BRAF mutation is associated with aggressive tumor characteristics of PTC, such as extrathyroidal extension, lymph node and/or distant metastases, and advanced tumor stage at presentation [1, 22, 23] see Box 11.1. Additional evidence for more aggressive tumor behavior comes from the analysis of tumor stage. A significant proportion of BRAF-mutation positive tumors present at the advanced stage of the disease when compared with tumors that harbor the RET rearrangement, RAS mutation, and those with no mutations [1]. This can be attributed to the fact that BRAF mutation-positive patients are older and have a higher incidence of extrathyroidal extension and lymph node metastasis, the combination of which yields a higher tumor stage and consequently, less-favorable prognosis. Other studies have found an association between BRAF mutation and distant metastasis [10, 24]. More importantly, BRAF mutation has been found to be an independent predictor for resistance to radioactive iodine therapy and other treatment failures, and tumor recurrence even in patients with low-stage disease and as an independent risk factor for tumor-related death [4, 25–29].

The MAPK pathway plays a major role in tumor development and progression of PTC. Consequently, the MAPK pathway is a potentially effective therapeutic target for PTC. In recent years, a lot of work has been done to investigate the therapeutic potential of suppressing the pathway in human cancers. For thyroid cancer, recent effort has been particularly directed toward testing the therapeutic potential of knocking down the MAPK pathway signaling aberrantly activated by BRAF mutation. RAF kinase inhibitor AAL881 has been demonstrated to inhibit MEK and ERK phosphorylation and also induce apoptosis preferentially in cells that harbor BRAF V600E mutation when compared with wild-type ones, possibly because of better inhibitory activity against BRAF(V600E) [30]. Another inhibitor of RAF kinase activity, LBT-613 is also thought to hold promise for most forms of PTC. It blocks RAF-dependent MEK and ERK phosphorylation and also potently blocks MEK phosphorylation in human thyroid cancer cell lines with BRAF V600E mutations [31]. Efforts have also been made to develop strategies targeting MEK with anticancer drugs to suppress the MAPK pathway signaling. MEK inhibitor CI-1040 inhibits cell proliferation in thyroid cancer cells harboring BRAF V600E mutation [32].

The various small molecule inhibitors of activated BRAF serine/threonine kinase that have been developed are categorized on the basis of their mechanism of action. Type I inhibitors target a highly conserved motif containing a catalytic aspartate residue in the so-called “DFG-in” conformation, leading to interactions with the kinase hinge region and ATP binding site of the activated form of the kinase while Type II inhibitors bind to the ATP binding site itself and an adjacent hydrophobic pocket created by the activation loop. Type I inhibitors with preferential binding to the kinase domain of BRAF in the active conformation demonstrate greater inhibitory potency against the BRAF V600E mutant kinase than the wild-type [33]. In thyroid cancer cells where, RAF1 (also known as CRAF) expression is thought to be minimal, selective targeting of the mutant BRAF most associated with aggressive disease is a highly attractive therapeutic option [34]. Vemurafenib is a potent kinase inhibitor of BRAF V600E whose effect has been studied in patients with malignant melanoma. It inhibits proliferation and ERK phosphorylation in cell lines bearing activating mutations in codon 600 in a dose-dependent manner [35]. Preclinical studies in PTC have mimicked the experience with melanoma [36]. Regorafenib inhibits both wild-type and mutant BRAF and has been studied in a phase 1 clinical trial of continuous daily dosing that has been previously reported [37]. There was no objective response in any of the five thyroid cancer patients in the trial.

