Analytical validity of a microRNA‐based assay for diagnosing indeterminate thyroid FNA smears from routinely prepared cytology slides

Abstract

BACKGROUNDThe majority of thyroid nodules are diagnosed using fine‐needle aspiration (FNA) biopsies. The authors recently described the clinical validation of a molecular microRNA‐based assay, RosettaGX Reveal, which can diagnose thyroid nodules as benign or suspicious using a single stained FNA smear. This paper describes the analytical validation of the assay.METHODSMore than 800 FNA slides were tested, including slides stained with Romanowsky‐type and Papanicolaou stains. The assay was examined for the following features: intranodule concordance, effect of stain type, minimal acceptable RNA amounts, performance on low numbers of thyroid cells, effect of time since sampling, and analytical sensitivity, specificity, and reproducibility.RESULTSThe assay can be run on FNA slides for which as little as 1% of the cells are thyroid epithelial cells or from which only 5 ng of RNA have been extracted. Samples composed entirely of blood failed quality control and were not classified. Stain type did not affect performance. All slides were stored at room temperature. However, the length of time between FNA sampling and processing did not affect assay performance. There was a high level of concordance between laboratories (96%), and the concordance for slides created from the same FNA pass was 93%.CONCLUSIONSThe microRNA‐based assay was robust to various physical processing conditions and to differing sample characteristics. Given the assay's performance, robustness, and use of routinely prepared FNA slides, it has the potential to provide valuable aid for physicians in the diagnosis of thyroid nodules. Cancer Cytopathol 2016;124:711–21. © 2016 Rosetta Genomics. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

Ancillary molecular testing of indeterminate thyroid nodules

“…These markers have been tested both individually and in panels even though only some of them (BRAF V600E mutation and RET/PCT rearrangements) show high specificity and likelihood of a malignant diagnosis in mutated thyroid lesions. Molecular thyroid tests that are commercially available in the United States are: 1) ThyroSeq (University of Pittsburgh Medical Center/Cytopath Biopsy Lab, Pittsburgh, PA), 2) Afirma Gene Expression Classifier (GEC; Veracyte, South San Francisco, CA), 3) RosettaGX Reveal (Rosetta Genomics, Philadelphia, PA), and 4) ThyGenX and ThyraMIR (both from Interpace Diagnostics, Parsippany, NJ) . Whereas the Thyroseq and ThyGenX tests have high positive predictive value (PPV) and negative predictive value (NPV) wherein high PPV helps as a “rule‐in malignancy” test, the Afirma GEC with its high NPV helps as a “rule‐out malignancy” test mostly for indeterminate thyroid lesions .…”

“…This recent acknowledgement of the role of molecular testing formalizes their use in defining risk stratification of patients with thyroid nodules. In this review, we discuss the clinical implications of the different molecular tests in the indeterminate Bethesda categories . It is important to note that the literature to date generally supports employing a combination of morphology and molecular testing to best guide the clinical or surgical approach for patients diagnosed with indeterminate thyroid nodules (Table ).…”

Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears

“…The type of cytological stain used did not affect the classification performance. 27 The classification method used for this miRNA-based assay, named RosettaGX Reveal, combines several LDA steps along with a KNN-based classifier. The performance of the training set is summarised in table 3.…”

Molecular testing for thyroid nodules: Review and current state

“…MiRNAs act as negative regulators of gene expression and may impact cellular processes that lead to carcinogenesis. Because miRNA expression may be dysregulated in thyroid cancer, this represents an additional approach to molecular test development with high sensitivity and high specificity …”