The role of different molecular markers in thyroid epithelium transformation: functional studies and possible clinical implications.

ABSTRACT Background. Papillary thyroid cancer (PTC) is the most common (~90%) endocrine malignancy. The first manifestation of the thyroid cancer is through thyroid nodules and the most sensitive and specific diagnostic tool to detect malignancy in patients with thyroid nodules is fine-needle aspiration biopsy (FNAB). Nevertheless, sometimes it is not efficient enough to give a specific diagnosis leading to the so called diagnoses of indeterminate or suspicious lesions for PTC which ranges from 20 to 30% of cases. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. Recent studies conducted in our laboratories have shown a significant highly decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones, and it was demonstrated to be effective as a new biomarker in the preoperative diagnosis of thyroid tumors. Aim: The aim of the present study is mainly to investigate thoroughly the role of the c-KIT gene in thyroid cancerogenesis, and to characterize in details the c-KIT signaling pathway and the cause of its down-regulation in thyroid cancer. Another aim of this present study is to identify other molecular markers in order to improve the cytological diagnosis and to better understand the mechanisms underlying thyroid epithelium transformation. Methods: We have collected 169 pre-operative thyroid Fine Needle Aspirate (FNA) sample. All 169 FNA samples analyzed in this study were molecularly characterized for the presence of the V600E BRAF mutation in exon 15. SNP analysis, methylation analysis and various gene expression analyses were conducted in order to clarify c-Kit role in thyroid neoplastic trasformation. Gene expression computational models (Neural Network Bayesian Classifier, Discrimination Analysis) were built, together with ROC curves and PCA (Principal Component Analysis) to distinguish a malignant/benign status and BRAF status. Finally a panel of 84 Human Tyrosine Kinases gene array was amplified on 8 benign samples and 12 malignant samples. Results: 64/103 malignant samples carried the V600E mutation while all 66 benign samples were wild type for BRAF exon15. The results of the analysis related to c-KIT function support our hypothesis that this receptor controls a differentiation pathway in thyrocytes. Methylation biochemal process and 146b/222 miRNA expression account for part of the c-KIT dowregulation. The Bayesian Artificial Neural Network and Discriminant Analysis, made of 4 gene (KIT, TC1, miRNA222, miRNA146b) showed a very strong predictive value (94.12% and 92.16% 7 respectively) in discriminating malignant from benign patients and it is interesting to notice that Discriminant Analysis showed a correct classification of 100.00 % of the samples in the malignant group, and 95.00 % by BNN. This same model defines two clearly different genetic background related to BRAF mutational status. In the panel of 84 Human Tyrosine Kinases gene array we found in three (malignant vs benign; V600E vs benign; WT malignant vs benign) of the four conducted comparisons, four genes (ALK, CSK, HCK e MSTR1) in common that had a significantly altered expression. Conclusion: The results of this research support the idea that c-KIT is driving a thyroid cell differentiation pathway, which results altered in thyroid neoplasm transformation. In the same study a 4 gene model was build able to discriminate with high probability between benign and malignant FNAs. The model is proposed to be added to the routinely BRAF diagnostic test in order to improve FNA diagnostic accuracy solving the problems of the nodules that otherwise would remain suspicious. Moreover the present study shows clearly how the presence of the BRAF V600E mutation is accompanied by a unique genetic scenario in which sets of genes specifically discriminate the mutational and wild-type status. Several tyrosine kinase genes showed statistically significant differential expression between malignant and benign thyroid nodules.