Molecular Alterations in Adrenal and Extraadrenal Pheochromocytomas and Paragangliomas
Josephine Nefkens Institute
Rotterdam, The Netherlands
R.R. de Krijger
University Hospital Rotterdam
Rotterdam, The Netherlands
Paraganglia are dispersed neuroendocrine organs, existing predominantly as anatomically discrete
bodies, characterized by catecholamine- and peptide-producing secretory cells derived from the neural
crest. One group of paraganglia is aligned to the sympathoadrenal and the other to the parasympathetic
autonomic nervous system. Sympathetic paraganglia are distributed along the pre- and paravertebral
sympathetic chains and follow the sympathetic innervation of the pelvic and retroperitoneal organs. They
are not generally known by individual names, and their locations are variable and ill-defined.
Exceptions are the adrenal medulla and the organs of Zuckerkandl, located at the origin of the inferior
Parasympathetic paraganglia are almost exclusively located in the distribution of cranial and thoracic
branches of the glossopharyngeal and vagus nerves. The principal glossopharyngeal paraganglia are the
tympanic paraganglia in the wall of the middle ear and the carotid bodies.
Pheochromocytomas arise from chromaffin cells, also called pheochromocytes, of the sympathetic
paraganglionic tissue. Pheochromocytomas mostly occur within the adrenal medulla, but ~10% are
extra-adrenal. Due to inappropriate catecholamine secretion, most pheochromocytomas give rise to
hypertension. Malignancy, occurring in approximately 10% of the patients, cannot be predicted. Also,
10% of PCC patients have a positive family history for associated cancer syndromes, but predisposing
germline mutations have also been found in up to 24% of apparently sporadic PCC patients. 1
Paragangliomas originate from neural crest-derived chief cells in the paraganglia that are aligned to
the parasympathetic nervous system. The carotid body is the most frequent location of paragangliomas,
followed by the jugulotympanic paraganglia. The tumors are slowly growing, highly vascularized, and
mostly benign, but metastatic spread is found in ~10% of patients. A positive family history is
present in 10 to 50% of the patients, but genetic predisposition may also be present in a considerable
minority of isolated patients. 2 An interesting observation is a markedly increased incidence of carotid
body paragangliomas in people living permanently under hypoxic conditions. 3
Pheochromocytomas (PCC) and paragangliomas (PGL) both arise from neural crest-derived, neuroendocrine
precursor cells and share several histopathological features.(4) Furthermore, concurrence of the tumors
in the same patient has been described. However, genetic predisposition to PCC or PGL is mostly achieved
through different hereditary cancer syndromes. Concurrent inheritance of both tumor types is only found
in the familial paraganglioma syndrome and Carneys triad. Other differences between sympathetic and
parasympathetic tumors are the frequency of hormone secretion (90% vs. 5%)
and the genetic aberrations encountered in both tumor types.
MOLECULAR STUDIES: The hereditary perspective
The identification of the genes involved in PCC and PGL predisposition improved our understanding of
the pathogenesis of these tumors and created new starting points in unraveling the pathogenetic
mechanisms in sporadic and syndrome-related tumorigenesis. Until now, 1 oncogene and 5 tumor suppressor
genes are known to be involved PCC and/or PGL pathogenesis. However, although germline mutations are
found in nearly all familial tumors and in a considerable subset of apparently sporadic PCCs (24%) or
PGLs (32%), somatic mutations in all these genes are relatively uncommon (~1-15%) in the sporadic
forms of these tumors.
Germline point mutations of the
RET proto-oncogene (chromosomal locus 10q11) are responsible for the
inheritance of Multiple Endocrine Neoplasia type 2 (MEN2), which is classified into three subtypes:
MEN2A, FMTC (familial medullary thyroid carcinoma), and MEN2B, all characterised by the presence of
medullary thyroid carcinoma in nearly 100% of cases. MEN2A and MEN2B have an equally increased risk for
PCC (occurring in 50% of patients). Somatic mutations of RET are infrequent
(15%) in PCC and mutations of RET-ligands very rare. 5 However, RET is overexpressed in the majority of
sporadic PCCs. 6
As a receptor tyrosine kinase, RET can activate a variety of intracellular signaling pathways,
including RAS/ERK, phosphatidylinositol 3-kinase (PI3K)/AKT, and phospholipase C pathways. Understanding
the molecular basis of RET signaling in pheochromocytes will help to clarify the role of wild-type RET
overexpression in PCC tumorigenesis.
