BRAF V600E Mutations in Papillary Thyroid Carcinoma: Their Relation to Clinical Features and Oncologic Outcomes in A Single Cancer Centre Experience

Introduction BRAF V600E is one of the most common mutations in Papillary Thyroid Cancer (PTC). Its clinical correlation has been extensively studied with contradictory results. The aim of this study is to evaluate the oncological impact of BRAF V600E mutation on a cohort of Middle Eastern PTC patients treated at a single institute. Methods Patients with histologically conrmed PTC that were treated surgically between 2006 to 2015 were included in the study. Formalin xed paran embedded tumor blocks were sectioned and tested for BRAF V600E mutation. Short- and long-term oncological outcomes were collected. Results 128 patients (68% females) were included with a mean age of 38 years (±13.8). Median follow-up was 50 months. BRAF V600E mutation was found in 71% of patientsI The BRAF negative tumors were signicantly larger than the BRAF positive (3.47 cm versus 2.31 cm respectively, P = 0.009). All other clinicopathological characteristics were comparable between BRAF V600E mutation positive and negative groups. The two groups showed similar 5-year Disease-free (P= 0.37) and Overall survival rates (P = 0.94). Conclusion BRAF V600E mutation did not affect loco-reginal recurrence, distant metastasis, overall and disease-free survival. These results support the diversity of BRAF V600E signicance among various ethnicities.


Introduction
Thyroid cancer, with its most prevalent papillary subtype, is the most common malignant endocrine tumor worldwide 1 . In Jordan, thyroid cancer ranked ninth among all cancers with an incidence of 2.9% in 2001.
This incidence raised to 4.3% in 2013 making thyroid cancer the 4th most common malignancy in Jordan 2 . This change is also re ected by a similar increase worldwide. Papillary thyroid cancer (PTC) subtype had the lion's share of this increase and accounted for approximately 85 to 88% of all thyroid cancer diagnoses. The incidence of other subtypes remained unchanged 3 .
The reason behind this increase in incidence is controversial. A few have attributed this trend towards an abundant clinical surveillance, improved diagnostics such as high resolution thyroid ultrasound, bedside availability of such imaging modalities, an expansion in their indications, along with an increasing use of ne needle aspiration biopsy 4 .
An increased detection of occult papillary thyroid cancer on pathologic examination of thyroid glands removed for benign conditions and reported as thyroid cancer cases to national cancer registries may also contribute towards the increasing incidence of thyroid cancer 5 .
Despite the increasing incidence, the mortality rates for well differentiated thyroid cancer remained relatively stable. The 30-year disease-speci c survival rates can exceed ninety ve percent. In-spite-of that, a subset of patients with metastatic disease have a glimmer outcome with a 5-year survival as low as 56% 6 . Although treatment is often curative, there is a 15% recurrence rate over the course of 10 years, and approximately 10% of patients die as a result of disease progression 7,8 .
BRAF mutations, are the most common genetic events in thyroid cancer with the highest incidence in PTC. Various studies reported an incidence ranging between 29 to 88% of all PTC cases 9 . BRAF V600E is the most common of BRAF mutations. Its clinical correlation with PTC has been extensively studied with contradictory results [10][11][12][13] . The aim of this study is to evaluate the incidence and clinical impact of BRAF V600E mutation on a cohort of PTC treated at a single institute with disease free & overall survival as the primary end point.

Methods
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 ne needle aspiration cytology (US-FNAC) was used for evaluation.
Tumor-node-metastasis (TNM) staging was de ned 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 con rmed 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 xed para n 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 puri ed 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 ve somatic mutations in the BRAF gene including: V600E, V600E complex (V600Ec), V600D, V600K, and V600R.

Statistical analysis
Patients characteristics, clinical, pathological ndings, 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 Ttest 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. Signi cance was de ned as P value less than 0.05. Statistically signi cant factors on univariate analysis were included in the multivariate model.

Patient's characteristics
A hundred and twenty-eight patients were included with a mean age of thirty-eight years (±13.8) at the time of diagnosis. Forty-one (32%) patients were men and eighty-seven (68%) were women. Patients were followed for a median of fty-month post-surgical resection.
The mean size of the primary tumour was 2.6 cm (±2.2). Eighty-six percent of the patients were staged as stage I PTC. Of the 128 patients with conventional PTC, BRAF V600E mutation was found in 91 (71%) patients. Table 1 lists the characteristics of the 128 patients with conventional PTC included in the study.  With a median follow-up of fty months, the two groups showed similar 5-year Disease-free survival (DFS) (P = 0.37) (Fig. 1) and Overall survival (P = 0.94) (Fig. 2).

Risk Factors Affecting The Disease-free And Overall Survival
To assess the effect of several risk factors on survival time in patients with PTC, the disease-free and overall survival analysis was extended using univariate and multivariate Cox regression model. The BRAF status, gender, family history, concomitant multinodular goiter, Hashimoto Thyroiditis, multifocality, multicentricity, and thyroglobulin level post-operatively, and extent of neck dissection were entered into the model. Female gender was the only factor signi cantly associated with improved disease-free survival on univariate (Hazard Ratio: 0.637, P = 0.05). None was associated with improved survival on multivariate analysis. (Table 3). As for the overall survival (Table 4); Patients diagnosed below the age of 50 were signi cantly associated with improved overall survival on univariate (Hazard ratio 0.031, P = 0.001) and multivariate analysis (Hazard ratio 0.037, P = 0.003).

Discussion
In this study, we examined the clinicopathological factors associated with BRAF V600E mutation. We also explored the relationship between BRAF V600E and the oncological outcomes in PTC. The BRAF V600E mutation is considered a speci c diagnostic and prognostic marker in PTC. V600E is a point mutation at codon 600 of BRAF gene. This causes a constitutive activation of the BRAF kinase and an uncontrolled activation of the MAP Kinase signalling pathway.
Previous publications have linked BRAF mutation with worse prognostic features. This includes age at diagnosis, male sex, multifocal tumor, and advanced TNM stage 10,11,14−18 . Xing et al reported a signi cant association between BRAF mutation, adjacent structures invasion, and lymph node metastasis 10 . Other reports found BRAF mutation to independently predict Central lymph nodes metastasis 18 . At multivariate analysis of BRAF V600E mutation showed an independent correlation with worst outcome.
Moreover, the survival curves of PTC patients showed a worse survivor in the BRAF V600E-mutated group 19 .
These reported outcomes are in contradiction with the result of our current study. Other large retrospective studies were consistent with our ndings and failed to corroborate the above reported associations 12 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.