BRAF,NRAS Mutation And Ultrasound Features Of Papillary Thyroid Carcinoma And Its Lymph Node Metastasis

Part one: A study of ultrasound features,contrast-enhanced ultrasound,and elastography in thyroid papillary carcinoma and lymph node metastasesBackground and objectiveThyroid cancer is one of the most common malignant tumors,and papillary thyroid carcinoma(PTC)accounts for 80%-90%,which includes many subtypes,such as conventional PTC(C-PTC),follicular variant of PTC(FV-PTC)and Tall cell variant of PTC(TCV).Etc.The most common metastatic site of PTC is cervical lymph node with the metastasis rate up to 50%-70%.Ultrasound is superior to other modalities in evaluating thyroid abnormality.The application of ultrasonography makes the clinical detection rate of thyroid cancer gradually increase,while ultrasound could detect suspicious lymph node metastasis and preoperative localization.Besides,contrast-enhanced ultrasound(CEUS)and elastography are valuable in diagnosing lymph node metastasis of thyroid cancer.Therefore,this study will analyze the ultrasonic features,contrast-enhanced ultrasound and elastography of the subtypes of PTC and its lymph node metastasis,to evaluate the difference of ultrasound features of PTC subtypes and to determine the accuracy of ultrasound in diagnosis of lymph node metastasis of thyroid cancer via ultrasonic features,CEUS and elastography.Methods1.Selected patients who underwent surgery and confirmed as PTC by pathologically in Henan Cancer Hospital from September,2014 to December,2016.Then,a total of 178 PTC patients with three common subtypes such as C-PTC,FV-PTC and TCV were selected,including 40 males and 138 females,and 66 cases were pathologically diagnosed with cervical lymph node metastasis,112 cases without metastasis.The patient’s clinical and imaging data were collected,including age,gender,tumor size,surgical mode,TNM stage and ultrasonic features.2.Statistical analysis the differences in the clinical data of each PTC subtype(C-PTC,FV-PTC and TCV)patients;and analysis the ultrasonic features of each subtype patients.3.According to pathological results,assessment the accuracy of ultrasonic features,CEUS and elastography in diagnosing PTC lymph node metastasis.Results1.In the 178 cases,c-PTC,FV-PTC and TCV was 124(69.7%),35(19.7%)and 19(10.6%),respectively.There were no statistical differences in clinical data between each PTC subtype patients.2.Ultrasonography showed significant differences in marginal and internal structures between each subtype,and the margin of FV-PTC tended to be well-defined,and the margin of another two subtypes tended to be ill-defined,the ratio of ill-defined were 42.9%,54.8% and 78.9%,respectively(P<0.05);Solid ratio of the internal component of C-PTC and TCV were higher than the FV-PTC,the ratio of solid and predominately was 85.5%,73.7% and 68.6%,respectively(P <0.05);while,there were no statistical differences in other ultrasonic features.3.Ultrasonic features showed significant differences in size,A/T,calcification,and blood flow between lymph node metastasis and non-metastatic(P<0.05);while,no statistical differences in internal composition,echogenicity,margin and halo.4.The sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)of ultrasonic features in diagnosing PTC lymph node metastasis was 72.7%,91.1%,82.8% and 85.0%,respectively.The sensitivity,specificity,PPV and NPV of CEUS in diagnosing PTC lymph node metastasis was 75.5%,92.0%,84.7% and 86.6%,respectively.The sensitivity,specificity,PPV and NPV of elastography in diagnosing PTC lymph node metastasis was 78.8%,92.0%,85.2% and 88.0%,respectively.Conclusions1.There are significant differences in internal composition and margin between each PTC subtype.2.Ultrasonic features,contrast-enhanced ultrasound,and elastography have a high value in diagnosing cervical lymph node metastases in papillary thyroid carcinoma.Part two: Analysis of BRAF and NRAS mutation in papillary thyroid carcinoma and lymph node metastasis Background and objectiveBRAF mutation is a common genetic variation in PTC,leading to abnormal activation of MAPK signaling pathway,which