RNA is favourable for analysingEGFRmutations in malignant pleural effusion of lung cancer

Abstract

Malignant pleural effusion (MPE) is a useful specimen allowing for the evaluation of EGFR status in nonsmall cell lung cancer (NSCLC). However, direct sequencing of genomic DNA from MPE samples was found not to be sensitive for EGFR mutation detection.To test whether EGFR analysis from RNA is less prone to interference from nontumour cells that have no or lower EGFR expression, we compared three methods (sequencing from cell-derived RNA versus sequencing and mass-spectrometric analysis from genomic DNA), in parallel, for EGFR mutation detection from MPE samples in 150 lung adenocarcinoma patients receiving firstline tyrosine kinase inhibitors (TKIs).Among these MPE samples, EGFR mutations were much more frequently identified by sequencing using RNA than by sequencing and mass-spectrometric analysis from genomic DNA (for all mutations, 67.3 versus 44.7 and 46.7%; for L858R or exon 19 deletions, 61.3 versus 41.3 and 46.7%, respectively). The better mutation detection yield of sequencing from RNA was coupled with the superior prediction of clinical efficacy of first-line TKIs. In patients with acquired resistance, EGFR sequencing from RNA provided satisfactory detection of T790M (54.2%).These results demonstrated that EGFR sequencing using RNA as template greatly improves sensitivity for EGFR mutation detection from samples of MPE, highlighting RNA as the favourable source for analysing EGFR mutations from heterogeneous MPE specimens in NSCLC.

Survival of lung adenocarcinoma patients with malignant pleural effusion

“…The processing of samples (i.e. from sampling to freezing) took ,2 h as previously reported [4]. RNA was extracted from whole-cell lysate using Tri-reagent (Molecular Research Centre, Inc., Cincinnati, OH, USA) and Qiamp RNA Mini Kit (Qiagen) according to the manufacturer's protocol.…”

“…Cytology-proven MPEs caused by lung adenocarcinoma were all included and analysed for EGFR mutations. In our previous studies on EGFR mutations, materials from 225 patients were examined [3,4].…”

“…Testing cancer cells for epidermal growth factor receptor (EGFR) gene (EGFR) mutation status may aid in predicting the response of EGFR-tyrosine kinase inhibitor (TKI) therapy [3]. Our previous reports have demonstrated that direct sequencing using cell-derived RNA from cell pellets of centrifuged MPEs was a sensitive detection method for EGFR mutation, without using the complicated procedures necessary to isolate cancer cells [3,4]. This is because inflammatory and mesothelial cells within MPEs have considerably lower EGFR expression in comparison with the overexpression of EGFR in nonsmall cell lung cancer (NSCLC) cells [4][5][6].…”

“…This is because inflammatory and mesothelial cells within MPEs have considerably lower EGFR expression in comparison with the overexpression of EGFR in nonsmall cell lung cancer (NSCLC) cells [4][5][6]. The differential expression enriches mutant EGFR from tumour cells and minimises the dilution of wild-type EGFR content from nontumour cells [4].…”

“…Our previous reports have demonstrated that direct sequencing using cell-derived RNA from cell pellets of centrifuged MPEs was a sensitive detection method for EGFR mutation, without using the complicated procedures necessary to isolate cancer cells [3,4]. This is because inflammatory and mesothelial cells within MPEs have considerably lower EGFR expression in comparison with the overexpression of EGFR in nonsmall cell lung cancer (NSCLC) cells [4][5][6]. The differential expression enriches mutant EGFR from tumour cells and minimises the dilution of wild-type EGFR content from nontumour cells [4].…”