Thyroid cytology smear slides: An untapped resource for ThyroSeq testing

Abstract

Background Molecular testing of thyroid nodules with indeterminate fine‐needle aspiration (FNA) cytology is commonly used to guide patient management and is typically performed on freshly collected FNA samples. In this study, the authors evaluated the performance of the ThyroSeq test in cytology smear slides. Methods Air‐dried Diff‐Quik (DQ)‐stained and alcohol‐fixed Papanicolaou (Pap)‐stained smears were used to determine required cellularity and sensitivity of mutation detection and to compare ThyroSeq v3 Genomic Classifier (GC) results obtained in cytology smears and fresh FNA samples from the same nodules. Results ThyroSeq testing of 31 cytology smears revealed that 25 smears (81%) were adequate for ThyroSeq analysis, including 14 Pap‐stained smears (100%) and 11 DQ‐stained smears (65%), whereas 6 DQ‐stained smears (35%) failed RNA sequencing. The overall accuracy for detecting molecular alterations was 98%, with 100% concordance for mutations and gene expression alterations, 96% concordance for fusions, and 94% concordance for copy number alterations. Cytology smears were adequate for ThyroSeq analysis when at least 200 to 300 cells were present in 1 to 3 slides. ThyroSeq detected all studied mutations down to 5% allele frequency and BRAF mutations down to 1% allele frequency. Testing of smears yielded a positive ThyroSeq GC result in all nodules originally classified as positive. Conclusions Thyroid FNA cytology smear slides with adequate cellularity can be successfully used for ThyroSeq GC testing in approximately 80% of cases, with an even higher success rate in Pap‐stained smears. Compared with FNA samples collected into preservative solution, 94% to 100% of different genetic alterations could be accurately detected in smears, validating cytology smears as an alternative for ThyroSeq testing in patients with indeterminate thyroid cytology.

Thyroseq v3, Afirma GSC, and microRNA Panels Versus Previous Molecular Tests in the Preoperative Diagnosis of Indeterminate Thyroid Nodules: A Systematic Review and Meta-Analysis

“…Based on the readings of the full-text articles, we excluded 28 articles for the following reasons: only enrolled nodules with benign test results (n = 4) ( 78 – 81 ) or suspicious test results (n = 1) ( 82 ), evaluated nodules with benign or malignant cytology (n = 2) ( 83 , 84 ), did not perform surgery and consequently did not provide reference standard in nodules with benign test results (n = 7) ( 85 – 91 ), an overlap of the participants with other studies (n = 8) ( 92 – 99 ), used freshly collected FNA samples as the reference standard (n = 1) ( 100 ), unavailable statistical analysis (n = 4) ( 13 , 22 , 101 , 102 ), and unavailable full-text article (n = 1). Finally, 40 articles met initial eligibility criteria and were systematically reviewed and abstracted.…”

Real-world, Prospective, and Multicenter Validation of a microRNA-based Thyroid Molecular Classifier

“…Although ThyroSeq v3 is under validation for FNA smear slides, the available results are limited to only 31 samples with different performance depending on slide staining (Papanicolau 100%, Diff-Quick 65%, and overall 81%). 25 Furthermore, FNA smear slides were not used for the prospective study validation. 19…”

“…For physicians, there is no guarantee that the new puncture will have the same cellular composition as the previous sample that indicated the need for molecular testing or that the logistics chain will maintain the sample at the appropriate temperature and ensure timely delivery. Although ThyroSeq v3 is under validation for FNA smear slides, the available results are limited to only 31 samples with different performance depending on slide staining (Papanicolau 100%, Diff-Quick 65%, and overall 81%) 25. Furthermore, FNA smear slides were not used for the prospective study validation 19.…”

Molecular Tests for Risk-Stratifying Cytologically Indeterminate Thyroid Nodules: An Overview of Commercially Available Testing Platforms in the United States

“…For thyroid FNAs classified as Bethesda III or IV, nucleic acid for ThyroSeq testing can be extracted from cellular material collected directly into nucleic acid preservative (provided by the vendor), formalin-fixed paraffin-embedded (FFPE) cellblock preparations, and more recently, cells on direct smear slides [58]. ThyroSeq's limited gene expression analysis serves as quality control steps, confirming adequate thyroid follicular cell sampling as well as identifying aspirates that are not of thyroid follicular cell origin (e.g., medullary thyroid carcinoma (MTC), parathyroid, metastatic tumors).…”