Molecular analysis for a panel of mutations has significant diagnostic value for all categories of indeterminate cytology and can be helpful for more effective clinical management of these patients.

BACKGROUND: Fine-needle aspiration (FNA) cytology is a common approach to evaluating thyroid nodules, although 20% to 30% of FNAs have indeterminate cytology, which hampers the appropriate management of these patients. Follicular (or oncocytic) neoplasm/suspicious for a follicular (or oncocytic) neoplasm (FN/SFN) is a common indeterminate diagnosis with a cancer risk of approximately 15% to 30%. In this study, the authors tested whether the most complete next-generation sequencing (NGS) panel of genetic markers could significantly improve cancer diagnosis in these nodules. METHODS: The evaluation of 143 consecutive FNA samples with a cytologic diagnosis of FN/SFN from patients with known surgical outcomes included 91 retrospective samples and 52 prospective samples. Analyses were performed on a proprietary sequencer using the targeted ThyroSeq v2 NGS panel, which simultaneously tests for point mutations in 13 genes and for 42 types of gene fusions that occur in thyroid cancer. The expression of 8 genes was used to assess the cellular composition of FNA samples. RESULTS: In the entire cohort, histologic analysis revealed 104 benign nodules and 39 malignant nodules. The most common point mutations involved the neuroblastoma RAS viral oncogene homolog (NRAS), followed by the Kirsten rat sarcoma viral oncogene homolog (KRAS), the telomerase reverse transcriptase (TERT) gene, and the thyroid-stimulating hormone receptor (TSHR) gene. The identified fusions involved the thyroid adenoma associated (THADA) gene; the peroxisome proliferator-activated receptor g (PPARG) gene; and the neurotrophic tyrosine kinase, receptor, type 3 (NTRK3) gene. Performance characteristics were similar in the retrospective and prospective groups. Among all FN/SFN nodules, preoperative ThyroSeq v2 performed with 90% sensitivity (95% confidence interval [CI], 80%-99%), 93% specificity (95% CI, 88%-98%), a positive predictive value of 83% (95% CI, 72%-95%), a negative predictive value of 96% (95% CI, 92%-100%), and 92% accuracy (95% CI, 88%-97%). CONCLUSIONS: The current results indicate that comprehensive genotyping of thyroid nodules using a broad NGS panel provides a highly accurate diagnosis for nodules with FN/SFN cytology and should facilitate the optimal management of these patients.

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.

ObjectiveDNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.DesignOver 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/GNAS mutations by Sanger sequencing.ResultsKRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively).ConclusionsIn contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/PIK3CA/PTEN alterations is a useful preoperative marker for advanced neoplasia.