Detection of EGFR Mutations in Archived NSCLC Cytologic Specimens
Detection of EGFR Mutations in Archived NSCLC Cytologic Specimens
Mutations of the epidermal growth factor receptor (EGFR), particularly deletional mutations (DEL) in exon 19 and L858R in exon 21, are reportedly correlated with clinical outcome in patients with non-small cell lung cancer (NSCLC) receiving the EGFR tyrosine kinase inhibitors gefitinib and erlotinib, suggesting that detection of EGFR mutations would have an important role in clinical decision making. We established and validated an easy, inexpensive, and rapid method for detecting DEL and L858R from cytologic material by high-resolution melting analysis (HRMA). Dilution for sensitivity studies revealed that DEL and L858R were detectable in the presence of at least 10% and 0.1% EGFR-mutant cells, respectively. We analyzed 37 archived cytological slides of specimens from 29 patients with advanced NSCLC and compared the results with direct sequencing data obtained previously. Of 37 samples, 34 (92%) yielded consistent results with direct sequencing, 2 were false negative, and 1 was indeterminate. The sensitivity of this analysis was 90% (19/21) and specificity, 100% (15/15). These results suggest that HRMA of archived cytologic specimens of advanced NSCLC is useful for detecting EGFR mutations in clinical practice.
Increased expression of epidermal growth factor receptor (EGFR) has been reported in carcinomas of various organs, including of the lung, and has been shown to have a crucial role in tumor progression. Gefitinib (Iressa, AstraZeneca, Osaka, Japan) is an orally active, selective EGFR tyrosine kinase inhibitor that binds to the adenosine triphosphate binding pocket of the kinase domain and blocks downstream signaling pathways. Clinical phase 2 studies have demonstrated gefitinib antitumor activity in patients with advanced non-small cell lung cancer (NSCLC). Although some of these studies have shown that the rate of response to gefitinib is higher in women, patients with adenocarcinoma, patients who have never smoked, and Japanese and East Asians, no predictive molecular marker had been discovered until April 2004, when somatic mutations in the kinase domain of EGFR were suggested to be correlated with gefitinib sensitivity. Thereafter, several studies revealed a strong association between EGFR mutations and clinical outcome in parameters such as response rate, time to progression, and overall survival in consecutive NSCLC patients treated with gefitinib.
Many types of EGFR mutation have been identified. They are concentrated in exons 18 to 21 of EGFR, close to the region encoding the adenosine triphosphate binding pocket, and about 90% of patients with EGFR mutations have mutations in 2 hotspots: in-frame deletions including amino acids at codons 747 to 749 (DEL) in exon 19 and a missense mutation at codon 858 (L858R) in exon 21.
The mutational status of EGFR, especially DEL and L858R, is a strong predictor of gefitinib sensitivity, and detection of such mutations would provide patients and physicians with important information for optimal choice of therapy. Therefore, analysis of a sufficient number of tumor samples in good condition and direct sequencing after laser capture microdissection (LCM) is considered the "gold standard" for detecting EGFR mutations. However, this approach is not necessarily practical for clinical use for a number of reasons. First, tumor samples with a large volume and in good condition are difficult to obtain in most cases of advanced NSCLC. Second, LCM and direct sequencing require special instruments and are time-consuming and costly. Therefore, it is necessary to establish practical and precise methods for detecting EGFR mutations from easily obtainable diagnostic samples, which usually contain a small number of tumor cells and a large number of normal cells.
The real-time reverse transcription-polymerase chain reaction (PCR) assay has been reported for detection of EGFR mutations. In this method, many samples can be genotyped within a few hours without the need for post-PCR sample manipulation, although expensive fluorescence-labeled probes and restriction enzymes are needed. A new inexpensive dye, SYBR Green I, has been developed, but this limits the melting resolution because of dye redistribution during melting.
Recently, studies have validated the usefulness of high-resolution melting analysis (HRMA) using LCGreen I dye for mutational analysis, and another study has validated analysis using cytologic samples for c-kit. The advantages of this approach are that labeling of either primer with dye is not needed and PCR amplification and melting analysis can be performed in the same capillary tube, minimizing sample handling and reducing the possibility of error and sample contamination. HRMA is easy, rapid, and inexpensive to perform and has considerable potential for mutation detection in clinical practice.
We report a new method for detecting DEL and L858R from archival Papanicolaou-stained cytologic slides by HRMA. We validated the method by comparing the results with direct sequencing data from specimens surgically resected from the same patients. We also performed a titration assay to evaluate the lower limit of the proportion of tumor cells for detection of EGFR mutations by using a mixture of wild-type (WT) and EGFR-mutant lung cancer cell lines.
