The Value of Mutational Profiling of the Cytocentrifugation Supernatant Fluid from Fine Needle Aspiration of Pancreatic Solid Mass Lesions
Georgios Deftereos, Sydney D Finkelstein, Uma Krishnamurti, Yulin Liu, Jan F Silverman, Candy Binkert, Beth Ujevich, Alok Mohanty. Allegheny General Hospital, Pittsburgh, PA; RedPath Integrated Pathology, Inc., Pittsburgh, PA; The Western Pennsylvania Hospital, Pittsburgh, PA
Background: Fine needle aspiration (FNA) of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of slide based atypical cells has been reported as an aid for a more definitive diagnosis. The aim of this study is to evaluate how the molecular analysis of the cell-free DNA can help reducing sampling variability.
Design: FNA smears from 19 pancreatic solid masses were made. The remaining aspirate was diluted for preparation of cytocentrifuged slides or cell blocks. The supernatant fluid underwent DNA extraction and mutational analysis. DNA was extracted from 2 ml of the cytocentrifugation supernatant fluid and the DNA quantity measured by optical density. qPCR was used to determine DNA amplifiability. Aliquots of the extracted DNA underwent 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). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were similarly analyzed and compared.
Results: 5/19 cases cytologically confirmed as malignant showed detectable mutational change both in the microdissected slide based cytology cells and the cytocentrifugation supernatant fluid. While most mutations detected were present in both microdissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity of detection. Mutations present in both types of specimens were concordant affecting the same parental allele or KRAS point mutation genotype. The proportion of DNA mutated for individual markers (clonality) was higher in the supernatant fluid than in microdissected cells.
Conclusions: 1. The cytocentrifugation supernatant fluid contains adequate levels of amplifiable DNA suitable for mutation detection and characterization. 2. The finding of additional detectable mutations at higher clonality indicates that the supernatant fluid may be enriched with tumor DNA. 3. The molecular analysis of the supernatant fluid could serve as an adjunct method to reduce sampling variability, especially in cases with a high clinical suspicion for malignancy and limited number of atypical cells in the smears.
Tuesday, March 20, 2012 8:15 AM
Platform Session: Section F, Tuesday Morning