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.

Molecular analysis of EGFR gene in different types of tumor material from NSCLC patients

“…Angulo et al [35] and Marchetti et al [36] analyzed FFPE material and reported mutation frequencies of 16.2% and 24.1% respectively, a detection rate relatively close to our FFPE set. Therefore we assume that our mutation detection approach is not the reason why such a low mutation frequency was detected in cytological smear samples.…”

“…As reported in literature, generally preferred tumor material for molecular EGFR analysis and detection of somatic mutations is FFPE tumor tissue [35,36]. However, in case of lung tumors, availability of such material may be limited due to tumor location and many technical limitations.…”

“…We may speculate that this patient had greater-than-usual benefit from biological treatment. Although some studies have reported cases of double mutants, they always involved combination one activating mutation and one resistance mutation [35,40]. Double mutants are usually only found in cell lines, especially substitutions in exon 21 (p.L858R) and in exon 20 (p.T790M).…”

High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

“…Such selection of patients with NSCLC promoted a better treatment strategy for both high and low EGFR mutations abundant tumors. [30][31][32][33] Further quantification of staining scores in mutation-positive NSCLC has been assessed, and high staining score has been associated with a significant longer progression-free survival in mutation-positive patients as compared with low staining score. 31 A study in which the randomization to EGFR tyrosine kinase inhibitors would be based on the method used for demonstrating an activating EGFR mutation might give clinical relevant information regarding the significance of the amount of EGFR-mutated cells.…”

Molecular Detection of EGFR Mutation in Lung Cancer Using Bioinformatics Database with PCR Technique

“…This is due to the large number of total EGFR mutations (over 1,300 EGFR mutation entries in COSMIC) and partly explains the ever-growing importance of EFGR mutations in lung cancer (11). The mutation which selected in this research was A763_Y764insFQEA in exon 20.…”