Molecular Diagnostic Analysis of Supernatant Fluid from Fine Needle Aspirate, Bile Duct Brushing and Effusion Cytology Specimens
William W Bivin, Jan F Silverman, Sydney D Finkelstein, Yulin Liu, Alok Mohanty, Beth Ujevich, Candy Binkert, Uma Krishnamurti. Allegheny General Hospital, Pittsburgh, PA; RedPath Integrated Pathology, Inc., Pittsburgh, PA
Background: Fine needle aspiration (FNA), bile duct brushing and body cavity fluid specimens can be limited by low cellularity and sampling variability. Molecular analysis can complement the cytologic findings in challenging cases. In this study we evaluated a novel technique performing DNA mutational profiling on the cytocentrifugation supernatant fluid collected during cytology preparation but is usually discarded.
Design: A variety of cytology specimens (45 FNAs of solid organs, 10 pancreatic/biliary duct brushings and 23 body cavity fluids) underwent standard cytology processing which included centrifugation to separate cells of interest. DNA was extracted from 2 ml of the centrifugation supernatant fluid. DNA quantity was measured by optical density and quality by qPCR. Mutational analysis followed 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). Microdissection of corresponding stained cytology smears and/or cytocentrifugation cellular slides were similarly analyzed and compared.
Results: In each organ system and sampling format, the cell-free supernatant fluid contained adequate levels of amplifiable DNA for analysis. Known non-neoplastic specimens (n=58) did not show false positive detection of mutations. In contrast, all positive malignant specimens had detectable mutational change. There was a high degree of mutational profile concordance for mutations detected in the microdissected stained cytology. More importantly, mutation detection was consistently superior in the supernatant fluid (mean 6.7 mutations) compared to that in the microdissected cells (mean 4.2 mutations, p<.001), both in number of total mutations as well as extent of mutation clonality.
Conclusions: 1.) Centrifugation supernatant fluid provides for an abundant and robust source of DNA for molecular analysis. 2.) The greater extent and clonality of detectable mutational change in the supernatant fluid supports it being enriched with neoplastic DNA when cancer is present. 3.) Mutations detected in the supernatant fluid are concordant with that detected in neoplastic slide based cells. 4.) All these findings make the supernatant fluid a reliable source for mutational analysis and helps overcome the limitations of low cellularity and sampling variability in FNA biopsy, bile duct brushing and effusion cytology specimens.
Monday, March 19, 2012 1:00 PM
Poster Session II # 289, Monday Afternoon