Fine needle aspiration (FNA) cytology is currently the most reliable diagnostic test in the evaluation of thyroid nodules [38, 39]. An estimated 20–30 % of FNA specimens present with equivocal cytological findings and are thus classified as indeterminate. Lesions in the indeterminate category include follicular lesions of undetermined significance/atypia of undetermined significance (FLUS/AUS), follicular neoplasm/suspicious for follicular neoplasm and suspicious for malignancy [40, 41]. Preoperative planning of optimal surgical management in patients with these cytologically indeterminate lesions may be challenging, with significant management implications ranging from delay in definitive surgery to unnecessary surgical intervention. To avoid this dilemma, a number of studies have evaluated the use of cytological preparations for BRAF V600E molecular analysis and they have shown that it improves the accuracy of cytologic diagnosis of thyroid nodules [42–48]. In view of the high specificity of BRAF mutation in the detection of PTC, indeterminate lesions with BRAF mutation are triaged for total thyroidectomy with or without central lymph node dissection. This eliminates the need for second surgery in up to 40 % of patients with indeterminate cytologic diagnosis. For patients with follicular neoplasm and suspicious for malignancy diagnoses and are BRAF mutation negative, lobectomy with or without intraoperative consultation is indicated. For BRAF mutation negative FLUS/AUS lesions, patients may be followed up conservatively for 6–12 months unless there are other concomitant risk factors [42, 43, 49]. It has been shown that the presence of preoperative detection of BRAF V600E mutation is perhaps the only available preoperative clinical parameter, which independently predicts central compartment lymph node metastasis in patients with PTC. This further supports BRAF mutation testing in the preoperative setting thereby guiding the extent of initial surgical management for patients with PTC [50–52].

Exon 15 nucleotide alterations account for approximately 90 % of all BRAF mutations in thyroid cancer and the additional 10 % of mutations involve exon 11. Ideally, a comprehensive diagnostic assay should be optimized to detect mutations within both these regions. However, for diagnostic purposes, more than 95 % of clinically relevant mutations can be covered by assays examining exon 15, particularly the V600E mutation. In clinical practice, almost all available methodologies are PCR-based assays and essentially any type of tissue or cytological material can be used for BRAF mutation testing. BRAF mutation testing requires a much higher analytical sensitivity and specificity in dealing with cytological specimens where the tumor cells may be a minor population in a background of non-neoplastic cells. While PCR-Sanger sequencing, allelic-specific amplification, melting curve analysis [53, 54], real-time PCR are common laboratory methods, BRAF mutation detection methods that have the highly desired sensitivity and specificity appropriate for testing minimal tissue or cytological samples include shifted termination primer extension assay (STA), single-strand conformation polymorphism (SSCP) analysis, pyrosequencing, and coamplification at low denaturation temperature—PCR (COLD-PCR) [55].

Single-strand conformation polymorphism (SSCP) analysis detects randomly distributed mutations. It is the traditional method for high-sensitivity detection of a known point mutation [56–58]. When combined with follow-up Sanger sequencing of abnormal SSCP products, the method is able to direct variant mutations within the amplicon with high sensitivity and specificity. DNA is extracted and amplified using primers flanking BRAF exon 15. PCR products are then analyzed by polyacrylamide or capillary gel electrophoresis. The SSCP band pattern is compared to that of a positive BRAF V600E DNA control sample, for interpretation. If abnormal bands are present but do not match those of the positive control, the abnormal SSCP bands are purified from the gel and Sanger sequencing is then performed to identify the variant mutations of exon 15 [55]. The drawback of this method is that it does not allow identification of the precise nucleotide change and requires an additional complementary method. In addition, the method is also labor intensive.

More recently, the use of VE1 immunohistochemistry in the assessment of BRAF mutational status in thyroid core needle biopsy has been investigated [59]. The results demonstrated good concordance between immunophenotypical VE1 expression and V600E mutational status. Homogeneous BRAF immunohistochemical staining occurs in the vast majority of cases and absent/faint staining for BRAF correlates with the lack of BRAF V600E mutation. However, only strong immunohistochemical staining is highly correlated with the BRAF mutation in thyroid carcinomas including papillary, poorly differentiated, and anaplastic histological types. Moderate staining cannot be relied on and molecular testing should be performed to confirm the presence of BRAF mutations [60].