| Thyroid carcinoma is the most common malignancy of the endocrine system.Its incidence has increased significantly during the past decades and it is currently the seventh most common malignancy diagnosed in women.Thyroid carcinomas are classified histologically as papillary,follicular,anaplastic,or medullary,with papillary thyroid carcinoma(PTC)accounts for more than 85%of all thyroid cancer.Papillary thyroid carcinoma is defined by the World Health Organization(WHO)as a malignant epithelial tumour showing evidence of follicular cell differentiation and characterized by distinctive nuclear features。The distinctive nuclear feature of the PTC includes enlarged oval nucleus,nuclear overlap,nuclear grooves and intranuclear pseudoinclusions.The typical nuclear is clearing.PTC is diagnosed almost solely based on the above feature of the nucleus,and these cells accounts for the most part of the tumor.Affected by the specialized skill and the subjectivity,it is difficult for the clinical pathologist to identified the typical nuclear of the PTC,even for the specialist.The molecular diagnostic techniques have been applied to the diagnosis of PTC,however,the clinical value of it is limited,as the price is too high and the specificity also needs to be improved.So,it becomes the searching for the immunohistochemial markers used for diagnosing the papillary thyroid carcinoma with high specificity and commonly used is becoming the concern of the researchers.PTC is generally an indolent disease and has a favorable prognosis in most affected patients,however,there is a subset of patients present with recurrence and/or evidence of metastatic disease.Although factors such as gender,age,size and number of the tumor and TNM tend to correlate with the prognosis of PTC,the patients with the same TNM also have different prognosis.This difference may be due to the effect of different molecular mechanism.Immunohistochemical markers related with the prognosis may be useful for risk stratification and treatment with the patients.Standard treatment of PTC usually includes primary surgery,thyroid-stimulating hormone suppressive therapy,and ablation of the thyroid remnant with radioactive iodine(RAI).Despite the generally good prognosis of thyroid carcinoma,about less than 10%of patients will develop metastatic disease,which fails to respond to RAI,exhibiting a more aggressive behavior.BRAF mutations are found in a wide range of cancers.Moreover,the mutation is associated with aggressive tumor characteristics or poor prognostic factors in most.The BRAF V600E mutation is the most common genetic change in patients with PTC,being observed in 29-83%of all cases.Treatment with Bay 43-9006 can inhibit BRAF stimulated DNA synthesis and cell proliferation,induce apoptosis in PTC cells harboring BRAF mutation.Many different methods for BRAF mutation analyses have been developed,including Sanger sequencing,pyrosequencing,high-resolution melting analysis,and allele-specific polymerase chain reaction(PCR)-based methods.A major drawback of these methods is that they are expensive,labor and time intensive,not always available,and may be difficult to implement in clinical settings.An immunohistochemical(IHC)approach for the detection of BRAF mutations in PTC is certainly a complementary method to molecular biology analysis.Immunohistochemistry with mutation-specific antibodies become mandatory for detecting the mutated protein in clinical settings.The presence of multiple foci of PTC is a common clinical finding.Multifocality was correlated with recurrence and lymphnode metastasis/distant metastasis.So far,the origin of these foci is unsettled.They may be intraglandular metastases from a single primary tumor,or each tumor may arise from a distinct progenitor cell.Evidence from previous studies has lent support to both arguments.Clearly defining the genetic relationships among the multifocal PTC lesions could have important surgical,therapeutic,and prognostic implications.In addition,understanding the nature of tumor multifocality can serve to further our understanding of the genetic basis of tumor progression in PTC.Objective:1.To investigate the value of immunohistochemical markers in pathological diagnosis and its correlation with prognosis of PTC.2.To compare the detection of the BRAFV600E mutation in PTC by molecular biology and by IHC.Evaluate the usefulness of using IHC to detect the BRAFV600E mutation in PTC.3.To analysis the clonal origin of mutifocal PTC to better understanding the mechanism and provide evidence for treatment of PTC.Methods:1.By immunohistochemical method called MaxVision,ER,PR,CK19,E-cadherin,CyclinDl and ki67 were examined respectively in 66 PTC and 25 nodulated struma cases.IHC was scored on intensity,proportion scale.IHC positive tumors were stratified into intensity categories.Estimate their clinical value in distinguishing PTC and nodulated struma.The categories were assessed for the clinicopathologic variables including age,extrathyroidal extension,lymphovascular invasion,and lymph node metastases of PTC.2.The BRAF V600E was detected in 40 PTCs by Quantitative Real-time PCR and immunohistochemical method.Evaluated the association between BRAF V600E expression and various clinicopathologic parameters.Analized the relationship between protein expression and gene copy number of BRAF V600E.3.The clonal origin of 89 female mutifocal PTCs were analyzed using X-chromosome inactivation studies based on the differential methylation patterns of active and inactive of HUMARA alleles.4.Analyses were carried out using SPSS 16.0 statistical software and the significant P-value was set at 0.05.Results:1.The expression of ERa was identified in 37 cases of PTC(56.1%)and 4 cases of nodulated struma(16%).ERa expression was significantly elevated in PTC(X2=11.755,P<0.05).The proportion of ERa expression is about 5-75%and 2-10%the scores