Research Of Thyroid Cancer Molecular Marker Based On Xinjiang Population

Objective: Thyroid disease is a high incident endocrinal disease caused by diverse factors and represents such clinical manifestations as nodular goiter, Hashimoto’s thyroiditis, thyroid adenoma(benign)and thyroid cancer(malignant), threatening the health and life of more than three hundred million people in the world. Thyroid cancer is one of the most common endocrine malignancies in China, occurred in one of ten malignant cancers of Chinese women, rapidly increased in last three decades. Xinjiang is multi-ethnic region of China,the increasing trend of the number of thyroid cancer patients from various ethnic groups treated in major hospitals has been noticeable, however, the incidence of thyroid disease, specially the thyroid cancer in Xinjiang has not been reported so far.Thyroid cancer is a kind of malignant tumor of epithelial cell origin, generally represents the papillary thyroid cancer, follicular thyroid carcinoma, undifferentiated thyroid carcinoma and medullar thyroid carcinoma, with the ratio of 80-90% for papillary thyroid cancer.Medical imaging is the main method for the diagnoses of the cancer, but often fails in early detection of many tumors without distinct clinical symptoms, and always leads to unsuitable treatment because of difficulties in distinguishing benign and malignant tumors with similar clinical features.To study the thyroid cancer by molecular genetics, molecular biology and immunology, to understand the molecular mechanism of the cancer development, to discover the molecular markers for early diagnosis or discrimination of the begin from malignant tumor, is a cutting edge to establish molecular diagnostic profile, effective prevention and clinical treatment of the cancer.Therefore, in the first part of this study, we will analyze the case information of patients treated in the hospital in last eight years, and understand the development state of thyroid diseases or thyroid cancer in Xinjiang in different aspects such as the morbidity trend, gender, age, and ethnic groups.In the second part, we will search for candidate genes specific or related to adenocarcinoma confirmed in previous reports, screen the up-and down-regulated gene profile in thyroid cancer at protein expression level based on cases treated in Xinjiang, and evaluate the role as biomarkers for thyroid disease development. In the third part, we will focus on genes down-regulated or lost in protein expression according to the data generated from the“second part”, identify molecular markers hypermethylated in, and specific to, papillary thyroid cancer by analysis of candidate gene methylation at promoter region using Sequenom MassARRAY platform for quantitative analysis of methylated DNA. Method:1.We collected the information of 6071 case according to the archive of the first affiliated hospital of Xinjiang medical university from 2002～2009, including 5865 cases of thyroid begin lesions and 206 cases of thyroid cancer, with age range of 1～91, 1707 cases of male, 4364 cases of female, 4191 cases of Han, 1160 cases of Uyghur, 810 cases of other ethnic groups. SPSS13.0 and Microsoft Excel 2007 were used for statistical analysis, x~2 with significance atα=0.05.2.195 formalin fixed and paraffin-embedded thyroid specimens were collected from the period of 2004～2010, in tissue bank of pathology department of first affiliated hospital of Xinjiang medical university, divided into 112 cases of thyroid cancer, 10 cases of nodular goiter, 15 cases of Hashimoto’s thyroiditis, 26 cases of thyroid adenoma, 32 cases of normal tissue. Eleven thyroid cancer-specific up-or down-regulated candidate genes CDH1, MGMT, TIMP3, ESR1, DAPK1, RARB, APC, CDKN2A and MSX1 were selected for the analysis of protein expression level of single gene by immunohistochemical S-P method with specific antibodies. 3.High quality tissue DNA was prepared from 49 papillary thyroid cancer tissue specimens and from 23 normal tissue specimens using the improved genomic DNA extraction method resulted from the pilot experiment. PCR primes specific to CpG Island of promoter region of 5 types of candidate genes were designed for the quantitative analysis of methylation in tissue DNA by Sequenom MassARRAY platform.Result:1.The incidence of the thyroid cancer has markedly increased from 2002～2009,the ratio of thyroid cancer cases was very low (3.39%) compared to benign lesion(s96.61%)representing mainly the Graves’ disease and nodular goite(r48 and 4 0%, respectively). Papillary thyroid cancer(81%) and folicular cancer(14%)were the main types of thyroid cancer. The morbidity of thyroid disease has significant difference among various age ranges, and in gender with a male to female ratio of 1/2.56(P <0.05).The ratio of Han and Uyghur ethnic group in all cases was dominant ones, but no difference was found in the distribution of both groups in all disease types.The ratio between female to male significantly higher than the ratio of Han’s both in thyroid disease or in thyroid cancer.2.