The Role Of Molecular Characteristics In Recognition Of Radioiodine-refractory Differentiated Thyroid Cancer

Part I:Association between TERT promoter mutation,BRAF mutation,and radioiodine-refractory character in distant metastatic differentiated thyroid cancerTelomerase Reverse Transcriptase(TERT)promoter mutation has been reported to be associated with aggressive characteristics in differentiated thyroid cancer(DTC).The first part examined the status of TERT promoter mutation in distant metastatic DTC(DM-DTC),and evaluated the correlation between TERT mutation and radioactive iodine-131(RAI)uptake,as well as that between TERT mutation and therapy response.TERT promoter and B-Raf proto-oncogene(BRAF)V600E mutation were retrospectively examined in primary tumors of 66 DM-DTC patients.Stimulated thyroglobulin(sTg)changes,RAI uptake status(avid or non-avid),and other imaging evidence were analyzed to evaluate therapy response.After a median follow-up of 46.5 months(interquartile range,29.0 to 70.5 months),therapy response was classified as refractory and disease control.The prevalence of TERT mutations was 22.73%(15/66),of which C228T mutation was more prevalent(13/15)than C250T mutation(2/15).Rising sTg was noticed in 93.33%(14/15)of TERT mutation group.While in cases with both mutations negative,78.12%(25/32)presented with decreased sTg.TERT mutation closely correlated with poor RAI therapy response(P<0.001),and all 15 patients were classified as refractory to RAI with a positive predictive value of 100%at the end point of follow-up.TERT mutation was associated with older mean age at diagnosis(P<0.001),larger mean tumor diameter(P=0.013),and more likelihood of both BRAF mutation coexistence(P=0.044)and refractory to RAI(P<0.001).In the 36 cases received imaging semi-quantitative analysis,it was found that TERT promoter mutation significantly correlated with non-RAI-avidity,with a much lower mean tumor/background(T/B)ratio(obtained from post RAI therapy whole-body scanning)than TERT wild-type(P<0.001).And DM-DTC patients with TERT mutation were more likely to lose RAI-avidity at initial RAI therapy than those with only BRAF mutation(8/8vs.5/11,Fisher’s exact test,P=0.018).TERT promoter mutation closely associates with non-RAI-avidity in DM-DTC,and when comparing with BRAF mutation,TERT mutation manifested a worse negative influence on RAI uptake.It could also be used as a predictive marker to early identify refractory to RAI.Part II:Association between miR-125a-3p and radioiodine-refractory character of distant metastatic differentiated thyroid cancerTo date,studies on microRNA(miRNA)related to radioactive iodine(RAI)uptake of DTC lesions is still confined to in vitro cell level.No miRNA expression profile of RAIR-DTC tissues could be referred.The main purpose of the second part is to evaluate miRNA expression profile difference of DM-DTC patients according to their RAI therapy response,in order to provide the molecular basis for the recognition of RAIR-DTC.This study retrospectively selected 99 cases of patients with DM-DTC,who underwent at least two courses of RAI therapy.The median follow-up time was 51.2 months after initial therapy(total thyroidectomy+131I therapy).At the end of follow-up,all patients were divided into two groups,radioiodine refractory(RAIR)or disease control,in terms of post RAI therapy whole-body scanning and serological reaction.We collected paraffin embedding tissues of these patients,and extracted total RNA.After quality assessment,88 cases were enrolled in subsequent experiments,of which 56 cases received miRNA microarray hybridization,and the other 32 was regarded as an independent study cohort used for further quantitative PCR verification.Eventually,hsa-miR-125a-3p was validated to overexpress in RAIR-DTC group and target on NIS mRNA.After transfecting miR-125a-3p mimic to DTC cell line B-CPAP,both NIS mRNA and protein expression were significantly decreased when compared with its negative control group.TSHR mRNA expression was decreased,but no significant decrease was observed on its protein level.Neither TG nor TPO expression level was significantly influenced.In addition,131I uptake ratio of B-CPAP cell declined significantly.Similarly,after transfecting miR-125a-3p inhibitor to DTC cell line B-CPAP,both NIS mRNA and protein expression were significantly increased.TSHR mRNA expression was increased,but no significant increase was observed on its protein level.Neither TG nor TPO expression level was significantly influenced.In addition,131I uptake ratio of B-CPAP cell increased significantly.In all,according to abovementioned evidence,hsa-miR-125a-3p could decline 131I uptake of DTC cell by inhibiting the expression of NIS,and associate with tumorigenesis of RAIR-DTC.Part III:Preablative stimulated thyroglobulin correlates to new therapy response system in differentiated thyroid cancerStudies suggested a potential value of preablative stimulated Tg(ps-Tg)on predicting the recurrent and persistent diseases of differentiated thyroid cancer(DTC),whereas its correlations with therapeutic response remain uncertain.The main purpose of the third part was to establish the correlation between ps-Tg and therapy response proposed by 2015 American Thyroid Association(ATA)guidelines and calculate a cut-off ps-Tg threshold for predicting a poor response.Patients who underwent total thyroidectomy and radioactive iodine(RAI)therapy in a University hospital participated in this retrospective study.Totally,452 DTC patients were followed for a median of 38 months and were divided into three groups in terms of preablative stimulated thyroglobulin(ps-Tg)level:Groupl:<1 ng/ml(n=82),Group2:1～10 ng/ml(n=173),Group3:≥10 ng/ml(n=197).Clinical outcomes were assessed based on response to therapy re-staging system,dividing response into excellent(ER),indeterminate(IDR),biomedical incomplete(BIR),and structural incomplete(SIR).Therapeutic responses could be obviously distinguished by different ps-Tg strata.SIR was identified in none of Group 1,1.73%of Group 2,and 42.74%of Group 3,respectively(x2=13.037,P<0.001).A cut-off value of ps-Tg at 26.75 ng/ml was obtained by receiver operating characteristic(ROC)curve for differentiating SIR from either ER,IDR,or BIR.The area under curve(AUC)was 0.947 and negative predictive value(NPV)was 96.99%.Ps-Tg was an independent predictive variable of SIR(OR:42.312,P<0.001).Ps-Tg has a great performance in predicting therapeutic response and providing incremental value for decision making of RAI therapy,especially for patients with high ps-Tg level.