Experimental Study On Circulating BRAF Mutant Gene Combined With Other Detection And Their Role In The Diagnosis And Prognosis Of Papillary Thyroid Carcinoma

Object:To investigate the prevalence and clinicopathological significance of BRAF mutation, RET rearrangement and the expression of CK19, HBME-1, Galectin-3, E-cadherin in thyroid papillary carcinomas. Meanwhile, a wonderful objective evidence could be found to serve as molecular diagnosis and evaluation of prognosis and treatment in targeted-gene. This paper also detected the frequency of gene mutation efficiently so as to explore a kind of efficient, sensitive and specific method of detection, nucleic acid samples were performed in biopsy samples from thyroid carcinoma blood plasm.Method:PARTⅠ90 thyroid tumors were enrolled in this study which included 15 nodular goiters with papillary hyperplasia,10 follicular tumor,5 follicular thyroid carcinoma and 60 papillary thyroid carcinoma. Total RNA was isolated from the tumor tissues and blood plasm and reversely transcribed to cDNA. BRAFT1799A mutation genes were amplified by PCR and confirmed by sequencing.The clinicopathological significance of BRAFT1799A mutation genes was analyzed. PART IITotal RNA and DNA of tumor and para-tumor tissues of 236 papillary thyroid carcinoma in paraffin-embedded specimen were isolated. BRAF gene coding sequence were amplified by PCR with DNA as template and sequenced. RET/PTC 1 and RET/PTC3 fusion genes were amplified by PCR and confirmed by sequencing. The interaction, exclusivity and clinicopathological significance of rearrangement, BRAF mutation and RET/PTC rearrangement were analyzed.PART III269 thyroid tumors were enrolled in this study which included 10 nodular goiters with papillary hyperplasia,10 follicular tumor,8 follicular thyroid carcinoma,5 normal thyroid tissue and 236 papillary thyroid carcinoma. By manual tissue chip technique, this is done by using a needle to biopsy a standard histological section and placing the core into an array on a recipient paraffin block. The expression of CK19, HBME-1, Galectin-3 and E-cadherin was examined by SP immunohisochemical stain(IHC) in thyroid carcinoma, thyroid adenoma and nodular goiter with papillary hyperplasia. Compared with the expression BRAFT1799A in thyroid carcinoma. To evaluate whether CK19, HBME-1, Galectin-3, E-cadherin immunohistochemical analysis and the expression BRAFT1799A can reliably differentiate benign thyroid lesions from papillary carcinomas and the panel can be useful to help classify thyroid lesions.PARTⅣIn this section, We collected 1286 cases of paraffin-embed tissue of papillary thyroid carcinoma. All of the samples were taken from the Jiangsu province tumor hospital from 1980 to 2009 and all were diagnosed and classified by two pathological Professors referring to WHO standard. We analysed the association between clinical character and prognosis.Result:PARTⅠBRAF mutation was detected in 26 of 60 PTC(43.3%) and in 9 of 22 PTC with circulating nucleic acid. All mutations were heterozygous BRAF mutation that lied in nucleotide position 1799 of exon 15. The mutation was not found in 5 FTC,15 nodular goiters with papillary hyperplasia,10 follicular tumor and 12 normal thyroid tissue. Statistical analysis demonstrated that the relationship between PTC tissues and circulating nucleic acid in BRAF mutation was significant(P<0.05). However, there was no significant difference between PTC without regional lymph node metastasis and PTC with regional lymph node metastasis.