Category: Thyroid


Saturday, Apr 27
10:30 AM – 10:45 AM

Objective :

Molecular testing (MT) of thyroid nodules diagnosed as indeterminate on fine-needle aspiration (FNA) cytology is increasingly used to devise patient management. The FNA samples collected into a dedicated preservative solution represent an ideal sample type for MT. However, in some cases, the on-site FNA smears are the only specimen type available for MT.  In this study, we evaluated the analytical performance of ThyroSeq V3 GC in on-site FNA smears prepared for cytopathologic interpretation. 

Methods :

ThyroSeq v3 GC analysis was performed on 33 clinical routinely prepared air-dried Romanowsky-type (Diff-Quik®) stained or alcohol-fixed, Papanicolaou-stained (PAP) slides. The slides were soaked in xylene for 1-3 days and coverslips removed. Nucleic acids (DNA and RNA) were isolated using QIAcube (Qiagen) extractor. Additional validation of ThyroSeq test was performed on reference cytology slides (Horizon Diagnostic) used in a Worldwide Ring Trial Study (Malapelle, et al 2017 Cancer Cytopathol) and harbored mutations in KRAS, NRAS, BRAF, and PIK3CA genes at various allelic frequency (10%, 5%, 1% and 0%).

Results :

The on-site FNA smears consisting of ~200 to 1000 cells per slide from 14 thyroid nodules were analyzed. Adequate DNA results for mutations, copy number alterations and RNA results for gene fusions and gene expression were found in 93% (13/14) and 79% (11/14) nodules respectively. Two samples with insufficient RNA results had low cellularity (200-300 cells) and one sample that failed both DNA and RNA sequencing had >500 thyroid cells but showed highly degraded nucleic acids. ThyroSeq detected all mutations at 3-5% and BRAF at 1% of allele frequency in reference cytology slides. The turnaround time for ThyroSeq results was 1-3 days longer due to additional preparatory steps when cytology smears, including air-dried and alcohol-fixed, as compared to FNA samples collected into preservative solution.

Discussion :

Molecular testing is increasingly used to improve clinical management of patients with indeterminate cytology nodules. This study provides first evidence that ThyroSeq test can be performed using routine on-site FNA smears and reports the rate of successful testing of DNA and RNA isolated from these smears. 

Conclusion :

The results of this study show that ThyroSeq v3 GC testing can be effectively performed employing routinely prepared cytology smears with adequate cellularity (at least 200-300 cells). Specifically, we found DNA analysis was informative in  93% of cases and RNA analysis in 79% of cases and demonstrated accurate detection of mutations. Additional studies are needed to provide more extensive validation of cytology smears adequacy for ThyroSeq testing. 


Lindsey Tolino

ThyroSeq Coordinator
University of Pittsburgh Medical Center

Pathology Coordinator

Marina N. Nikiforova

Director, Molecular and Genomic Pathology Lab
University of Pittsburgh

Dr. Nikiforova is a Professor of Pathology and Director of the Molecular & Genomic Pathology Laboratory at UPMC where she also serves as the Director of Molecular Oncology Services. She has published 159 peer reviewed articles and serves on multiple committees in the field of Molecular Genomics, including the Association of Molecular Pathology, the College of American Pathologists, and the American Thyroid Association.

Giancarlo Troncone

University of Naples Federico II


Abigail Wald

University of Pittsburgh Medical Center

Program Manager
Division of Molecular and Genomic Pathology

Zubair Baloch

Professor of Pathology
University of Pennsylvania Medical Center

Dr. Baloch received his medical degree from Liaquat Medical University in Sind, Pakistan. After receiving his PhD in 1991 he joined the pathology residency at Hahnemann University Hospital. This was followed by fellowships in surgical pathology at Memorial Sloan Kettering Cancer Center, New York and cytopathology at the University of Pennsylvania Medical Center, Philadelphia. Dr. Baloch began his career as an assistant professor in the Department of Pathology & Laboratory Medicine at UPENN medical center in1997 and is currently a professor of pathology and laboratory medicine there. He has authored more than 200 peer reviewed publications in leading pathology journals on the subject of endocrine and head and neck pathology; and book chapters and monographs. Dr. Baloch has delivered numerous lectures, workshops and short courses both at the national and international meetings including ASC, USCAP, ASCP, CAP, ECC and IAP. Dr. Baloch has coauthored 3 books and has served as co-editor of the most recent “Milan System for Reporting Salivary Gland Cytopathology”. Dr. Baloch has also served as the chair of the terminology and morphologic criteria committee of the 2007 NCI sponsored thyroid FNA initiative which led to the development of “Bethesda Thyroid FNA Classification Scheme”. Dr. Baloch also served on the international panel which recommended the renaming of encapsulated follicular variant of papillary thyroid carcinoma to non-invasive follicular tumor with papillary like nuclear features (NIFTP). Dr. Baloch is executive editor of Diagnostic Cytopathology and member of the editorial board of Cancer Cytopathology, Cytojournal, Endocrine Pathology and Journal of American Society of Cytopathology.
Dr. Baloch has served as the chair of ASCP annual meeting scientific program committee (2014-2018), member of USCAP education committee, ASC executive board, ASCP committee for continuing professional development, Papanicolaou Society scientific program committee, American Association of Endocrinologist thyroid nodule guideline task force and American Thyroid Association laboratory test committee.

Yuri E. Nikiforov

Vice Chair, Dept of Pathology.
UPMC, Pennsylvania

Dr. Nikiforov is Professor of Pathology and Vice Chair at the Department of Pathology, University of Pittsburgh where he also serves as Director of the Division of Molecular & Genomic Pathology. He has published over 170 peer-reviewed articles and over 15 book chapters, and is a senior editor of the textbook “Diagnostic Pathology and Molecular Genetics of the Thyroid.” His research program is funded by the National Institute of Health and focused on molecular genetics of thyroid cancer, thyroid cancer diagnostics, and molecular mechanisms of chromosomal rearrangements. He is an elected member of the American Society for Clinical Investigation and a recipient of the 2007 Van Meter Award from the American Thyroid Association, 2014 Light of Light Foundation Honorary Award, 2016 Aldo Pinchera Award from the Italian Thyroid Carcinoma Observatory, and 2017 Sidney H. Ingbar Distinguished Lectureship Award the American Thyroid Association.