Von Hippel-Lindau (VHL) disease is a
dominantly inherited cancer syndrome characterised by predisposition to multiple tumors of mesenchymal
and neural crest-derived organs. Mutations or deletions in the VHL gene
(3p25) have been identified in the germline of nearly all tested individuals with VHL disease and can
also be found in ~8% of apparently sporadic PCC. 7 Genotype-phenotype correlations have been
observed such that specific genetic abnormalities can result in four clinical subtypes with different
tumor-specific susceptibilities. Ten to 34% of all VHL patients develop PCC, and 96% of these patients
harbor missense mutations as opposed to VHL-patients with renal cell carcinoma who frequently harbor
deletions or nonsense mutations. 8
Somatic VHL mutations are present in a small subset of sporadic PCCs,
with a trend towards increased frequency in malignant PCCs. Loss of the wild-type allele in tumors with
a somatic VHL alteration and detectable pVHL immunoreactivity in most PCCs,
indicate that promoter hypermethylation is uncommon and support the hypothesis that some retention of
pVHL function is necessary in VHL-related and sporadic PCC development.
Despite the absence of VHL mutations, the signaling pathway in which pVHL
has a place might well be abrogated in sporadic PCC as upstream or downstream targets of VHL may be affected. pVHL interacts in a tissue-specific manner with many cellular
proteins and is involved in regulation of angiogenesis, extracellular matrix formation, and plays a role
in the cell cycle.
Several VHL target genes have recently been detected, including
hypoxia-inducible (VEGF, PAI-1) and hypoxia-independent targets (e.g. Cyclin D1, CDK6, and CD59
glycoprotein precursor). Analysis of pVHL mutants associated with PCC susceptibility (V188L) suggest
that hypoxia inducible factor (HIF) dysregulation and loss of pVHL-mediated suppression of cyclin D1 are
not necessary for PCC tumorigenesis. 9
There is a well-known, but poorly
understood, association between the human hereditary disorder neurofibromatosis type 1 (NF1) and PCCs.
It is estimated that PCCs develop in about 1% of NF1 patients, accounting for approximately 5% of all
pheochromocytomas. NF1 is transmitted by autosomal dominant inheritance, apparently via a single
loss-of-function allele of the NF1 gene. The NF1 tumor suppressor gene encodes a GTP-ase-activating protein, neurofibromin,
that functions primarily as a ras negative regulator. Loss of neurofibromin
or deletion of wild-type NF1 alleles has been demonstrated in syndrome related and sporadic PCCs. 10
mutations in succinate dehydrogenase subunit A (SDHA) cause Leigh syndrome,
a clinically and genetically heterogeneous disorder resembling other mitochondrial and Krebs cycle
defects, mutations in SDHB, SDHC, and SDHD predispose to tumors of sympathetic and parasympathetic paraganglionic
tissue. Germline mutations in these genes are found in virtually all familial PGL patients, in patients
with multifocal PGL and/or PCC, and in a 8-32% of apparently sporadic PGL patients. 11, 12
The mitochondrial succinate dehydrogenase enzyme complex II is involved in the citric acid cycle and
the aerobic respiratory chain. Loss of complex II enzymatic activity leads to a high expression of
hypoxic-angiogenic responsive genes such as vascular endothelial growth factor (VEGF) and endothelial PAS
domain protein 1 (EPAS1/HIF2α). 13 Together with the observation of increased PGL incidence in
people living permanently under hypoxic conditions, this suggests that hypoxia is of major importance in
PGL tumorigenesis and to a lower extent in PCC development. However, somatic mutations in the SDH
subunits are rarely found, leaving the pathogenesis of a considerable proportion of PGLs unraveled.