(P>0.05). BRAF mutation in PTC was not correlated with age. gender, histological type, but correlated with TNM staging and the tumor diameter.PARTⅡRET rearrangement, which included 55 RET/PTC 1 and 9 RET/PTC3 was only detected in 27.12%(64 of 236) of PTC and was not observed in non-PTC thyroid tumor and para-tumor normal tissues. No RET/PTC 1 and RET/PTC3 were detected in one tumor tissue. The patient group of age≤20 years had the highest frequency (57.78%) of RET rearrangement among the groups of age≤20 years,20-40 years (22.0%) and≥40 years(16.48%). RET/PTC 1 positive patients were more easily to suffer from another benign thyroid disease simultaneously. RET/PTC3 positive patients had a higher frequency of extrathyroidal extension and advanced T classification. Among 236 cases of papillary thyroid carcinoma,103 cases of BRAF mutation(43.64%).20 cases (8.47%) of BRAF mutation positive patients were found with another genetic aberration and the frequeney of multi-genetic aberrantions of RET rearrangement. The age of gene rearrangement positive patients was younger than that of the negative patients. BRAF was the only genetic aberration in patients of age≥60 years(80.65%). The frequency of gene rearrangement and BRAF mutation decreased with the age. BRAF mutation, RET rearrangement,male,age,multifocality and advanced T classification were all identified as risk factors of leveⅡ-Ⅴlymph node involvement in the multivariate analysis.PARTⅢ1. results of tissue chip6 tissue chips(12×15)were made. Dots of tissue chip lined up in order, uniformed in size and no shifting.7 tissue dots were absence.The significant tissue structures were watched in the other dots. The haematoxylin-eosin(HE) staining was uniformity and no dropping, moving and wrinkling. The immunohistochemical results showed that the positive location was accurate and the background was clear.2. The result of immunohistochemistry2.1 Expression of CK19 in various lesions of thyroidSamples of 10 thyroid adenomas,10 nodular goiters with papillary hyperplasia and 236 thyroid papillary carcinomas were stained immunohistochemically with CK19. CK19 staining was noted in 1/10 of thyroid adenomas,3/10 of nodular goiters with papillary hyperplasia and 97.88%(231/236) of papillary carcinomas. We found statistically significant differences between thyroid benign lesion and papillary carcinomas (P<0.05). The sensitivity, specificity and overall accuracy of CK19 were 97.88%,80.0%,96.48%,respectively.2.2 Expression of HBME-1 in various lesions of thyroid Samples of 1 thyroid adenomas,1 nodular goiters with papillary hyperplasia and 204 thyroid papillary carcinomas were stained immunohistochemically with HBME-1. 2 of thyroid benign lesion was tiny positive, but 87 of papillary carcinomas were strong positive. We found statistically significant difference between thyroid benign lesion and papillary carcinomas(P<0.05). HBME-1 examination in thyroid lesions had a sensitivity of 86.44%, specificity of 90.0%, and overall accurary of 86.71%.2.3 Expression of Galectin-3 in various lesions of thyroidImmunohistochemical staining for Galectin-3 was performed on 2 thyroid adenomas,3 nodular goiters with papillary hyperplasia and 226 thyroid papillary carcinomas.20.0% of thyroid adenoma and 30.0% of nodular goiters with papillary hyperplasia was positive. On the other hand,95.76% of thyroid papillary carcinoma was positive. The differences between thyroid papillary carcinomas and thyroid adenoma and between thyroid papillary carcinomas and nodular goiters with papillary hyperplasia were both statistically significant (P＜0.05). Galectin-3 examination in thyroid lesions had a sensitivity of 95.76%,specificity of 80.0%, and overall accurary of 94.53%.2.4 Expression of E-cadherin in various lesions of thyroid269 thyroid lesions were examined by immunohisto-chemistry with E-cadherin including 10 thyroid adenomas,10 nodular goiters with papillary hyperplasia and 236 thyroid papillary carcinomas.16.95% of papillary carcinomas expressed E-cadherin as well as 90.0% of thyroid adenomas and 80.0% of nodular goiters with papillary hyperplasia. We found statistically significant difference between thyroid benign lesion and papillary carcinomas(P<0.05). The sensitivity, specificity and overall accurary of E-cadherin were 83.05%,85.0%,83.2%, respectively.3. There was no significant correlation of CK19,HBME-1,Galectin-3 and E-cadherin in thyroid papillary carcinomas.4. No significant relationship were found between the expression of CK19,HBME-1,Galectin-3 and E-cadherin and clinical pathological features,such as age, sex, tumor size, local infiltration, metastasis. PARTⅣThe follow up meso-age is 9 years. The total survival rate of 5,10,15,20 years were 95.87%,92.04%,87.33%,82.13%. Male, age, multifocality, tumor