ETD system

Electronic theses and dissertations repository


Tesi etd-04042014-183814

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