BRAFV600E hot spot mutation in thyroid carcinomas: first Moroccan experience from a single-institution retrospective study

Background The incidence of thyroid cancer is increasing worldwide at an alarming rate. BRAFV600E mutation is described to be associated with a worse prognostic of thyroid carcinomas, as well as extrathyroidal invasion and increased mortality. Objective To our knowledge, there are no reported studies neither from Morocco nor from other Maghreb countries regarding the prevalence of BRAFV600E mutation in thyroid carcinomas. Here we aim to evaluate the frequency of BRAFV600E oncogene in Moroccan thyroid carcinomas. Methods In this Single-Institution retrospective study realized in the Anatomic Pathology and Histology Service in the Military Hospital of Instruction Mohammed V ‘HMIMV’ in Rabat, we report, using direct genomic sequencing, the assessment of BRAFV600E in 37 thyroid tumors. Results We detected BRAFV600E mutation exclusively in Papillary Thyroid Carcinomas ‘PTC’ with a prevalence of 28% (8 PTC out 29 PTC). Like international trends, Papillary Thyroid Carcinomas ‘PTC’ is more frequent than Follicular Thyroid Carcinomas ‘FTC’ and Anaplastic Thyroid Carcinomas ‘ATC’ (29 PTC, 7 FTC and 1 ATC). Conclusion Our finding gives to the international community the first estimated incidence of this oncogene in Morocco showing that this prevalence falls within the range of international trends (30% to 90%) reported in distinct worldwide geographic regions.


