The Cytocentrifugation of Supernatant Fluid from Thyroid Nodule Fine-Needle Aspirates Provides Analyzable DNA Suitable for Molecular Analysis
Shahid J Bokhari, Jan F Silverman, Sydney D Finkelstein, Uma Krishnamurti, Yulin Liu, Beth Ujevich, Candy Binkert, Alok Mohanty. Allegheny General Hospital, Pittsburgh, PA; RedPath Integrated Pathology, Pittsburgh, PA
Background: Thyroid fine needle aspiration (FNA) biopsy specimens can be among the more challenging specimens in cytology to reach a definitive diagnosis due to the often subtle and overlapping features of non-neoplastic (hyperplasia), benign (follicular adenoma) and malignant (follicular and papillary carcinoma) neoplasms. Molecular targets for specific lesions (point mutation, translocation, loss of heterozygosity) are known. Therefore, our study focused on a pilot assessment to determine whether the cell-free part of the FNA sample would have sufficient free DNA for molecular analysis, possibly aiding in the differentiation of the aforementioned classes of thyroid lesions, while at the same time submitting the available cellular material for cytologic examination.
Design: Smears and cytocentrifugued FNA needle-rinsings from 16 thyroid nodules were studied. DNA was extracted from 2 ml of the cytocentrifugation supernatant fluid and the DNA quantity/quality measured by optical density/qPCR. Mutational analysis was performed using PCR/capillary electrophoresis for a broad panel of markers (KRAS, BRAF point mutation by sequencing, microsatellite fragment analysis for loss of heterozygosity(LOH) of 16 markers at 19, 3p, 5q, 9p, 10q, 17p, 17q, 21q, 22q). Microdissection of stained cytology smears and/or cytocentrifugation cellular slides were similarly analyzed and compared.
Results: All thyroid FNA needle-rinse fluids contained abundant amplifiable DNA suitable for broad panel mutational analysis. The DNA level in the cell-free supernatant rinse fluid ranged from 3.7-83.9 ng/ul; DNA level in the supernatant did not necessarily correlate with morphologic assessment of smear cellularity. Oncogene point mutations and LOH allelic imbalance mutations present in the microdissected cells of the stained cytology smears were also identified in all corresponding cell-free needle rinse supernatant fluids. Of note, the DNA level was higher in malignant nodules (mean 47.2 ng/ul) compared to non-neoplastic disease (mean 12.4 ng/ul).
Conclusions: The cytocentrifugation supernatant fluid of thyroid FNA specimens contains adequate levels of amplifiable DNA for broad panel mutational analysis in both the cellular and paucicellular specimens. The cytocentrifugation supernatant fluid, currently not utilized, is available for complementary molecular analysis, and could provide additional useful information, especially in equivocal cases.
Monday, March 19, 2012 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 315, Monday Morning