The study protocol was approved by the hospital ethics committee and written consent were obtained from patients’ parents.This study was approved by the ethical and scientific review board of the First Affiliated Hospital of Zhengzhou University (2019-KY-0202). We confrm that all methods were performed in accordance with the relevant guidelines and regulations. We included 169 children (40 males and 129 females), ranging in age from 6 to 18 years, who had received their first surgical treatment in our hospital and in whom postoperative histopathology showed papillary thyroid carcinoma. There was no history of radiation exposure in these patients. The following data were recorded in each patient: age, sex, tumor size, unilateral/bilateral involvement, presence/absence of diffuse microcalcification and multifocal, extrathyroidal extension, Tumor Nodes Metastasis (TNM) stage, Thyroid-Stimulating Hormone (TSH) level, treatment received, and findings on follow-up. All cases were diagnosed and managed in accordance with the 2015 American Thyroid Association (ATA) guidelines for the diagnosis and treatment of pediatric thyroid nodules and differentiated thyroid cancer. All histologic slides were reviewed by two independent pathologists according to the 2010 American Joint Committee on Cancer (AJCC) Guidelines, Edition 7.
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
The mutational analysis was performed on formalin-fixed, paraffin-embedded tumor tissue from the thyroid resection specimens. Three to six pieces of unstained sections (10 μm thick) from the tumor were deparaffinized and macro-dissected. DNA was extracted using a Qiagen tissue DNA extraction kit according to the manufacturer’s protocol (QIAamp DNA FFPE Tissue Kit, QIAGEN, Germany).
BRAF exon 15 was amplified using a BRAF mutation detection kit. Briefly, 5 μL DNA was added to 35 μL of the amplification system. The upstream primer sequence of BRAF was 5 '-ATGCTTGCTCTGatagGAA-3' while the downstream primer sequence was 5 '-GCATCTCAGGGCCAAA-3'. Polymerase Chain Reaction (PCR) amplification was then performed. The amplification conditions were as follows: pre-denaturation at 95°C for 15 min; then denaturation at 94°C for 30 sec; annealing at 64°C for 20 sec; extension at 72°C for 30 sec for 35 cycles, the last 72°C extension being for 10 min. BRAF amplification products were purified and sequenced by the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Each sample was tested at least three times to ensure the repeatability of sequencing results.
The Chi-square test was used to assess whether BRAFV600E mutations were associated with age, sex, unilateral/bilateral involvement, presence/absence of diffuse microcalcification and extrathyroidal extension, multifocal/unifocal involvement, TNM stage, TSH level, treatment, and findings on short-term follow-up. Student’s t test and Mann-Whitney test were used appropriately for continuous numerical data, after applying the Shapiro-Wilk test for normality. Continuous data were summarized as median and range or mean ± standard deviation. All statistical calculations were performed using the SPSS Statistics 22.0 (IBM Inc., Armonk, New York, USA). A p value of <0.05 was considered statistically significant.