Gene Expression (mRNA) Markers for Differentiating between Malignant and Benign Follicular Thyroid Tumours

Distinguishing between follicular thyroid cancer (FTC) and follicular thyroid adenoma (FTA) constitutes a long-standing diagnostic problem resulting in equivocal histopathological diagnoses. There is therefore a need for additional molecular markers. To identify molecular differences between FTC and FTA, we analyzed the gene expression microarray data of 52 follicular neoplasms. We also performed a meta-analysis involving 14 studies employing high throughput methods (365 follicular neoplasms analyzed). Based on these two analyses, we selected 18 genes differentially expressed between FTA and FTC. We validated them by quantitative real-time polymerase chain reaction (qRT-PCR) in an independent set of 71 follicular neoplasms from formaldehyde-fixed paraffin embedded (FFPE) tissue material. We confirmed differential expression for 7 genes (CPQ, PLVAP, TFF3, ACVRL1, ZFYVE21, FAM189A2, and CLEC3B). Finally, we created a classifier that distinguished between FTC and FTA with an accuracy of 78%, sensitivity of 76%, and specificity of 80%, based on the expression of 4 genes (CPQ, PLVAP, TFF3, ACVRL1). In our study, we have demonstrated that meta-analysis is a valuable method for selecting possible molecular markers. Based on our results, we conclude that there might exist a plausible limit of gene classifier accuracy of approximately 80%, when follicular tumors are discriminated based on formalin-fixed postoperative material.

47.2 years, with a median of 45.5 years (range: 14-72 years). Eight subjects (among them 3 diagnosed with FTC) underwent primary total thyroidectomy, 15 underwent lobectomy, and the remaining 5 underwent thyroid lobectomy with partial or subtotal resection of a contralateral lobe. The mean and median tumour diameter were 27 mm and 23 mm, respectively (range: 10-55 mm). However, the tumour diameter was unknown in 3 patients. Two patients with FTA were also diagnosed with low risk papillary thyroid cancer. Complementary radioiodine (RAI) ablation was carried out in both patients who achieved complete remission. For the FTC patients, distant metastases (bones and lungs) were present at diagnosis in 2 patients, whereas one subject had local recurrence and distant metastases (mediastinum and lungs) 8 years after the initial diagnosis. Two were RAI-refractory, while RAI treatment with a cumulative activity of 500 mCi resulted in FTC stabilisation for another patient. One remaining FTC patient achieved complete remission after surgery and RAI ablation. The mean time to follow-up in the whole group was 3.7 years (range: 0-12 years).

Validation study
Forty patients were diagnosed with follicular thyroid adenoma (FTA subgroup) and 31 with follicular thyroid carcinoma (FTC subgroup).
The FTA subgroup included 35 women and 5 men, with a mean age at diagnosis of 46.3 years and a median of 45 years (range: 19-79 years). Twenty-seven subjects underwent thyroid lobectomy, and 12 underwent total thyroidectomy. No information regarding the extent of surgery was identifiable for one patient. The median and mean tumour diameter were 27.5 mm and 30.2 mm, respectively (range: 10-80 mm). Twelve patients demonstrated no tumour recurrence, and one patient was diagnosed with nodular goitre in a contralateral thyroid lobe. For 27 remaining subjects there were no data related to the further course of the disease. The mean time to follow-up was 2 years (range 0-9 years).
The FTC subgroup included 21 women and 10 men, with a mean age at FTC diagnosis of 57 years, and a median of 59 years (range 24-83 years). Four patients were diagnosed with widely invasive FTC. The mean tumour diameter was 38.3 mm, with a median of 33.5 mm (range 15-110 mm).
However, in 13 cases, no information regarding the tumour was provided. T1, T2, T3, and T4 features were diagnosed in 4, 11, 10, and 1 patient, respectively. Five remaining subjects were staged as Tx.
Multifocal tumour growth was observed in 11 subjects. Four patients demonstrated lymph node metastases (N1) and 5 distant metastases at FTC onset. Twenty-four patients underwent total thyroidectomy; among them primary total thyroid resection was carried out in 10 subjects and two stage procedure in 14 subjects. Six patients underwent surgery primarily due to nodular goitre a few years earlier (all of them finally had total thyroidectomy). Palliative surgery was performed in one remaining patient. All but one were treated with RAI. Seventeen subjects received therapeutic RAI activity only once, whereas 7 subjects required 2 courses of RAI ablation. Six patients with disseminated disease were treated with RAI 3 times or more. Seven patients were RAI refractory. Two subjects additionally underwent external beam radiation (one patient as a palliative procedure). One patient was given sorafenib. Finally, complete remission was observed in 18 patients, asymptomatic hyperthyroglobulinaemia in 3 subjects, partial regression in 1 subject, stable disease in 2 patients, and progressive disease in 6 patients. One patient died due to FTC. The mean time to follow-up was 7.4 years (range: 0-13 years).    The number of samples describes the samples in the high-throughput gene expression experiments. The number of genes means the number of distinct genes for which it was possible to assign an Entrez ID.   Table S5. Primer design for RAS mutation profiling. Three first primers marked "_FF" were used for freshly frozen materials and all regions containing 3 mutation sites were amplified. Other primers were used for the FFPE materials.