Introduction
Thyroid cancer is the commonest malignant endocrine tumour. Follicular thyroid cells (thyrocytes) represent the major part of thyroid cells, and tumors derived from these cells are classified depending on their histological and genetic characterization, in Follicular Thyroid Carcinomas 'FTC', Papillary Thyroid Carcinomas 'PTC' and undifferentiated Anaplasic Thyroid Carcinomas 'ATC' 1 . PTC is the major form of thyroid carcinomas accounting more than 80%; and BRAF V600E hot spot mutation is the most frequent genetic alteration of the MAPK pathway in PTC 1 ; 2 ; 3 ; 4 ; 5 ; 6 ; 7 . RAF proteins (A, B and C), serine threonine kinases, are key regulators of the mitogen-activated protein kinase (MAPK) pathway. BRAF mutations have been identified in various cancers with high frequency in melanomas (50-60%) and thyroid cancers (30-90%); and BRAF mutations are mostly V600E substitution 4 ; 8 ; 9 ; 10 ; 11; 12 ; 13 ; 14 ; 15 .
A thymidine-to-adenosine transversion at exon 15 nucleotide 1799 (T1799A) of the BRAF gene, causing a valine-to-glutamic acid change in codon 600 of the BRAF protein, the most common mutation of BRAF, leads to a constitutive activation of MEK/ERK pathway independently of RAS activation. BRAF V600E mutation is associated with a high level of ERK signature because the mutated form of BRAF V600E does not respond to the negative feedback of ERK 5; 16 . Several studies have investigated the clinical significance of BRAF V600E mutation in thyroid carcinomas and its role as diagnostic and prognostic marker remains unclear 1 ; 17 ; 18 .
Molecular assessment of BRAF V600E mutation, frequently found in classical forms of PTC and rare in follicular variant forms of PTC (FVPTC), might in some cases distinguish between PTC and FVPTC overcoming the ambiguity of the histological and cytological diagnosis 1 ; 19 . Also, the presence of BRAF V600E mutation might discriminate between benign and malignant thyroid nodules during a fine-needle biopsy (FNB) cytological examination 1 ; 20 . In fact, molecular detection of BRAF V600E , as an early event in thyroid tumorigenesis, could be particularly helpful for clinicians to improve diagnosis of thyroid nodules sampled by FNB and classified by cytology as indeterminate for malignancy or AUS/FLUS "atypia of undetermined significance/follicular lesion of undetermined significance. Although the single mutational testing for BRAF V600E has high specificity for thyroid malignancy, the 2015 ATA guidelines do not recommended the systematic use of the single molecular status of BRAF; and a mutational panels (RAS, BRAF, RET/PTC, ..) might be most helpful for appropriate and individual management of thyroid carcinomas (Haugen BR et al., 2015 ATA guidelines). Concerning the clinical use of BRAF V600E as prognostic marker, conflicting conclusions are deduced from the literature 1 ; 18 . Owing to the ability of BRAF V600E oncogene to predispose thyroid tumors to dedifferentiation, and knowing that thyroid de differentiation leads to the resistance to radioiodine therapy (I131) of thyroid tumors, BRAF V600E is thereby considered as one of the most powerful prognostic marker for thyroid carcinomas. PTC tumors harboring BRAF V600E show often a loss/decrease expression of the Natrium Iodide Symporter (NIS) which plays a central role in the treatment of thyroid cancer by radioiodine therapy (I131); and PTC-BRAF V600E tumors are reported to be refractory to radioiodine therapy 5 ; 6 ; 21 ; 22 . In the same way, a retrospective study of 1849 patients followed and treated for PTC concluded that BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC 4. In addition, several studies have shown that BRAF V600E oncogene is associated with extrathyroidal invasion, dedifferentiation, loss of radioiodine avidity, and resistance to radioiodine therapy 5 ; 6 ; 7 ; 21 ; 22 ; 23 . However, other groups did not find association between BRAF V600E and worse prognostic and mortality in thyroid cancer 12; 17 ; 24 ; 25 . Using a single stranded conformation polymorphism followed by direct sequencing, any association between BRAF V600E mutation and tumor aggressiveness has been observed 17; and using pyrosequencing, Barbaro D et al., deduced that BRAF V600E mutation is not associated with a worse prognosis.
According to the literature, this apparent discrepancy, concerning the clinical significance of BRAF V600E mutation, could be explained by numerous parameters including particularly 1) the detection method used for BRAF V600E testing and 2) the activation threshold of ERK signalling pathway in thyroid tumors harboring BRAF V600E . Unlike direct sequencing of BRAF V600E , the quantitative sequencing approach 'pyrosequencing' that allow the determination of the ratio of BRAF V600E /BRAFwt is usually advocated for thyroid tumors management 26; 25 . Higher prevalence of BRAF V600E mutation has been observed with pyrosequencing compared to direct sequencing 27;28 ; and the authors recommended the quantitative preoperative analysis of BRAF V600E by pyrosequencing, which could refine the PTC risk stratification. Marotta V et al., discussed the limitation of qualitative BRAF V600E determination and highlighted the additional value of the quantitative detection of BRAF V600E mutation that could correlate the presence of BRAF V600E to the threshold of mutated alleles associated with a poor prognostic 18 .
The incidence of thyroid cancer, especially PTC, is increasing worldwide at an alarming rate ; and by 2019, papillary thyroid cancer will double in incidence and become the third most common cancer in women in the United States of America 29 ; 30 . Thereby, thyroid cancer is increasingly a major public health issue, particularly for women. According to the Casablanca registry (2005-2007 ; Morocco) and Rabat Cancer Registry (2006-2008 ; Morocco), thyroid malignancies are classified respectively in the third (6.7 per 100 000 persons) and in the fifth range (3.9 per 100 000 persons) 31 ; 32 ; 33 . This alarming incidence illustrate that thyroid cancer is one of the most frequent female cancers in Morocco. The population-based cancer registry of Casablanca and Rabat are the two principal cancer registries in Morocco including different types of useful data (global incidence and mortality of each cancer, Age-standardized Incidence and Mortality, survival analysis…). There are no reported studies neither from Morocco nor from other Maghreb countries regarding the prevalence of BRAF V600E mutation in thyroid carcinomas. In this retrospective Single-Institution study, we analyzed the prevalence of BRAF V600E hot spot mutation in 37 human thyroid tumors blocks archived in the Anatomic Pathology and Histology Service in the Military Hospital of Instruction Mohammed V 'HMIMV' in Rabat, Morocco. We found that, like international trends, this mutation occurs exclusively in PTC tumors and that its prevalence (28%) falls within the range of international trends.

