Genetic and molecular characterization of follicular thyroid tumors : diagnostic and prognostic aspects

Sammanfattning: Follicular thyroid carcinoma (FTC) is the second most common thyroid malignancy and it sometimes metastasize to lungs and bone. A substantial subset of these tumors lacks an identified driver event and no well-established prognostic and diagnostic markers exist. Also, to distinguish this malignant tumor from benign follicular thyroid adenoma (FTA) and from follicular tumor with uncertain malignant potential (FT-UMP) patients need to undergo a diagnostic hemithyroidectomy for histopathological analysis. The aim of this thesis was to further clarify the molecular and genetic background of follicular thyroid tumors to facilitate diagnosis and prognostication. In Paper I, high frequencies of TERT promoter mutations, copy number (CN) gains, mRNA expression and hypermethylation in FTC were identified. Almost all tumors with TERT mRNA expression had one of the TERT alterations (mutation, CN gain or hypermethylation) which suggests that not only TERT promoter mutation can augment expression. Remarkably, similar high frequencies of these alterations were observed in FT-UMP but not in FTA. Given the intimate association of TERT alterations to a worse clinical outcome, this suggests that subsets of FT-UMPs do have malignant potential. Furthermore, these alterations could be used to pinpoint FTC with a poorer prognosis and to distinguish between FTA and FTC/FT-UMP. Paper II focused on TERT promoter mutated FT-UMPs and the possible link to malignant potential. A clinical follow-up of 51 cases of FT-UMPs was performed. In total, 16% of these cases had a TERT promoter mutation and 37% of these mutated cases recurred with metastatic disease. The disease-free survival for the mutated FT-UMP patients was similar as for the TERT promoter mutated minimally invasive FTC (miFTC), but significantly shorter than TERT wildtype miFTC. These results indicate that FT-UMPs with TERT promoter mutations harbor malignant potential. Treatment and follow-up strategies should therefore be similar as for malignant tumors. In Paper III, whole-exome sequencing (WES) was used to search for novel FTC driver events and to identify genetic signatures that could distinguish between the FTC histological subtypes: widely invasive FTC (wiFTC), encapsulated angioinvasive (eaiFTC) and miFTC. Recurrent mutations were identified in TSHR, DICER1, EIF1AX, KDM5C, NF1, PTEN, and TP53. Interestingly, there was no difference in the mutational burden across the subtypes. However, higher mutational burden was a risk factor for worse clinical outcome independent of histologic subtype. In Paper IV, the occurrence of DICER1 alterations and how this gene is regulated in FTC and the related Hürthle cell carcinoma (HCC) was explored. Mutations were rare but did occur in subset of young patients. However, there was a general downregulation in carcinomas compared with adenomas. The DICER1 mRNA expression was strongly correlated with the transcription factor GABPA mRNA expression suggesting an interaction. In vitro experiments verified that GABPA binds to the DICER1 promoter and regulates its expression. Furthermore, reduced DICER1 expression stimulated cell proliferation and disrupted the miRNA machinery. In Paper V, whole-genome sequencing and transcriptome sequencing was used to identify novel driver events in clinically aggressive FTC and HCC. A recurrent mutation (p.E518K) in the DGCR8 gene, a miRNA processing subunit, was identified. This particular mutation is known to cause early onset familial multinodular goiter and other tumors when present in germline DNA. In an extended cohort, no further mutations were evident in thyroid carcinomas, however, the expression was significantly reduced in FTC compared with FTA, suggesting an important role in tumorigenesis. The DGCR8 mutated cases and subsets of cases with low DGCR8 expression showed a specific miRNA profile and appeared in the same cluster in a hierarchical cluster analysis. Furthermore, this caused downregulation of specific mRNAs and miRNAs that could be involved in progression and metastasis of these tumors. Copy number analysis revealed recurrent gains on chromosomes 4, 6, and 10. The transcriptome analysis showed that the study cohort of 13 tumors amassed in two principal clusters. In conclusion, TERT alterations in follicular tumors can be used for diagnostic and prognostic purposes, and genetic alterations that disrupt the miRNA machinery seem to constitute an important mechanism in these cancers.