The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions.
Fine-needle aspiration (FNA) of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. The aim of this study was to evaluate how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. Twenty-three FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the microdissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both microdissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in microdissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicates that supernatant fluid may be enriched with tumor DNA. Molecular analysis of the supernatant fluid could serve as an adjunct method to reduce sampling variability and increase diagnostic yield, especially in cases with a high clinical suspicion for malignancy and limited number of atypical cells in the smears.
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Georgios
G
Deftereos
RedPath Integrated Pathology, Inc., Pittsburgh, PA, USA.
Sydney D
SD
Finkelstein
RedPath Integrated Pathology, Inc., Pittsburgh, PA, USA.
Sara A
SA
Jackson
RedPath Integrated Pathology, Inc., Pittsburgh, PA, USA.
Eric M G
EM
Ellsworth
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Uma
U
Krishnamurti
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Yulin
Y
Liu
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Jan F
JF
Silverman
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Candy R
CR
Binkert
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Beth A
BA
Ujevich
Department of Pathology and Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA, USA.
Alok
A
Mohanty
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D014408:Biomarkers, Tumor / Q000235:genetics*; D044963:Biopsy, Fine-Needle; D002498:Centrifugation; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D020022:Genetic Predisposition to Disease; D006801:Humans; D019656:Loss of Heterozygosity; D042282:Microdissection; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins