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Pheochromocytomas and paragangliomas are rare neuroendocrine catecholamine producing tumors with varied clinical presentations, biochemistries and genetic makeup. These features outline the complexity and the difficulties in studying and understanding the oncogenesis of these tumors. The study of families with genetically inherited mutations in pheochromocytoma susceptibility genes has greatly enhanced our understanding of the pathophysiology and mechanisms of oncogenesis of the disease, and consequently changed our clinical approach. Several molecular pathways and mutations in their important regulatory proteins have been identified. Such mutations are responsible for the dysregulation of metabolic pathways involved in oxygen and nutrient sensing, apoptosis regulation, cell proliferation, migration and invasion. The knowledge derived from the study of these pathways will be fundamental in the future clinical management of these patients. As a rare disease that often masks its clinical presentation, the diagnosis is frequently missed and a high level of suspicion is required. Management of this disease requires a multidisciplinary team approach and has been discussed along with advances in its treatment.
Pheochromocytoma is a rare catecholamine-producing neuroendocrine tumor derived from the chromaffin cells in the adrenal medulla (1). The embryological origin of these cells from the neural crest outlines the peculiar development and pathogenesis of these tumors. Catecholamine producing tumors arising from extra-adrenal cells are known as paragangliomas. These tumors share overlapping characteristics that span histopathology, epidemiology, and even molecular pathobiology, but also have many differences in terms of their behavior, aggressiveness and metastatic potential, and they can be either sporadic or be present in multiple members of the same family (2). The study of familial syndromes of pheochromocytoma has been very important in understanding the pathogenic mechanisms involved in both familial as well as sporadic forms. Several susceptibility genes have been established as playing a central role in the pathogenesis of both pheochromocytomas and paragangliomas (3). At the moment ten different genes have been implicated in the pathogenesis of these tumors: the von Hippel-Lindau (VHL) tumor suppressor gene, the Rearranged during transfection (RET) proto-oncogene, the neurofibromatosis type 1 (NF1) tumor suppressor gene, genes encoding for the four subunits (A, B, C and D) of the succinate dehydrogenase (SDH) complex, a gene encoding the enzyme responsible for flavination of the SDHA subunit (SDHAF2), and finally the genes TMEM127 and the MAX which have been recently described. Genetic studies suggest that other genes may be involved in the pathogenesis of these tumors and investigations are under way to uncover novel mutations in “sporadic” cases.
According to more recent publications, up to 30-32 % of these tumors are genetically inherited (4) and it is estimated that this number will rise in the future as new susceptibility genes are discovered. Indeed, it is generally accepted that certain susceptibility genes yet to be discovered may not ‘at first look’ seem to run in families due to their very low penetrance inspite of passing down several generations.
Hereditary pheochromocytomas and paragangliomas can be divided into two clusters based on transcription profile revealed by microarray analysis. One cluster is the tumors with VHL and SDHx mutant genes and the other cluster contains tumors with RET and NF1 mutant genes (5, 6). Sporadic tumors were surprisingly represented in both clusters. As newer genes were discovered further microarray studies tried to classify them into these two clusters and mutations in KIF1Bbeta, TMEM127 and MAX clustered with RET/NF1 and SDHAF2 and SDHA clustered with SDHx/VHL (7).
In the following discussion, we summarize the current knowledge on these two clusters.
Susceptibility genes and molecular pathways: the gateway to a better understanding of the pathophysiology of pheochromocytoma.
