Bile Duct Brushing Cytology Molecular Evaluation: Comparative Analysis of the Slide Based Cytology and Cell-Free Supernatant Fluid for Mutational Change
Claudia Velosa, Sydney D Finkelstein, Uma Krishnamurti, Yulin Liu, Jan F Silverman, Candy Binkert, Beth Ujevich, Alok Mohanty. Allegheny General Hospital, Pittsburgh, PA; West Penn Hospital, Pittsburgh, PA; RedPath Integrated Pathology, Pittsburgh, PA
Background: Bile duct brushing cytology plays a prominent role in confirming the presence of extrahepatic biliary tract malignancy. However, its value is limited by its relatively low sensitivity. Some of the factors that influence the accuracy of cytologic diagnosis are attributed to specimen adequacy, inflammation and sampling variation. We explored an alternative approach that is not dependent upon slide based cellularity, but rather uses the cell-free supernatant fluid accrued during cytology processing.
Design: 8 cases underwent analysis (5 benign, 3 malignant). The bile duct brushes were placed in saline and fixative after preparation of direct smears. Cytospin smears and a corresponding cell-free supernatant fluid were prepared. Both types of specimens underwent molecular analysis with comparison of mutational profiles. DNA, extracted from 2 ml of the cytocentrifugation supernatant fluid, was quantified by optical density and amplified 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). In selected cases, microdissection of stained cytology smears and/or cytospin smears were similarly analyzed and compared. Cytology and surgical pathology was used to define outcome status.
Results: 3 of 8 (37.5%) of the samples were hypocellular; however, DNA level (3.3-68.9 ng/ ul) was sufficient to be examined. DNA quantity was significantly higher in cases of malignancy (ave. 45.1 ng/ul) compared with non-neoplastic (ave. 5.5 ng/ml, p<.001) cases. No mutational change was present in the supernatant fluid of non-neoplastic specimens. All cases of malignancy demonstrated mutational change (3-8 mutations). Analysis of the supernatant fluid showed equal or additional mutations compared to that of the microdissected stained cytology smear.
Conclusions: 1. Mutational changes were detected in supernatant fluid of all malignant compared with benign cases. 2. The presence of mutated free DNA in the extracellular fluid rather than microdissected slide based cells specimen could serve as a valuable alternative to support a malignant diagnosis, especially when the specimen has limited cellularity.
Monday, March 19, 2012 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 61, Monday Morning