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

Abstract

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. Modern Pathology (2014) 27, 594-601; doi:10.1038/modpathol.2013.147; published online 20 September 2013Keywords: cytology supernatant; FNA; KRAS; LOH; mutational analysis; pancreas Pancreatic cancer rates have been recently reported to be on the rise. 1 Moreover, the lack of early symptomatic manifestation of disease usually results in lack of early detection. The overall dismal prognosis combined with the rising incidence make pancreatic cancer a growing public health concern. 2 With the American Cancer Society estimating that there will be 45 220 newly diagnosed cases of pancreatic cancer in 2013 and an estimated 38 460 pancreatic cancer-related deaths in the same year, 3 pancreatic cancer is becoming the fourth leading cause of cancer-related death among men and the third leading cause of cancer-related death among women in the United States. 3 Moreover, the 5-year survival remains around 5%, essentially unchanged in the last three deca...

Detection of BRAF mutation in the cytocentrifugation supernatant fluid from fine-needle aspiration of thyroid lesions may enhance the diagnostic yield

“…One possibility for this discrepancy lies in the nature of the neoplastic cell population. It is conceivable that some tumors may have a higher cellular turnover (i.e., increased mitosis, apoptosis, and necrosis), resulting in the shedding of DNA (cell-free DNA)[18222324] in the interstitial fluid of the tumor, which is then caught only in the supernatant. Therefore, the lack of BRAF mutations in the cellular component of these samples could be likely due to sampling issues (i.e., cancer cells with BRAF mutations were missed by the FNA needle in the fourth pass, which is the sample submitted to the reference laboratory, resulting in a negative cellular DNA analysis while the tumor cells with the BRAF mutations were obtained from the first three FNA passes and therefore were present in the supernatant fluid leading to a positive supernatant DNA analysis).…”

“…Our findings of detecting BRAF mutations in FNA supernatants of PTC are in agreement with the results of a recent study, which demonstrated that the use of FNA supernatant specimens from patients with pancreatic cancers for molecular testing may outperform cellular DNA analysis in certain situations. [18] We attempted to quantify the DNA amounts in the supernatant; however, the majority of samples showed the amount of DNA was below the linear range of NanoDrop 1000 (Thermo Scientific, Wilmington, DE) even though 87% of our samples had enough amplifiable DNA for the detection of BRAF mutation. Of note, we only used 200 µl of about 25–50 ml supernatant for DNA extraction; therefore the yield of DNA could potentially be increased if a greater amount of supernatant was used for DNA extraction.…”

“…However, at times, paucicellular or acellular FNA specimens may fail to provide adequate material for mutation testing. [18] We hypothesize that enough DNA may be present in the FNA supernatant to conduct mutation testing and that BRAF mutation analysis using supernatant may increase the diagnostic yield of the FNA.…”

Optimizing Workflows and Processing of Cytologic Samples for Comprehensive Analysis by Next-Generation Sequencing: Memorial Sloan Kettering Cancer Center Experience

“…Specifically for the ThinPrep slide, a small aliquot of pelleted material is transferred into the standard PreservCyt fixative jar (Hologic) per manufacturer's protocol and processed accordingly. Based on a recent study showing a substantial amount of nucleic acid in the supernatants of FNA needle rinses after cell pelleting, 25 we have modified our process to preserve the CytoLyt supernatants, material that has traditionally been discarded in the majority of cytopathology laboratories, for molecular studies. In addition, any unused cell suspension from the PreservCyt container is also saved.…”

Molecular testing of residual cytology samples: Rethink, reclaim, repurpose

“…12 Postcentrifuged supernatant fluid from FNA needle rinses has been known to yield substantial amounts of DNA that are sufficient for assays such as microsatellite fragment analysis for loss of heterozygosity as well as hotspot-based mutational analysis by PCR/capillary electrophoresis and pyrosequencing. [13][14][15][16][17] A recent study by our group demonstrated that supernatant fluid samples from FNA needle rinses can provide adequate DNA for NGS and droplet digital PCR to provide clinically relevant genomic information for a variety of solid tumors, including lung carcinoma, melanoma, and colorectal adenocarcinoma. 18 Furthermore, another recent study has demonstrated the feasibility of using supernatant DNA fluid from patients with lung cancer for tumor genotyping by mutational analysis.…”