In the past a link between high altitude chronic hypoxia and an increased prevalence of hyperplasia of the carotid bodies as well as a higher prevalence of paragangliomas compared to those living at sea level has been observed. This association and the fact that the carotid body plays a central role in oxygen sensing made researchers believe that oxygen sensing might play a role in the tumorigenesis of paragangliomas (8). The basis underlying this association seems to be confirmed with the pseudo hypoxia hypothesis for tumorigenesis caused due to VHL and SDHx mutations. Mutations in these genes lead to accumulation and stabilization of hypoxia inducible factor alpha. Hypoxia Inducible Factor (HIF)-α heterodimerizes with HIF-β and acts as an active transcription factor which induces a variety of genes involved in cellular and tissular adaptation to hypoxia (angiogenesis, metabolism and survival). The HIF-α is hydroxylated by prolyl hydroxylases (more specifically EGLN1) under normoxic conditions into hydroxylated HIF-α. This is recognized by the VHL protein and marked for degradation in the proteasome. If the VHL gene is mutated the protein is not formed and HIF-α cannot be degraded and it accumulates. Succinate dehydrogenase enzymes convert succinate to fumarate and its mutation leads to accumulation of succinate. Succinate is a competitive inhibitor of the prolyl hydroxylase EGLN1 and leads to accumulation of stable HIF-α. Therefore VHL and SDHx mutations both lead to induction of genes that would have also been induced via hypoxia and lead to tumor development by different mechanisms through the same pathway (7, 9). A mutation in the prolyl hydroxylase domain-2 (PHD2) gene has recently been implicated in a case of recurrent paragangliomas and erythrocytosis. This mutation affects PHD2 function and stabilizes HIF-α proteins. Also loss of heterozygosity could be indicative that the PHD2 could be a tumor-suppressor gene (10). (Figure 1)
Patient with the Von Hippel-Lindau disease, an autosomal dominant familial cancer, are prone to develop several types of tumors, including renal cell carcinoma and pheochromocytoma. Tumors of vascular origin are frequently a common clinical presentation in these patients. The VHL gene, located on the chromosome sub-band 3p25-26, encodes for the VHL protein, whose best characterized function is the ubiquitination and consequent degradation via the proteasome of the hypoxia inducible factor alpha (HIF- α) protein (11). In patients with VHL disease, the loss of pVHL function lead to lack of HIF- α ubiquitination and degradation and consequent upregulation of several hypoxia inducible genes.
The presence or absence of pheochromocytoma is one of the criteria to classify the VHL disease as a type 1 (absence) or type 2 (presence), with the subtype 2C classification reserved for families with pheochromocytoma only. Most of the VHL-related pheochromocytomas have a loss of the wild type allele as a second hit, according to the Knudson's two hit hypothesis of tumor suppressor genes. Pheochromocytomas developing in VHL syndrome have several differences compared to the sporadic tumor, including the multifocal location and the earlier onset; more over they tend to have a noradrenergic phenotype (12).
More recently, pheochromocytoma susceptibility has been associated with mutations in the succinate dehydrogenase (SDH) gene. Succinate Dehydrogenase is both part of the Electron Transport Chain (ETC, complex II) and the Krebs cycle, and its sub-units are encoded in the nucleus and imported to the mitochondria (13). THE SDHx gene encodes four subunits: SDHA, SDHB, SDHC and SDHD. SDHA (flavinated by the SDHAF2 protein in the complex) and SDHB (iron-sulfur protein) hold catalytic function and are bound to the inner mitochondrial membrane by the SDHC and SDHD subunits.
Mutations in these subunits cause the complete loss of enzymatic activity in the complex and lead to oncogenesis by upregulation of pro-angiogenic response genes (13). In particular mutation in the SDHB subunit has been associated with a high likelihood of malignancy (14). Interestingly, electron channeling through this complex does not result in H+ gradient build up and its function is believed to primarily reduce the amount of reactive oxygen species (ROS) that normally leaks from aerobic respiration. Pheochromocytomas represent excellent examples of the link between mitochondrial function and oncogenic proteins in the promotion of cancer development and the metabolic adaptation of tumor cells, combining increased glycolysis with a defective mitochondrial respiration.
The first association made between SDH and tumorigenesis was established by Baysal et al (15) when they investigated a hereditary paraganglioma. They were able to map a gene designated as SDHD in the chromosome band 11q23 and later confirmed to code for the subunit D of the SDH complex. Shortly after, mutations in genes that code for sub-units B (SDHB) and C (SDHC) were identified as genes of susceptibility. The familial paragangliomas syndromes (PGL) type 1 to 4 have been found to be associated with gene mutations in these SDH subunits namely – PGL-1 (SDHD), PGL-2 (SDHAF2/SDH5), PGL-3 (SDHC) and PGL-4 (SDHB) (16, 17). Missense, frame-shift and nonsense mutations predisposing to pheochromocytoma have been identified in all the subunits of the SDH complex, including more recently in the SDHA subunit (18) and in the SDH5/SDHAF2 gene, identified in patients with head and neck paragangliomas (19). Dopamine and norepinephrine or dopamine alone characterize the biochemical phenotype in these patients (20).
