Phd in HUMAN BIOLOGY AND MEDICAL GENETICS

Thesis title: Thyroid cancer diagnosis through a dual component molecular assay applied to fine-needle aspiration cytology

Purpose: FNA (fine-needle aspiration) cytology reliably establishes the diagnosis of benign or malignant nodule in 70 to 80% of all cases. However, the remaining 20 to 30% of nodules falls into one of the indeterminate categories. Many patients undergo diagnostic surgery, typically lobectomy, which could be avoided for the most patients with benign nodules. On the other hand, this surgical approach is suboptimal for many patients with cancer, who would need additional surgery to complete the thyroidectomy. The aim of this study was to evaluate the performance of a novel dual-component molecular assay as an ancillary molecular method for resolving indeterminate thyroid nodule cytology. This assay involves next-generation sequencing (NGS)- based detection of mutations in 23 thyroid cancer related genes and digital polymerase chain reaction (PCR) evaluation of the expression levels of an microRNA strongly associated with thyroid cancer. Methods: Analytic validity of the assay has been performed on various sample sources that can be used as starting material, including liquid based FNA samples, FNA samples collected in nucleic acid preservative solution, FNA stained smears, and Fresh-Frozen and FFPE thyroid tissue samples. Moreover, clinical validity of the dual-component molecular assay has been performed in a retrospective cohort of thyroid nodules from patients underwent thyroid surgery. Results: All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228x in liquid-based FNA to 95% in FNA stained smears and coverage uniformity ranging from 86% in FFPE to 95% in FNA collected in preservative solution, with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2–5% mutant allele frequency. Used alone, mutation analysis in the cytologically indeterminate thyroid nodules displayed high sensitivity (89%) and NPV (96%), while the miR-146b-5p assay offered high specificity (93%) and PPV (93%). Combined use of both analyses improved panel performance by eliminating false-negative results. Conclusions: This validation study of a novel dual-molecular assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types (including FNA from different sources and FF and FFPE) and can increase the diagnostic accuracy of thyroid cytology by reducing the number of nodules that will be classified as indeterminate and increasing those that can be reliably classified as benign.