High sensitivity detection of epidermal growth factor receptor mutations in the pleural effusion of non‐small cell lung cancer patients

Abstract

Epidermal growth factor receptor (EGFR) mutations are a strong determinant of tumor response to gefitinib in non-small cell lung cancer (NSCLC). We attempted to elucidate the feasibility of EGFR mutation detection in cells of pleural effusion fluid. We obtained 24 samples of pleural effusion fluid from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were used for detection. EGFR mutation status was determined by a direct sequencing method (exons 18-21) and by the Scorpion Amplified Refractory Mutation System (ARMS) method. EGFR mutations were detected in eight cases. Three mutations were detected by both methods, and the other five mutations were detected by Scorpion ARMS alone. The mutations were detected by both methods in all four partial responders among the seven patients who received gefitinib therapy. Direct sequencing detected the mutations in only two of four cases with partial response. These results suggest that the DNA in pleural effusion fluid can be used to detect EGFR mutations. The Scorpion ARMS method appears to be more sensitive for detecting EGFR mutations than the direct sequencing method. (Cancer Sci 2006; 97: 642-648) L ung cancer is a major cause of cancer-related mortality worldwide and is expected to remain a major health problem for the foreseeable future.(1) Targeting the epidermal growth factor receptor (EGFR) is one appealing strategy for the treatment of non-small cell lung cancer (NSCLC), because EGFR has been found to be expressed, sometimes strongly, in NSCLC.(2) Mutations of EGFR tyrosine kinase in NSCLC and hyper-responsiveness to gefitinib, a selective EGFR tyrosine kinase inhibitor, have been reported. (3,4) These mutations consist of small in-frame deletions or substitutions clustered around the ATP-binding site in exons 18, 19 and 21 of EGFR, and increase the affinity of the enzyme for ATP and gefitinib. Some investigators have subsequently found that EGFR mutations are a strong determinant of tumor response to EGFR tyrosine kinase inhibitor.(5-7) Approximately 90% of the NSCLC-associated EGFR mutations in two reports consisted of two major EGFR mutations (E746_A750del in exon 19 and L858R in exon 21). (5,8) These investigators used surgical tissue to detect the EGFR mutations in their trials. As it is often difficult to obtain a tumor sample from patients with inoperable NSCLC, a method of detecting mutant EGFR, especially the two major mutations, in other specimens needs to be established.Malignant pleural effusion is a common complication of lung cancer and is present in approximately 15% of lung cancer patients (9) and in 10-50% of patients at the time of diagnosis.Approximately one-half of NSCLC patients with pleural effusion are initially positive cytologically, and most of the effusions are ultimately determined to be malignant. As sampling of pleural effusion fluid is usually easy, non-invasive and repeatable, we hypothesized that tumor cells in the pleural effusion fluid of NSCLC patients are a source of useful infor...

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma

“…Recently, several highly sensitive methods, such as mutantenriched assays, the PCR-invader method, cycleave-PCR assay, Scorpion-Amplified refractory mutation system assay, TaqMan PCR assay, denaturing high-performance liquid chromatography method, high resolution melting assay, highresolution chipCE assay, immunohistochemistry (IHC) assay, and the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method, have been reported for the detection of EGFR mutations (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Some of these techniques have quite high sensitivity, but they are still not ideal for routine clinical use in general hospitals or outpatient clinics due to various reasons such as long turn-around times and complexity.…”

Association of epidermal growth factor receptor (EGFR) gene mutations with EGFR amplification in advanced non‐small cell lung cancer

“…We previously showed that the Scorpion-amplification-refractory mutation system (ARMS) is a sensitive technique for the detection of EGFR mutations in tumor specimens such as pleural effusion fluid or tissue obtained by transbronchial needle aspiration. (28)(29)(30) In the present study, we evaluated EGFR mutation status in small tumor specimens from patients with advanced NSCLC both by direct sequencing and by Scorpion-ARMS and compared the sensitivity of these methods for the detection of EGFR mutations. Furthermore, we determined EGFR copy number by FISH analysis in paired tumor specimens and examined its relationship to EGFR mutation.…”

EGFR mutations predict a favorable outcome for malignant pleural effusion of lung adenocarcinoma with Tarceva therapy

“…Soh et al (38) and Gow et al (39) reported an EGFR mutation rate of 24.5% (13 out of 53) in MPEs of lung adenocarcinoma. Kimura et al (40) found a 13% (3 out of 23) EGFR mutation rate by direct sequencing of EGFR mutations in MPEs of lung adenocarcinoma, of which 9.1% (1 out of 11) were females and 10% (1 out of 10) were never smokers. Overall, the EGFR mutation rate of MPEs ranged from 9.1 to 68.4%.…”