Efficient detection of EGFR, KRAS, and BRAF mutations in cell-free DNA from pleural effusions

e19151 Background: Detection of EGFR gene alterations is critical for predicting the response to tyrosine kinase inhibitors in patients with non-small-cell lung cancer. Analysis of other biomarkers might also be mandatory in the future, requiring more biological material. In routine practice, molecular testing is usually performed on tissues such as bronchial or transthoracic biopsies and surgical resections. We investigated the feasibility of using both cytology cell blocks and cell-free DNA from pleural effusions. Methods: Pleural effusion samples were centrifuged to form a pellet, fixed in formaldehyde, and processed as a cell block. DNA was extracted from 10-µm sections after paraffin removal using the Forensic kit and an iPrep system (Invitrogen). Cell-free DNA was extracted from the centrifugation supernatants using the QIAamp Circulating Nucleic Acid kit (Qiagen). Detection of molecular alterations were performed by allele-specific PCR (EGFR p.L858R mutation, BRAF p.V600E mutation), fragment size analysis (EGFR exon 19 deletions), and Sanger sequencing (KRAS mutations, codons 12 and 13). Results: Between January 2010 and December 2012, we tested 65 cell pellets (32 women and 33 men) and collected 34 paired supernatants. With the cell pellets, all the EGFR status were successfully determined (13 mutations; 20%), 9 mutations of KRAS on the 42 cases sequenced (17.6%), and 2 BRAF mutations on the 50 samples tested (4%) were found. We next investigated the potential use of the cell-free DNA from supernatants. Quantification by real-time PCR revealed significant amounts of DNA (mean = 306 ng extracted from 2 ml). EGFR, KRAS and BRAF tests were contributive for all these samples: 2 EGFR p.L858R mutations, 6 KRAS mutation (p.G12C, n=2; p.G12D, n=2; p.G13C, n=1 and p.G12F, n=1) and 1 BRAF p.V600E mutation were found. Most interestingly, the molecular status of the cell-free DNAs perfectly matched those obtained with the corresponding cell pellets. Conclusions: Pleural effusions provide high quality material for molecular testing and are suitable samples for routine practice. Cell-free DNA can also be successfully tested thus saving material for morphological / immunohistochemical / FISH analysis.