Advances in BRAF gene mutations in papillary thyroid carcinoma

ZHANG Xu-xu, MA Su-mei,2?

1. The First Clinical Medical College, Lanzhou University, Lanzhou 730000, China;

2. Department of Ultrasound, The First Hospital of Lanzhou University, Lanzhou 730000, China

Keywords:Papillary thyroid carcinoma Neoplasm invasiveness Proto-oncogene proteins BRAF Telomerase reverse transcriptase

ABSTRACT Thyroid cancer is the most common endocrine system tumor. Ultrasound guided fine needle puncture (FNA) can identify benign and malignant thyroid nodules. However, due to the limitation of cytological detection, some thyroid nodules are difficult to distinguish benign and malignant. BRAF gene mutation is a common human oncogenic mutation and the highest mutation frequency in papillary thyroid carcinoma. The combination of FNA and BRAF gene detection can significantly improve the diagnostic rate of benign and malignant thyroid nodules and make up for the deficiency of single diagnosis of cytology. Moreover, while the incidence of thyroid cancer is growing rapidly worldwide, its mortality remains stable. The problem of overdiagnosis and overtreatment of thyroid cancer is becoming more and more obvious.However, due to the limitations of current studies on BRAF genes, its prognostic value for papillary thyroid carcinoma remains controversial. Therefore, in order to reduce the adverse effects of overdiagnosis and treatment, the relationship between gene and tumor biological behavior needs further study in the future.

1. Introduction

Thyroid cancer is the most common endocrine system tumor [1],accounting for about 95% of all endocrine tumors. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Its incidence is increasing worldwide [2]. Mainly due to the clinical application of various auxiliary diagnostic techniques, especially high frequency ultrasound instrument, and the improvement of people's health consciousness makes the detection rate of PTC increase obviously. It is beneficial to clinical management with the help of ultrasound imaging signs. With the clinical need to differentiate benign and malignant thyroid nodules, only through hierarchical management can not meet the clinical needs. Fine needle aspiration (FNA) is the most commonly used tool to distinguish benign and malignant thyroid nodules before operation.The Bethesda system for reporting thyroid cytopathology is a normative interpretation in conjunction with FNA [3]. However,because of the limitations of cytology, about a third of thyroid nodules do not determine their benign or malignant [4], Therefore,other methods are needed to assist in the identification of benign and malignant. BRAF gene mutation is the most common mutation in PTC. The combined FNA of BRAF mutation detection can improve the diagnostic rate of PTC [5]. However, BRAF the value of gene mutation in predicting prognosis is still controversial. This article reviews the research progress of BRAF gene mutation in PTC.

2. General characteristics of BRAF gene mutations

BRAF mutation was the first found in malignant melanoma by Davies and his colleagues in 2002 [6]. This mutation was subsequently found in thyroid cancer and is widely studied. BRAF gene is a proto-oncogene. BRAF gene encode serine/threonine kinase BRAF, a family of RAF protein kinases. It is a mitogenactivated protein kinase (MAPK) intracellular effector of the signaling cascade. It helps to transmit signals from outside the cell to the nucleus. A signal transduction pathway called a MAPK pathway, important for growth and cell proliferation, differentiation,cell motility and apoptosis [7], is one of the most important oncogenic signaling pathways associated with human tumors. BRAF V600E mutation is the most common mutation site in thyroid cancer.The mutation results in the conversion of valine (V) to glutamic acid (E). BRAF V600E mutations are closely related to PTC. The mutation rate of thyroid cancer varies greatly. Previous studies have shown that the mutation rates BRAF V600E in thyroid cancer ranged from 29 to 83 percent [8]. Compared to the west, Asian countries seem to have higher mutation rates [9,10], and this may be related to the difference in iodine intake [11], race and other factors.

