EGFRvIII is an alternatively spliced form of the EGF receptor (EGFR) that is the result of the deletion of exons 2 to 7 removing 801 bp from the extracellular domain of the receptor. It has been found to be a constitutively active and ligand independent version of the EGFR that is highly oncogenic. Although absent in nearly all normal human tissues, it is present in many solid tumors and thus is an ideal target for anti-cancer therapies. However, there have been conflicting reports on the prevalence of EGFRvIII in different tumors. We examined the difficulties associated with EGFRvIII detection and have devised a one-step, highly sensitive RT-PCR method for detecting EGFRvIII mRNA from total tumor RNA. Despite the bias of PCR towards smaller fragments, we observed that EGFRvIII is ∼1000 fold less efficiently amplified than wt EGFR , which is most likely due to the G-C content of the EGFRvIII segment. Moreover, total RNA differentially inhibits amplification of EGFRvIII as compared to EGFR leading to an overall decreased efficiency of 105 to 106 fold vs. wt EGFR. To establish a highly sensitive one step RT-PCR method for EGFRvIII detection, we systematically examined several regions of the EGFR cDNA sequence and empirically identified a primer set that robustly amplifies a 186 bp fragment corresponding to EGFRvIII. Nevertheless, in tumor samples we found that amplification of wt EGFR interfered with EGFRvIII detection. To overcome the competitive effect of the wt EGFR sequence, primers with dideoxy-C termini were designed from sequences in exons 2 and 7 to inhibit amplification of wt EGFR. These ddC primers further increased sensitivity of EGFRvIII detection. To overcome the high G-C content and RNA secondary structure present in EGFRvIII, we found the addition of DMSO to a final concentration of 10% enhanced amplification. It was also discovered that a slight degradation of the total RNA further increased amplification efficiency. Using this method, 56% (13/23) of primary breast carcinomas, 52% (25/48) of ovarian tumors, and 56% (9/16) of colon cancers were found positive for the presence of EGFRvIII which is comparable to published results obtained by immunohistochemistry. The elucidation of the difficulties associated with detection of EGFRvIII and the development of a highly sensitive, one-step method for its detection will help to resolve any discrepancies in the presence of EGFRvIII in various tumors.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]