Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: Rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry

Abstract

Background

Recently, we have reported that EGFR mutation-specific antibodies performed well in immunohistochemical analysis, with good sensitivity. We investigated whether this method could detect non-small-cell lung cancer (NSCLC) carrying EGFR mutations in malignant effusions and cerebrospinal fluid (CSF), comparable to the peptide nucleic acid–locked nucleic acid (PNA–LNA) PCR clamp assay. Furthermore, we compared activating EGFR mutations between primary and recurrent NSCLC.

Patients and methods

Twenty-four patients with NSCLC effusions and CSF were examined by immunocytochemistry using antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21. The PNA–LNA PCR clamp assay was used to detect the E746-A750 deletion at exon 19, L858R mutation at exon 21, and T790M mutation at exon 20.

Results

We were able to identify EGFR mutations in NSCLC effusion and CSF with a sensitivity of 100% (5/5) using the anti-delE746-A750 antibody and 100% (8/8) using the anti-L858R antibody. Furthermore, in samples without these EGFR mutations, immunocytochemistry with the two specific antibodies identified 91% (10/11) as negative for both the deletion and the point mutations in EGFR. Activating EGFR mutations decreased in recurrent NSCLC compared with primary NSCLC, and the T790M mutation was detected in recurrent NSCLC of patients receiving gefitinib treatment.

Conclusions

Identification of EGFR mutations is important for patients with primary and recurrent NSCLC. Rapid and sensitive immunocytochemistry using mutation-specific antibodies to detect EGFR mutations will be useful for diagnosing responsiveness to EGFR-targeted drugs.

Introduction

Lung cancer is the leading cause of cancer deaths worldwide. Non-small-cell lung cancer (NSCLC) is the major type of lung cancer and is classified into three histological types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [1], [2]. The majority of patients with NSCLC are in an advanced stage at the time of clinical and/or histological diagnosis, and less than 30% of patients undergo surgical resection [3]. Epidermal growth factor receptor (EGFR) is one of the members of the Erb-B family of receptors and is composed of an intracellular binding domain, a transmembrane segment and an intracellular tyrosine kinase domain. Since the introduction of the EGFR tyrosine kinase inhibitor gefitinib, and its approval for clinical use in the treatment of advanced NSCLC [4], subsequent studies have shown a significant association between the presence of EGFR-activating mutations in lung tumors and their sensitivity to both gefitinib and another EGFR tyrosine kinase inhibitor, erlotinib. Most of these mutations occur in exon 19, such as delE746-A750, and the L858R point mutation in exon 21, in the tyrosine kinase domain [5], [6], [7]. In contrast, several studies have reported the appearance of a threonine-to-methionine substitution at amino acid position 790 (T790M) in EGFR in NSCLC that acquires resistance to gefitinib after treatment, whereas almost no T790M mutations have been found in NSCLC before gefitinib treatment [8].

Cancer with effusions in the serosal (peritoneal, pleural, and pericardial) cavity is usually considered an incurable systemic disease with a high risk of distant metastasis. Malignant pleural effusions can be observed in patients with various types of neoplasm, but most frequently in lung adenocarcinoma [9]. Malignant pleural effusion is often present in patients with advanced disease, such as stage IV NSCLC, and these patients cannot undergo surgical resection of the primary NSCLC [10]. Therefore, approaches that examine molecular biological markers from non-surgical tissue biopsy samples or cytological diagnostic samples, such as bronchial brushing or effusions in NSCLC patients may identify clinically relevant signatures that will be helpful [11]. Several authors have demonstrated mutation-specific antibodies recognizing the delE746-A750 and L858R mutations, which can be used to identify the EGFR status of tumor samples and provide a simple immunohistochemical method for diagnosing EGFR mutations in human tissue [12], [13], [14], [15]. We have previously also demonstrated the use of EGFR mutation-specific antibodies in immunohistochemistry, and their application to the diagnostic screening of NSCLC patients and their responsiveness to EGFR-targeted drugs [16]. Although mutations in p53, K-ras and EGFR have been detected in pleural effusion samples [17], [18], immunostaining analysis of EGFR mutations has not yet been performed using cytological samples.

In this study, we investigated the presence of EGFR exon 19 delE746-A750 and the exon 21 L858R point mutation in malignant effusion and cerebrospinal fluid (CSF) using EGFR mutation-specific antibodies, and examined the correlation between the EGFR mutation status by the peptide nucleic acid–locked nucleic acid (PNA–LNA) PCR clamp assay and the expression of EGFR mutation-specific antibodies. Furthermore, we compared the activating EGFR mutation status, including T790M mutation, between primary and recurrent NSCLC.