BRAF and KRAS mutations in papillary thyroid carcinoma in the United Arab Emirates.

Background Papillary thyroid carcinoma (PTC) is the most common malignant thyroid neoplasm comprising 80–90% of all thyroid malignancies. Molecular changes in thyroid follicular cells are likely associated with the development of PTC. Mutations in serine/threonine-protein kinase (BRAF) and Rat sarcoma viral oncogene homolog (RAS) are commonly seen in PTC. Methods In total, 90 cases of PTC are randomly selected from archive paraffin blocks and 10μm sections were cut and processed for DNA extraction. BRAF V600E mutation and 8 types of KRAS mutations were investigated using Real Time PCR. Results BRAF V600E mutation was identified in 46% of PTC while KRAS mutations were seen in 11% of PTC. There was significant correlation between BRAF V600E mutation and PTC larger than 5cm in diameter, positive surgical margin and lymph node metastasis. BRAF V600E mutation was significantly higher in patients with less than 55-year of age than those more than 55-year of age. BRAF V600E mutation was significantly higher in patients with family history of thyroid cancer than those without. There was no significant difference in BRAFV600E mutation between males and females, PTC classic and follicular variants, unifocal and multifocal PTC. There was a significant higher percentage of BRAF V600E mutation in classic PTC than papillary microcarcinoma variant. There was no significant age, gender, histologic type, tumor size, lymph node metastasis, tumor focality, and surgical margin status differences between KRAS mutated and non-mutated PTC. Conclusion BRAF V600E and KRAS mutation are seen in a significant number of PTC in the UAE. BRAF mutation is significantly correlated with large tumor size, positive surgical margins and lymph node metastasis suggesting an association between BRAF V600E mutation and tumor growth and spread.

Introduction Thyroid cancer is the 3 rd most common cancer among UAE citizens and the 2 nd most common cancer among females in the UAE [15]; hence identification of the molecular changes may have impact on the diagnosis and treatment of PTC. In this study, we evaluate the frequency of BRAF V600E and KRAS mutations in PTC and their correlation with clinical and pathological changes. This is the first study on BRAF V600E and KRAS mutations in PTC in the United Arab Emirates.

Collection of specimens
In total, 90 formalin fixed paraffin-embedded (FFPE) tissue blocks from surgically removed thyroid specimens, during the period 2011-2016, were randomly collected from the Department of Pathology, Tawam Hospital, Al Ain, United Arab Emirates. Three-um sections were prepared from selected blocks and stained with hematoxylin and eosin (H&E) stain. All sections were examined microscopically by a pathologist who participates in this study to be sure that the sections contain significant area of PTC (>50%of neoplastic cells). One ten-um section containing tumor-rich areas was taken from each block and was put in a separate labeled Eppendorf tube. New blade was used in cutting each block to prevent tissue contamination from case to case.
The protocol of the present study conformed to the ethical guidelines of the World Medical Association, Declaration of Helsinki, and was approved by Al Ain Medical District Human Research Ethics Committee (THREC-438). Patients or their caregivers signed a written consent allowing using their anonymous material for research purposes.

Histopathological classification of selected cases
H&E stained sections of selected cases were reviewed and classified according to the 4 th edition of WHO classification of tumors of thyroid gland [3] by a pathologist participated in this project. Tumors were called classic if they show predominant papillary growth with classic papillary nuclear features. Tumors were called papillary microcarcinoma if they show predominant papillary or follicular growth with classic papillary nuclear features and have a size of �1cm in greatest dimension. Tumors were called follicular variant if they show predominant follicular growth with classic papillary nuclear features. Follicular variant has two subtypes; the encapsulated with invasion, when the tumor is encapsulated and there is invasion of the capsule, while the infiltrative subtype when the tumor lack the capsule and shows infiltration of the stroma.

Collection of demographic data of selected cases
The demographic data and the clinical information were extracted from the electronic medical files of the subjects with the identified papillary thyroid carcinoma. The collected data include age at diagnosis, gender, body mass index (BMI), tumour size, thyroiditis, focality, family history of thyroid cancer, exposure to external radiation, smoking and post surgical TNM staging.

