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.

Mutational profiling of sporadic versus toxin-associated brain cancer formation: initial findings using loss of heterozygosity profiling.

The role of environmental and occupational toxin exposure as a cause of or contributing factor for cancer development and progression is incompletely understood. A unique signature of specific mutational change to discriminate toxin-exposed from sporadic cancer is generally sought but not often encountered. We report an approach to better understand cancer causality based on the measurement of the cumulative DNA damage (via loss of heterozygosity) over a defined genomic region (chromosome 3) that is applicable to archival, fixative-treated tissue and cytology specimens of cancer. Our method was applied to (1) a cohort of 10 brain tumor subjects (9 gliomas, 1 hemangioblastoma) with potential exposure to chlorinated solvents and (2) a control cohort of sporadic brain cancer controls (7 gliomas, 1 hemangioblastoma). We show that brain tumors arising in potentially toxin-exposed subjects bear a significantly higher level of passenger LOH mutations compared to sporadic cancer controls. The methodology utilized tissue microdissection, PCR amplification and capillary electrophoresis (fragment analysis for LOH determination, DNA sequencing for specific point mutations), and examined a panel of 15 microsatellite markers distributed along both arms of chromosome 3 that aimed at capturing passenger mutational change accrued during stages of clonal expansion of neoplastic cells. This proof-of-principle study using mutational profiling for passenger LOH mutational damage provides support for the utility of this approach and further studies in order to differentiate between genotoxin-associated versus sporadic (unexposed) cancer development.