EGFR is a member of the ErbB family of receptor tyrosine kinases, which also includes HER2/neu, HER3, and HER4[15
]. Activating mutations in the tyrosine kinase domain, involving mainly exons 19 and 21, play an important role in lung oncogenesis and tumor progression and are related to the clinical efficacy of EGFR TKIs such as gefitinib or erlotinib[5
]. Analysis of these mutations has become an important tool for targeted therapy in lung cancer3
, and in recent years many efforts have been made to find a more specific and sensitive methodology to detect them[10
]. Nevertheless, these techniques are relatively expensive for routine use in clinical laboratories, and depend on the quality of the samples. IHC is a standardized assay of simple methodology and high sensitivity and specificity, and the development of specific antibodies against EGFR mutation proteins might be useful for the diagnosis and treatment of lung cancer.
In 2009 Yu et al [12
] first generated two mAbs against E746-A750del and L858R point mutation from New Zealand rabbits and evaluated them by Western blotting, immunofluorescence and IHC. They tested these antibodies in a series of cell lines and in tumor tissues from patients with primary NSCLC, with known and unknown EGFR mutations, comparing the IHC results with DNA sequencing. They found that IHC with these mutation-specific antibodies for EGFR mutations showed a sensitivity of 92% and a specificity of 99%.
Recently, five studies[14
] examined the presence of EGFR mutations in NSCLC by IHC using the same two rabbit mAbs and reported sensitivity ranging from 36% to 100% and specificity ranging from 94% to 99% (Table ). Kato et al [20
] analyzed 70 gefitinib-treated NSCLC patients. Although a high sensitivity and specificity for these mAbs were described, IHC staining was not significantly correlated with overall survival. A very exhaustive analysis of the role of EGFR in NSCLC was recently reported by Ilie et al [19
]. They assessed EGFR status in a tissue microarray (TMA) of 61 lung adenocarcinomas by IHC, fluorescent in situ hybridization (FISH) and direct sequencing and compared their results with those of conventional methods performed on whole-tissue sections. The authors reported a specificity of 92% for the mAb against the E746-750 deletion. Kawahara et al [21
] reported a sensitivity of 83% for the L858R mutation antibody and 79% for the exon 19 deletion antibody. Brevet et al [14
] reported a sensitivity of 84.6% and a specificity of 98.9% for E746-A750 and a sensitivity of 95.2% and a specificity of 98.8% for L858R. Kitamura et al [22
] reported a sensitivity of 36% and a specificity of 97% for L858R and a sensitivity of 40% and a specificity of 99% for E746-A750. In the present study, we found a sensitivity of 100% and a specificity of 100% for the L858R exon 21 mutation antibody and a sensitivity of 63% and a specificity of 100% for the 15-bp deletion antibody. Table summarizes the clinicopathological characteristics of patients and the findings of seven studies examining EGFR mutations by IHC, including the present study.
Patient characteristics and EGFR mutation status in seven studies examining EGFR mutations by IHC. (Blank cells indicate that information is not available.)
Although the most common EGFR mutations are the 15-bp ELREA deletion in exon 19 and the L858R substitution in exon 21[2
], other less frequent deletions have been identified[4
]. Using DNA sequencing, Yu et al [12
] detected only two cases with uncommon deletions in exon 19; E746-T751 del stained positive and L747-A750 negative for IHC. In the present study, we had 12 samples with uncommon deletions in exon 19 (9-bp, 12-bp, 18-bp, 21-bp and 24-bp) and 2 samples with the uncommon exon 21 L816Q mutation. In these samples, IHC for both mutation-specific antibodies was not able to detect the alteration.