Genetic and molecular background of pheochromocytoma and paraganglioma

Sammanfattning: Pheochromocytomas (PCC) and abdominal paragangliomas (PGL), collectively denoted PPGL, are neuroendocrine tumors (NET) with a highly diverse genetic and molecular etiology, arisen in the adrenal medulla and abdominal paraganglia respectively. The tumors often cause cardiovascular symptoms due to the high production of catecholamines, and malignancy occurs in 10% of the PCCs and 30% of the PGLs. The genetic background of the PPGL disease constitutes of hereditary mutations in a growing list of susceptibility genes, although a large subset of the sporadic tumors still has an unknown etiology. This thesis work aimed to further characterize the genetic and molecular background of PPGL, in order to contribute to better diagnosis, prognosis and treatment for the patients. In the first study we investigated the role of susceptibility gene promoter methylation in PPGL. By studying the gene expression and assessing the promoter methylation levels, it was found that the VHL gene is epigenetically inactivated in PPGL. Subsequently, in order to investigate the established susceptibility genes in a large cohort, we used a targeted sequencing approach in the second study and found that next-generation sequencing is fast and cost-effective method for mutational screening of PPGL. Additionally, the NF1 gene was found to be the most frequently mutated gene, and in the third study we used the NF1 mutational status obtained in the second study to investigate if immunohistochemistry could be used as a screening tool for NF1 mutations. We found a strong sensitivity but poor specificity for the method and therefore recommend genetic screening as the most efficient tool to identify NF1 patients. The list of PPGL susceptibility genes is constantly growing, and one of the latest genes verified is the HRAS gene. In the fourth study we screened a large cohort of PPGL for mutations in the HRAS gene and compared with the overall gene expression obtained using a mRNA microarray approach. Taken together with HRAS mutations in the literature, the overall HRAS mutation frequency was calculated to 5.2% in PPGL. The gene expression profiling showed that the HRAS mutated tumors clustered together with the NF1- and RET-mutated tumors that are associated with activation of kinase pathways. With the mutational information obtained in the second study, we were able to screen PCCs without known genetic drivers. In the fifth study, these tumors underwent whole-exome sequencing, detecting recurrent mutations in the KMT2D gene. After screening of a verification cohort, a total KMT2D mutation frequency of 14% was established. In the sixth study we screened PGLs for mutations in the KMT2D gene. All PGLs exhibited wild-type however KMT2D gene over-expression was observed in PGLs compared to normal adrenal samples. These results would imply dysregulation of methyltransferase as a novel disease mechanism in PPGL. In summary, the studies included in the thesis have increased the knowledge of the genetic and molecular background of PPGL. The results may therefore in the long run contribute to better diagnosis, prognosis and development of future treatment options for the patients.