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.

Institutional prevalence of malignancy of indeterminate thyroid cytology is necessary but insufficient to accurately interpret molecular marker tests

“…It is important to note that these predictive values are calculated with the estimated sensitivity and specificity of single studies, each of them with limited sample size and wide confidence intervals, which could differ significantly from the true performance. This might be especially relevant in the calculations made for ThyroSeq v2 because the test metrics used for these calculations are derived from a single institution, nonblinded study, whereas for Afirma and miRInform, these parameters are derived from multicenter and blinded studies (8,9,10,11). For example, the original single-center, nonblinded study of the sevengene oncogene panel (upon which miRInform was based) performed rather better than in the more recent multicenter, blinded study, emphasizing the fact that singlecenter results lack external validation (9,16).…”

“…The estimated NPV and PPV for Afirma, miRInform, and ThyroSeq v2 were calculated based on the sensitivity and specificity reported in extant publications, using Bayes' theorem, whereas 95% confidence intervals were developed using the Student's t-distribution. Specifically, to assess the predicted performance of the Afirma assay (Veracyte Inc., South San Francisco, CA, USA), we used the data published by Alexander and coworkers (8); for miRInform (Asuragen Inc), we used the data published by Beaudenon-Huibregtse and coworkers (9) (sensitivity and specificity for Bethesda III and IV categories were calculated from their Table 2, because they were not provided in the text); and for ThyroSeq v2, we used the data published by Nikiforov and coworkers (10,11) We then reviewed our own institutional experience with miRInform, which was in use between July 2012 and October 2014. Because this was a retrospective study, we recognized that the pathologists reading the histology were aware of both the cytological diagnosis and the oncogene panel result at the time of histological interpretation.…”

Noninvasive Follicular Variant of Papillary Thyroid Carcinoma and the Afirma Gene-Expression Classifier

“…Thus, it is not surprising that most of the carcinomas resected on the basis of a suspicious Afirma result are indolent FVPTCs, since Afirma testing is being performed on nodules associated with an AUS/FLUS or SFN diagnosis on FNA. For this same reason, many of the carcinomas resected in the setting of a preceding indeterminate FNA and a mutation detected by Thyroseq are RAS mutation-positive FVPTCs (26,27). The extent of initial surgery that should be performed for a nodule with an AUS/FLUS or SFN cytologic diagnosis and a suspicious Afirma result has not been established.…”

Papillary Thyroid Carcinoma: Association Between Germline DNA Variant Markers and Clinical Parameters

“…Notably, novel assays such as ThyroSeq (CBLPath, Rye Brook, NY) allow tumor DNA to be tested from material obtained by fine-needle aspiration. These tests provide good sensitivity and specificity for characterizing cytology samples with atypia of undetermined significance/ follicular lesion of undermined significance (25). The lack of data assessing the usefulness of germline variants in the clinical stratification of PTC patients prompted us this investigation of the potential association of germline SNPs and the expression of their potential target genes with clinical features.…”