cases carrying multiple mutations of these 6 exons are exceedingly rare.
In view of the importance of c-KIT and PDGFRA in GIST, tyrosine kinase inhibitors such as imatinib have been used to treat GIST. Imatinib resistance, found in a proportion of patients with GIST, correlates with mutations of specific exons in c-KIT. Specifically, patients with tumors carrying mutations of exon 9 are more likely to show resistance, whereas those with tumors carrying mutations of exon 11 are more likely to show a good response. Mutations of exons 12 and 18 of PDGFRA also correlate with good responsiveness to imatinib, except for a point mutation in amino acid 842 in PDGFRA exon 18, which confers absolute resistance. Thus, detection and analysis of gene mutations of c-KIT and PDGFRA have become a standard of care for patients diagnosed with GIST.
With regard to the detection method, most of the previously published studies used DNA sequencing, which is a relatively labor-intensive and time-consuming procedure. A more efficient method for detecting gene mutations is that of denaturing high-pressure liquid chromatography (DHPLC), which has been used extensively to rapidly screen for gene mutations in research settings. The other major advantage of DHPLC over DNA sequencing is that interpretation of the generated data is less subjective and labor-intensive. Because the vast majority of GISTs carry only 1 mutation in the 6 implicated exons, it would be more cost-effective if DNA sequencing were performed only to confirm and analyze mutations that are initially detected by DHPLC.
To our knowledge, there are only a small number of publications describing the use of DHPLC to detect c-KIT and PDGFRA mutations in GIST. In most of these studies, the focus was not related to the validation of the DHPLC assay, and therefore, these studies are usually restricted to specific c-KIT exons or a relatively small sample. Furthermore, only 1 of these studies used a well-defined single population cohort.
In this study, we aimed to validate the clinical diagnostic usefulness of DHPLC in detecting mutations of c-KIT and PDGFRA in 54 consecutive cases of GIST that were derived from a well-defined population.
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