Prediction For The Risk Of Lymph Node Metastasis And Clinical Application Of Microwave Ablation In CN₀ Papillary Thyroid Microcarcinoma

ObjectiveThyroid carcinoma?TC?is a kind of malignant carcinoma with rapid-increasing incidence.The annual number of new TC cases is about567,000.Over-diagnosis and over-treatment are hot issues in TC.More than 50%of the new cases of TC are papillary thyroid microcarcinoma?PTMC?.The overall prognosis of PTMC is satisfied.The postoperative recurrence rate and disease-related mortality rate are 0.36-7.4%and0-1.0%,respectively.How to scientifically manage and give individualized treatment to reduce overtreatment is the current clinical focus of the researches.Clinically,the PTMC case detected with negative lymph node metastasis by ultrasonography and CT is called cN₀-PTMC.In fact,the incidence of cervical lymph node metastasis in cN₀-PTMC case is as high as 15.3-49.2%.The clinical weight of PTMC accounts an important part in thyroid diseases.For c N₀-PTMC,the key to its clinical controversy is how to accurately assess the risk of lymph node metastasis,which can guide the implementation of individualized treatment:for high-risk cN₀-PTMC,to select surgical resection with central lymph node dissection,and for mid-low risk cN₀-PTMC,to select the appropriate surgical subtotal resection or non-surgical resection treatment cases?such as microwave ablation,radiofrequency ablation and clinical follow-up observation?.The mechanism of tumorigenesis and development is very complex,which is the result of the interaction of multiple genes.We study the aspects of gene and molecular combined with high-resolution ultrasound image to predict the risk of cervical lymph node metastasis in patients with cN₀-PTMC,overcoming the defect of failing to evaluate the ability of cervical lymph node metastasis via traditional imaging methods,especially in the central cervical region,and then to introduce the individualized treatment scheme of cN₀-PTMC?scope of surgical resection,minimally invasive ablation and follow-up observation?,and explore the clinical application of non-surgical treatment like micro-ablation in the mid-low risk cN₀-PTMC.Methods1.Study on the relationship between BRAF gene and ultrasonographic features in papillary thyroid microcarcinomaThe cases were from 291 patients that underwent thyroid carcinoma resection and cervical lymph node dissection in the Affiliated Hospital of Jiangsu University during May 2014 to December 2017.The thyroid lesions in all cases were larger than 5 mm in diameter,and specimens were taken by US-guided fine needle aspiration?FNA?before surgical resection.The exclusion criteria were as follows:?a?the cases larger than1cm in diameter;?b?the cases without FNA-BRAF gene analysis results;?c?the cases without complete ultrasound imaging data.Finally,116cases of PTMC were enrolled in this part of the study.The patient's complete clinical and pathological data were collected,including age,gender,with or without Hashimoto's thyroiditis,BRAF^V600E600E mutation detection and postoperative pathology of cervical lymph node.?1?Analysis of ultrasound imaging characteristics of papillary thyroid microcarcinoma116 cases of papillary thyroid microcarcinoma were analyzed separately,and ultrasound image features such as size,number location,echo,margin,calcification,taller or wider,capsule of the nodule,envelope invasion and distance from the envelope were analyzed.?2?Analysis of BRAF gene mutation in 116 cases of papillary thyroid microcarcinomaThe cells of PTMC were obtained from ultrasound-guided FNA.After obtaining the conditions and primers for PCR amplification of exon15 of BRAF gene,QIAamp-DNA-FFPE tissue kit?QIAGEN?was used to extract genomic DNA.And the exon 15 was amplified by PCR using DNA direct sequencing,and then sequenced in an ABI Prism 3730Genetic Analyzer?Applied Biosystems,Foster,California?.The cycle sequencing reaction and sequence reading were terminated on the kit to see if there was a BRAF^V600E mutation.?3?Analysis of the relationship between BRAF^V600E mutation and ultrasound imaging characteristicsWe analyzed the ultrasound imaging features of the papillary thyroid microcarcinoma and the BRAF^V600E600E gene mutation results.The continuous variables were used the Student test and the Chi-square test was used to analyze the categorical variables.Multivariate regression analysis was used to assess the relationship between ultrasound image characteristics and BRAF^V600E600E mutations.Two-tailed P<0.05 indicated a statistically