It is hard to predict the biologic behaviour of the GIST. Its prognosis may vary according to tumor localization, size, morphologic type, mitotic activity and the presence of metastasis[3].

c-KIT and PDGFRA are the members of tyrosine kinase receptor family[1]. KIT and PDGFRA gene mutations are the major genetic alterations seen in GIST[1]. In 90% of the tumors, one of these genetic mutations is present[4,5]. KIT and PDGFRA gene mutations are not only associated with specific cell morphology, histologic phenotype, metastasis and prognosis, but they are also highly crucial in determining the therapeutic targets. The detection of KIT and PDGFRA gene mutations in GISTs enabled the application of imatinib, sunitinib and other tyrosine kinase inhibitors in the treatment of the tumors.

Table 1: Cinical and morphological features of 29 GIST cases that were analysed for mutation

The amplification of KIT and PDGFRA target exons
Using designed primers for KIT gene exons 11, 9, 13, 17 and PDGFRA gene exons 18, 12, 14, all of the target exons from extracted DNA of the cases were amplified with the polymerase chain reaction (PCR). Designed primers for target gene exons and details of the PCR method is shown below (Table 2 and Table 3). In 6 cases out of 17, none of the exons were amplified in PCR reaction.

Table 2: The primers designed for target exon amplification of KIT and PDGFRA genes

Table 3: The details of PCR method applied for target exon amplification of KIT and PDGFRA genes

Mutation analysis of the amplicons
In 11 cases out of 17, KIT gene exons 11, 9, 13, 17 and PDGFRA gene exons 18, 12, 14 were amplified in PCR reaction. For mutation screening in these amplicons;

a) The heteroduplexes formed by amplicons and their wild type homologous were electrophoresed and their agarose gel patterns were examined after surveyor DNA restriction enzyme reaction (SURVEYOR Mutation Detection Kit for Standard Gel Electrophoresis. Transgenomics, USA).

b) Conformation diversity of single strand DNA of the cases and their wild type homologous were comparatively evaluated using their polyacrylamide gel patterns[7,8].

Of 11 cases searched for mutation, in 4 cases, mutation findings were detected (Figure 1). These mutations were confirmed by sequence analysis and mutation types were identified (Figure 1).

Fig 1. The description of the mutations detected in the series. (I): Comparison of polyacrylamide gel patterns showing conformation diversity of single strand DNA of the cases and their wild type homologous; (II): Agarose gel patterns of the heteroduplexes formed by amplicons and their wild type homologous after ‘surveyor' DNA restriction enzyme reaction
A: Adenine; T: Thymine; G: Guanine; C: Cytosine; WT: Wild Type; M: Mutation related fragment; bp: base pair

In spite of various modifications, in 6 cases out of 17, amplification was not successful in PCR reaction. Also, in all these six cases, phenol/chloroform/isoamyl alcohol method was used for DNA extraction. The reason for unsuccessful DNA extraction in this method may be insufficient elimination of PCR inhibitors, such as DNA polymerase enzyme inhibitors.

Mutations were detected in 4 of 11cases, in which all target exons were successfully amplified. In three cases out of 4, mutation was in KIT gene, whereas it was in PDGFRA gene in one case. In literature, the mutation rates vary between 30-95% for KIT gene, and 5-20% for PDGFRA gene[1,4,9-11]. KIT and PDGFRA encodes homologous of tyrosine kinase receptor proteins. Both of them are pericentromerically located on 4q12[4]. Tyrosine kinase receptor controls cell proliferation, differentiation and adaptation. Mutations of KIT and PDGFRA genes in GISTs may cause uncontrolled cell proliferation and inhibition of apoptosis by producing continuous signals without ligand of the receptor. In decreasing frequency, these mutations are seen in exons 11, 9, 13 and 17 for KIT gene, and in exons 18, 12 and 14 for PDGFRA gene[3,12,13]. In our study, all 3 cases having KIT gene mutation were seen in exon 11, and only one case with PDGFRA gene mutation was in exon 18. Mutations of KIT and PDGFRA genes are closely related to each other in terms of histological phenotype, prognosis as well as treatment choice (5). KIT exon 11 mutation is the most common one seen in GISTs and it appears as deletion, point mutation and duplication of 3' end. Exon 9 mutations are usually duplication of 6 nucleotide sequences. Exon 13 and 17 mutations are almost always point mutations and their frequency is less than 1-2%[3,5]. In this study, two of the 3 KIT mutations are deletion and the remaining one is single nucleotide alteration type mutation. More than 80% of PDGFRA gene mutations are found to be in exon 18. They are point mutations characterized by replacement of aspartic acid for valine amino acid. Exon 14 and 12 mutations are also commonly missense point mutations. PDGFRA mutation detected in this study is a point mutation characterized by replacement of thymine for adenine in codon 842.

KIT exon 11 mutation is reported to be more commonly seen in GISTs having large size, high mitotic activity and malignant biologic behavior (14-16). Duplicative mutations in exon 11 are found to be more common in gastric GISTs and these tumors have spindle cell phenotype[17,18]. Gastric GISTs having exon 13 mutation have worse prognosis[4,19-24].

PDGFRA mutation is closely related with gastric GISTs. This mutation has been detected in more than 95% of gastric GIST. In such cases, epithelioid or mixed phenotype is usually seen and they have better prognosis[4,25]. Many studies revealing the close relation of PDGFRA mutation with epithelioid morphology and gastric localization have been reported[22,24,26]. Most of the cases having mutant KIT proteins are sensitive to imatinib treatment. However GISTs having exon 17 mutation are resistant to treatment. GISTs with exon 9 mutation are less responsive to treatment than those with exon 11 mutation[3,27]. GISTs with PDGFRA exon 18 mutation are resistant to imatinib treatment[3,28]. Because of the limited number of the mutation detected cases in this study, the relationship between morphologic features, biologic behavior, treatment response and mutational details was not possible to evaluate.

In conclusion, most common genetic alterations seen in GISTs are on the KIT and PDGFRA genes and detection of the mutations in these genes is not only related to morphologic features, metastatic potential and prognosis but also important in determining the specific treatment protocol[29]. Therefore, mutation analysis of KIT exons 9, 11, 13,17 and PDGFRA exons 12, 14, 18 should be performed in GISTs after the histopathological diagnosis. For this purpose, a method also has been proposed in this study.

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