The clustering was performed with 3830 genes
The clustering was performed with 3830 genes. The specificity of the antibody was validated by western blot analysis Results In gene expression analysis QPRT was recognized as differently IL6ST indicated between follicular thyroid adenoma and follicular thyroid Inolitazone dihydrochloride carcinoma. QPRT protein could be recognized by immunohistochemistry in 65% of follicular thyroid carcinomas including minimal invasive variant and only 22% of follicular adenomas. Summary Consequently, QPRT is definitely a potential fresh marker for the immunohistochemical screening of follicular thyroid nodules. Background Differentiated thyroid carcinomas display an incidence of approximately 1% of all human being malignancies [1]. In the group of endocrine malignant tumours they form, however, the largest entity. Differentiated thyroid carcinomas are a heterogeneous group composed of papillary, follicular (FTC) and medullary thyroid carcinoma [2]. In contrast to papillary carcinoma, which usually can be very easily diagnosed by its characteristic growth pattern und nuclear features, FTC can appear cytologically identical to follicular thyroid adenoma (FTA). In these cases only the growth pattern distinguishes between benign and malignant thyroid tumours. According to the grade of invasion, FTC can be subdivided in widely invasive FTC and minimal invasive FTC. These show a different clinical behaviour [3]. Histologically, minimal invasive FTC as well as widely invasive FTC are usually well differentiated tumours, lacking cytological atypia. The diagnosis of FTC is based on histological findings such as angioinvasion and/or invasion that penetrates the full thickness of the tumour-surrounding capsule [4]. To what extend these criteria are fulfilled in special cases may remain a matter of interpretation and Inolitazone dihydrochloride provides a high inter- and even intraobserver variability [5,6]. In order to establish additional criteria for FTC molecular techniques such as sequencing and FISH [7,8] were applied. These had limited value in discriminating FTC from FTA. RAS point mutations were evident in FTC as well as FTA, and chromosomal rearrangements (PAX8/PPAR-rearrangement) were seen in some FTC and FTA with a preference of FTC [9-11]. The aim of our study was the discovery of new helpful immunohistochemical markers for the detection and definition of FTC. Inolitazone dihydrochloride Methods Material Tissue of 4 FTA, 4 minimal invasive FTC and 4 widely invasive FTC was divided in two parts each. One part of the specimens was fixed in 4% buffered formalin and embedded in paraffin. The other part was snapfrozen in liquid nitrogen and stored at -80C. qRT-PCR: Fresh frozen material from 4 FTA and 4 FTC was used. Tissue from 149 patients was Inolitazone dihydrochloride available for immunohistochemistry for a retrospective study. 77 of these showed FTA and 72 FTC. Huerthle cell tumours were not included in this study. Western Blotting was performed by using fresh frozen tissue of 3 FTC and 3 FTA. The tissue of these 3 FTC was also taken for gene expression analysis. Moreover, a prospective study of QPRT-expression with staining of 149 solitary thyroid nodules was undertalen. Of these 149 nodules, 75 were FTA, 51 nodular goiter, 9 oxyphilic FTA, 7 minimal invasive FTC and 7 others (Graves’ disease, papillary thyroid carcinoma, diffuse goiter, or no nodule). All specimen were originally submitted for diagnostic purposes and studied in accordance with national ethical principles and in compliance with the Helsinki declaration. Informed consent for the use of fresh frozen material in gene expression analysis was obtained from the patients. The study was approved by the ethics committee of the university hospital Frankfurt/Main. RNA-extraction RNA-extraction from fresh frozen tissue was performed using the RNAeasy Kit (Quiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. RNA quantity was measured using GeneQuant II photometer (Amersham Pharmacia Biotech, San Francisco, USA). Gene expression analysis Four biological replicates of FTC and FTA were used for gene expression profiling. Briefly, DIG-labeled cRNA was generated using 1 g total RNA per sample for amplification and labeling conducted according to manufacturer’s instructions (Applied Biosystems RT-IVT.