Research On Specific Protein Markers For The Differential Diagnosis Of Papillary Thyroid Carcinomas And Thyroid Borderline Lesions

[Background]Thyroid carcinoma(TC)is the most common malignant tumor of endocrine system which usually occurs in female patients.Papillary thyroid carcinoma(PTC)is the most common malignant carcinoma in TC and papillary thyroid microcarcinoma(PTMC)is a subtype of PTC.At present,pathologists genarally distinguish benign lesion from malignant tumor by histopathological tests.It's difficult to accurately differentiate thyroid carcinom and bordline lesion only through histopathological tests because of the ambiguous morphological features of bordline lesion.With the development of the molecular biology and proteomics,more researches have focused on biomarkers of thyroid carcinoma in which a few researches involved in thyroid bordline lesion.Usually,CK19,Galectin-3 and TPO were important markers of thyroid tumors.[Aim]We analyzed and identified the different proteins expression between PTC and thyroid borderline lesion to look for potential new biomarkers.We evaluated CK19,Galectin-3 and TPO antibody in PTC and bordline lesion to investigate their diagnostic usefulness.[Methods]The total protein extracts from frozen thyroid samples(PTC,PTMC,borderline lesions and normal control tissues)were profiled with two-dimensional electrophoresis.Then the differential protein spots were revealed by PDQUEST 7.3 software,and identified by MALDI-TOF-TOF/MS and Swiss-prot database searching.These differential proteins were classified by PANTHER analysis.After constructing the tissue microarray(TMA),10 differently expressed proteins of these spots which had good repeatability and high relative content were further validated by immunohistochemistry in TMA.Immunohistochemistry was used to detect the expressions of CK19,Galectin-3 and TPO in 50 cases of PTC,71 cases of borderline lesions and 35 cases of normal thyroid tissues.The relationships between their expressions and clinicopathologic features of PTC were analyzed.[Results]A set of 43 differentially expressed spots significant for discriminating between the sample groups were found,and 29 proteins were identified which included CK8,CK18,HSP60 etc.Immunohistochemistry showed that all 10 proteins were located in cytoplasm.Also,CTSB,ANXA5 and CRT were located in membrane.8 proteins(CTSB,ANXA,y-Actin,GSTP1,CRT,HSP60,CK8 and CK18)were validated to be overexpressed in PTC compared with borderline lesions and normal tissues.Sametime,these proteins were validated to be overexpressed in borderline lesions with normal tissues.2 proteins(CNDP2 and PDIA3)were validated to be overexpressed in PTC and borderline lesions compared with normal tissues and no statistical differences in PTC compared with borderline lesions.When ANXA5,HSP60,CK8 and CK18 were combined discretionarily,the area under curve(AUC)of the combination of HSP60,CK8 and CK18 was 93.3%,the largest in all combinations,in distinguishing borderline lesions and normal tissue;the AUCs of the combinations of ANXA5 and HSP60,ANXA5 and HSP60 and CK8,ANXA5 and HSP60 and CK18,HSP60 and CK8 and CK18,ANXA5 and HSP60 and CK8 and CK18 were 100%in distinguishing PTC and normal tissue;the AUC of the combination of ANXA5 and HSP60 and CK8 and CK18 was largest in all combinations in distinguishing PTC and borderline lesions.CK19 and Galectin-3 showed higher expressions in PTC than borderline lesions and normal tissues.In comparison,TPO showed lower expression in PTC than borderline lesions and normal tissues.By the combined application of CK19 and TPO,the sensitivity,specificity,diagnostic accuracy rate were 88%,91.3%and 89.9%,respectively.In borderline lesions,the expression of CK19 was positively related to that of Galectin-3.Also,the expressions of CK19 and Galectin-3 were negatively related to that of TPO.In PTC,the expression of CK19 was positively related to that of Galectin-3,and the expression of CK19 was negatively related to that of TPO.In PTC,the expression of CK19 in different age,gender,tumor size,multifocality,lymph node metastasis,distant metastasis and clinicopathologic stage had no statistical differences.The expressions of Galectin-3 and TPO were related to gender,but there were no relationships within expression of Galectin-3 and other clinicopathologic features.Also,there were no relationships within expression of TPO and other clinicopathologic features.[Conclusions]The study showed that proteomics analysis is useful to find new biomarkers for PTC and thyroid borderline lesion.Expression patterns of these differently expressed proteins can be further validated by immunohistochemistry.The newly identified proteins may eventually serve as early diagnostic markers for PTC.We have concluded that combinations ofANXA5,HSP60,CK8 and CK18 have high diagnostic values in distinguishing PTC,border lesion and normal tissue.We have concluded that as important reference markers,combination of CK19,Galectin-3 and TPO can improve the diagnostic accuracy for PTC and borderline lesion.