3. Detection of BRAF gene mutations

Gold standard for detecting BRAF status is direct detection of tissues by Sanger sequencing or next generation sequencing.Although direct sequencing is accurate and intuitive, the price is high, the operation is tedious, time-consuming and laborious.Not conducive to clinical promotion. In addition, there are also immunohistochemical methods based on BRAF gene expression protein products and polymerase chain reaction (PCR) indirect detection methods. There were 72 cases of paraffin embedded in formalin after operation. Evaluation of mutation status by immunohistochemical methods of BRAFV600E antibodies and sanger sequencing suggests that, immunohistochemistry is a highly sensitive, specific and accurate method, have high application value in routine pathology laboratory [12]. However, in FNA specimens,compared with gene sequencing, direct use of immunohistochemical methods on cell smears has a higher risk of false positives [13]. Kim and others detected BRAF mutations in real-time PCR compared with next generation sequencing, the results showed that real-time PCR detection of BRAF V600E mutations had similar diagnostic sensitivity and specificity to next generation sequencing, and the real-time PCR detection method is more convenient and faster.Moreover, the concentration of template DNA is also lower than that direct sequencing [14]. Therefore, BRAF V600E mutation by PCR detection has high clinical application value.

4. Relationship between BRAF gene mutation and PTC invasiveness

Due to the growing problem of overdiagnosis and treatment of thyroid cancer, by looking for risk factors related to invasiveness to predict PTC prognosis of patients is still actively explored. It is very important to find one or more biomarkers to specifically identify malignant tumors. In thyroid cancer, at present, one of the most promising and controversial biomarkers is BRAFV600E gene mutation. Since BRAFV600E mutations have high specificity(99%) and high positive predictive value [15], by evaluating the BRAFV600E status of thyroid nodules, helps to improve PTC diagnosis, reduce unnecessary diagnostic surgery [5,16]. However, the identification of invasive pathological features PTC BRAF V600E gene mutations is still controversial. Some studies have shown that, BRAF V600E gene mutations are associated with PTC extrathyroidal invasion, pathological features, lymph node metastasis,local recurrence [8,17-19], are independent risk factors for PTC poor prognosis. Additionally, Wang et al. showed in a multicenter retrospective study, and in patients with BRAF V600E mutations,male sex is an independent risk factor for PTC poor clinical prognosis [20]. However, these findings have not been repeatedly verified in studies in different countries [21-23]. BRAF mutation determination has some correlation with PTC. But there may be some errors in the clinical application of lymph node metastasis and local recurrence, according to the BRAFV600E mutation state,whether the surgical method can improve the clinical prognosis needs further verification.

5. Relationship between coexistence of BRAF and TERT gene mutations and PTC invasiveness

Due to BRAF limitations of single mutation detection,tumorigenesis is also a process of polygenic involvement. Therefore,and there are studies that hope to combine other mutations associated with PTC, jointly predict the prognosis of PTC patients.The correlation between telomerase reverse transcriptase (TERT)promoter mutation and thyroid cancer has been confirmed [24].TERT is the catalytic protein subunit of telomerase, and it has the function of maintaining chromosome integrity and genome stability[25]. Telomerase is well expressed in germ cells and stem cells, and is inhibited in somatic cells. When a TERT mutation occurs, can make cells gain unlimited proliferation potential and lead to cancer [26].The results of Lee et al show that compared with the TERT promoter and the BRAF V600E group, both mutations were significantly associated with older and later tumor stages [27]. Trybek et al.through rigorous monitoring of 568 PTC patients, PTC patients with BRAF V600E and TERT mutations found to be associated with poor prognosis and clinical course, and may help predict poor therapeutic response, recurrence and poor prognostic [28]. Therefore, the mechanism by which BRAFV600E and TERT promoter mutations can enhance PTC invasiveness has been studied. One of the conjectures is that BRAF mutation induces upregulation of several ETS transcription factors. Then, TERT promoter mutations may be associated with initiating carcinogenesis by creating ETS binding sites and increasing TERT expression. Besides, molecular pathways activated by BRAF V600E mutations are further enhanced by TERT promoter mutations or TERT expression. The co-existence of these two mutations enhances the invasiveness of cancer nodules [29].There is also a conjecture that BRAF V600E/MAP kinase pathway phosphorylates and activates FOS, FOS as a transcription factor binding to GABPB promoters increases GABPB expression and driving GABPA-GABPB complex formation, the latter selectively binds and activates TERT mutant promoter and upregulates TERT expression. Elevated TERT as a tumor protein, and strongly promote the invasive behavior of cancer cells and tumor development [30].Both conjectures are related to synergy, eventually leading to increased TERT expression. On the current status of research, the detection of BRAF V600E and TERT promoter mutations indicates poor clinical outcomes, and it is helpful to better treat thyroid cancer patients. Except for TERT promoter mutation, BRAF mutation combined with PIK3CA mutation, TP53 mutation, AKT1 mutation are also associated with aggressive behavior [15,31]. Colombo et al. detected 19 common genetic changes simultaneously in 208 patients with PTC, through genome background analysis, accurate risk stratification of thyroid nodules can not depend on single gene changes, mutation density and genetic heterogeneity also have great influence on clinical characteristics and disease outcomes [32]. Lee et al. performed multiple genetic tests PTC selected patients with high risk of recurrence, also found that most patients have more than one genetic variant [33].