GENOME WIDE ANALYSES
The absence or low incidence of somatic mutations in the RET, VHL, NF1, and SDHD,
SDHB, or SDHC genes in sporadic tumors called
for a broader approach in the search for clues to PCC and PGL tumorigenesis. From LOH studies, losses of
chromosomal regions 1p, 3p, 11p, 17p, and 22q were known to occur in PCC. Comparative genomic
hybridization analysis, a genome-wide screening method to detect DNA copy number changes, revealed
frequent loss of chromosomal region 1p and 3q in sporadic and MEN2- and NF1-related PCCs, but not in
VHL-related PCCs. 14-16 In the latter, frequent loss of 3p (the VHL locus) is significantly accompanied
by losses of chromosome 11. We found a correlation between malignancy and losses of 6q; also losses of
17q were more common in these tumors. The CGH studies also revealed a similar pattern of aberrations in
adrenal and extra-adrenal PCCs, but a totally different profile in PGLs. Copy number alterations in
these tumors are uncommon. The only frequent aberration is loss of 11q with a differential profile
between familial and sporadic PGL (86% vs. 22%). 17 Also, LOH analysis did
not reveal additional loci of interest.
MARKERS OF MALIGNANCY
The search for predictive markers, especially in PCCs, has been the aim of many studies and has
included, among others, studies on hormone excretion, nuclear volume, DNA plo´dy, gene mutations and
expression. However, these markers have at best shown a general relation to prognosis and provide no
definitive information for the patient. 18-20
Genetically engineered mouse models have been used to study
the mechanisms underlying the carcinogenesis of a wide variety of human cancers. Such a model has been
lacking for studies of pheochromocytoma development until recently. 21, 22 Furthermore, a considerable
number of mouse and rat models that develop PCCs have been described in the literature. Some animal
knockout and transgenic models resemble hereditary syndrome-related PCC in human, whereas other animal
models reveal new starting points for human PCC research.
Microarray CGH/cDNA array
Over 1100 publications have described the use
of comparative genomic hybridization (CGH) to analyze the pattern of copy number alterations in cancer,
but very few of the genes affected are known. This is partly due to the limitations of current methods
for mapping alterations. With Comparative Genomic Hybridization (CGH) analysis only large deletions
(>5Mb) can be detected and Loss Of Heterozygosity (LOH) analysis is limited by inaccurately mapped,
and insufficient numbers of markers. One of the applications that can facilitate fine mapping of genetic
alterations is the high-resolution CGH or DNA arrays, which will allow high resolution mapping of genetic
alterations in this region, and facilitate identification of minimal regions and genes of interest.
Furthermore, our understanding of pathogenetic mechanisms in the tumorigenesis of PCC and PGL will
importantly increase by characterizing gene expression and identifying pathways involved in their
The pathogenesis of adrenal and extra-adrenal tumors of the sympathetic paraganglionic system is
different from parasympathetic PGLs as is evidenced by different genetic predisposition and differential
genomic aberrations. In the latter, hypoxia induced by abrogation of mitochondrial complex II function
or by living under constant hypoxic conditions is of major importance, whereas complex II dysfunction is
infrequently involved in PCC development.
From human and animal PCC studies, a picture emerges in which .
To date, reliable determinants of malignant behaviour are lacking, but modern
molecular techniques may help to elucidate mechanisms of PCC and PGL progression in order to identify
- Neumann HP, Bausch B, McWhinney SR, Bender BU, Gimm O, Franke G, et al. 2002 Germ-line
mutations in nonsyndromic pheochromocytoma. N Engl J Med 346:1459-66.
- Baysal BE, Willett-Brozick JE, Lawrence EC, Drovdlic CM, Savul SA, McLeod DR, et al. 2002
Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck
paragangliomas. J Med Genet 39:178-83
- Saldana MJ, Salem LE, Travezan R 1973 High altitude hypoxia and chemodectomas. Hum Pathol
- Komminoth P, de Krijger RR, Tischler AS 2002 Paraganglia and the Adrenal Medulla. In:
LiVolsi VA, Asa SL (eds) Endocrine Pathology. Churchill Livingstone, Philadelphia:149-169
- van der Harst E, de Krijger RR, Bruining HA, Lamberts SW, Bonjer HJ, Dinjens WN, et al. 1998
Prognostic value of RET proto-oncogene point mutations in malignant and benign, sporadic
phaeochromocytomas. Int J Cancer 79:537-40
- Le Hir H, Colucci-D'Amato LG, Charlet-Berguerand N, Plouin PF, Bertagna X, de Franciscis V, Thermes
C 2000 High levels of tyrosine phosphorylated proto-ret in sporadic phenochromocytomas. Cancer
- van der Harst E, de Krijger RR, Dinjens WN, Weeks LE, Bonjer HJ, Bruining HA, Lamberts SW, Koper
JW 1998 Germline mutations in the vhl gene in patients presenting with phaeochromocytomas. Int J
- Sims KB 2001 Von Hippel-Lindau disease: gene to bedside. Curr Opin Neurol 14:695-703.
