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

Abstract

Determination of epidermal growth factor receptor (EGFR) mutations has a pivotal impact on treatment of non-small-cell lung cancer (NSCLC). A standardized test has not yet been approved. So far, Sanger DNA sequencing has been widely used. Its rather low sensitivity has led to the development of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR PCR kit, Qiagen, UK; reference method). For immunohistochemistry, antibodies against exon19 deletions (clone 6B6), exon21 mutations (clone 43B2) from Cell Signaling Technology (Boston, USA) and EGFR variantIII (clone 218C9) from Dako (Copenhagen, DK) were applied. Protein expression was evaluated, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score 40. Specificity of exon19 antibody was 98.8% (95% confidence interval ¼ 95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval ¼ 94.4-99.4%). Sensitivity of exon19 antibody was 63.2% (95% confidence interval ¼ 38.4-83.7%) and of exon21 antibody was 80.0% (95% confidence interval ¼ 44.4-97.5%). Seven exon19 and four exon21 mutations were false negatives (immunohistochemistry negative, RT-PCR positive). Two exon19 and three exon21 mutations were false positive (immunohistochemistry positive, RT-PCR negative). One false positive exon21 mutation had staining score 300. The EGFR variantIII antibody showed no correlation to EGFR mutation status determined by RT-PCR or to EGFR immunohistochemistry. High specificity of the mutation-specific antibodies was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC. Refinement of sensitivity for the mutation-specific antibodies is warranted to improve molecular diagnosis using EGFR immunohistochemistry.

Evaluation of epidermal growth factor receptor mutations based on mutation specific immunohistochemistry in non-small cell lung cancer: A preliminary study

“…Other mutations such as TKI resistance exon 20 mutations cannot be detected by these two antibodies. Though suboptimal sensitivity for L858R mutation precludes use of these antibodies as ascreening tool 23 , these may potentially be used at the time of diagnosis due to their high specificity and positive predictive value (though based on low number of cases), and for the obvious advantages described earlier especially in Indian setting. Moreover, IHC is useful to overcome some of the obstacles that are unavoidable in molecular testing which include low cellularity in small biopsies and contamination of tumour by normal tissue.…”

“…IHC scoring : The IHC staining score was based on the staining intensity and percentage positivity (0-100%) of cells in the membrane and/or cytoplasm of tumour cells as previously described 23 24 .…”

Prognostic implications of immunohistochemistry markers for EGFR-TKI therapy in Chinese patients with advanced lung adenocarcinoma harboring EGFR mutations

“…These findings are consistent with those of previous reports using the same mutant-specific antibodies (clone 6B6 and clone 43B2). 9 , 11 , 18 – 20 Thus, the excellent specificity of IHC using the two mutation-specific antibodies is clearly demonstrated.…”

Molecular Diagnostics in Non-Small Cell Lung Carcinoma

“…However, the sensitivity of the exon 19 deletion antibody in particular is limited to the most common five amino acid deletion and is unreliable for detection of other deletions involving that motif. [60][61][62][63][64] In general, use of EGFR mutant-specific IHC as a sole basis for therapy selection is discouraged. 6 BRAF BRAF kinase domain (KD) mutations occur in 5% of lung adenocarcinomas, of which about half are V600E substitutions.…”