DNA isolation
Genomic DNA was isolated from each sampled tissue sections with the REPLI-g FFPE Kit (Qiagen, Hilden, Germany) for direct whole genome amplification of DNA from FFPE tissue according to the manufacturer's instructions. Briefly, 1x FFPE lysis solution was prepared and 100 μl was added to the tissue section and mixed and centrifuge briefly. The samples were incubated at 95˚C for 10 min to melt the paraffin followed by cooling down the sample to room temperature. Then 2μl of Proteinase K was added to each sample and mixed and centrifuge briefly. Each sample was then incubated for 60 min at 60˚C and then for a further 10 min at 95˚C. Then, each 10μl of the lysed tissue section was transferred into a new micro centrifuge tube. The FFPE master mix was prepared as per manufacturer instructions on ice and vortex and centrifuge briefly. Then, 10μl FFPE master mix was added to 10 μl DNA from the lysed tissue then mixed and centrifuged briefly. Then the samples were incubated at 24˚C for 30 min. Then the reaction was stopped by incubation at 95˚C for 5 min followed by cooling down to 4˚C using a thermal cycler. Finally, the samples were incubated at 30˚C for 8 h (highyield reaction) then stopping the reaction by incubation at 95˚C for 10 min. The amplified DNA was stored at -20˚C until required for downstream applications.

Quantification of DNA
Quantifiler™ Trio DNA Quantification, Kit Catalog number: 4482910 was used to quantify the total amount of amplifiable human DNA in the sample. For the Quantifiler™ Trio DNA Quantification Kit: the Quantifiler™ Trio Primer Mix and Quantifiler™ THP PCR Reaction Mix was mixed as per manufacturer's instructions. The PCR mix was vortexed and centrifuged briefly. The 2 μL of gDNA was added to the applicable wells. The reaction plate was sealed with the Optical Adhesive Cover and care was taken to remove bubbles. The plate was centrifuged at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles. A total of 90 samples were processed for DNA isolation. The concentration of gDNA was determined using Nanodrop instrument using nuclease free water as blank solvent.

Determination of KRAS/ BRAF mutation
GenoScreen KRAS/BRAF Real Time PCR Kit (DiagCor Bioscience Inc. Ltd, Hong Kong) was used for a qualitative assessment intended for the detection of eight KRAS mutations in codon 12 and 13, and one BRAF mutation in codon 600 using real-time PCR assay. The GenoScreen KRAS/BRAF Real Time PCR Kit is developed based on PCR amplification of mutant DNA with specific primers, detected by real time polymerase chain reaction (PCR) technology. Detection of target amplified product (amplicon) is achieved by the cleavage of dual fluorescent dye labeled oligonucleotide probes during the quantitation of mutant DNA.

Procedure
All the PCR reagents were pipetted mix and spin down before use. The final PCR reaction (volume 10 μL) was prepared according to the manual. The recommended reaction component volumes to amplify DNA for KRAS/BRAF PCR Master Mix: 5μL, Primer Mix: 3μL, Template (DNA/ H2O): 2μL. From the 8 μL of PCR mixture (containing KRAS/BRAF PCR Master Mix and Primer Mix) aliquoted into each PCR reaction and added the appropriate amount of DNA template suggested and finally the reaction volume was top up to 10 μL with DNase Free Water if necessary. The mixture was then spin down and placed in real time PCR thermal cycler, QuantStudio3 (QS3). The reporter was select "FAM", "NFQ-MGB" for Quencher and "ROX" for passive reference. Amplification Profile was set at 95˚C, 10 min/1 cycle and amplification at 95˚C, 15 sec/ 50cycle finally 60˚C, 1 min FAM channel.

Data analysis and interpretation
The data was analyzed after setting the threshold for FAM signals. The cycle threshold (Ct) value was set at 1/20 of each individual marker's highest fluorescence point for the run (i.e. FAM). The FAM signal from the positive and negative assay was used to determine the validity of the real time run. The positive assay gave the Ct values between 24-38, and Negative Control assay was Ct value greater than 45

Statistical analysis
The statistical analysis was computer assisted using SPSS for windows version 20 (SPSS Inc, Chicago, USA). Student's t-test was used to compare continuous variables. Quantitative variables were analyzed with the chi-squared test and correlations of ordinal variables using the Spearman rank correlation coefficient and Chi-square (Fisher's exact) test. P value <0.05 were considered significant. Where appropriate numerical data were presented as the mean ±SD.

Demographic data
In total, 90 cases of PTC were analyzed in this study. The mean age was 41.21 ± 13.94, the mean BMI was 29.18 ± 6.01, and the female to male ratio was 2.33. Family history of thyroid cancer was seen in 13% of cases, while family history for other neuroendocrine tumors was seen in 1% of cases. History of exposure to external radiation was seen in 3% of cases. Only 8% of cases were smokers (Table 1).