- Zatyka M, da Silva NF, Clifford SC, Morris MR, Wiesener MS, Eckardt KU, Houlston RS, Richards FM,
Latif F, Maher ER 2002 Identification of cyclin D1 and other novel targets for the von
Hippel-Lindau tumor suppressor gene by expression array analysis and investigation of cyclin D1 genotype
as a modifier in von Hippel-Lindau disease. Cancer Res 62:3803-11.
- Gutmann DH, Geist RT, Rose K, Wallin G, Moley JF 1995 Loss of neurofibromatosis type I (NF1)
gene expression in pheochromocytomas from patients without NF1. Genes Chromosomes Cancer 13:104-9
- Dannenberg H, Dinjens WN, Abbou M, Van Urk H, Pauw BK, Mouwen D, Mooi WJ, de Krijger RR 2002
Frequent germ-line succinate dehydrogenase subunit D gene mutations in patients with apparently sporadic
parasympathetic paraganglioma. Clin Cancer Res 8:2061-6.
- Baysal BE, Rubinstein WS, Taschner PE 2001 Phenotypic dichotomy in mitochondrial complex II
genetic disorders. J Mol Med 79:495-503
- Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C, Kerlan V, Plouin PF, Rotig A, Jeunemaitre
X 2002 Functional consequences of a SDHB gene mutation in an apparently sporadic pheochromocytoma.
J Clin Endocrinol Metab 87:4771-4.
- Dannenberg H, Speel EJ, Zhao J, Saremaslani P, van der Harst E, Roth J, et al. 2000 Losses of
chromosomes 1p and 3q are early genetic events in the development of sporadic pheochromocytomas. Am J
- Edstrom E, Mahlamaki E, Nord B, Kjellman M, Karhu R, Hoog A, Goncharov N, Teh BT, Backdahl M, Larsson
C 2000 Comparative genomic hybridization reveals frequent losses of chromosomes 1p and 3q in
pheochromocytomas and abdominal paragangliomas, suggesting a common genetic etiology. Am J Pathol
- Lui WO, Chen J, Glasker S, Bender BU, Madura C, Khoo SK, Kort E, Larsson C, Neumann HP, Teh BT
2002 Selective loss of chromosome 11 in pheochromocytomas associated with the VHL syndrome. Oncogene
- Dannenberg H, de Krijger RR, Zhao J, Speel EJ, Saremaslani P, Dinjens WN, Mooi WJ, Roth J, Heitz PU,
Komminoth P 2001 Differential loss of chromosome 11q in familial and sporadic parasympathetic
paragangliomas detected by comparative genomic hybridization. Am J Pathol 158:1937-42.
- van der Harst E, de Herder WW, de Krijger RR, Bruining HA, Bonjer HJ, Lamberts SW, van den Meiracker
AH, Stijnen TH, Boomsma F 2002 The value of plasma markers for the clinical behaviour of
phaeochromocytomas. Eur J Endocrinol 147:85-94
- van der Harst E, Bruining HA, Jaap Bonjer H, van der Ham F, Dinjens WN, Lamberts SW, et al.
2000 Proliferative index in phaeochromocytomas: does it predict the occurrence of metastases? J Pathol
- de Krijger RR, van der Harst E, van der Ham F, Stijnen T, Dinjens WN, Koper JW, Bruining HA, Lamberts
SW, Bosman FT 1999 Prognostic value of p53, bcl-2, and c-erbB-2 protein expression in
phaeochromocytomas. J Pathol 188:51-5.
- Powers JF, Evinger MJ, Tsokas P, Bedri S, Alroy J, Shahsavari M, Tischler AS 2000
Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice. Cell Tissue Res
- Smith-Hicks CL, Sizer KC, Powers JF, Tischler AS, Costantini F 2000 C-cell hyperplasia,
pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type
2B. Embo J 19:612-22.