Basic and translational studies of follicular thyroid neoplasia

Thyroid tumors occur frequently in the general population and although the majority represents benign follicular adenomas (FTA), thyroid cancer constitutes 1% of all malignancies worldwide. Among the differentiated cancers, three main types are recognized, including the papillary and follicular forms (PTC and FTC) that originate from the follicular thyrocytes, and the medullary carcinoma that arises from the C-cells. The undifferentiated form, i.e. anaplastic carcinoma (ATC), is a highly aggressive cancer of sometimes unknown cellular origin. The overall goal of this thesis was to investigate the alternative genetic pathways leading to follicular thyroid tumorigenesis and thereby identify clinically relevant biomarkers. A large panel of primary non-medullary thyroid tumors of different histopathological diagnoses as well as human thyroid cell lines were analyzed. In Paper I, cytogenetic analyses of primary thyroid tumors and of the established thyroid cancer cell lines DRO, ARO and CGTH W- 1 were performed. Five of the 16 primary tumors revealed an abnormal karyotype including a FTC with a somatic translocation involving chromosomes 2, 5 and 10 and a PTC with a balanced translocation t(3;15). In Paper II, a panel of 87 follicular thyroid tumors was screened for the presence of PAX8PPARgamma chromosomal translocation. Interphase fluorescence in situ hybridization (iFISH), RTPCR followed by sequencing and Western blotting were used for the detection of the PAX8PPARgamma fusion on the DNA, RNA and protein level, respectively. The rearrangement was detected in 10 of 34 (29%) FTCs and in one of 20 (5%) atypical FTAs, but not in any of the 20 regular FTAs or 13 ATCs studied. The findings suggest that PAX8-PPARgamma occurs frequently in FTC and is highly suggestive of a malignant tumor. Subsequently, the functional consequences of PAX8-PPARgamma were explored (Paper III). Using highdensity oligonucleotide arrays, the gene expression profiles of FTCs bearing a PAX8-PPARgamma fusion were compared with FTCs lacking this fusion. Unsupervised clustering and multidimensional scaling analyses showed that PAX8-PPARgamma positive FTCs have a highly uniform and distinct gene expression signature and are likely to constitute a distinct biological entity. Notably, a large number of ribosomal protein and translation-associated genes were concurrently under-expressed in the FTCs with the fusion. In Paper IV, the role of Ras and its pro-apoptotic effectors NORE1A and RASSF1A in FTC development was investigated. Dramatically reduced mRNA expression of NORE1A was evident in all PAX8-PPARgamma positive FTCs, while RAS mutations and PAX8-PPARgamma fusion were mutually exclusive events. RASSF1A expression was reduced in the majority of the FTCs analyzed. In Paper V, expression of the PTEN tumor suppressor gene was analyzed in 87 sporadic thyroid tumors. Complete loss of PTEN mRNA expression was evident in six of the tumors and the transcriptional silencing of PTEN was significantly associated with the anaplastic subtype. No association was observed between the expression, loss of heterozygosity, and mutation status of PTEN in the 3 3 cases in which these parameters were compared. In Paper VI, 26 potential markers of malignancy were evaluated in 75 follicular thyroid tumors by TaqMan quantitative RT-PCR. A combination of five genes (TERT, TFF3, PPARgamma, EGR2 and CITED1) could accurately predict aggressive FTC, while two genes (TERT and TFF3) specifically detected malignant tumors