Sensitive molecular testing methods can demonstrate NSCLC driver mutations in malignant pleural effusion despite non-malignant cytology

Daniel P. Steinfort, Sevastjan Kranz, Anthony Dowers, Leakhena Leas, Voula Dimitriadis, Kym Pham, Arthur Hsu, Steven Bozinovski, Louis B. Irving, Paula Loveland, Michael Christie


Malignant pleural effusion (MPE) may be diagnosed by cytologic evaluation of pleural fluid, though false negative results can occur. Pleural effusions may provide a source of tumour material for genotyping in lung cancer patients. Detection of MPE may be improved through use of highly sensitive molecular techniques. We identified five patients with non-small cell lung cancer (NSCLC) with initial pleural fluid samples that were non-malignant on cytology, but were subsequently clinically confirmed to have MPE. Tumour mutation status was confirmed via routine testing of diagnostic clinical specimens. Cytologically negative pleural fluid cell-block specimens were analysed by amplicon-based parallel sequencing (APS) for somatic mutations commonly detected in NSCLC, and selected cases by improved and complete enrichment CO-amplification at lower denaturation temperature PCR (ICECOLD PCR) for known mutations. Mutations were detected in three out of three (sensitivity 100%) cytologically non-malignant pleural fluids from patients with a known mutation: two patients with known Kirsten rat sarcoma (KRAS) mutation demonstrated the same KRAS mutation in their pleural fluids by APS, both at approximately 2% mutant allele frequency. In one patient with a known KRAS mutation, ICECOLD PCR detected the same KRAS variant at 0.7% frequency. No mutations were detected in patients with wild-type findings from reference samples (specificity 100%). Sensitive DNA sequencing methods can detect cancer-driver mutations in cytologically non-malignant pleural fluid specimens from NSCLC patients with MPE. Our findings demonstrate the feasibility of sensitive molecular diagnostic techniques for improvement of diagnostic assessment of pleural effusions in patients with lung cancer.