size, lymph node metastasis and advanced T classification were all identified as risk factors in the monovariate analysis. Male, age, tumor size, lymph node metastasis, metastasis and advanced T classification were all identified as risk factors in the multivariate analysis.Conclusion:PARTⅠBRAF mutation was detected in 9 of 22 PTC with circulating nucleic acid. Statistical analysis demonstrated that the relationship between PTC tissues and circulating nucleic acid in BRAF mutation was significant(P<0.05). However, there was no significant difference between PTC without regional lymph node metastasis and PTC with regional lymph node metastasis.(P>0.05). BRAF mutation in PTC was not correlated with age,gender,histological type, but correlated with TNM staging and the tumor diameter. The frequency of BRAF mutation was decreasing with the increasing age. While BRAF mutation was the only genetic aberration of papillary thyroid carcinoma in patients of age≥60 years (80.65%).PARTⅡOnly two RET rearrangements,RET/PTC 1 and RET/PTC3 were detected in Chinese patients with papillary thyroid carcinoma in the present study. The patients with age<20 years have the highest frequency of RET rearrangement among the four age groups (57.78%). RET rearrangement is associated with LevelⅡ-Ⅴlymph node metastasis. Male,young age,multifocality,advanced T classification and RET rearrangement were indentified as the risk factors of lateral neck lymph node metastasis. Serious pre-operative scanning and intensive post-operative follow up should be performed to the patients with the risk factors. RET/PTC 1 and RET/PTC3 have different role in the progression of thyroid cancer,and RET/PTC 1 may be associated with thyroiditis. RET rearrangement and BRAF mutation can be detected in one tumor sample simultaneously,especially in patients with young age.The predictive value of molecular changes as a diagnostic and target therapeutic markers will be improved by screening RET rearrangement and BRAF mutation and other possibly involved genes together.PARTⅢ1. The sensitity of CK19 examination in thyroid lesions was higher than the other markers, therefore CK19 was the most sensitive for the distinction between thyroid benign lesion and papillary carcinomas.2. In terms of specificity,HBME-1 was the most specific marker for the differential diagnosis of thyroid carcinoma.3. In terms of overall accuracy, CK19 was the most accurate for the distinction between thyroid benign lesion and papillary carcinomas.4. The combination of Galectin-3 and CK19 increased the specificity and overall accuracy up to 80.0% and 96.82%.A two-marker antibody panel with Galectin-3 and CK19 was the most efficient for the distinction between benign and malignant thyroid lesion.The panel can be useful to help classify thyroid lesions.5. A three-marker antibody panel with Galectin-3,HBME-1 and CK19 was the best and decreased sensitity and overall accuracy by 66.7%and 88.5%,although the specificitywas 100%.So the panel three markers may not be helpful in the differential diagnosis between benign lesions and papillary carcinomas.6. There was probably no significant correlation of CK19,HBME-1,Galectin-3 and E-cadherin in the carcinogenesis and development thyroid papillary carcinomas.7. No significant relationship were found between the expression of CK19, HBME-1,Galectin-3 and E-cadherin and clinical pathological features, such as age,sex,tumor size,focal infiltration.8. Galectin-3 protein expression in PTC was correlated with lymph node metastasis. But E-cadherin protein expression in PTC was correlated with TNM staging and lymph node metastasis. The diagnostic value of protein changes will be improved by screening Galectin-3 protein expression and E-cadherin protein expression.9. Manual tissue chip technique is simple,cost-effective,and reliable.This technique can provide a highly efficient, high-through put mechanism for some research. PARTⅣGender, age, multifocality, tumor size, lymph node metastasis and advanced T classification were all identified as risk factors in the PTC prognosis. The PI value was important in estimation of PTC prognosis.