A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry

Abstract

The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.

Figure 1. EGFR mutational results for (A) direct sequencing, and (B) the Therascreen EGFR Mutation Test Kit.

The electropherogram depicts an EGFR insertion V769_D770 insASV in exon 20 (sequence obtained from reverse primer) and the amplification graph corresponds to the same tumour analyzed with the Therascreen EGFR Mutation Test Kit. The low signal intensity corresponding to the mutated sequence for the EGFR deletion and insertion can be confused with background noise, which makes interpretation more difficult, while the interpretation of the result of the Therascreen EGFR Mutation Test was unequivocal.

Figure 2. Limit of detection of the Therascreen EGFR Mutation Test Kit in comparison with direct sequencing.

Serial dilutions of DNA from EGFR mutant and wild-type FFPE tumours were used to compare the relative sensitivities of both methods. (A), the Therascreen EGFR Mutation Test Kit was able to detect an EGFR mutation when the DNA from the mutant tumour represented 1% of the total DNA in half of the analyzed tumours. Sample S08-3853, which harbours an exon 19 deletion, is shown as an example. (B), the Therascreen EGFR Mutation Test Kit was able to identify EGFR mutation in a 5% dilution of the total DNA in all the tumours analyzed. Sample S09-397, which harbours an exon 19 deletion, is shown as an example. (C), at least 30% mutant DNA was necessary in a background of wild-type DNA to detect EGFR mutations by direct sequencing in most of the analyzed tumours. Sample S08-3853 is shown as an example. Percentages indicate the proportion of DNA from a mutant tumour relative to DNA from a wild-type tumour. The ΔCt cut-off value to detect the presence of an EGFR exon 19 deletion is provided by the manufacturer. It is derived from cell lines and synthetic constructs.

Figure 3. Immunohistochemical detection of (A) EGFR exon 19 E746-A750 deletion, and (B) exon 21 L858R point mutation.

The images show a non-small cell lung carcinoma with an E746-A750 mutation stained with the specific anti- E746-A750del antibody (200x), and a non-small cell lung carcinoma carrying the L858R mutation stained with the specific anti-L858R antibody (200x).