①Immunohistochemical and statistical analysis showed that the protein express level (positive rate) of CDH1, MLH1, TIMP3 and DAPK1 genes was significantly decreased from the group of normal tissues to nodular goiter, Hashimoto’s thyroiditis, thyroid adenoma and thyroid cancer(P <0.05), and in contrast, the alteration trend of ESR1, MGMT and RARβprotein expression remarkably increased with statistical significance. But no difference was found for APC.②From the clinicopathological point of view, the downregulation of CDH1 protein expression or the upregulation of ESR1 were closely associated with the prognosis and types of the cancer, whereas the downregulation of DAPK1 or upregulation of RARβcorrelated with lymphnode metastasis(P<0.05).③Analysis focused on the papillary thyroid cancer showed significant difference of CDH1, MLH1, TIMP3, DAPK1, ESR1, MGMT and RARβprotein expression between papillary thyroid cancer and the normal control,(P<0.05).Additionally, the positive rate of CALCA protein expression was significantly higher in papillary thyroid cancer compared to normal control, but no difference was found for CDKN2A and MSX1.④As tumor specific genes of thyroid cancer , CDH1 and MLH1, MLH1 and TIMP3, RARβand ESR1, RARβand MGMT or CDKN2A/p14ARF and CALCA at protein expression level to various exten(t0.4<r <0.7,P<0.01). (5) For ESR1, RARβand MGMT as a single gene, high sensitivity (64.1, 76.2 and 62.2%, respectively), specificity(83.3, 66.7, 83.3%) and accuracy(67.1, 65.8, 74.7%)was estimated in protein detection by immunohistochemistry, but this was not the case for other genes examined. The analysis of the combined detection level showed very high sensitivity, specificity and accuracy for three genes ESR1/RARβ/MGMT(67.6、77.8、69.2%)at protein level, promising a very good (ideal) combination, whereas the pair wise combination of CDH1, MLH1, TIMP3 and DAPK (41.1, 20.9, 37.8%)was medium or low, and the combinations like TIMP3/CDH1(46.5,25.0,43.2%),TIMP3/MLH1(44.1,25.0,40.8%)were still promising.⑤ESR1, RARβand MGMT had high sensitivity (64.1, 76.2, 62.2%), specificity(83.3, 66.7, 83.3%) and accuracy(67.1, 65.8, 74.7%)as a single gene, but this was not the case for other genes examined. The analysis of the combined detection showed very high sensitivity, specificity and accuracy for three genes ESR1/RARβ/MGMT(67.6,77.8,69.2%)at protein expression level, promising an very good (ideal) combination, whereas the pair wise combination of CDH1, MLH1, TIMP3 and DAPK (41.1, 20.9, 37.8%)was medium or low, and the combinations like TIMP3/CDH1(46.5,25.0,43.2%),TIMP3/MLH1(44.1,25.0,40.8%)were still promising.3. We prepared high quality genomic DNA with the improved method for DNA extraction from paraffin-embedded specimens. By analysis of quantitatively methylation level using Sequenom MassArray approach, we found target CpG fragment methylation of TIMP3 at the promoter region specific to papillary thyroid cancer with statistical difference, in comparison to the normal control (P<0.05),but no difference was shown for RARB, CALCA,CDH1 and MLH1. Further analysis of TIMP3 for single CpG site methylation indicated that the methylation of CpG-7/CpG-8 combination and CpG-9 was quantitatively higher in cancer tissue DNA than in the normal, and associated with the degree of clinical stages(P<0.05 or P<0.01).Conclusion:1. An increasing trend in morbidity was a common feature of thyroid disease in Xinjiang population, high in women patients than in men, earlier age in women patient than in men (malignant)with only a minimal proportion of malignancy compared to the predominance of benign lesions. Papillary thyroid cancer and the Grave’s diseases are the most common types occurred as malignant cancer and benign disease, respectively.Although there was no age or ethnic difference in all thyroid disease types in Xinjiang,whereas there was significant difference at constituent ratio of clinical types of thyroid cancer between the Uyghur and Han,at the same time the ratio of gender of patient in Uyghur higher than in Han.2. (1)The upregulation of CDH1,MLH1,TIMP3 and DAPK1 protein expression or downregulation of ESR1,MGMT and RARβmay be a molecular marker profile of thyroid cancer, and among them, the alteration of CDH1,MLH1, TIMP3,DAPK1,ESR1,MGMT,RARβand CALCA protein level was more specific to papillary thyroid cancer than the other cancer types. (2)The change in CDH1,ESR1,DAPK1and RARβprotein expression may be a important marker for the estimation of prognosis, cancer types or lymphnode metastasis. An association of the alteration trend in protein expression was estimated for CDH1,MLH1,TIMP3,RARβ,ESR1,MGMT,CDKN2A and CALCA. (3)The combined detection with three genes ESR1,RARβand MGMT at protein level may promise a very high sensitivity, specificity and accuracy for the diagnosis of thyroid cancer, whereas the combination of TIMP3with CDH1or MLH1 may also promise a high detection rate of the cancer. 3. The methylation of TIMP3 at promoter region may be an epigenetic event accounting for the downregulation or loss of protein expression specific to papillary thyroid cancer, but not the case for such genes as RARB, CALCA, CDH1 or MLH1.