Mutational Analysis of Cytocentrifugation Supernatant Fluid of Pleural Fluid Provides an Independent Means To Differentiate Benign from Neoplastic Disease
Shweta Patel, Allan R Smith, Yulin Liu, Uma Krishnamurti, Shahid J Bokhari, Candy Binkert, Beth Ujevich, Sidney D Finklestein, Alok Mahonty, Jan F Silverman. Allegheny General Hospital, Pittsburgh, PA; RedPath Integrated Pathology, Pittsburgh, PA
Background: The definitive diagnosis of pleural fluid cytologic specimens can prove difficult, especially when attempting to exclude cancer. To increase both sensitivity for cancer detection and negative predictive value to exclude malignancy, we analyzed cell-free DNA isolated from the cytocentrifugation supernatant of pleural fluid as an aid to discriminate between reactive and malignant pleural fluid specimens.
Design: Non-neoplastic (n=13) and malignant (n=4) pleural fluids underwent standard cytology fixation and preparation. Cytology diagnosis served as the gold standard for this initial feasibility assessment of mutational analysis. DNA was extracted from 2 ml of the cytocentrifugation supernatant fluid followed by mutational analysis using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterogeneity of 16 markers at 19, 3p, 5q, 9p, 10q, 17p, 17q, 21q, 22q). Similar mutational analysis was performed on DNA extracted from microdissected corresponding stained cytology cells.
Results: Mutational profiling of the cell free cytocentrifugation supernatant fluid contained abundant (over 5.3 ng/ul), amplifiable (qPCR Ct below 30 cycles) DNA even in relatively hypocellular pleural fluid specimens. Using the criterion of the presence of at least one detectable mutation to indicate the presence of neoplasia, molecular analysis accurately discriminated between benign versus malignant pleural fluid cytology specimens. The mutational profile of the supernatant fluid DNA matched closely that derived from microdissected stained cytology cells. Furthermore, in three of four cancer samples (75%), the supernatant contained additional mutations not detectable in the microdissected cells.
Conclusions: The cytocentrifugation supernatant fluid is an excellent source of representative DNA to test for the presence or absence of cancer-associated molecular changes in pleural fluid specimens. Through the use of a broad panel of markers, cancer versus non-cancer status could be determined by molecular analysis using supernatant fluid that typically would be discarded. This non-competing molecular anlysis on supernatant fluid could potentially improve the sensitivity of cancer detection in pleural fluid and other body cavity fluid specimens and help determine the pathobiologic etiology of effusion specimens.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 265, Wednesday Morning