IntroductionGastrointestinal stromal tumours (GIST) are mesenchymal neoplasms that usually carry an activating mutation in KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes with predictive and prognostic significance. We investigated the extended mutational status of GIST in a patient population of north-western Greece in order to look at geopraphic/genotypic distinctive traits.Patient and methodsClinicopathological and molecular data of 38 patients diagnosed from 1996 to 2016 with GIST in the region of Epirus in Greece were retrospectively assessed. Formalin-fixed paraffin-embedded tumours were successfully analysed for mutations in 54 genes with oncogenic potential. Next generation sequencing was conducted by using the Ion AmpliSeqCancer Hotspot Panel V.2 for DNA analysis (Thermofisher Scientific).ResultsAmong 38 tumours, 24 (63.16%) and seven (18.42%) of the tumours harboured mutations in the KIT and PDGFRA genes, respectively, while seven (18.42%) tumours were negative for either KIT or PDGFRA mutation. No mutations were detected in five (13.16%) cases. Concomitant mutations of BRAF and fibroblast growth factor receptor 3 (FGFR3) genes were observed in two patients with KIT gene mutation. Two patients with KIT/PDGFRA wild-type GIST had mutations in either KRAS or phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) genes. There was no significant survival difference regarding the exonic site of mutation in either KIT or PDGFRA gene. The presence of a mutation in pathway effectors downstream of KIT or PDGFRA, such as BRAF, KRAS or PIK3CA, was associated with poor prognosis. Adverse prognosticators were also high mitotic index and the advanced disease status at diagnosis.ConclusionsWe report comparable incidence of KIT and PDGFRA mutation in patients with GIST from north-western Greece as compared with cohorts from other regions. Interestingly, we identified rare mutations on RAS, BRAF and PIK3CA genes in patients with poor prognosis.
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and are characterized by mutations in the proto-oncogene KIT (c-kit). To date, the detection of genomic alterations of the c-kit gene has been based mostly on direct sequencing. However, sequencing is an expensive and time-consuming approach. Since the technology of WAVE DNA Fragment Analysis System (Transgenomic, Inc., Worcester, MA) (dHPLC) is available in our laboratory, we decided to evaluate its use. Sixteen patients with small/large intestine, stomach tumors were included in the study. Immunohistochemical evaluation was performed on formalin-fixed, paraffin-embedded specimens with the polyclonal antibody CD117 for the KIT protein. After DNA extraction and isolation from paraffin-embedded sections, a nested PCR approach was applied to amplify sequences of exon 11 of the c-kit gene. dHPLC and the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Bedford, MA) were used respectively for screening and identification of genomic alterations. Immunohistochemical analysis revealed strong and diffuse KIT expression in each of the 16 paraffin-embedded sections examined. dHPLC analysis in two temperatures showed the presence of genomic alterations in 8 out of 16 (50%) samples examined. Subsequently, sequence analysis of exon 11 in those samples revealed c-kit alterations in only 8 out of 16 (50%) samples. These were five deletions, one of which was an in-frame deletion one-point mutation and one insertion. Furthermore, the sensitivity of both methods was compared by using different mixtures of a wild-type and a sample with a deletion in exon 11. dHPLC was shown to be able to detect genomic alterations in all four different sample mixtures, whereas with sequence analysis genomic alterations were detected only in the 1:2 and 1:4 sample mixtures. In conclusion, we showed that dHPLC is an efficient and accurate, as well as a more sensitive, method for screening of genomic alterations in exon 11 of the c-kit gene, compared to sequence analysis.
This is the first report to reveal a high prevalence of novel MEN1 gene mutations among Greek MEN1 patients with apparent absence of genotype-phenotype correlation. Because of the small number of patients examined, the high prevalence detected might be a chance phenomenon.
AIM: The aim of this study was to further delineate the extent and nature of mutations in the BRCA1 and BRCA2 genes, responsible for hereditary breast and ovarian cancer in Greek families. MATERAILS & METHODS: Genomic DNA was isolated from whole peripheral blood of patients referred to our center for mutation analysis of the BRCA1 and BRCA2 genes. Patients were included on the basis of affected family members, types of cancer present in the family and age at diagnosis of breast cancer in the proband. Families were subdivided into high, medium and low risk depending on the number of affected family members, types of cancer diagnosed in the family and age at diagnosis of affected family members. In total, 675 families have been analyzed by our group in the past 4 years. Mutation analysis in all cases included sequencing of the coding region and the splice sites of the two genes. In addition, MLPA analysis was carried in 585 of the patients. RESULTS: In total, a pathogenic mutation has been identified in 12% of the 675 patients analyzed. Of the 78 mutations identified in total, 13 (17%) were large genomic rearrangements. These were deletions of exons 8, 20, 23, 23-24 and the entire BRCA1 gene, in addition to a duplication of exons 3-8 of the BRCA1 gene. As far as BRCA2 is involved deletions of exons 3, 15 and the entire BRCA2 gene were detected. All deletions were confirmed by use of other MLPA probe sets and/or relative quantitation by Real Time PCR. Of the rearrangements identified, two, namely deletions of exon 20 and exons 23-24 of the BRCA1 gene were identified in more than one unrelated families. In addition, the recurrent mutations 5382insC and G1738R, which have been previously identified as founder mutations in the Greek population, were identified in multiple unrelated analyzed families. CONCLUSIONS: Our results indicate that different large genomic rearrangements account for an important proportion (17%) of the mutations in the BRCA1 and BRCA2 genes, in Greek families at risk of carrying a germline mutation as judged by family / personal history. The use of the available technologies for the identification of such mutational events is therefore necessary when carrying out complete analysis of the genes in high risk families of Greek background. Citation Format: Angela Apessos, Eirini Papadopoulou, Vassiliki Metaxa-Mariatou, Konstantinos Agiannitopoulos, Christos Markopoulos, Vasileios Venizelos, Grigorios Xepapadakis, Maria Vasilaki-Antonatou, Antonios Keramopoulos, Nikolaos Bredakis, Aristeidis Tsiftsoglou, Georgios Kesisis, Stylianos Kakolyris, Nikolaos Touroutoglou, Ioannis Natsiopoulos, Konstantinos Papazisis, Georgios Nasioulas. Different genomic rearrangements account for 17% of BRCA1/2 mutations in Greece [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-03-08.
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