This gene expression cluster is linked together by the activation of kinase signaling pathways driven by oncogenes. Activation of receptor tyrosine kinases (RTK) leads to downstream activation of several intracellular pathways through a series of consequent phosphorylation events. RET and NF1 mutations lead to activation of the RAS/RAF/MAPK and the PI3K/AKT/mTOR signaling pathways. TMEM127 mutant tumors clusters with the RET/NF1 group and they enhance mTOR activity independent of the above two kinase pathways. Microarray expression analysis of KIF1Bbeta mutant tumors also groups with RET/NF1 tumors though its potential role in kinase pathways is not yet known (7).
The study of families with MEN2 A and B, which develop pheochromocytomas among other tumors, allowed the discovery of mutations in the RET (Rearranged during Transfection) proto-oncogene. The proto-oncogene RET is a tyrosine kinase receptor primarily expressed in neural crest cells (including parasympathetic and sympathetic ganglion cells) and urogenital cells (22). The RET gene was originally found as a novel gene rearrangement within the NIH-3T3 fibroblast cell line following transfection with DNA from human lymphomas (23). The name was subsequently used to designate the receptor tyrosine kinase within the fused oncogene. The RET gene resides in the pericentromeric region of the chromosome 10q11.2 and comprises 21 exons (23, 24).
The Ret protein is a transmembrane receptor with an extracellular ligand-binding portion containing four cadherin like repeats, a cysteine rich domain and a calcium-binding site. Interestingly, such organization makes RET a remote member of the cadherin cell adhesion protein superfamily (25). These phospho-tyrosine residues are important docking sites for several intracellular adaptor proteins, making the RET receptor a versatile platform for signal transduction. Downstream targets include STAT3, Grb2, Grb7 and Grb10, Shc, PLC-gamma and Src, among others, allowing the activation of major intracellular pathways, including the PI3K/AKT/mTOR, Ras/ERK and JNK pathways. Of note, Src is important for RET signaling through focal adhesion kinase (FAK), which is an essential mediator of tumor cell migration and metastasis (26).
Gain-of-function point mutations in RET, causing ligand-independent activation of the gene product, is the initial oncogenic event in the hereditary cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), which recognize three subtypes based on clinical presentation: 1) MEN 2A; 2) MEN 2B; and 3) familial medullary thyroid carcinoma (FMTC). Trisomy 10 with duplication of the mutant RET allele, loss of wild-type RET allele and tandem duplication with amplification of the mutant RET, are among the “second hit” mechanisms identified that favor tumor development in these patients (27). Pheochromocytoma develops in about 50% of patients with MEN 2A and MEN 2B. There is a strong correlation between the position of the RET mutation and the clinical phenotype. For example, in MEN 2A pheochromocytomas occur more frequently in patients with RET mutations in codon 634 and less frequently when the mutation involves codons 618, 620 or 791 (28).
Loss-of-function mutations in the RET gene are responsible for a different congenital disorder, known as Hirschsprung's disease (or aganglionic megacolon) in which normal enteric nerves are absent(29). The occurrence of this disease reinforces the importance of RET in directing cells of the neural cell crest (which later become ganglionic cells) during development, and, as an important disease-causing gene.
Several groups have suggested that mutant RET in MEN 2B activates additional aberrant signaling pathways, which account for the more aggressive phenotype in patients with this syndrome compared to patients affected by MEN 2A. Interestingly, also RET mutations in MEN 2A occur mainly in the extracellular domain cysteine residue, causing alterations in receptor dimerization, while MEN 2B RET mutation are predominantly located in the intracellular domain, resulting in activation of different signaling pathways and consequent expression of different target genes. Expression microarray analysis supports these conclusions (30). Patients with MEN 2 syndrome have pheochromocytomas that secrete predominantly epinephrine (20).