Patients and methods Samples
Formalin-fixed paraffin-embedded (FFPE) thyroid tumor blocks: This retrospective study is carried out according to the approval local ethical committee of the Faculty of Medicine and Pharmacy in Rabat. We have included all available thyroid tumors blocks showing at the minimum 50% of estimated tumor cell percentage after HE staining 'Hematoxylin and Eosin'. All thyroid cancer cases are arising from follicular origin and operated at the Military Hospital of Instruction Mohammed V 'HMIMV' in Rabat between January 1999 and December 2012. After collection of all available clinical informations from registries, and after estimation of the tumor cell percentage by the experimented Pathologists from Anatomic Pathology and Histology Service in the HMIMV of Rabat, only 37 thyroid tumors responding to our inclusion criteria (availability and tumor cell percentage) are chosen for genomic DNA extraction. BCPAP Cell line: derived from human PTC (heterozygous for BRAF V600E ) is cultured as described in Dulbecco's modified Eagle's medium (DMEM) (4.5 g/l glucose) (Life Technologies) supplemented with 10% (vol/vol) FCS (Life Technologies) and penicillin/streptomycin (100 mg/ml; Life Technologies) as previously described 22 .

Genomic DNA extraction
After collection of 4 sections of FFPE tissue in Eppendorf tubes (5 µm/section), samples were deparaffinized and digested with proteinase K at 56°C overnight. Genomic DNA was extracted using Bioline kit according to manufacture's protocol; and the quality of extracted DNA was checked using the Qubit fluorometer. It is important to note that the experimented pathologists make one staining HE before and after cutting sections for genomic DNA to be sure that extracted genomic DNA is from the part of tumor blocks containing at the minimum 50% of estimated tumor cell percentage.
The PCR products are purified using the Exo-SAP prior sequencing with the Big Dye Terminator sequencing kit (Applied Biosystems, Foster City, CA). It is important to note that PCR products were bidirectionally sequenced using primers complimentary to the Forward and Reverse tags. The products were analyzed on an automated 3730 DNA Analyzer (Applied Biosystems). Sequence reading and alignment were performed with the SeqScape1 software (Applied Biosystems). The positive samples for BRAF V600E are confirmed with another experimentation (PCR and sequencing). DNA extracted from human thyroid BCPAP cell line (BRAF V600E / V600E ) is used as positive control for BRAF V600E detection.

Exploration of data from The Cancer Atlas Genome (TCGA)
Based on the conventional classification, several subtypes of PTCs are grouped in the same group. Recently, a comprehensive multiplatform analysis of homogenous cohort of 496 PTCs developed from The Cancer Atlas Genome (TCGA) has been performed, and allows reclassification of papillary thyroid cancers into molecular subtypes (5). This cohort offers a better understanding and clustering of PTC disease based on Thyroid Differentiation Score (TDS), BRAF-RAS Score (BRS), downstream signalling pathway activated by each pathogenic mutation, and risk assessment. We explore this large cohort of PTC samples from TCGA concerning the prevalence of BRAF V600E mutation and the risk of tumor recurrence already performed from TCGA.

Results and discussion
Trends by sex and age and Histological types: After clinical registries analysis, checking the availability of tumor blocks and estimation of tumor cell percentage in each available FFPE block, only 37 cases were included in this study. The results summarized in Table 1 show that thyroid carcinomas is more frequent in female confirming what was reported in literature (around three times more thyroid cancers in women than in men) 33 ; 34 ; 35 . Furthermore, thyroid cancer is diagnosed in older age in men compared to women (50.9±6.1 Vs 42.6±2.1) ( Table 1). Based on histological type classification, we evaluate the frequency of PTC, FTC and ATC in this cohort from the Anatomic Pathology and Histology Service of the HMIMV in Rabat which is at our knowledge the first experience from Morocco. We find that PTC is more frequent accounting for 78% followed by FTC (19%) and finally ATC (3%) ; and this profile distribution is in concordance with the international trend 1.

