The human thyroid cancer cell lines BCPAP, Cal-62, 8505C, and ML-1 were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and maintained as recommended. All cell lines were authenticated by short tandem repeat profiling. HiYield Genomic DNA Mini kits were purchased from Real Biotech Corp. (Taipei, Taiwan). DNeasy Blood & Tissue kits and QIAquick polymerase chain reaction (PCR) purification kits were purchased from Qiagen (Valencia, CA, USA). The AccuPower PCR premix was purchased from Bioneer (Daejeon, Korea). Media and cell culture reagents were purchased from Gibco (Grand Island, NY, USA).

BCPAP cells originating from PTC, Cal-62 and 8505c cells originating from ATC, and ML-1 cells originating from follicular thyroid cancer (FTC) cells, were used for DNA extraction. Genomic DNA was isolated from thyroid cancer cells using HiYield Genomic DNA Mini kits.

Thirty-five fresh-frozen PTC tissues from surgically removed thyroid samples were collected at the Asan Bio-Resource Center and were used to isolate genomic DNA following Institutional Review Board approval (2013-0539). These tissues were selected randomly from the tissue lists at the Asan Bio-Resource Center. Genomic DNA was isolated from 10 mg fresh-frozen PTC tissue using the DNeasy Blood & Tissue kit according to the manufacturer's instructions.

Genomic DNA extracted from thyroid cancer cells and fresh-frozen PTC tissues was amplified using PCR and sequenced to evaluate the C228T and C250T TERT promoter mutations. The primers are listed in Table 1. Each reaction mixture contained 10 mM Tris (pH 9.0), 250 µM of each deoxynucleotide triphosphate, 1.5 mM MgCL₂, 30 mM KCl, 1 µL each primer, 200 ng extracted DNA, and 1 U DNA polymerase (Bioneer) in a final volume of 20 µL. The amplification protocol consisted of initial denaturation at 94℃ for 5 minutes, 34 cycles of denaturation at 94℃ for 30 seconds, annealing at 65℃ for 30 seconds, extension at 72℃ for 30 seconds, and a final extension at 72℃ for 7 minutes. A single major PCR product was confirmed by 2% agarose gel electrophoresis, and the size of the product was 164 bp. Each PCR product was purified using a QIAquick PCR purification kit and sequenced using a DNA analyzer (PRISM 373A, Applied Biosystems, Foster City, CA, USA). The BRAF V600E mutation was analyzed as reported previously [9].

We used four different thyroid cancer cells from different origins to evaluate TERT promoter mutations. BCPAP cells originating from PTC presented the CC228229TT TERT promoter mutation (Fig. 1A) and 8505C cells originating from ATC presented the C250T and C253T TERT promoter mutations (Fig. 1B). However, no TERT promoter mutations were detected in Cal-62 from ATC or ML-1 from FTC (data not shown).

We evaluated TERT promoter mutations in PTC tissues of 35 patients. The baseline characteristics of the patients are described in Table 2. Median patient age was 45 years and 28 patients (80%) were female. Median tumor size was 2.2 cm, and most of the primary tumors had extrathyroidal extension. Cervical lymph node (LN) metastasis occurred in 26 patients (74%) including four (11%) with lateral cervical LN metastasis. During the median 5.5-year follow-up, seven patients (20%) had recurrence in the lateral cervical neck LN and one patient had distant lung metastasis.

Of the 35 PTCs, only one (2.8%) had the C228T TERT promoter mutation (Fig. 2A). No C250T mutation was found in any of the subjects (Fig. 2B). Twenty (57.1%) had the BRAF V600E mutation.

A 75-year-old female patient with the C228T TERT promoter mutation also harbored the BRAF V600E mutation and had a large primary tumor (4.2 cm) with central cervical LN metastasis. She had recurrence at the lateral cervical neck LNs and underwent reoperation. She had a high anti-thyroglobulin anti-body level without structurally persistent disease at the last evaluation.