This is a retrospective chart review study approved by the Institutional Review Board (IRB) at King Hussein Cancer Center (KHCC). IRB (Ref: 15KHCC101). The KHCC IRB is guided by the principles described in the World Medical Association’s Declaration of Helsinki (1964) and its amendments.
Because of the retrospective nature of the study and the lack of personal or clinical details of participants that compromise anonymity, consent was waived and the study was approved by King Hussein Cancer Center Institutional Review Board (IRB). The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Study Cohort And Tumor Samples
Patients with primary thyroid carcinomas were analyzed between the years of 2006 to 2015.
In all cases, curative hemithyroidectomy or total thyroidectomy with or without neck dissection was performed. Radio-active Iodine was given according to institution guidelines.
All patients were regularly followed with physical examinations, thyroid function tests and neck ultrasonography every 6 to12 month after the initial surgery. If suspicious thyroid nodules or lymph nodes were found, ultrasound-guided fine needle aspiration cytology (US-FNAC) was used for evaluation.
Tumor-node-metastasis (TNM) staging was defined based on the eighth edition of the American Joint Committee on Cancer (AJCC) staging system.
The study was reviewed and approved by the local institution review board at King Hussain Cancer Center KHCC.
Molecular Testing For Somatic Genetic Changes
All the retrieved Hematoxylin and Eosin (H&E) stained sections for the cohort cases were reviewed separately by two experienced histopathologists in endocrine pathology. The pathology diagnoses were reviewed and confirmed to be papillary thyroid carcinoma.
The most appropriate slide for BRAF molecular testing was determined based on the percentage of primary thyroid tumor and lymph node metastasis if present. Ten percent was deemed as minimal accepted tumor percentage on the selected slides. Five sections of approximately 5 to 10 micrometer thickness were sectioned from the formalin fixed paraffin embedded (FFPE) tumor tissue corresponding to the selected slides. Sectioned tissue was collected in Eppendorf tubes with the appropriate labelling. The deoxyribonucleic acid (DNA) was extracted and purified using the QIAamp(r) DNA Mini Kit (Qiagen). Samples were assessed for DNA concentration and purity using the NanoDrop(r) ND-1000 spectrophotometer. BRAF mutation testing was performed using therascreen(r) BRAF RGQ PCR Kit on the QIAGEN Rotor-Gene Q MDx instrument, that is designed to detect five somatic mutations in the BRAF gene including: V600E, V600E complex (V600Ec), V600D, V600K, and V600R.
Statistical analysis
Patients characteristics, clinical, pathological findings, and clinical outcomes were collected in a retrospective manner. Data was analysed using the software package SPSS 24 (Chicago, Illinois, USA). Results were expressed as medians and interquartile ranges (IQR) or mean and standard deviation (SD). Comparison between the two groups was performed using the χ2 test for categorical variables and the T-test for continuous variables. Survival functions were compared using the non-parametric Kaplan-Meier estimator. Clinical and pathological predictors of overall and disease-free survival were analyzed using univariate and multivariate Cox proportional-hazards models. Significance was defined as P value less than 0.05. Statistically significant factors on univariate analysis were included in the multivariate model.