BRAF Gene Mutation Based Recurrent Risk Analysis And Target Therapy Optimization In Papillary Thyroid Carcinoma

BackgroundThyroid carcinoma?TC?is the most common endocrine malignancy and the incidence is rather high in middle-aged women.Differentiated thyroid carcinoma?DTC?,including papillary thyroid carcinoma?PTC?and follicular thyroid carcinoma?FTC?,is the most common histopathology type of TC.For most DTC patients,after radical surgery combined with thyroid stimulating hormone?TSH?suppression therapy and appropriate radioactive iodine therapy,the 10-year survival rate can reach 90%.However,a small amount of DTC patients develop tumor recurrence,or become resistance to radioactive iodide therapy,or develop distant metastasis to lung,bone and brain,which is one of the leading cause of death in TC and one of the most important factors affecting the overall prognosis of patients with thyroid carcinoma.BRAF^V600Emutation is the most important and common molecular event in PTC,and has been reported to be closely associated with large tumor diameter,extraglandular invasion,lymph node metastasis or other invasive clinicopathological features in PTC with poor prognosis.In recent years,it has been found that the higher proportion of PTC with BRAF mutation presents bilateral and multifocal,which indicated the malignant biological behavior of tumor and significantly increase the risk of tumor recurrence.In addition,the status of BRAF mutation in PTC patients affected the value of male sex and older age in predicting the risk of recurrence and disease free survival.Patients with male sex and older age developed higher risk of tumor-related death,however,there was no correlation in patients with wild type BRAF.Telomerase reverse transcriptase?TERT?promoter mutation was also associated with highly invasive clinicopathological features of PTC and played an important role in predicting the risk of tumor recurrence and tumor-related death.It has been reported that the coexistence of TERT promoter mutation and BRAF mutation was helpful to predict those PTC with highest malignant behavior.Vemurafenib,a specific inhibitor of BRAF mutation,is effective in the treatment of advanced malignant melanoma with BRAF mutation and has been used in clinical practice.Clinical trials of vemurafenib in thyroid carcinoma have also demonstrated antitumor effects in patients with BRAF^V600E-mutant metastatic or unresectable PTCs refractory to radioactive iodine.However,compared with other BRAF mutant malignancies,the overall response rate of vemurafenib in TC is relatively low?29%?and drug resistance is easy to occur.Mirroring the discrepancy in clinical response rates to vemurafenib between BRAF-mutant thyroid carcinoma and melanoma,the majority of thyroid cancer cell lines demonstrate decreased sensitivity to vemurafenib in vitro.It has been reported that the reactivation of MAPK signaling,the high expression of ERBB family proteins and activation of PI3K/Akt/m TOR and other signaling pathways may be responsible for the BRAF inhibition resistance in thyroid malignancy,but it has not yet been fully elucidated.Until now,the most study cohort of BRAF mutation analysis in the Chinese population has a sample size of several hundreds.Based on the clinicalpathlogical characteristics and follow-up data of more than 1000 cases of thyroid carcinoma,combined with TERT promoter mutations analysis,our study was intended to systematically explore the role of BRAF mutation in predicting the malignant behavior and recurrence risk of papillary thyroid carcinoma.The purpose of this study is to optimize the treatment and follow-up strategy for patients with BRAF mutant PTC.At the same time,in view of targeted BRAF therapy resistance,exploring the potential mechanisms will provide scientific guidance for overcoming drug resistance and finally has a far-reaching significance with an improved overall prognosis of PTC.Section ?:Study on the role of BRAF mutation in predicting high-risk clinicopathological features and recurrence of papillary thyroid carcinomaObjective:Based on the clinicopathological characteristics and follow-up data of 1002 cases of thyroid papillary carcinoma,the value of BRAF^V600Emutation in predicting the high-risk clinicopathological characteristics and tumor recurrent risk of papillary thyroid carcinoma was systematically studied.Methods:The cases of thyroid papillary carcinoma confirmed by pathological examination postoperatively in the Department of Surgical Oncology of the first affiliated Hospital of Zhejiang University from January 2008 to June 2015 were retrospectively analyzed.According to the inclusion and exclusion criteria,an overall of 1002 cases were enrolled in our study and their clinicopathological features and follow-up information were collected.Genomic