Histologic types of PTC
Classic (conventional) PTC, which exhibits a predominant papillary pattern of growth with characteristic nuclear features of PTC, was the most common type comprising 46.6% (42) of the cases followed by microcarcinoma 30% (27) and follicular variant PTC comprising 23.4% (21).
Most of the papillary microcarcinomas 25 (93%) exhibit papillary pattern of growth similar to the classic PTC, and only 2 cases (7%) exhibit predominate follicular pattern of growth similar to follicular variant PTC. The follicular variant of PTC, which exhibit a predominant follicular pattern of growth with the characteristic nuclear features of PTC, has two subtypes. The infiltrative which comprises 45% (10) of the cases, and the encapsulated invasive subtype which comprises 55% (11) of the cases (Table 5).

Site of involvement
Right lobe was the most common site of PTC comprising 48% (43) of cases, while left lobe and both lobes were involved in 28% (24) and 21.2% (22) of cases respectively (Table 2).

Presence of capsule
Most of PTC were non-encapsulated and comprising 80% (72) of cases while encapsulated PTCs were seen in 20% (18) of the cases ( Table 2) (Fig 1).

Distant metastasis
Only one cases of PTC shows distant metastasis ( Table 3).

Age and BRAF V600E and KRAS mutations
BRAF V600E mutation was significantly higher in patients with less than 55-year of age than those higher than 55-year of age (P = 0.014) ( Table 5). There was no signification correlation between KRAS mutation and any age group (Table 5).

Gender and BRAF V600E and KRAS mutations
There was no signification correlation between BRAF V600E mutation and any gender group as well as there was no signification correlation between KRAS mutation and any gender group (Table 5).

Family history of thyroid cancer
There was a significant correlation between family history of thyroid cancer and BRAF V600E mutation (P = 0.036). There was no significant correlation between family history of thyroid cancer and KRAS mutation (Table 5).

History of smoking
There was no significant correlation between history of smoking and BRAF V600E and KRAS mutation (Table 5).

Exposure to radiation
There was no significant correlation between history of exposure to radiation and BRAF V600E and KRAS mutation (Table 5).

Histologic variant and BRAF V600E and KRAS mutations
The percentage of BRAF V600E mutation was higher in classic PTC than follicular variant PTC but it did not reach the statistical significant (P = 0.08). In addition, there was no significant difference in the percentage of KRAS mutation between the classic PTC and the follicular variant PTC. (Table 5). There was a significantly higher percentage of BRAF V600E mutation in classic PTC than papillary microcarcinoma (P = 0.0157), while there was no significant differences in the percentage of KRAS mutation between classic PTC and papillary microcarcinoma.

Tumor size and BRAF V600E and KRAS mutations
Tumor sizes above 4cm are significantly correlated with BRAF V600E mutation (P = 0.023). Any tumor size was not correlated with any KRAS mutation (Table 5).

Lymph node metastasis and BRAF V600E and KRAS mutations
There was a significant correlation between lymph node metastasis and BRAF V600E mutation (P = 0.002). There was no significant correlation between lymph node metastasis and KRAS mutation ( Table 5) (Fig 1).

Tumor focality and BRAF V600E and KRAS mutations
There was no signification correlations between BRAF V600E mutation and focality as well as there was no signification correlation between KRAS mutation and focality (Table 5).

Surgical margin and BRAF V600E and KRAS mutations
There was a significant correlation between positive surgical margin and BRAF V600E mutation (P = 0.43). There was no signification correlation between KRAS mutation and positive surgical margin (Table 5) (Fig 1).

Lymphovascular invasion and BRAF V600E and KRAS mutations
There was no correlation between lymphovascular invasion and BRAF V600E or KRAS mutations (Table 5) (Fig 1).

