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Genetic Alterations in Pheochromocytoma and Paraganglioma
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pheochromocytomas and paragangliomas are neuroendocrine tumors that arise from neural crest-derived cells of the adrenal medulla and the extra-adrenal paraganglia. They cause hypertension due to an abnormally high production of catecholamines (mainly adrenaline and noradrenaline), with symptoms including recurrent episodes of headache, palpitations and sweating, and an increased risk of cardiovascular disease. Malignancy in the form of distant metastases occurs in 10-15% of the patients. The malignant cases are difficult to predict and cure, and have a poor prognosis. About a third of pheochromocytomas and paragangliomas are caused by hereditary mutations in a growing list of known susceptibility genes. However, the cause of the remaining, sporadic, tumors is still largely unknown. The aim of this thesis project has been to further characterize the genetic background of pheochromocytomas and paragangliomas, with a focus on the sporadic tumors.

First, we investigated the role of the genes known from the familial tumors in the sporadic form of the disease. By studying mutations, copy number variations, DNA methylation and gene expression, we found that many of the known susceptibility genes harbor somatic alterations in sporadic pheochromocytomas. Particularly, we found that the NF1 gene, which plays an important role in suppressing cell growth and proliferation by regulating the RASMAPK pathway, was inactivated by mutations in more than 20% of the cases. The mutations occurred together with deletions of the normal allele and were associated with a reduced NF1 gene expression and a specific hormone profile. We also detected activating mutations in the gene EPAS1, which encodes HIF-2α, in a subset of sporadic cases. Microarray analysis of gene expression showed that several genes involved in angiogenesis and cell metabolism were upregulated in EPAS1-mutated tumors, which is in agreement with the role of HIF-2α in the cellular response to hypoxia. In order to comprehensively investigate all the known susceptibility genes in a larger patient cohort, we designed a targeted next-generation sequencing approach and could conclude that it was fast and cost-efficient for genetic testing of pheochromocytomas and paragangliomas. The results showed that about 40% of the sporadic cases had mutations in the tested genes. The majority of the mutations were somatic, but some apparently sporadic cases in fact carried germline mutations. Such knowledge of the genetic background can be of importance to facilitate early detection and correct treatment of pheochromocytomas, paragangliomas and potential co-occurring cancers, and also to identify relatives that might be at risk. By sequencing all the coding regions of the genome, the exome, we then identified recurrent activating mutations in a novel gene, in which mutations have previously only been reported in subgroups of brain tumors. The identified mutations are proposed to cause constitutive activation of the encoded receptor tyrosine kinase, resulting in the activation of downstream kinase signaling pathways that promote cell growth and proliferation.

In summary, the studies increase our biological understanding of pheochromocytoma and paraganglioma, and possibly also co-occurring cancers in which the same genes and pathways are involved. Together with the findings of other scientific studies, our results may contribute to the development of future treatment options.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. , 64 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1439
Keyword [en]
Pheochromocytoma; paraganglioma; genetics; sequencing; cancer; mutation
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-114806DOI: 10.3384/diss.diva-114806ISBN: 978-91-7519-145-4 (print)OAI: oai:DiVA.org:liu-114806DiVA: diva2:792555
Public defence
2015-04-09, Berzeliussalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2015-03-04 Created: 2015-03-04 Last updated: 2016-04-07Bibliographically approved
List of papers
1. Integrative genomics reveals frequent somatic NF1 mutations in sporadic pheochromocytomas
Open this publication in new window or tab >>Integrative genomics reveals frequent somatic NF1 mutations in sporadic pheochromocytomas
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2012 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 21, no 26, 5406-5416 p.Article in journal (Refereed) Published
Abstract [en]

Pheochromocytomas are neuroendocrine tumors of the adrenal medulla which can occur either sporadically or in the context of hereditary tumor syndromes. Whereas the genetic background of hereditary pheochromocytomas is becoming rather well-defined, very little is known about the more common sporadic form of the disease which constitutes approximate to 70 of all cases. In this study, we elucidate some of the molecular mechanisms behind sporadic pheochromocytoma by performing a comprehensive analysis of copy number alterations, gene expression, promoter methylation and somatic mutations in the genes RET, VHL, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2, KIF1B, TMEM127 and MAX, which have been associated with hereditary pheochromocytoma or paraganglioma. Our genomic and genetic analyses of 42 sporadic pheochromocytomas reveal that a large proportion (83) has an altered copy number in at least one of the known susceptibility genes, often in association with an altered messenger RNA (mRNA) expression. Specifically, 11 sporadic tumors (26) displayed a loss of one allele of the NF1 gene, which significantly correlated with a reduced NF1 mRNA expression. Subsequent sequencing of NF1 mRNA, followed by confirmation in the corresponding genomic DNA (gDNA), revealed somatic truncating mutations in 10 of the 11 tumors with NF1 loss. Our results thus suggest that the NF1 gene constitutes the most frequent (24) target of somatic mutations so far known in sporadic pheochromocytomas.