significant difference.2.Study on BRAF^V600E detection combined with ultrasound imaging features for predicting the risk of central lymph node metastasis of cN₀-papillary thyroid microcarcinomaA retrospective analysis of 232 patients who underwent thyroidectomy and central lymph node dissection at the Affiliated Hospital of Jiangsu University from October 2015 to December 2017were performed.The inclusion criteria are as follows:?a?cases with ultrasound-guided FNA before surgery;?b?cases with complete ultrasound imaging features;?c?cases with complete postoperative pathological data and clinical data.The exclusion criteria are as follows:?a?cases with thyroidectomies;?b?cases larger than 10mm in diameter under ultrasound detection;?c?patients that reject BRAF^V600E analysis;?d?cases that lack ultrasound images.Finally,182 PTMC cases were eligible for the study,and clinical and pathological information such as age,gender,BRAF^V600E mutation,CLNM and with or without chronic lymphocytic thyroiditis were obtained from electronic clinical and pathological records.?1?analyzing the Ultrasound imagesEach nodule was evaluated by two experienced sonographers.They did not know the clinicopathological features and the BRAF gene results of FNA specimens.Then,retrospectively read the ultrasound image features of each PTMC and record them by standardized thyroid imaging report and data system?TI-RADS?.When there was a disagreement between the two sonographers,another senior doctor reviewed the characteristics of the ultrasound images and made a final decision.?2?BRAF^V600E mutation analysisThe way of the analysis was the same as above.?3?Statistical analysisStatistical analysis was performed by SPSS software?version 19.0,SPSS,USA?.The T-test was used to compare continuous variables,the chi-square test and the fisher test were used to compare categorical variables.Two-tailed P?0.05 is considered statistically significant.3.Safety and clinical efficacy of microwave ablation for middle-low risk cN₀-PTMCRetrospective analysis of 32 cases of mid-low risk cN₀-PTMC under microwave ablation in the Affiliated Hospital of Jiangsu University from May 2014 to June 2018 were performed.All the selected patients met the following criteria:?a?ultrasound-guided FNA cytology results were PTC,single-shot,no other suspicious nodules on the ultrasound images;?b?PTMC lesions that had a maximum diameter of no more than 10 mm;?c?PTMC lesions did not break the capsule,invade the trachea,Esophageal and surrounding muscles and vessels;?d?no signs of cervical lymph node metastasis on ultrasound images;?e?cases with wild-type BRAF^V600E600E mutation;?f?patients with TSH,FT3 and FT4 in normal range;?g?patients refused surgeries and follow-up observation after fully informed the risk of microwave ablation.Exclusion criteria:?a?two cases with follow-up time<6 months;?b?one recurrent case after thyroid resection;?c?one case that received resection after ablation;?d?one undiagnosed case which was classified into grade-5 by TI-RADS system before ablation was reviewed for 3 grade.Finally,27 cases of PTMC microwave ablation cases were included in this part of statistical analysis.?1?Ultrasound equipmentThe real-time monitoring Color Doppler ultrasound should have the contrast-enhanced function.?2?Microwave ablation equipment?a?microwave source?microwave therapeutic apparatus?at 2450MHz or 915MHz,commonly used 2450MHz and the power was 15-45W,commonly used 35W;?b?microwave ablation needle?ablation antenna?,hard fissure water-cooled ablation needle,which had the advantage of simple puncture,less central carbonization zone,high temperature resistance,anti-adhesion and not easy to damage.The diameter was 12-18G,commonly used 16G.?3?Power and time of Microwave ablationThe microwave ablation power was 25-35W.By adjusting the power and ablation time,the ablation range was completely covered by the tumor and the ablation range was expanded to 2-5 mm of the tumor edge.While ensuring adequate thermal ablation of the thyroid nodules,it was generally necessary to gradually adjust from small to large to avoid heat transfer to adjacent surrounding tissues.?4?The process of microwave ablationPatients were placed in the supine position,routine disinfection,drape,ultrasound-guided local subcutaneous infiltration anesthesia with lidocaine,anesthesia with lidocaine and saline and isolation of anterior thyroid anesthesia,and fine needle injection of normal saline under ultrasound monitoring to the inside and outside of the thyroid