Molecular analysis of GISTs: evaluation of sequencing and dHPLC.
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.
Metaxa-Mariatou
V
V
Molecular Biology Department Research Center HYGEIA "Antonis Papayiannis," Maroussi, Athens, Hellas, Greece.
Papadopoulos
S
S
Papadopoulou
E
E
Passa
O
O
Georgiadis
Th
T
Arapadoni-Dadioti
P
P
Leondara
V
V
Nasioulas
G
G
D000368:Aged; D000369:Aged, 80 and over; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005770:Gastrointestinal Neoplasms / Q000473:pathology*; D041981:Gastrointestinal Tract / Q000166:cytology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D017422:Sequence Analysis, DNA; D017154:Stromal Cells / Q000166:cytology*