Impact of the Multi-Gene ThyroSeq Next-Generation Sequencing Assay on Cancer Diagnosis in Thyroid Nodules with Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance Cytology

Abstract

Background: Fine-needle aspiration (FNA) cytology is a common approach to evaluate thyroid nodules. It offers definitive diagnosis of a benign or malignant nodule in the majority of cases. However, 10-25% of nodules yield one of three indeterminate cytologic diagnoses, leading to suboptimal management of these patients. Atypia of undetermined significance/follicular lesion of undermined significance (AUS/FLUS) is a common indeterminate diagnosis, with the cancer risk ranging from 6% to 48%. This study assessed whether a multi-gene next-generation sequencing (NGS) assay can offer significant improvement in diagnosis in AUS/FLUS nodules.

Methods: From May 2014 to March 2015, 465 consecutive FNA samples with the cytologic diagnosis of AUS/FLUS underwent prospective molecular testing using the ThyroSeq v2.1 panel. The panel included 14 genes analyzed for point mutations and 42 types of gene fusions occurring in thyroid cancer. In addition, eight genes were assessed for expression in order to evaluate the cell composition of FNA samples. Ninety-eight (21%) of these nodules had definitive surgical (n = 96) or nonsurgical (n = 2) follow-up and were used to determine the assay performance.

Results: Among 465 AUS/FLUS nodules, three were found to be composed of parathyroid cells and 462 of thyroid follicular cells. Of the latter, 31 (6.7%) were positive for mutations. The most frequently mutated genes were NRAS and HRAS, and overall point mutations in seven different genes and five types of gene fusions were identified in these nodules. Among 98 nodules with known outcome, histologic analysis revealed 22 (22.5%) cancers. ThyroSeq v2.1 was able to classify 20/22 cancers correctly, showing a sensitivity of 90.9% [confidence interval (CI) 78.8-100], specificity of 92.1% [CI 86.0-98.2], positive predictive value of 76.9% [CI 60.7-93.1], and negative predictive value of 97.2% [CI 78.8-100], with an overall accuracy of 91.8% [CI 86.4-97.3].

Conclusions: The results of the study demonstrate that the ThyroSeq v2.1 multi-gene NGS panel of molecular markers provides both high sensitivity and high specificity for cancer detection in thyroid nodules with AUS/FLUS cytology, which should allow improved management for these patients.

Figures

FIG. 1.

Schematic representation of the study flow and main findings.

FIG. 2.

Microscopic appearance of one of the NRAS-positive nodules histopathologically diagnosed as a hyperplastic nodule. (A) Low-power view showing a moderately thick capsule (arrow) and a nodule (top) composed predominantly of large follicles with abundant colloid intermixed with small follicles (hematoxylin and eosin [H&E], 40×). (B) In focal areas, small follicles are lined by cells with nuclear enlargement, irregular nuclear contours, and some chromatin clearing (arrows) (H&E, 200×). (C) Small follicular structures are focally positive for HBME-1 by immunohistochemistry (arrows), whereas most of the adjacent large follicles are negative (HBME-1 immunostain, 100×). Color images available online at www.liebertpub.com/thy

FIG. 3.

Negative predictive value (NPV; red line) and positive predictive value (PPV; blue line) of ThyroSeq v2.1 in thyroid nodules with atypia of undetermined significance/follicular lesion of undermined significance cytology found in this cohort with a cancer prevalence of 22.5% (black dotted line) and expected in a range of cancer prevalence rates between 6% and 48% (yellow dotted lines). Color images available online at www.liebertpub.com/thy

FIG. 4.

Predicted NPV and PPV of ThyroSeq v2.1 compared to the Afirma gene expression classifier test in AUS/FLUS nodules based on the sensitivity and specificity of ThyroSeq (solid lines) identified in this study and of Afirma (dashed lines) reported by Alexander et al. (14). Color images available online at www.liebertpub.com/thy