gland,effectively separate the recurrent laryngeal nerve,trachea and esophagus from the thyroid capsule more than 1cm.Under ultrasound monitoring,avoiding the sternocleidomastoid,visible blood vessels,carotid artery and internal jugular vein.Using pave-moving ablation technique,the ablation needle was inserted into the center of the PTMC lesion,and the 25-35W power was ablated to cover 2-5 mm outside the edge of the lesion.Contrast-enhanced ultrasound confirmed whether the ablation range completely covered the lesion.If there would be a suspicious region without ablation,re-ablated it.When ablation was completed,recording the ablation time,ablation power,general condition of the patient?symptomatic treatment if necessary?,and intraoperative pain score.Results1.Study on the relationship between BRAF gene and ultrasonographic features in papillary thyroid microcarcinoma?1?Overall clinical characteristicsIn 116 cases of PTMC,the average age of patients was 43.66±12.11years old,and the average tumor size was 7.28±1.58 mm?5-10 mm?.114cases of PTMC with solid structure accounted for 98.3%and 22 cases of marked hypoechoic PTMC accounted for 19.0%.There were 75 calcified nodules,of which 72 microcalcifications accounted for 77.0%and three large and coarse calcified nodules accounted for 2.6%.56 PTMCs with an taller feature accounted for 48.3%and 75 with noisy edges accounted for 64.7%.34 patients?34/116,29.3%?had a background in Hashimoto's thyroiditis.There were 28 patients with lymph node metastasis,accounting for 24.1%of 116 patients.70 cases with BRAF^V600E mutations accounted for 60.3%?70/116?of the cases.?2?The relationship between BRAF^V600E gene and ultrasound imaging features and clinicopathological featuresPTMC of multifocal features of lesions in the BRAF^V600E600E mutation group and BRAF^V600E wild group were 58.6%?41/70?and 17.4%?8/46?,respectively,with statistical significance?P<0.000?.The taller than wide shape features were 65.4%?46/70?and 21.7%?10/46?in the BRAF^V600E600E mutation group and the BRAF^V600E wild group,respectively?P<0.000?.Other ultrasound imaging features,such as edge,hypoechoic,and very low echo were not statistically significant between the BRAF^V600E gene mutation group and the BRAF^V600E gene wild group?P>0.05?.In the clinicopathological features,28 cases of lymph node metastasis were found in the BRAF^V600E mutation group and the gene wild type group:23 cases?23/70,32.9%?and 5 cases?5/46,10.9%?,respectively,with statistical difference.?P=0.008?.Other clinical features,such as gender,age,and Hashimoto's thyroiditis,were not statistically different in the BRAF^V600E mutation group and the non-mutation group.?3?Multivariate regression analysis of BRAF^V600E gene mutation and ultrasound imaging characteristics and clinicopathological featuresIn multivariate regression analysis results,multifocal,taller than wide shape and lymph node metastasis were characteristic indicators for predicting BRAF mutation,and their odds ratio?OR?were:3.681,3.181and 4.615,respectively,and the p values were:0.031,0.041,and 0.009.2.Study on BRAF^V600E detection combined with ultrasound imaging features for predicting the risk of central lymph node metastasis of cN₀-papillary thyroid microcarcinoma:?1?Overall characteristics distributionAmong 182 patients,suspicious ultrasound findings included solid components,marked hypoechoic,microcalcification,irregular/lobulated edges and taller feature,seen at 98.9%,17.6%,59.3,69.8%and 45.6%of patients with PTMC,respectively.55?30.2%?patients of PTMC had chronic lymphocytic thyroiditis.There were 87 cases with BRAF^V600E600E mutation,accounting for 47.8%of 182 cases.?2?CLNM distribution characteristics of cN₀-PTMC patients with different clinical pathology and ultrasound characteristicsThe positive rate of CLNM with BRAF^V600E600E mutation was 64.8%,and the positive rate of CLNM without BRAF^V600E mutation was 35.2%?P<0.001?.Among the ultrasound features of PTMC,tumor size?P=0.027?,multifocality?P=0.009?and marked hypoechoic?P=0.028?were significantly associated with the presence of CLNM.Other ultrasound features,including solid components,microcalcifications,marginal irregularities or lobulations and taller than wide shape,were not associated with CLNM?P>0.05?.?3?Multivariate logistic regression analysis of PTMC with CLNMMultivariate logistic regression analysis showed independent correlation between clinical pathology and ultrasound features and CLNM positive risk.Features of tumor>7mm?OR=3.636,95%CI,1.671-7.914?,p=0.001),marked