BRAF V600E mutation
We have used DNA from BCPAP cell line, which are BRAF V600E homozygous, to validate BRAF V600E detection method (data not shown). The presence of BRAF V600E in the FFPE blocs is detected using the same method. Figure 1 shows electropherogram of BRAF wild-type PTC and heterozygous BRAF-mutated PTC (Fig.1). In our study, BRAF V600E hot spot mutation is detected exclusively in PTC and any mutation has been found in FTC (Table 2). This first report from this single-institution study in Morocco is in concordance with international literature concerning the specificity of BRAF V600E for PTC compared with FTC 1 ; 8 . This mutation can occur rarely in follicular variant of PTC 'FVPTC' 19 , but in our retrospective study all PTC have only a classical forms. According to the literature and unlike the most common BRAF mutation observed in PTC, K601E mutation in the Exon 15 of BRAF has been described in some cases of both classical FTC 36 and FVPTC 19; and in our study any BRAF K601E was detected in FTC (Table 2). . The prevalence of BRAF V600E mutation in our study (28%, Table 2) falls within the range of international trends (30% to 90%) reported in distinct worldwide geographic regions. These results could indicate no apparent geographic specificity regarding the molecular statut of BRAF V600E mutation in our cohort. Other studies must be carried out including different regions from Morocco, focused on a large cohort and using pyrosequencing even if a good concordance (94%) between the both sequencing methods (direct sequencing vs pyrosequencing) has been reported 25 .
In this retrospective study, 5 PTC patients from 29 showed lymph node metastasis (Table 2) in which 3 tumors are harboring BRAF V600E mutation. This finding is in concordance with the already reported association between BRAF V600E oncogene and the worse clinical prognostic of PTC as well as the extrathyroidal invasion 4 ; 5; 7 . We explore a large cohort of 496 PTCs from The Cancer Atlas Genome (TCGA) 5 and we observe that BRAF V600E mutation is more prevalent in thyroid tumors when the risk of recurrence is not low (Fig.2). This observation confirms the reported positive correlation between the presence of this hot spot mutation and the poor prognosis and aggressively of thyroid tumors (BRAF V600E positive). However, we observe also the wild type form of BRAF when the risk of recurrence is not low (Fig.2) highlighting the need of the identification of supplementary marker(s), downstream BRAF V600E , that could refine the clinical use of BRAF V600E in the management of thyroid carcinomas. In fact, the PTC tumors harbouring BRAF V600E (PTC-BRAF V600E ) are heterogeneous and this mutation is detected in two subgroups of PTC showing different TDS (Thyroid Differentiation Score) and distinct level of ERK signature 5 . Interestingly, the more aggressive PTCs-BRAF V600E are highly dedifferentiated (TDS is negative) and showed a high level of activation of ERK 5 . Recently, Azouzi et al., demonstrated that BRAF V600E down-regulated NIS in two human thyroid tumor cell lines through a redox mechanism involving the NADPH oxidases NOX4 22 . The exploration of data from TCGA showed that NOX4 is positively correlated with 1) dedifferentiation of PTCs-BRAF V600E , and with 2) ERK activation 22 .

BRAF
V600E mutation is detected in PTC with low and high risk of recurrence and its prevalence is more important in PTC with poor prognosis.
Taken together, more deep investigations are needed to clarify how thyroid dedifferentiation, ERK activation level and NOX4 expression level could be used in the clinical routine as a panel of markers for the individual management of thyroid carcinoma harbouring BRAF V600E mutation.
Although the importance of this first study in Morocco concerning the non-negligible prevalence of BRAF V600E oncogene in thyroid tumors, studies including more hospital centers from different geographic region are needed to precise the national prevalence of BRAF V600E in thyroid tumors as well as its possible clinical significance.