DNA was extracted from 1002 fresh frozen tumor tissues of patients with thyroid papillary carcinoma.The BRAF^V600Emutation and TERT promoter mutation were confirmed by Sanger sequencing.Statistical methods were used to analyze the correlation between mutations and clinicopathological characteristics or recurrence risks.Results:In 1002 cases of thyroid papillary carcinoma,the mutation rates of BRAF^V600Eand TERT promoter was 72.7%and 1.7%,respectively.Papillary thyroid carcinoma with BRAF^V600Emutation had a significantly higher incidence of bilateral?P=0.01?and multifocal lesions?P=0.017?but a lower incidence of Hashimoto's thyroiditis?P<0.0001?.Bilateral and multifocal lesions were suggested to have more aggressive behavior but Hashimoto's thyroiditis had no value in predicting malignant phenotype in papillary thyroid carcinoma.TERT promoter mutations was associated with older age,male sex,larger tumor size,capsular invasion and extrathyroidal extension and advanced tumor stage.Those PTC patients harboring both TERT promoter and BRAF mutations had more aggressive tumor behaviors and higher recurrent risk.Bilateral lesions?P<0.04?,tumor size more than 10 mm?P<0.004?and TERT promoter mutation?P<0.049?were independent risk factors for recurrence in all PTC patients.Bilateral lesions and extraglandular infiltration suggested a higher risk of recurrence in patients with mutant BRAF,but the value is limited in patients with wild-type BRAF.Larger tumor size indicated a higher risk of recurrence in patients with wild-type BRAF but limited value in patients with mutant BRAF.Conclusion:BRAF mutation was helpful to identify bilateral and multifocal features of thyroid papillary carcinoma,which were also demonstrated as aggressive behavior characteristics.PTC patients with BRAF mutation had a higher risk of recurrence if they harbored TERT promoter mutation simultaneously.In patients with BRAF mutation,bilateral tumor and extraglandular infiltration were independent risk factors for recurrence.Section ?: Study on the role of VCAM-1 during BRAF inhibition resistance in thyroid carcinomaObjective:In order to explore the potential molecular biological events related to BRAF inhibition resistance,we compared the transcriptional expression profile of BRAFV600 E mutated thyroid cancer cell line BCPAP after vemurafenib treatment by a high-throughput RNA sequencing.Vascular cell adhesion molecular-1?VCAM-1?was identified to be highly up-regulated during BRAF inhibition.The present study was designed to explore the role of VCAM-1 during BRAF inhibition resistance and its biological function in thyroid carcinoma.Methods:RNA sequencing was performed on the Illumina Hi Seq 4000.Cell viability was determined by CCK-8 assays,cell apoptosis and cell cycle assays were done to evaluate the anti-cancer efficacy of vemurafenib with knockdown of VCAM-1 by si RNAs transfection in a BRAF mutant papillary thyroid cancer cell line?BCPAP?.Meanwhile,the cytotoxic activity of vemurafenib was also assessed in a BRAF mutant anaplastic thyroid cancer cell line?FRO?with overexpression of VCAM-1 by lentiviral vectors transfection.The potential molecular mechanism underlying the up-regulation of VCAM-1 was studied by pathway inhibitors,and the biological function of VCAM-1 in thyroid cancer was also studied in vitro.Additionally,the correlation between clinicopathological features and the expressions of VCAM-1 in 50 pairs of thyroid papillary carcinoma samples was analyzed to further evaluate the role of VCAM-1 in thyroid carcinoma.Results:VCAM-1 was significantly up-regulated by vemurafenib in both time-and dose-dependent manners.Knockdown of the induced VCAM-1 augmented the anti-tumor effects of vemurafenib.Meanwhile,overexpression of VCAM-1 reduced the sensitivity to vemurafenib.Further investigation showed that phosphatidylinositol3-kinase?PI3K?-Akt-mammalian target of rapamycin?m TOR?pathway was activated during BRAF inhibition.Co-treatment with Akt signaling inhibitor MK2206 decreased the induced expression of VCAM-1 during BRAF inhibition.This combination further improved the efficacy of vemurafenib.Moreover,although VCAM-1 did not facilitated the proliferation,apoptosis and clone formation ability of thyroid cancer cells,it promoted migration and invasion in thyroid cancer in vitro,which was also indicated in thyroid cancer patients.Conclusion:The current study is the first to demonstrate that VCAM-1 is up-regulated in thyroid cancer cells treated with vemurafenib and contributes to vemurafenib resistance in BRAF-mutant thyroid cancer cells.In addition,it plays a role in tumor invasion and metastasis in thyroid cancer.Targeting the PI3K-Akt-m TOR pathway mediated VCAM-1 response may be an alternative strategy to sensitize BRAF-mutant thyroid cancers to vemurafenib.