Related Experiment Videos
Reverse transcriptase polymerase chain reaction as a reliable method to detect epidermal growth factor receptor exon
K Worm1, P Dabbagh, K Schwechheimer
1Universität-Gesamthochschule Essen, Institut f. Neuropathologie, Germany.
Abstract:
Epidermal growth factor receptor (EGFR) gene amplification has been reported to occur in diverse carcinoma types such as lung, ovarian, and breast carcinomas and in glioblastomas. A 801-bp in-frame deletion close to the aminoterminus of the receptor protein has been found to occur more or less frequently within at least three of these tumor entities. We studied EGFR gene alterations using the polymerase chain reaction and EGFR gene expression of 65 astrocytic tumors (51 glioblastomas World Health Organization [WHO] IV, five anaplastic astrocytomas WHO III, and nine astrocytomas WHO II). EGFR gene amplification, as determined by Southern blotting using a full-length cDNA probe, was observed in 22 of 51 glioblastomas (43%) but in none of the grade II astrocytomas. Two of five anaplastic astrocytomas at WHO III showed a considerable degree of EGFR amplification but, according to the neuroradiological data, these two tumors had to be considered as glioblastomas. The most frequently found genetic alteration was the 801-bp deletion near the receptor aminoterminus comprising a complete loss of exon 2 to exon 7 (del2-7). We showed that RT-PCR is superior to Southern blot analysis in detection of this type of deletion and can be assigned to 9 of 38 (24%) glioblastomas examined. Expression of a EGF receptor protein was enhanced in most of the tumors with gene amplification. However, 5 of 18 tumors that express a receptor protein in the absence of EGFR gene amplification also showed elevated levels of EGFR gene expression. In addition to the full-length receptor protein, a signal in the 140-kDa range was observed in 17 of 35 glioblastomas (49%). This fragment may correspond to the truncated de12-7 receptor protein or might be due to proteolysis of the full-length receptor protein.
Insights
Epidermal growth factor receptor (EGFR) gene amplification and a specific deletion (del2-7) are common in glioblastomas. These alterations affect EGFR protein expression and may indicate tumor progression.
Area of Science:
- Neuro-oncology
- Molecular Biology
- Cancer Genetics
Background:
- Epidermal growth factor receptor (EGFR) gene amplification is prevalent in various carcinomas, including glioblastomas.
- A specific 801-bp deletion (del2-7) near the EGFR aminoterminus is observed in several tumor types.
Purpose of the Study:
- To investigate EGFR gene alterations (amplification and del2-7) and their expression in astrocytic tumors.
- To compare RT-PCR and Southern blotting for detecting EGFR deletions.
Main Methods:
- Polymerase chain reaction (PCR) and Southern blotting were used to analyze EGFR gene amplification in 65 astrocytic tumors.
- RT-PCR was employed to detect the 801-bp deletion (del2-7) and assess EGFR gene expression.
Main Results:
- EGFR gene amplification occurred in 43% of glioblastomas (WHO IV) but not in grade II astrocytomas.
- The 801-bp deletion (del2-7) was detected in 24% of glioblastomas using RT-PCR, which proved superior to Southern blotting for this alteration.
- Enhanced EGFR protein expression was noted in tumors with gene amplification, and a 140-kDa fragment, possibly truncated EGFR, was observed in 49% of glioblastomas.
Conclusions:
- EGFR gene amplification and the del2-7 deletion are significant genetic alterations in glioblastomas.
- RT-PCR is a more sensitive method for detecting the del2-7 EGFR deletion.
- Altered EGFR expression and potential truncated forms are associated with glioblastoma development.