Place, publisher, year, edition, pages
Oxford University Press (OUP): Policy B, 2012
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-87462 (URN)10.1093/hmg/dds402 (DOI)000312505000002 ()
Note

Funding Agencies|University of Linkoping||Swedish Cancer Society||Cancer Society in Stockholm||Swedish Research Council||

Available from: 2013-01-18 Created: 2013-01-18 Last updated: 2017-12-06
2. Frequent EPAS1/HIF2 alpha exons 9 and 12 mutations in non-familial pheochromocytoma
Open this publication in new window or tab >>Frequent EPAS1/HIF2 alpha exons 9 and 12 mutations in non-familial pheochromocytoma
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2014 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 21, no 3, 495-504 p.Article in journal (Refereed) Published
Abstract [en]

Pheochromocytomas are neuroendocrine tumors arising from the adrenal medulla. While heritable mutations are frequently described, less is known about the genetics of sporadic pheochromocytoma. Mutations in genes involved in the cellular hypoxia response have been identified in tumors, and recently EPAS1, encoding HIF2 alpha, has been revealed to be a new gene involved in the pathogenesis of pheochromocytoma and abdominal paraganglioma. The aim of this study was to further characterize EPAS1 alterations in non-familial pheochromocytomas. Tumor DNA from 42 adrenal pheochromocytoma cases with apparently sporadic presentation, without known hereditary mutations in predisposing genes, were analyzed for mutations in EPAS1 by sequencing of exons 9 and 12, which contain the two hydroxylation sites involved in HIF2a degradation, and also exon 2. In addition, the copy number at the EPAS1 locus as well as transcriptome-wide gene expression were studied by DNA and RNA microarray analyses, respectively. We identified six missense EPAS1 mutations, three in exon 9 and three in exon 12, in five of 42 pheochromocytomas (12%). The mutations were both somatic and constitutional, and had no overlap in 11 cases (26%) with somatic mutations in NF1 or RET. One sample had two different EPAS1 mutations, shown by cloning to occur in cis, possibly indicating a novel mechanism of HIF2a stabilization through inactivation of both hydroxylation sites. One of the tumors with an EPAS1 mutation also had a gain in DNA copy number at the EPAS1 locus. All EPAS1-mutated tumors displayed a pseudo-hypoxic gene expression pattern, indicating an oncogenic role of the identified mutations.

Place, publisher, year, edition, pages
BioScientifica, 2014
Keyword
EPAS1; HIF2A; pheochromocytoma; mutation; copy number; gene expression
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-112842 (URN)10.1530/ERC-13-0384 (DOI)000344787700030 ()24741025 (PubMedID)
Note

Funding Agencies|University of Linkoping; Swedish Cancer Foundation; Swedish Research Council; Cancer Society in Stockholm; StratCan at Karolinska Institutet

Available from: 2015-01-08 Created: 2014-12-17 Last updated: 2017-12-05
3. Rare Germline Mutations Identified by Targeted Next-Generation Sequencing of Susceptibility Genes in Pheochromocytoma and Paraganglioma
Open this publication in new window or tab >>Rare Germline Mutations Identified by Targeted Next-Generation Sequencing of Susceptibility Genes in Pheochromocytoma and Paraganglioma
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2014 (English)In: Journal of Clinical Endocrinology and Metabolism, ISSN 0021-972X, E-ISSN 1945-7197, Vol. 99, no 7, E1352-E1360 p.Article in journal (Refereed) Published
Abstract [en]

Context: Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in 1 of 14 identified genes. Objective: This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes. Design: A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for a mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1B beta, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. Results were verified in tumor and constitutional DNA with Sanger sequencing. Results: In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 nonfamilial tumors, 32 mutations were identified in 28 of the samples (41%), including germline mutations in EGLN1, KIF1B beta, SDHA, SDHB, and TMEM127 and somatic mutations in EPAS1, KIF1B beta, MAX, NF1, RET, and VHL, including one double monoallelic EPAS1 mutation. Conclusions: Targeted next-generation sequencing proved to be fast and cost effective for the genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harbored mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1B beta, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127 and could be considered for genetic testing of these patients.

Place, publisher, year, edition, pages
Endocrine Society, 2014
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-111616 (URN)10.1210/jc.2013-4375 (DOI)000342341000026 ()24694336 (PubMedID)
Note

Funding Agencies|University of Linkoping; LiU Cancer Network; Swedish Research Council; Cancer Society in Stockholm; Swedish Cancer Society; StratCan at Karolinska Institutet; Genetics Services Unit at the Wisconsin National Primate Research Center [P51RR000167/P51OD011106]

Available from: 2014-10-27 Created: 2014-10-27 Last updated: 2017-12-05
4. Activating FGFR1 mutations in sporadic pheochromocytoma
Open this publication in new window or tab >>Activating FGFR1 mutations in sporadic pheochromocytoma
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2015 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Pheochromocytomas are neuroendocrine tumors of the adrenal glands that cause hypertension. More than a third of the cases are associated with hereditary mutations in a growing list of susceptibility genes, some of which are also somatically altered in sporadic pheochromocytomas. However, for the majority of sporadic pheochromocytomas, a genetic explanation is still lacking. Here we investigated the genomic landscape of sporadic pheochromocytomas with whole-exome sequencing of 16 paired tumor and normal DNA samples, and discovered on average 33 non-silent somatic mutations per tumor. One of the recurrently mutated genes was FGFR1, encoding the fibroblast growth factor receptor 1, which was recently revealed as an oncogene in pilocytic astrocytoma and childhood glioblastoma. Including a subsequent analysis of a larger cohort, activating FGFR1  mutations were detected in three of 80 sporadic pheochromocytomas (3.8%). Gene expression microarray profiling showed that these tumors clustered with NF1- RET- and HRAS-mutated pheochromocytomas, indicating activation of the MAPK and PI3K-AKT signal transduction pathways. The results advance our biological understanding of pheochromocytoma and suggest that somatic FGFR1 activation is an important event in a subset of these tumors.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-114805 (URN)
Available from: 2015-03-04 Created: 2015-03-04 Last updated: 2015-03-04Bibliographically approved

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