Analytical Performance of a Gene Expression Classifier for Medullary Thyroid Carcinoma.

Analytical Performance of a Gene Expression Classifier for Medullary Thyroid Carcinoma.

Thyroid. 2016 Sep 7;

Authors: Pankratz DG, Hu Z, Kim SY, Monroe RJ, Wong MG, Traweek ST, Kloos RT, Walsh PS, Kennedy GC

Abstract
BACKGROUND: To demonstrate the analytical validity of an RNA classifier for medullary thyroid carcinoma (MTC).
METHODS: Fresh-frozen tissue specimens were obtained from commercial sources, and MTC diagnoses were confirmed by histopathology review. De-identified patient fine-needle aspiration biopsies (FNABs) and whole blood from normal donors were obtained. Total RNA was extracted, amplified, and hybridized to custom microarrays for gene expression analysis. Gene expression data was normalized and classified via a machine learning algorithm. Positive control materials were produced from MTC tissues and tested across multiple experiments and laboratories. 27 MTC tissue specimens were used to evaluate the sensitivity of the MTC classifier. Gene expression data from tissues and FNABs were used to model classifier response to mixtures of MTC samples with normal thyroid tissue, a benign thyroid nodule, a Hürthle cell adenoma and whole blood. Select mixture conditions were confirmed in vitro. Assay tolerance to RNA input variation (5-25ng) and genomic DNA contamination (30% by mass) was evaluated. The intra- and inter-run reproducibility, and inter-laboratory accuracy of MTC classifier results were characterized.
RESULTS: MTC classifier sensitivity of 96.3% (95% CI: 81.0%-99.9%) was determined retrospectively using 27 MTC confirmed tissue specimens. One false negative result in a necrotic tissue implicated sample necrosis in reduced classifier sensitivity. Dilution modeling of MTC samples with normal or benign tissues showed consistent detection of MTC down to 20% sample proportions, with in vitro confirmation of 20% analytical sensitivity. Classifier tolerance to RNA input variation (5-25ng), genomic DNA contamination (30% by mass), and an interfering substance (blood) was demonstrated with 100% accurate classifier results under all tested conditions. The maximum observed run-to-run score difference for a single FNAB sample was ~1 unit, compared to the average score difference between 38 MTC and non-MTC FNABs of ~32 units. MTC classifier results for 20 tissues processed from total RNA in two different laboratories showed 100% concordance.
CONCLUSIONS: The MTC classifier, offered as part of the routine molecular testing of cytology-indeterminate thyroid nodules, demonstrates robust analytical sensitivity, specificity, accuracy and reproducibility.

PMID: 27605259 [PubMed - as supplied by publisher]

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