Novel NGS Test May Enhance Diagnosis and Management of Indeterminate Thyroid Nodules

Katherine Hasal, MS
Published: Sunday, Nov 02, 2014

Dr. Shih-Min Cheng

Shih-Min Cheng, PhD

A next-generation sequencing (NGS) assay has shown promise as a new tool to aid in the diagnosis and treatment of indeterminate thyroid nodules, according to research presented at the 2014 American Thyroid Association (ATA) Annual Meeting.

“We developed a novel NGS-based assay designed to provide targeted mutation detection to aid in the diagnosis of indeterminate thyroid nodules from fine needle aspirate [FNA] and formalin-fixed paraffin-embedded [FFPE] tissue samples,” said lead author Shih-Min Cheng, PhD, of Quest Diagnostics in San Juan Capistrano, California.

Although FNA cytology provides a definitive diagnosis of either malignant or benign for the majority of thyroid nodules, about 25% of nodules have indeterminate cytomorphology. Based on draft 2014 ATA Guidelines, two categories of FNA cytology results may be clinically managed by conducting repeat FNA or molecular testing. Samples are categorized as atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS) in approximately 7% of cases and carry a risk of malignancy of 5% to 10%. Another 5% to 10% of cytology results are classified as follicular or Hürthle cell neoplasm (FN) or suspicious for FN (SFN) with a risk of malignancy of 20% to 30%. It is these two categories of indeterminate FNA cytology results for thyroid nodules that are particularly challenging for clinicians to decide how to appropriately manage.

The NGS panel for indeterminate thyroid nodules was based on a seven-gene polymerase chain reaction (PCR)–based assay that was then expanded to include additional molecular markers based on new gene mutations and gene translocations identified in the Cancer Genome Atlas (TCGA) or recent literature publications.

The types of specimen used in this study included indeterminate thyroid nodules obtained through FNA or FFPE or from lobectomy/thyroidectomy specimens (FFPE). DNA was extracted from thyroid FNA and FFPE samples and was quantified fluorometrically. The assay performed well with limited biopsy/tissue materials, as the mutational panel minimum DNA input was 1 to 10 ng, and it carried a 5% analytic assay sensitivity.

A two-step multiplex PCR was used to enrich the targeted exonic regions of 13 specific genes, including AKT1, BRAF, CTNNB1, EIF1AX, GNAS, HRAS, KRAS, NRAS, PIK3CA, PTEN, RET, TP53, and TSHR, plus the TERT promoter region. “The targeted amplification protocol covers all known hot-spot mutations,” said Cheng. Mutation frequency cutoff for clinical sample reporting was set at 5% to 10%.

After purification, the amplicons were sequenced using the Illumina MiSeq desktop sequencing platform. A TruSeq amplicon workflow was used to analyze coverage and detect mutations, and a positive control with known mutation frequencies was included in each run to ensure optimal assay performance. Data were analyzed using MiSeq analysis software, Quest Sequencing Analysis Pipeline (QSAP), and Integrative Genomics Viewer. A computer simulation of the assay calculated a clinical assay sensitivity of 89% or higher.

Using as little as 1 ng of DNA in the assay, the system was able to detect single-nucleotide variants and insertions and deletions to a 5% sensitivity level. Mutations in a cell line control mixture were also detected by the panel at the expected frequencies. Indeterminate FNA and FFPE tissue samples that had previously been tested for BRAF and RAS mutations were also sequenced, and all previously identified mutations were identified with no false-negative results. However, this panel assay also detected mutations in the TERT promoter and others that had not been identified initially.

Overall, the NGS panel confirmed all BRAF and RAS mutation–positive results from the current assay and increased the mutation detection rate in FNA specimens. Cheng cautioned that these results are preliminary and that clinical validation is ongoing, however he is pleased with the outcomes thus far.

“Importantly, the assay [we developed] meets the challenges of limited DNA amounts and variable quality often associated with FNA and FFPE clinical specimens. The approach offers the opportunity to streamline the characterization of genomic alterations in thyroid cancers to aid in thyroid cancer diagnosis,” concluded Cheng.


Cheng S, Lew D, Barlan C, et al. Development of a clinical targeted next-generation sequencing test for indeterminate thyroid nodules. Presented at: Annual Meeting of the American Thyroid Association; October 29 – November 2, 2014; Coronado, CA. Abstract 125.

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