Investigation Of Molecular Mechanism In Thyroid Cancer And Its Application Value In Clinical Practice

BackgroundThyroid cancer is one of the most common endocrine malignancies and has became the fifth common malignancy of American women.Papillary thyroid cancer(PTC)and follicular thyroid cancer(FTC),classified as differentiated thyroid cancer(DTC),are the major histopathological types.The 10-year survival rate of DTC patients can reach 90%after surgery assisting with TSH suppression therapy and I131 radiation.However,a small part of patients suffers from disease progression in the early stage,tumor recurrence after surgery or resistance to I131 radiation.Therefore,investigating the underlying molecular mechanism of thyroid cancer can provide effective biomarkers to identify aggressive cases in the early stage and promising therapeutic targets for treatment.A number of genetic and epigenetic alterations are occurred and accumulated in the carcinogenesis and progression of thyroid cancer.Besides,the molecular mechanisms of different histopathological types of thyroid cancer are variant.For example,RET associated-signaling is the most important mechanism for medullary thyroid cancer(MTC),MAPK signaling activated by BRAF mutation contributes to the carcinogenesis of PTC,and mutations in RAS,PTEN and PIK3CA can triggerPI3K pathway inducing FTC.Previous researches demonstrated that 60%-70%thyroid cancer had more than one molecular alterations,which may be regarded as promising biomarkers for diagnosis,risk stratification,treatment strategy and prognostic prediction.Great progression has been achieved in the molecular mechanism of thyroid cancer,but the majority of researches have been focused on nuclear genome and the study in mitochondrial genome is rare.Mitochondrion is an important organell taking part in bioenergetic metabolism,aging and apoptosis.Even in hypoxia condition,most cancer cells are dependent on glycolysis for energy,which may be caused by mitochondrial dysfunction.Mitochondrial DNA is a 16569bp,double chain and circular DNA coding 13 pepitides,2 rRNA and 22 tRNA for mitochondrial respiration,it also contains an uncoding region(D-loop region)regulating the transcription and translation of mitochondrial DNA.Previous studies demonstrated that mtDNA mutations and copy number changes were common in various cancers,and deleterious mtDNA mutations can severely hamper the electron transfer chain and overproduce reactive oxygen species contributing to the proliferation and progression of cancer.However,the studies of mitochondrial characteristics in thyroid cancer are limited and mainly focused on oncocytic thyroid neoplasm and D-loop region.This study aimed to comprehensively characterize the mitochondrial genome in thyroid cancer,including mtDNA mutation,mitocopy,haplotype and mtSNP.Besides,we identified potentially pathogenic mtDNA mutations associated with thyroid cancer and further analyzed their relationship with clinicopathological features and prognosis.We also evaluated the application value of BRAFV600E and TERT promoter mutations in diagnostic,risk stratification and therapeutic strategy.Section Ⅰ:A comprehensive characterization of mitochondrial genome in papillary thyroid cancerBackground:Nuclear genetic alterations have been widely investigated in papillary thyroid cancer(PTC),however,the characteristics of the mitochondrial genome remain uncertain.Methods:We sequenced the entire mitochondrial genome of 66 PTCs,16 normal thyroid tissues and 376 blood samples of healthy individuals.Results:Abstract:There were 2508 variations(543 sites)detected in PTCs,among which 33 variations were novel.Nearly half of the PTCs(31/66)had heteroplasmic variations.Among the 31 PTCs,28 specimens harbored a total of 52 somatic mutations distributed in 44 sites.Thirty-three variations ircluding seven nonsense,11frameshift and 15 non-synonymous variations selected by bioinformatic software were regarded as pathogenic.These 33 pathogenic mutations were associated with older age(p = 0.0176)and advanced tumor stage(p = 0.0218).In addition,they tended to be novel(p = 0.0003),heteroplasmic(p = 0.0343)and somatic(p = 0.0018).The mtDNA copy number increased in more than two-third(46/66)of PTCs,and the average content in tumors was nearly four times higher than that in adjacent normal tissues(p<0.0001).Three sub-haplogroups of N(A4,B4a and B4g)and eight single-nucleotide polymorphisms(mtSNPs)(A16164G,C16266T,G5460A,T6680C,G9123A,A14587G,T16362C,and G709A)were associated with the occurrence of PTC.Conclusions:Here we report a comprehensive characterization of the mitochondrial genome and demonstrate its significance in pathogenesis and progression of PTC.This can help to clarify the molecular mechanisms underlying PTC and offer potential biomarkers or therapeutic targets for future clinical practice.Section Ⅱ:Diagnostic value of BRAFV600E-mutation analysis in fine-needle aspiration of thyroid nodules:a meta-analysisBackground:Fine-needle aspiration(FNA)is a reliable method for preoperative diagnosis of thyroid nodules;however,about 10%-40%nodules are classified as indeterminate.The BRAFV600E mutation is the most promising marker for thyroid FNA.This meta-analysis was conducted to investigate the diagnostic value of BRAFV600E analysis in thyroid FNA,especially the indeterminate cases.Methods:Systematic searches were performed in PubMed,Web of Science,Scopus,Ovid,Elsevier,and the Cochrane Library databases for relevant studies prior to June 2015,and a total of 88 studies were ultimately included in this meta-analysis.Results:Compared with FNA cytology,the synergism of BRAFV600E testing increased the diagnostic sensitivity from 81.4%to 87.4%and decreased the false-negative rate from 8%to 5.2%.In the indeterminate group,the mutation rate of BRAFV600E was 23%and varied in different subcategories(43.2%in suspicious for malignant cells[SMC],13.77%in atypia of undetermined significance/follicular lesion of undetermined significance[AUS/FLUS],and 4.43%in follicular neoplasm/suspicious for follicular neoplasm[FN/SFN]).The sensitivity of BRAFV600E analysis was higher in SMC than that in AUS/FLUS and FN/SFN cases(59.4%vs 40.1%vs 19.5%respectively),while specificity was opposite(86.1%vs 99.5%vs 99.7%respectively).The areas under the summary receiver-operating characteristic curve also confirmed the diagnostic value of BRAFV600E testing in SMC and AUS/FLUS rather than FN/SFN cases.Conclusions:Therefore,BRAFV600E analysis can improve the diagnostic accuracy of thyroid FNA,especially indeterminate cases classified as SMC,and select malignancy to guide the extent of surgery.Keywords:thyroid cancer,fine-needle aspiration,BRAFV600E mutation,meta-analysisSection Ⅲ:Association of telomerase reverse transcriptase promoter mutations with clinicopathological features and prognosis of thyroid cancer:a meta-analysisBackground:The clinicopathological and prognostic significance of telomerase reverse transcriptase(TERT)promoter mutations have been widely investigated in thyroid cancer;however,the results are still discrepant.Methods:Systematic searches were performed in PubMed,Web of Science,Scopus,Ovid,and the Cochran Library databases for relevant articles prior to April 2016.Mutation rates were synthesized by R statistical software.The odds ratio or standardized mean difference with 95%confidence interval was pooled by Stata.Results:A total of 22 studies with 4,907 cases were included in this meta-analysis.TERT promoter mutations tended to present in aggressive histological types including poorly differentiated thyroid cancer(33.37%),anaplastic thyroid cancer(38.69%),and tall-cell variant papillary thyroid cancer(30.23%).These promoter mutations were likely to exist in older patients and males and were well associated with larger tumor size,extrathyroidal extension,vascular invasion,lymph node metastasis,distant metastasis,advanced tumor stage,disease recurrence/persistence,and mortality.In addition,TERT promoter mutations(especially C228T)tended to coexist with BRAFV600E mutation,which indicated more aggressive tumor behavior.Conclusions:Therefore,TERT promoter mutations may be promising biomarkers for early diagnosis,risk stratification,prognostic prediction,and management of thyroid cancer.