249P - The cell-free fraction of pleural effusions is an important source of DNA suitable for molecular testing

Date 28 September 2014
Event ESMO 2014
Session Poster Display session
Topics Pathology/Molecular Biology
Translational Research
Presenter Audrey Vallee
Citation Annals of Oncology (2014) 25 (suppl_4): iv58-iv84. 10.1093/annonc/mdu326
Authors A. Vallee1, C. Sagan2, M.G. Denis1
  • 1Department Of Biochemistry And Molecular Biology, Nantes University Hospital, 44093 - Nantes/FR
  • 2Department Of Pathology, Nantes University Hospital, Nantes/FR

Abstract

Aim

Detection of EGFR and ALK alterations is critical for predicting the response of Non-Small Cell Lung Cancer patients to targeted therapy. Analysis of additional biomarkers (KRAS, BRAF, ERBB2…) might also be mandatory in the future, requiring more biological material. In routine practice, molecular testing is performed on formaldehyde fixed paraffin embedded tissues or cell pellets. We investigated the potential of cell-free DNA from pleural effusions to perform molecular testing.

Methods

Pleural effusion samples were collected and centrifuged. The pellets were fixed in formaldehyde, and processed as cell blocks. Supernatants were collected and frozen at −20°C until use. DNA was extracted from 10-µm sections of cell blocks after paraffin removal using the ChargeSwitch® Forensic Kit and an iPrep™ Purification Instrument (Life Technologies). Cell-free DNA was extracted from the supernatants (0.4 ml) using the iPrep™ PureLink® Virus Kit. Molecular alterations were detected 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

We tested 44 pleural fluids collected from 22 women and 22 men. Significant amounts of DNA were extracted from 0.4 ml of the cell-free fractions obtained following centrifugation (mean 1.3mg; median 428ng; range : 83ng - 9.3mg). DNA could not be amplified in only 1 case, and EGFR, KRAS and BRAF tests were contributive for the remaining 43 samples: 3 EGFR alterations (2 p.L858R mutations and 1 exon 19 deletion), 9 KRAS mutation (p.G12C, n = 3; p.G12D, n = 2; p.G12A, n = 1; p.G12F, n = 1; p.G12S, n = 1; and p.G13C, n = 1) and 3 BRAF p.V600E mutation were detected. Most interestingly, the molecular status of the cell-free DNAs perfectly matched those obtained with the corresponding cell pellets.

Conclusions

Pleural effusions contain significant amounts of cell-free DNA that can be easily and rapidly extracted. This material is suitable for molecular testing in routine practice, thus saving material for morphological, immunohistochemical and FISH analysis.

Disclosure

All authors have declared no conflicts of interest.