play a key role in the occurrence and development of PTC.In recent years,many studies have shown that BRAF mutation in PTC patients is associated with the pathological features and poor prognosis of PTC.The mutation frequency of NRAS mutations in PTC patients is lower than that of BRAF mutations,but some studies have found that the proportion of NRAS mutations in PTC is up to 30%.Accordingly,NRAS mutations is involved in the occurrence and development of PTC.The tissue specimens are commonly used to detect gene mutation.However,with the rise of liquid biopsy technology,the use of blood or other liquid biopsy for screening,diagnosis and prognosis of cancer patients has been more widely used.Therefore,the purpose of this study was to detect BRAF and NRAS mutations in the tissues and blood of patients with different PTC subtypes,and assess the differences in genetic variation in different subtype patients,and analysis the differences in BRAF and NRAS mutations between lymph node metastases and non-metastatic patients.This study provides a reliable biomarker for diagnosis of PTC lymph node metastasis;and investigates the feasibility of non-invasive blood mutation testing to replace tissue mutation detection by comparing the results of blood and tissue.Methods1.178 patients were chosen as subjects as described in Part one;10ml blood samples were collected before the surgery,and separated the plasma of them;tissue samples were collected after the surgery.2.DNA was extracted from samples to detect the BRAF and NRAS mutation by droplet digital PCR(dd-PCR).Mutation sites included 3 sites of BRAF and 6 sites of NRAS.3.Statistics the all mutation sites detection results in both of tissue and blood samples;analysis the differences in BRAF and NRAS mutations among three PTC subtype by statistical;analysis the differences in lymph node metastases and non-metastatic patients by statistical.4.Consistency of plasma and tissue assays was analyzed in comparison with the results of plasma and tissue tests.5.Correlation between ultrasonic features and gene mutations of the lymph nodes was analyzed by correlation analysis,and the accuracy of ultrasonography associated gene mutations in the diagnosis of PTC lymph node metastases was assessed.Results1.In the 178 PTC cases,74(41.6%)cases were BRAF mutation positive,and 21(11.8%)cases were NRAS mutation positive in tissue;41(23.0%)cases were BRAF mutation positive,and 13(7.3%)cases were NRAS mutation positive in plasm;In the plasma and tissue detection results,consistency in mutation was 0.28,consistency in wild type was 0.33,and dis-consistency in mutation was 0.39.2.The results of plasma and tissue detection showed that there were differences in BRAF mutations among three subtypes(P<0.05),but there was no significant difference in NRAS mutations.3.There were differences in BRAF mutations and NRAS mutations between lymph node metastasis and non-metastatic PTC patients;and a higher proportion of BRAF and NRAS mutations in patients with lymph node metastases(BRAF:62.2% vs 19.2% P<0.01;NRAS: 61.9% vs 33.8% P<0.05);BRAF and NARS mutations were associated with TNM stag in patients,mutation rate was higher in patients with late stage(BRAF: 51.4% vs 22.1% P<0.01;NRAS: 33.3% vs 12.7% P<0.05).4.BRAF mutations were related to the size of PTC nodules(P<0.05)and had no significant correlation with other ultrasonic features;Correlation between NRAS mutation and ultrasound of PTC had no statistical difference.5.After combining ultrasonic features with plasma BRAF and NRAS mutation in diagnosing PTC lymph node metastasis,the sensitivity,specificity,positive predictive value and negative predictive value was 84.8%,92.9%,87.5% and 91.2%,respectively.Conclusion1.Both BRAF mutation and NRAS mutation are relate with PTC subtypes,lymph node metastasis and TNM stage.2.Consistency between BRAF and NRAS in plasma and tissue detections is 0.61;and plasma gene mutation detection has reliable application value.3.Combination of ultrasonic features with plasma BRAF and NRAS mutation information are more accurate than sole ultrasonography in diagnosing PTC lymph node metastasis.