Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is one of the most frequent human malignancies, constituting about 80% of all lung tumors. NSCLC can be divided into genetic subsets on the basis of the activating mutations that they harbor; each of these subsets may correspond to patient cohorts that are likely to benefit from treatment with specific inhibitors[1].

Activating mutations in the epidermal growth factor receptor (EGFR), affecting hotspots within exons that code for the tyrosine kinase domain, can be found in 10-40% of NSCLC patients, mostly in adenocarcinomas, with the higher frequency observed in Asian patients[1, 2]. About 50% of mutated patients harbor in-frame deletions in exon 19, (around codons 746 to 750) and 35-45% show the substitution of leucine 858 by an arginine in the exon 21. The remaining mutants are insertions in exon 20 (5%) and uncommon substitutions spanning exons from 18 to 21, such as L861Q[3, 4].

These specific mutations are related to a higher sensitivity to the tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib[4‐7], whereas the EGFR T790 M mutation in exon 20 is observed in 50% of cases with acquired resistance to erlotinib and gefitinib[8] and has also been detected in 38% of patients with de novo drug resistance[9].

Molecular biology techniques, such as SARMS or direct automatic sequencing, are currently used to detect EGFR mutations in formalin-fixed, paraffin-embedded tissues (FFPET). In our experience, in-frame deletions in exon 19 are detected by fragment analysis of fluorescently labeled PCR products, and L858R mutations in exon 21 by TaqMan assay. Mutations are then confirmed by direct sequencing[10, 11]. However, the routine use of these methods in clinical laboratories is still often limited by financial and technical constraints. Moreover, their sensitivity depends on the quality and the quantity of tumoral cells in FFPET. In a previous study, we developed a highly sensitive molecular method for detecting EGFR mutations in NSCLC samples containing as few as eight tumor cells[10].

The development of antibodies that specifically detect mutant EGFR protein by IHC would be an easy pre-screening test to complement the molecular assays currently used for the assessment of EGFR mutations in NSCLC. Yu et al[12] have developed mutation-specific rabbit monoclonal antibodies (mAb) against EGFR with the E746_A750 deletion in exon 19 or the L858R point mutation in exon 21 for IHC application (Cell Signaling Technology Inc., Danvers, MA, USA).

In the present study, these two rabbit mAbs were used to assess EGFR mutations in five NSCLC cell lines and in tumor biopsies from 78 stage IV NSCLC patients. The results were then compared with those obtained by other molecular analyses[10, 11].

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, 9, 11]. 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, 16‐18]. 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, 19‐22] 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 4). 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 4 summarizes the clinicopathological characteristics of patients and the findings of seven studies examining EGFR mutations by IHC, including the present study.

Although the most common EGFR mutations are the 15-bp ELREA deletion in exon 19 and the L858R substitution in exon 21[2, 3], other less frequent deletions have been identified[4, 6, 8, 23]. 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.

IHC with the mutation-specific rabbit mAbs against EGFR is a simple and standardized assay which could prove useful as a first, quick screening of NSCLC patients. However, although these antibodies seem to be quite reliable for the detection of patients carrying the most common EGFR mutations[12], they were not able to detect other EGFR gene mutations, such as 9-bp, 12-bp, 18-bp, 21-bp or 24-bp deletions or the L861Q substitution[14]. In consequence, if the antibodies are to be used in clinical practice, molecular biology techniques will be needed to further analyze the IHC-negative patients. However, the generation of a refined panel of antibodies able to detect both the frequent and the uncommon EGFR exon 19 deletions and exon 21 mutations as well as the resistance mutation T790 M in exon 20 could lead to the universal application of IHC for detecting EGFR mutations in NSCLC patients, as part of the routine IHC work-up of lung adenocarcinomas.