Utility of microdissected cytology smears for molecular analysis of thyroid malignancy

Abstract

Background Molecular testing of thyroid fine‐needle aspirates has demonstrated value in cases of indeterminate cytology (Bethesda categories III, IV, and V) enabling optimized individual patient management leading to better outcomes with health economic benefits. For most molecular testing modalities, including mutational panels and classifier analyses, part or all of a dedicated needle aspiration pass is required to obtain an adequate sample for testing. Our analysis, which is based on a combination approach (mutation detection and microRNA classifier status), has documented clinical validity and utility when performed on thyroid fine‐needle aspirates placed directly into RNA preservative fluid. Here we show that the combination approach can be extended to microdissected stained cytology slides provides the physician greater opportunity to resolve cytological indeterminacy. Methods Extracted nucleic acid from needle aspirate and corresponding cytology preparations of 47 thyroid nodules were analyzed using identical methodology and results were compared. Results Of 94 molecular analyses (47 mutational analyses, 47 microRNA classifier assessments based on a validated 10 marker panel) only 5 samples showed discordant results. Conclusion These findings, together with supplementary work using archival specimens shows that the combination approach can be effectively applied to both direct aspirated thyroid nodule aspirates or to nucleic acid extracted from macrodissected and microdissected cytology slide smears, with the expectation of equivalent results. The advantages of both specimen sources, direct aspirate, and cytology slide smears are discussed.

Development and Analytical Validation of an Expanded Mutation Detection Panel for Next-Generation Sequencing of Thyroid Nodule Aspirates

“…The two sample types served as reference samples with known results from the focused panel chemistry where their testing performance is similar. 24 Both tests identified the same genetic alterations in the markers common to both panels (n Z 93, 82 mutations and 11 fusions) in 92 samples (one case was double positive for NRAS and PIK3CA), indicating 100% qualitative agreement between the two tests. Further breakdown of results by sample type (cytology slides and FNAs) is shown in Table 5.…”

“…TNA was isolated from stained cytology smear slides (Papanicolaou and Diff-Quik stain), as previously described using glass fiber absorption (Recoverall kit; Life Technologies). 24 Cell clusters were first microdissected from Diff-Quik or Papanicolaou stained cytology slides using a simple manual technique approach under stereomicroscopic visualization (CZ40 stereomicroscope; Zeiss, Thornwood, NY).…”

“…19,20 On this basis, it is believed that use of a combination molecular analysis best meets the diagnostic requirements for cytology indeterminate thyroid nodules, enabling the most accurate risk assessment to guide management decisions. 24 To further increase the diagnostic yield of the oncogenic panel and miRNA classifier combination tests, the mutational panel of the NGS-based section of the combination test was expanded to include the detection of sequence alterations of additional five cancer genes (total of 10 genes sequenced), both oncogenes and tumor suppressor genes, as well as 32 new fusion transcripts (Tables 1 and 2). The test was further analytically validated for its certification for clinical testing as a laboratory-developed test.…”

Thyroid cytology smear slides: An untapped resource for ThyroSeq testing

“…A combined use of ThyGenX (previous version of ThyGeNEXT) and ThyraMIR was recently validated in thyroid cytology smears 14 . In that study, a comparison of detecting mutations and gene fusions between freshly collected FNA samples and cytology showed 98% concordance, and 89% of microRNA classifier analysis results were concordant 14 . The study provided overall encouraging results for detecting mutations, gene fusions, and expression of microRNAs.…”

“…However, current diagnostic and therapeutic approaches require sensitive analyses of multiple genetic alterations. A combined use of ThyGenX (previous version of ThyGeNEXT) and ThyraMIR was recently validated in thyroid cytology smears 14 . In that study, a comparison of detecting mutations and gene fusions between freshly collected FNA samples and cytology showed 98% concordance, and 89% of microRNA classifier analysis results were concordant 14 .…”