PP22-4 Validation of a Targeted Next-Generation Sequencing Test for Indeterminate Thyroid Nodules in a Clinical Setting

Program: Abstracts - Orals, Poster Previews, and Posters
Session: SAT 270-310-Thyroid Neoplasia (posters)
Saturday, April 2, 2016: 1:15 PM-3:15 PM
Exhibit/Poster Hall (BCEC)

Poster Board SAT 283
Shih-Min Cheng*1, Kevin Qu1, Cindy Barlan1, Hai-rong Li1, Jared F. Taylor1, Andrew Grupe2, David A. Wolfson2, Alla Smolgovsky2, Heather R. Sanders1, Anna Gerasimova1, Quoclinh Nguyen1, Joseph J. Catanese1, Charles M. Strom1, Frederick K. Racke1, Frederic M. Waldman1, Richard E. Reitz1 and Feras M. Hantash1
1Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, 2Quest Diagnostics Nichols Institute, Alameda, CA
         Next-generation sequencing (NGS) has become a powerful cancer diagnostic technology, as it can detect multiple genomic alterations in a single assay from limited source material. We developed a novel NGS assay to provide targeted mutation and translocation detection to facilitate diagnosis of indeterminate thyroid nodules from fine needle aspirate (FNA) and formalin-fixed paraffin-embedded tissue (FFPET) samples. The panel includes genes that are commonly altered in thyroid cancers as identified by The Cancer Genome Atlas (TCGA) database.

          Total nucleic acid for this assay was extracted from thyroid FNA and FFPET samples and quantitated. A laboratory-developed multiplex PCR test was designed to target single-nucleotide variants and insertions/deletions in the hotspot regions of 14 genes.  Additionally, 32 translocations involving 8 gene targets were interrogated. Housekeeping and thyroid specific genes were included to ensure quality and adequacy of RNA material. Amplicons were then sequenced on a MiSeq. Analysis included a custom variant caller and translocation detection pipeline. A “no template” and positive controls were included in each run to ensure assay precision and accuracy.

          While the analytic sensitivity for variant calls was determined to be 2.5%, the clinical sensitivity was set at 5% for known hotspot mutations and 10% for others. The NGS assay was validated with as little as 3 ng DNA and 4 ng RNA input. A total of 59 specimens previously tested on a 7-gene molecular panel (BRAF and H-/K-/N-RAS mutations, and PAX8-PPARG and RET-PTC1/3 translocations) were analyzed using this NGS test. All previously identified mutations were detected. Additionally, we detected variants not identified with the 7-gene test, including mutations in GNAS, RET, TERT, and TSHR, as well as ALK and NTRK3 translocations. All normal thyroid tissue samples (n=13) and all except 2 benign thyroid nodules (n=11) were negative for any alterations. In both benign nodules, TSHR variants were detected at allele frequencies not associated with thyroid cancer.  22% (4 of 18) of AUS/FLUS (atypia of undetermined significance/follicular lesion of undetermined significance) samples and 60% (3 of 5) of FN/SFN (follicular neoplasm/suspicious for follicular neoplasm) samples tested positive by the NGS assay. Meanwhile, all malignant samples (8 of 8), and 75% (3 of 4) of suspicious for malignancy (SFM) samples resulted in positive findings by the NGS assay. BRAF V600E was the most prevalent alteration in the SFM and malignant samples (55%, 6 of 11).

          In conclusion, NGS assays have significant advantages over standard molecular assays as an aid in the diagnosis, prognosis, and management of indeterminate thyroid nodules. The assay overcomes some of the challenges caused by limited DNA/RNA material and offers the opportunity to streamline the characterization of genomic alterations in thyroid cancers.

Disclosure: SMC: Employee, Quest Diagnostics. KQ: Employee, Quest Diagnostics. CB: Employee, Quest Diagnostics. HRL: Employee, Quest Diagnostics. JFT: Employee, Quest Diagnostics. AG: Employee, Quest Diagnostics. DAW: Employee, Quest Diagnostics. AS: Employee, Quest Diagnostics. HRS: Employee, Quest Diagnostics. AG: Employee, Quest Diagnostics. QN: Employee, Quest Diagnostics. JJC: Employee, Quest Diagnostics. CMS: Employee, Quest Diagnostics. FKR: Employee, Quest Diagnostics. FMW: Employee, Quest Diagnostics. RER: Employee, Quest Diagnostics. FMH: Employee, Quest Diagnostics.

*Please take note of The Endocrine Society's News Embargo Policy at https://www.endocrine.org/news-room/endo-annual-meeting/pr-resources-for-endo