Molecular Analysis of Centrifugation Supernatant Fluid from Pancreaticobiliary Duct Samples Can Improve Cancer Detection

Abstract

Objective: We aimed to supplement microscopic examination 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 cytocentrifugation 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 mutations 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 supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. Conclusion: Brush cytology specimens yielded supernatant fluid enriched with DNA, probably from actively proliferating cells. Mutational profiling can enhance the cytologic evaluation and characterization of specimens suspected to contain pancreatic or bile duct cancer.

The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions

“…Notwithstanding, available data on cytology specimens have consistently demonstrated that microdissected stained cytology cells are especially suitable for mutational analysis and applicable to common cytology practice. 17,18,20,23,43 Furthermore, as most of the commonly used fixatives in cytology are alcohol based, and are not expected to induce significant DNA degradation, it would be reasonable to expect favorable results with these other methods of sample preparation of the supernatant fluid as well.…”

“…A panel of 16 microsatellite markers was used for this purpose. These markers targeted common sites for tumor-suppressor genes associated with pancreaticobiliary cancers and have previously undergone analytic and clinical validation for pancreaticobiliary disease as reported in prior studies, [16][17][18][19][20][21][22][23] and present at the following chromosomal locations: 1p (CMM1, Lmyc), 3p (VHL, OGG1), 5q (MCC, APC), 9p (CDKN2A, CDKN2B), 10q (PTEN, MXI1), 17p (TP53), 17q (NME1, RNF43), 21q, 22q (NF2) using quantitative fluorescent PCR/capillary electrophoresis.…”

“…[16][17][18][19][20][21][22][23] The use of these techniques nonetheless requires the presence of not only an adequate amount of cellular material, but also cellular material that shows cytomorphological atypia, in order to be selected for microdissection. In fact, paucicellular or acellular specimens not only do not provide optimal material for cytological evaluation but also, at times, fail to provide material for cell-based DNA mutational analysis.…”

“…However, our hypothesis is that the supernatant fluid that is left after cytocentrifugation does in fact contain cell-free tumor DNA, and this DNA may represent the extracellular space that surrounds the cancer cells and contains high-quality tumor cell-free DNA. 23 Furthermore, the utilization of the cytocentrifugation supernatant, normally discarded, can prove to be of utmost value in those cases where the cellular material is scarce or even absent, as tumor cell-free DNA can still be analyzed, without compromising any of the material routinely used for other diagnostic studies.…”

The added value of using mutational profiling in addition to cytology in diagnosing aggressive pancreaticobiliary disease: review of clinical cases at a single center

“…Some studies have also shown that microdissection-based mutational profiling of DNA can be clinically useful in diagnosing these malignancies [10-15]. Such molecular analysis is especially significant when morphologic methods are uncertain or not obtainable [18-22]. Like cytology, the use of mutational profiling has traditionally required cellular material that shows morphological signs of disease in order to extract DNA from microdissected areas of relevant cells.…”