Abstract and Introduction
Abstract
Mutations of the epidermal growth factor receptor (EGFR), particularly deletional mutations (DEL) in exon 19 and L858R in exon 21, are reportedly correlated with clinical outcome in patients with non-small cell lung cancer (NSCLC) receiving the EGFR tyrosine kinase inhibitors gefitinib and erlotinib, suggesting that detection of EGFR mutations would have an important role in clinical decision making. We established and validated an easy, inexpensive, and rapid method for detecting DEL and L858R from cytologic material by high-resolution melting analysis (HRMA). Dilution for sensitivity studies revealed that DEL and L858R were detectable in the presence of at least 10% and 0.1% EGFR-mutant cells, respectively. We analyzed 37 archived cytological slides of specimens from 29 patients with advanced NSCLC and compared the results with direct sequencing data obtained previously. Of 37 samples, 34 (92%) yielded consistent results with direct sequencing, 2 were false negative, and 1 was indeterminate. The sensitivity of this analysis was 90% (19/21) and specificity, 100% (15/15). These results suggest that HRMA of archived cytologic specimens of advanced NSCLC is useful for detecting EGFR mutations in clinical practice.
Introduction
Increased expression of epidermal growth factor receptor (EGFR) has been reported in carcinomas of various organs, including of the lung, and has been shown to have a crucial role in tumor progression. Gefitinib (Iressa, AstraZeneca, Osaka, Japan) is an orally active, selective EGFR tyrosine kinase inhibitor that binds to the adenosine triphosphate binding pocket of the kinase domain and blocks downstream signaling pathways. Clinical phase 2 studies have demonstrated gefitinib antitumor activity in patients with advanced non-small cell lung cancer (NSCLC). Although some of these studies have shown that the rate of response to gefitinib is higher in women, patients with adenocarcinoma, patients who have never smoked, and Japanese and East Asians, no predictive molecular marker had been discovered until April 2004, when somatic mutations in the kinase domain of EGFR were suggested to be correlated with gefitinib sensitivity. Thereafter, several studies revealed a strong association between EGFR mutations and clinical outcome in parameters such as response rate, time to progression, and overall survival in consecutive NSCLC patients treated with gefitinib.
Many types of EGFR mutation have been identified. They are concentrated in exons 18 to 21 of EGFR, close to the region encoding the adenosine triphosphate binding pocket, and about 90% of patients with EGFR mutations have mutations in 2 hotspots: in-frame deletions including amino acids at codons 747 to 749 (DEL) in exon 19 and a missense mutation at codon 858 (L858R) in exon 21.
The mutational status of EGFR, especially DEL and L858R, is a strong predictor of gefitinib sensitivity, and detection of such mutations would provide patients and physicians with important information for optimal choice of therapy. Therefore, analysis of a sufficient number of tumor samples in good condition and direct sequencing after laser capture microdissection (LCM) is considered the "gold standard" for detecting EGFR mutations. However, this approach is not necessarily practical for clinical use for a number of reasons. First, tumor samples with a large volume and in good condition are difficult to obtain in most cases of advanced NSCLC. Second, LCM and direct sequencing require special instruments and are time-consuming and costly. Therefore, it is necessary to establish practical and precise methods for detecting EGFR mutations from easily obtainable diagnostic samples, which usually contain a small number of tumor cells and a large number of normal cells.
The real-time reverse transcription-polymerase chain reaction (PCR) assay has been reported for detection of EGFR mutations. In this method, many samples can be genotyped within a few hours without the need for post-PCR sample manipulation, although expensive fluorescence-labeled probes and restriction enzymes are needed. A new inexpensive dye, SYBR Green I, has been developed, but this limits the melting resolution because of dye redistribution during melting.
Recently, studies have validated the usefulness of high-resolution melting analysis (HRMA) using LCGreen I dye for mutational analysis, and another study has validated analysis using cytologic samples for c-kit. The advantages of this approach are that labeling of either primer with dye is not needed and PCR amplification and melting analysis can be performed in the same capillary tube, minimizing sample handling and reducing the possibility of error and sample contamination. HRMA is easy, rapid, and inexpensive to perform and has considerable potential for mutation detection in clinical practice.
We report a new method for detecting DEL and L858R from archival Papanicolaou-stained cytologic slides by HRMA. We validated the method by comparing the results with direct sequencing data from specimens surgically resected from the same patients. We also performed a titration assay to evaluate the lower limit of the proportion of tumor cells for detection of EGFR mutations by using a mixture of wild-type (WT) and EGFR-mutant lung cancer cell lines.
