Malignant pleural effusions as an alternative to biopsies for the detection of EGFR TKI sensitizing mutations

Lung cancer is the cause of a fourth of all cancer-related deaths. About a third of all lung adenocarcinoma tumours harbour mutations on exons 18, 19, 20 and 21 of the epidermal growth factor receptor (EGFR) gene. Detection of these mutations must be both accurate and timely as patients with certain EGFR mutations have better clinical outcomes when treated with EGFR Tyrosine kinase inhibitors. In our study, we utilized malignant pleural effusions (MPEs) as liquid biopsies to detect EGFR mutations when tissue biopsies were unavailable. First, a direct sequencing approach was attempted leading to the detection of a common exon 19 deletion. We then tested the sensitivity of this method by sequencing the mutant DNA sample diluted with wildtype DNA. Signatures of this large deletion were detectable even in a 1 part mutant and 99 part wildtype mixture but with poor base-call confidence. MPEs consist of both wildtype inflammatory cells as well as mutant metastatic cells suggesting that this sequencing-based approach was going to limit our ability to detect single base-pair mutations. To mitigate this limitation of sequencing, an EGFR mutant-specific quantitative polymerase chain reaction (PCR) based assay was used on a further n=10 pleural effusion samples (1 non-malignant pleural effusion as an internal negative control). 5/9 (55.55%) samples harboured EGFR mutations with 2/9 (22.22%) being exon 19 deletions and 3/9 (33.33%) had the S768I exon 20 mutation. These data suggest MPEs may be utilized to detect mutations even before a biopsy is made available. The high frequency of S768I mutations seen in our study suggests that cancer cells harbouring these mutations may be superior in their ability to migrate, home or reside in pleural fluid.