Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Molecular Techniques Acta Cytologica 2012;56:439–447 DOI: 10.1159/000339638 Molecular Analysis of Centrifugation Supernatant Fluid from Pancreaticobiliary Duct Samples Can Improve Cancer Detection Sydney D. Finkelstein a Marluce Bibbo b David E. Loren b Ali A. Siddiqui b Charalambos Solomides b Thomas E. Kowalski b Eric Ellsworth a a RedPath Integrated Pathology, Inc., Pittsburgh, Pa., and b Thomas Jefferson University Hospital, Philadelphia, Pa., USA specimens yielded supernatant fluid enriched with DNA, probably from actively proliferating cells. Mutational profil- ing can enhance the cytologic evaluation and characteriza- tion of specimens suspected to contain pancreatic or bile duct cancer. Copyright © 2012 S. Karger AG, Basel Introduction Microscopic examination of pancreatic and bile duct brushing samples ranks among the most challenging ar- eas of cytology practice. Inflammatory states can pro- duce significant reactive cellular atypia which, further modified by cellular degeneration effects, overlaps fea- tures seen in neoplastic strictures. Similarly, the lack of an adequate number of intact representative cells in a brushing specimen may prevent accurate diagnosis. Both phenomena together make diagnosis difficult and limit diagnostic sensitivity for pancreatic and biliary cancer. While recent advances in endoscopic visualization (e.g. confocal microscopy) and sampling have improved sen- sitivity for cancer [1, 2], it continues to remain subopti- mal, ranging from 25–50% [3, 4]. Diagnostic modalities that improve both detection and exclusion of cancer requiring high positive and negative predictive values would aid in resolving this dilemma and lead to better management of patients. Key Words Bile duct stricture  Cytocentrifugation  Genotyping  KRAS  Loss of heterozygosity  Mutations  Pancreatic duct stricture  Supernatant Abstract Objective: We aimed to supplement microscopic examina- tion of biliary cytobrush specimens to improve sensitivity by mutational profiling of: (1) selected cells microdissected from cytology slides and (2) corresponding cell-free DNA in residual supernatant fluid. Study Design: From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from microdissected cells and 1–2 ml of cytocen- trifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and loss of heterozygosity (LOH) analysis of microsatellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. Results: Mutations were found in 25/28 patients with malignancy, and no muta- tions were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer. Mutational profiling of su- pernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malig- nancy over cytology alone. Conclusion: Brush cytology Received: February 8, 2012 Accepted after revision: May 21, 2012 Published online: July 25, 2012 Correspondence to: Dr. Sydney Finkelstein RedPath Integrated Pathology, Inc. 2515 Liberty Avenue Pittsburgh, PA 15212 (USA) Tel. +1 412 224 6100, E-Mail sdf  @  redpathip.com © 2012 S. Karger AG, Basel 0001–5547/12/0564–0439$38.00/0 Accessible online at: www.karger.com/acy