Thyroid nodules are among the most common endocrine complaints in the world, which sometimes represents a significant diagnostic challenge in differentiating malignant from benign lesions. Currently, morphological diagnosis methods, such as immunohistochemistry and cytology, play an important role in differentiating malignant from benign nodules. For instance, depending on the histomorphologic features of routine hematoxylin and eosin (H&E), cytology of FNABs often provides a definitive diagnosis and sound therapeutic guidance. However, a substantial number of cases cannot be diagnosed by this technique and many cases generate indeterminate results . Nowadays, the role of molecular markers in cancer diagnosis and treatment has been established, including thyroid cancer [25, 26]. For example, galectin-3, fibronectin-1, CK-19, CK-903, CITED1, Ret oncoprotein, TG, Ki67, anti-MAP kinase, p16, and the mesothelial cell surface protein HBME-1 have been used as preoperative diagnostic markers [27–32]. Unfortunately, none of them are specific for thyroid malignancies.
In recent years, testing for the BRAF
mutation has been extensively used to improve the diagnostic accuracy of thyroid malignancy in nodules, because of the high specificity of this mutation for PTC [12–14, 19, 25, 26, 33]. BRAF
mutation, which is the most common oncogenic genetic event found in thyroid cancer, particularly in PTC, results in constitutive and oncogenic activation of BRAF kinase in the MAPK signaling pathway . Through activating MAPK pathway, the BRAF mutation plays a fundamental role in the tumorigenesis of PTC and predicts its poor clinical outcomes [10, 11]. In this study, we tested the role of BRAF mutation in the diagnosis of malignant thyroid nodules. The data showed that the prevalence of BRAF mutation was more than 70%, and was highly specific for PTCs, as supported by our data that all BRAF
-positive cases were histologically diagnosed as PTC after thyroidectomy. These observations suggest that molecular testing for BRAF mutation has an important impact on the diagnosis of thyroid malignant nodules, particularly PTC, as well as on decisions concerning the extent of surgery, particularly in the cases with indeterminate fine-needle aspiration cytology. Generally, some patients with indeterminate cytological findings undergo total thyroidectomy, whereas others choose to receive hemithyroidectomy. The patients in the latter group who prove to have thyroid cancer are usually advised to undergo completion thyroidectomy. Thus, for thyroid nodule patients, particularly the patients with indeterminate cytological findings, preoperative evaluation of a specific molecular marker, such as BRAF mutation, would be greatly helpful. In addition, accumulated evidences have demonstrated that the patients with BRAF mutation show frequent recurrence and resistance to radioactive iodine therapy, BRAF mutation may also be used as a prognostic factor [34, 35]. BRAF mutation testing would thus be a very informative and useful tool in the management of thyroid nodules and cancers.
In addition to genetic factors, epigenetic events, such as aberrant gene methylation, play a critical role in thyroid tumorigenesis . DNA methylation is one of major mechanisms of inactivation of tumor-related genes, particularly tumor suppressor genes, in cancer cells. The advantages of gene methylation as a molecular marker for the detection and diagnosis cancer in biopsy specimens and non-invasive body fluids, such as serum, has led to many studies of application in human cancers, including thyroid cancer . Similarly, we hypothesize that there is potential value of gene methylation testing on FNABs as diagnostic markers to distinguish malignant from benign nodules. There are several reasons to support our hypothesis. First, DNA methylation commonly occurs in thyroid cancer . Second, in generate, the amount of DNA from cancer cells in FNABs is much greater than in serum samples. Third, DNA, unlike mRNA, is a stable molecule, offering the promise of greater test stability. Fourth, DNA methylation can be quantitatively analyzed by Q-MSP assay used in this study, which has become a well-established and widely available technique [18, 20, 22, 24]. In this study, we have confirmed the measurability of FNAB methylation markers and preliminarily defined the specificity and sensitivity of a panel of methylation markers for evaluating thyroid nodules. Using these five genes, CALCA, RAR-beta, DAPK1, TIMP3, and RASSF1A for differential diagnosis of thyroid nodules with appropriate cut-off values, we were able to identify positive gene methylation in 33.33-88.10% of malignant nodules, but the diagnostic specificities were all low, ranging from 75.68 to 83.78%. Although DAPK1 and RASSF1A genes exhibited significantly different levels of DNA methylation between malignant and benign nodules, their diagnostic accuracy was still poor. These findings suggest that quantitative detection of these methylation markers on FNABs has serious limitations in the diagnosis of thyroid cancer.
Notably, the analysis of BRAF mutation confirmed the previously reported inverse association with RASSF1A methylation [22, 24, 36, 37]. In addition, our data showed a positive correlation between DAPK1 methylation and BRAF mutation. To our knowledge this is the first evidence indicating a relationship between these two molecular alterations in thyroid cancer. Given that both of BRAF mutation and gene methylation play a key role in thyroid carcinogenesis and there is certain relationship between them, we presume that combined detection of these two molecular events on FNABs may improve the diagnostic accuracy of thyroid nodules. To this end, we performed BRAF mutation and DNA methylation assays in training and test groups, respectively, and demonstrated that, as compared with BRAF mutation and DNA methylation testing each alone, combined analysis of these two molecular events increased the diagnostic sensitivity and accuracy, with excellent specificity (94.44-100%).