are between 3-7 and 4 in PTC and nodulated struma respectively.2.The expression of PR was identified in 36 cases of PTC(54.5%)and 4 cases of nodulated struma(16%).PR expression was significantly elevated in PTC(χ2=6.722,P<0.05).The IHC scores of PR expression are between 3-8 and 4-6 in PTC and nodulated struma respectively.3.The expression of CK19 was identified in 65 cases of PTC(98.5%)and 16 cases of nodulated struma(64%).PR expression was significantly elevated in PTC,P<0.05.The IHC scores of CK19 expression are 8 and 5 in PTC and nodulated struma respectively.The proportion of CK19 expression is above 60%,while in nodulated struma it is 10%-1/3.4.The expression of E-cadherin was identified in 55 cases of PTC(88.3%)and 25 cases of nodulated struma(100%).E-cadherin expression was significantly elevated in nodulated struma(χ2=4.740,P<0.05).5.The expression of CyclinD1 was identified in 64 cases of PTC(97%)and 16 cases of nodulated struma(64%).CyclinD1 expression was significantly elevated in PTC,P<0.05.The proportion and intensity of CyclinD1 expression in PTC and nodulated struma are equivalent.6.The expression ratio of both ERa and PR was 30%and 8%in PTC and nodulated struma respectively.The expression ratio of both ERa and CK19 was 52.5%and 8%in PTC and nodulated struma respectively.The expression ratio of both ERa and CyclinDl was 45.5%and 4%in PTC and nodulated struma respectively.The expression ratio of ERa,PR and CK19 was 30.30%and 4%in PTC and nodulated struma respectively.The expression ratio of ERa,PR and CyclinD1 was 31.82%and 0%in PTC and nodulated struma respectively.The expression ratio of PR,CK19 and CyclinD1 was 45.45%and 8%in PTC and nodulated struma respectively.The difference between them is significant.7.In 38 female PTCs,the ERa positive rate of lesions more than 1cm and less than 1cm was 82.4%and 33.3%respectively.The difference was statistically significant.In 28 male PTCs,the ERa positive rate of lesions less than 1cm and more than 1cm is 91.7%and 25%respectively.The difference was statistically significant.8.The PR positive rate was significantly higher in female PTCs(57.9%)than that in male PTCs(28.6%).9.The E-cadherin positive rate was significantly higher in female PTCs(94.7%)than that in male PTCs(67.9%).10.The ki67 positive rate was significantly higher in male PTCs(10.7%)than that in female PTCs(0%).In 28 male PTCs,the ki67 positive rate of older than 45 years(30%)was significantly higher than that of less than 45 years old(0%).11.The positive rate was 85%(34/40)with quantitative Real-time PCR detection of BRAF V600E.The positive rate was 90%(36/40)with IHC detection of BRAF V600E mutant protein.The sensitivity and specificity of the BRAF V600E(VE1)is 100%and 66.67%.12.There was no difference between PTC with 45 years older and younger than 45 years,male and femal,lesions less than 1cm and more than 1cm,with and without lymphnode metastasis by molecular analysis of the BRAF V600E mution.13.When the expression of BRAF V600E mutant protein was determined as positive with the immunohistochemical scores more than 3,the positive rate was 50%and 36.63%in PTC of older and younger than 45 years respectively.There is no difference between them.14.The mean ACt value(4.72)with immunohistochemical sore of 1 was significantly higher than that(2.85)with immunohistochemical sore of 2.15.Extracted the genomic DNA of 89 cases nomal thyroid tissue beside the tumor.Two HUMARA allelic bands were detected after PCR amplification in 11 cases.Two bands were detected after being treated with HhaI in 6 cases out of the 11cases.16.Three out of the six cases of the multifocal PTCs exhibited a monoclonal pattern,two cases exhibited polyclonal pattern.Both monoclonal and polyclonal might exsist in female multifocal PTCs.Conclusions:1.As he expression positive rate and intensity of ERa,PR,CK19 and CyclinDl protein in PTC were significantly higher than that in nodulated struma.It might be helpful for diagnosis of PTC when more than 10%tumor cells exhibited moderate immunoexpression of ERa,or more than 25%tumor cells exhibited moderate immunoexpression of PR,or more than 80%tumor cells exhibited moderate immunoexpression of CK19 and CyclinDl.2.The tumor cells exhibited positive immunoexpression of ER,PR,CyclinD1 and CK19 would support for the diagnosis of PTC.3.It is specific for diagnosis of PTC that immunoexpression of E-cadherin,CyclinDl and CK19 are all exhibite positive.4.The ERa positive rate had a significant association with the lesion size.In female PTC,the ERa positive rate was significantly higher in lesion more than 1cm than that of less than 1cm.In male PTC,the ERa positive rate was significantly lower in lesion more than 1cm than that of less than 1cm.Estrogen/ER might improve tumor cell proliferation for female PTC patients,which leading the bad prognosis.For the male patients,the effect might differs.5.The PR positive rate had a significant association with agender of PTC.The PR positive rate was significantly higher in female PTC than male PTC,which might illustrate the mechanism of worse prognosis of female patients than male patients.6.Male,45 years older and ki67 positive expression might be the foctor related with the bad prognosis of PTC.7.Both Quantitative Real-time PCR and immunohistochemical method could be used to detect the BRAFV600E.The detection of BRAFV600E by immnohitochemical method with BRAFV600E(VE1)is highly sensitive and specific.8.Detection the expression BRAFV600E mutant protein by immunohistochemical method would predict the BRAFV600E mutation.9.The heterozygosity of HUMARA was 12.4%(11/89)in our study of female multifocal PTC.10.Three out of the six cases of the multifocal PTCs exhibited a monoclonal pattern,two cases exhibited polyclonal pattern.Both monoclonal and polyclonal might exsist in female multifocal PTCs. |
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