Discussion
Papillary thyroid carcinoma is the most prevalent type of thyroid cancer worldwide [16]. A lot of works have been done to identify fundamental mechanisms involved in the development of PTC [13,14,[16][17][18][19][20][21]. Many studies have shown a constant rise in the incidence of PTC in different countries all over the world over the last decades [17]. Jung et al. have shown the increase in thyroid cancer incidence during the last four decades is accompanied by a high frequency of BRAF mutations and a sharp Increase in RAS mutations [18]. Identifying molecular changes in PTC is an important step in understanding major mechanisms participate in its development as well as open new doors for early diagnosis and treatment. The MAPK pathway is an important intracellular signal transduction pathway that is required for maintaining cell proliferation, differentiation, and programed cell death in response to tyrosine kinase receptor (RTK) stimulation [19]. Moreover, it is a crucial player in the pathogenesis of PTC, as somatic mutations in its various components constantly drive the oncogenic process [20].
In this study we have identified KRAS mutation in 12% of PTC. We have identified 5 out of 8 investigated mutations in KRAS gene. Those mutations were identified in codon 12 and 13, and include KRAS_12V, KRAS_G12S, KRAS_G12D, KRAS_G13C and KRAS_G13D were seen in 7%, 2% (2), 1% (1), 1% (1), 1% (1) of PTC, respectively. In fact, to the best of our knowledge this is the first report of these mutations in KRAS gene in PTC using Real Time PCR.
In addition, there was no significant difference in the percentage of KRAS mutation between the classic PTC and the follicular variant PTC. This finding might be related to the low number of selected cases in this study.
The increase of the RAS mutations is explainable with a decrease of classic PTC variant and a sharp increase of follicular PTC variant in the last decades [18]. In our study, one quarter of the cases were pure follicular variant of PTC. Besides, the other three quarters were classic PTC, and although predominantly show classic papillary pattern, there are foci of follicular pattern as well seen in many of these PTCs, as part of histologic spectrum of classic PTC. This observation may explain the high rate of KRAS mutations in our study.
Variability in frequencies of RAS mutations reflects differences in samples, method of DNA extraction, detection method of mutations, and possible geographical difference in the pattern of RAS mutation in PTC.
RAS mutation is considered as an early molecular event in follicular cell oncogenesis that leads to a well-differentiated neoplasm and may progress to a de-differentiated tumor following the gaining of further mutations [23,24]. Knauf et al. have shown active RAS mutation can accelerate progression of cell cycle and promotes DNA damage by interfering with different cell cycle check points [24].
The percentage of BRAF mutation was higher in classic PTC than follicular variant PTC but it did not reach the statistical significant (P = 0.08). It is possible that a higher number of included cases will improve the results. We think that this is a limitation in our study. In addition, we have shown a significantly higher percentage of BRAF V600E in classic PTC than papillary microcarcinoma.
Moreover, we have also show a significantly higher percentage of BRAF V600E mutation in infiltrative follicular variant of PTC than the encapsulate with invasion subtype of follicular variant of PTC. These results suggest that BRAF V600E mutation is associated with tumor growth and spread [22,33,39].
Ugolini et al. have identified frequent BRAF V600E mutation in papillary thyroid microcarcinomas (PTMC) [41]. Knauf et al. have shown that BRAF V600E transgenic mice develops poorly differentiated PTC with aggressive behavior which confirms the oncogenic role of BRAF V600E mutation [42].
We also have shown a significantly higher frequency of BRAF V600E mutation in patients with ages younger than 55 years. Other studies [43][44][45] have shown a higher frequency BRAF V600E mutation among patients with ages higher than 55 years. We believe that this difference in the age pattern of BRAF V600E mutation is mainly due to the differences in patients samples between ours and these studies; as most of our PTC samples (70%) are from patients with ages younger than 55 years (Fig 2). In addition, a previous study in the UAE [16] have also shown more than three quarters of PTC cases were diagnosed below the age of 55 years. Geng et al. https://doi.org/10.1371/journal.pone.0231341.g002 PLOS ONE [46] have also shown a higher frequency of PTC with BRAF V600E mutation in pediatric age group of less than 10 years of age.
Moreover, we have shown a significant correlation between BRAF V600E mutation and PTC larger than 5cm in diameter and positive surgical margin suggesting an association between BRAF V600E mutation and an aggressive phenotype. This was also seen in other studies [47][48][49][50].
The association of a large size PTC with BRAF V600E mutation suggesting a critical rule of this mutation with cell proliferation. On the other hand, the clear associations of BRAF V600E mutation with positive surgical margin in this study points towards a crucial role of this mutation in increasing invasiveness of proliferating neoplastic cells. This was reported by Mesa et al., whom have shown conditional expression of BRAF V600E in thyroid cells markedly increased the Matrigel™ invasion of the transformed thyroid cells, which is more invasive than RET/PTC expressed cells [51].
We have shown a significant correlation between lymph node metastasis and BRAF V600E mutation, this finding supported by Carol et al. study [52] Lymphovascular invasion (LVI) is an important prognostic factor in in PTC and significantly associated with BRAF V600E mutation suggesting the presence of LVI should be considered as an indicator of aggressive clinicopathological features and patients with positive LVI should be followed up carefully for possible recurrence or metastasis [53]. In addition, BRAF V600E mutation is an independent predictor of lymph node metastasis in PTC [54,55].

Conclusion
BRAF V600E and KRAS mutation are seen in a significant number of PTC in the UAE. BRAF mutation is significantly correlated with large tumor size, positive surgical margins and lymph node metastasis suggesting an association between BRAF V600E mutation and tumor growth and spread.