However, data from Kuma Hospital in Japan, among the cases in which active surveillance surgery was abandoned for PTMC progression, BRAF gene mutations were not significantly different between stable disease group (61%), tumor enlargement group(70%) and lymph node metastasis group (80%), respectively. None of these advanced cases showed positive TERT promoter mutation[34]. PTMC that appear to be progressing do not have both BRAF and TERT mutations. But this can be explained by the latest concept of self-limited cancer. A self-limiting cancer is a conceptual of the natural history of thyroid cancer [35], and that self-limited cancer is truly malignant, but it doesn't develop into deadly cancer. First, A number of studies have shown that prophylactic resection PTMC does not change mortality. Second, Because PTMC also have the ability to proliferate and metastasize, but it doesn't develop into fatal cancer. Third, the high incidence of papillary carcinoma in young people suggests that the initial onset of thyroid cancer may occur in infancy. According to the concept of self-limited cancer, these advanced cases may still be self-limited thyroid cancer, rather than lethal thyroid cancer. Current studies suggest that tumorigenesis is a multifactorial effect, the result of multi-gene participation and multi-stage development, the expression of genes is also the process of the interaction of expression products in cells or between cells.The coexistence of BRAF and TERT gene mutations may be a switch to fatal thyroid cancer. The value of BRAFV600E evaluation PTC patient outcomes is controversial, could be related to genetic variation other than BRAFV600E not being detected. Moreover, the mutation rate of BRAF in PTC is about 29-83% [8], while the 20-year survival rate of PTC patients postoperative is 99% [36]. This difference also proves that BRAF gene mutation alone is unlikely to be a predictor of PTC poor prognosis. Therefore, it is not reliable to predict PTC invasive characteristics only by BRAF a gene mutation.In conclusion, BRAF mutation gene detection of thyroid nodules can be used to assist the diagnosis of malignant thyroid nodules.Nevertheless, BRAF V600E mutations remain controversial in evaluating the value of PTC invasiveness. This may be related to the potential genetic variation not being detected. Therefore, in order to reduce the adverse effects of excessive diagnosis and treatment on patients and promote the development of precision medicine and individualized treatment, more in-depth research is needed in the future to benefit patients more.

Acknowledgments: Thank Lanzhou Bureau of Science and Technology for the funding of this study.

Author Contributions: Zhang Xvxv is the executor of the experimental research of the project, collects the case data of the project, completes the data analysis and the writing of the first draft of the paper; Ma Sumei is the designer and responsible person of the project, instructs the experimental design, the data analysis, the thesis writing and the revision. Both authors read and agree to the final text.