Clinical significance of EGFR amplification and the aberrant EGFRvIII transcript in conventionally treated astrocytic gliomas

Abstract

The aim of this study was to evaluate the clinical value of assessing epidermal growth factor receptor (EGFR) amplification and the common 5' rearrangement of EGFR resulting in the EGFRvIII transcript in astrocytic gliomas. Data from 221 tumours were correlated with patient survival. The majority of previous studies evaluated amplification alone and provided contradictory results. Amplification was analysed by a densitometry of Southern blot analysis or quantitative polymerase chain reaction (PCR). EGFR transcripts were examined by reverse transcription PCR and subsequent sequencing. A ribonuclease (RNase) protection assay was carried out on a subgroup to confirm PCR results. Amplification of EGFR was found in 41% (65/160) of glioblastomas (GBs) and 10% (4/41) of anaplastic astrocytomas (AAs). The EGFRvIII rearrangement was identified in 54% (35/65) of GBs and 75% (3/4) of AAs with amplification, as well as in 8% (8/95) of GBs and 5% (2/37) of AAs without amplification (confirmed by RNase protection assay). There were no abnormalities of the EFGR or its transcript in grade II astrocytoma (AII). We found no significant association between EGFR amplification or rearrangement, and age or survival in the 160 GB patients. We noted a tendency towards decreased survival with any EGFR abnormality in the 41 patients with AAs. This was most marked in the five cases with the EGFRvIII transcript (p=0.069), but these were significantly older than those without (p=0.023). No abnormalities of EGFR were identified in AII patients. We conclude that neither EGFR amplification nor the presence of the EGFRvIII transcript predicts patient outcome in conventionally treated GBs. However, in AAs, although uncommon, EGFR aberrations appear to be associated with shorter survival.

Figure 1

A diagram showing the 5′ end of the wild-type EGFR transcript, coding for the extracellular domain, with all probes and primers used in the study. RPA=Ribonuclease Protection Assay; TM=sequence coding for the transmembrane domain. The arrowhead indicates the 3′ end of each primer.

Figure 2

A-D. A) The radiogram of blood (=B) and tumour DNA (=T) pairs on TaqI Southern blots probed with the pEP1 probe (encompasses 1000 bp upstream of EGFR exon 1, exon 1 itself and approximately 2000 bp of intron 1). Note the large signal difference between B and T DNA in cases GB23, GB45, GB47, GB242 and GB248 indicating EGFR amplification. Control probing of the same blots with other chromosome 7 and chromosome 2 probes showed loading of DNA to be approximately equal for each of the B and T pairs. B-C) PCR products using PC66 and PC1403 from some of the AA and GB cases. The case number is given above each lane together with EGFR amplification status (“+” for amplified; “−” for non-amplified). Note that while all tumours show a 923 bp product (indicating presence of the wild-type transcript) some also show a 122 bp product (indicative of the EGFRvIII transcript). A product of the EGFRvIII transcript is shown for GB6 (which had EGFR amplification) as well as in AA94, GB128 and GB13 (which did not fulfil our criteria for EGFR amplification). wt=wild-type; bp=base pair D) Discriminative RT-PCR using primers PC66 and PC1445. The RNA used was from the same cases shown in Figure 2C and demonstrates the specificity of the reaction with only GB13 showing a positive result (a 125 bp band) from this set of tumours.

Figure 3

Ribonuclease Protection Assay (RPA) demonstrating the presence of both the wild-type and the EGFRvIII transcript. A813 is a 4th passage of xenograft with an amplified and rearranged EGFR gene, which mainly express the EGFRvIII transcript. GB182 is a GB without amplification of the EGFR gene but that express the EGFRvIII transcript, as is demonstrated here. GB180 and AA108 are tumours with EGFR amplification that only express the wild-type transcript.