hypoechoic?OR=2.686,95%CI:1.080-6.678,P=0.002?,multifocal?OR=4.184,95%CI:1.707-10.258,P=0.002?and BRAF^V600E mutation?OR=5.339,95%CI:2.529-11.272,P<0.001?were independent risk factors for CLNM in clinically PTMC-negative patients?Table 3.2?.?4?BRAF^V600E mutation and ultrasound imaging characteristics predict central lymph node metastasisThe risk scores of each patient were calculated using the significant predictors identified in?3?above,and the risk scores were used to construct the ROC curve,area under the curve?AUC?was 0.755 with a sensitivity of 63.4%and a specificity of 80.2%.?5?Wild-type BRAF^V600E and ultrasonographic features predict non-lymph node metastasisUsing multivariate regression analysis to analyze the wild-type BRAF^V600E gene and low-risk predictors among ultrasound features of lymph node metastasis,the results showed tumor size?7 mm,non-marked hypoechoic,solitary lesion and wild type of BRAF^V600E gene had statistically predictive value for negative lymph node metastasis?P<0.05?.The OR values were 1.291,0.988,1.431 and 1.675,respectively.These factors were analyzed by ROC curve with AUC of0.716.3.Safety and clinical efficacy of microwave ablation for middle-low risk cN₀-PTMC:?1?General information of patients before operation27 patients with PTMC had 27 lesions,the average age of patients was 43.22±16.37 years old?21-61 years old?,including 8 males and 19females,and there were 2 cases had Hashimoto's thyroiditis.The average diameter of PTMC lesions was 7.2±3.3 mm?4.5-10.0 mm?and the volume was 0.13±0.08 ml?0.06-0.42 ml?.?2?Information of operation processThe dosage of 2%lidocaine was from 10-30ml,the average was 18±8ml.The intraoperative pain score was 1.71±0.65?range:0-4?and no severe pain occurred.In all 27 patients with PTMC microwave ablation,one case occurred venous hemorrhage in the neck.Immediately after ablation of the bleeding point by microwave ablation,hemostasis was stopped during the operation,and no serious bleeding such as neck hemorrhage or neck skin burn occurred.No case of laryngeal recurrent nerve damage.Of all the 27 cases,five cases were outpatients,and 22cases were hospitalized for one day.?3?Thyroid function after ablationIn all cases of ablation,levothyroxine sodium tablets were given for inhibition treatment three months after surgery,and TSH has decreased to<0.05?IU/L.There was no significant difference in thyroid-related hormone levels?FT3,FT4 and TSH?between one week before surgery and one month or three months after surgery?P>0.05?.?4?Follow-up of postoperative lesionsAll 27 PTMC lesions began to shrink in size at the 3rd month after ablation,and the volume reduction rate reached 48.6±11.6%.By the 6th month,the volume reduction rate reached 85.6±17.7%.27 PTMC nodules were completely absorbed in 18 cases after ablation,and 9 PTMC nodules were not completely absorbed.Of the 9 nodules that were not fully absorbed,7 cases were followed up for<9 months after ablation,one case for 13.5 months and one for 26 months.In all 27 cases,no postoperative lymph node recurrence was found after postoperative ultrasound and CT follow-up.One male patient had a new PTMC lesion?diameter 4.6 mm?at the 23rd months after ablation and was given re-ablation,no recurrence of thyroid or cervical lymph nodes was observed at 9 months follow-up.Conclusions?1?Among the PTMC population in China,the BRAF^V600E gene mutation rate was relatively high.The BRAF^V600E gene mutation in PTMC was significantly associated with multifocal tumors,taller than wide and cervical lymph node metastasis.?2?Tumor>7mm,significantly low echo,multifocal tumors and BRAF^V600E mutation were independent factors to predict cervical lymph node metastasis in patients with cN₀-PTMC.FNA-BRAF^V600E detection combined with ultrasound imaging features has a certain value to predict lymph node metastasis in patients with cN₀-PTMC.?3?Wild-type FNA-BRAF^V600E gene combined with ultrasound imaging features had a certain value to predict patients with cN₀-PTMC have no lymph node metastasis.?4?Microwave ablation for the treatment of mid-low risk cN₀-PTMC has good clinical safety and minimally invasive properties.?5?Microwave ablation for mid-low risk cN₀-PTMC can achieve local radical cure in the mid-term follow-up results.In general,for patients with c N₀-PTMC,preoperative FNA detection combined with ultrasound imaging features can predict the risk of cervical lymphatic metastasis,and ultrasound-guided microwave ablation for patients with mid-low risk cN₀-PTMC has good security and local radical cure.