The tumor suppressor gene Neurofibromatosis type 1 (NF 1) encodes for the protein neurofibromin, a GTPase-activating protein in the RAS signaling cascade and mTOR signaling pathway (31). In patients with mutation in this gene, in conjunction with other tumors, pheochromocytoma is present in up to 5% of cases and it is frequently diagnosed later in life (32). Neurofibromatosis is diagnosed clinically by a thorough physical examination or a positive family history so confirmation of diagnosis does not require genetic testing. Neurofibromatosis patients with pheochromocytomas produce both epinephrine and norepinephrine (20).
Studies on transgenic mice with NF1 mutations have clearly linked this gene with the development of pheochromocytoma. In addition, the only available mouse pheochromocytoma cells lines (the MPC cells and its derivative MTT cells) that we are using for pre-clinical experimentation were derived from this mouse model (33, 34).
Next generation sequencing technology and improvement in our understanding of the molecular basis of the disease, have allowed the discovery of newer pheochromocytoma susceptibility genes.
Screening more than one hundred samples from a large institutional collection of pheochromocytomas and paragangliomas, several germline mutation were identified in the TMEM127 gene (35), which encode for a transmembrane protein; although the function of TMEM 127 protein is not well characterized, a link with the mTOR pathway has been identified. Indeed, TMEM127 is involved in endosomal organelle dynamics, which may contribute to its modulation of mTOR kinase signaling (36). Interestingly, tumor samples from patient with TMEM127 mutations have an increased activation of mTORC1(37). Patients with TMEM127 mutation tend to have benign, bilateral adrenal pheochromocytomas; a clear association with development of other tumors in these patients still needs to be clarified.
Using exome sequencing, Comino-Mendez et al. (38) were able to identify MAX gene mutations in three independent familial cases of pheochromocytoma; the inactivating nature of the germline mutation points to a tumor suppression function for this gene. As mentioned before, the MAX (MYC-associated factor X) protein is a key component of the MYC-MAX-MXD1 cellular network, involved in several functions, including proliferation, differentiation and apoptosis. These tumors tend to be bilateral and are associated in 25% of cases with malignant behavior (39).
In summary, classification of pheochromocytoma into two clusters we described above is widely accepted to distinguish tumorigenesis amongst the multiple mutations observed. However, there is another proposed theory that different susceptibility genes converge into a single common pathway of tumorigenesis. According to this model the germline mutations in RET, VHL, NF1 and SDHx prevent apoptosis of the neuronal progenitor cells. Normally during embryogenesis as nerve growth factor becomes limiting, c-Jun becomes activated and induces the neuronal progenitor cells to undergo apoptosis (7).
The recognition that germline mutations in the genes described above are important in the pathogenesis and clinical presentation of patients with pheochromocytoma, provide a solid justification for genetic testing as an important part of patient management.
As new genes are identified as susceptibility genes for pheochromocytoma, this is going to be true even more so in the future. Clinical presentation, family history and histopathological and radiological characteristics of the tumor help guide the sequence in which genes must be tested.
Being a rare disorder with a relatively common presentation it can be difficult to decide which patients should be subjected to a work up for suspected pheochromocytoma. Patients with symptoms and signs suspicious for a pheochromocytoma are often tested to rule out the disease rather than being tested because of a high level of suspicion for the disease. Differentials that must be considered before and after testing are described in Table 1. Differential Diagnosis of Pheochromocytoma (40). Therefore a screening test with a high level of sensitivity is required so that negative tests provide confidence that the diagnosis has not been missed and repeat testing will not be necessary (1). Previously urinary or plasma catecholamines were measured to screen for pheochromocytomas. The test results are not reliable as catecholamine secretion can be intermittent thus causing false negative results.
A newer test measuring the urinary and plasma metanephrines has become the ideal test at present as these metabolites of the catecholamines are released continuously from the tumor independent of the time of catecholamine release. Also some tumors metabolize catecholamines within the tumor to free metanephrines and secrete them without directly secreting catecholamines. Thus plasma free metanephrines would offer better diagnostic sensitivity over other tests (41). Hence usage of plasma free metanephrines as first line biochemical testing for detecting a pheochromocytoma offers a number of advantages which are independence from fluctuating catecholamine levels (is an indicator of long term catecholamine levels), levels proportional to tumor mass, minimal interference from drugs and a simpler, less time consuming procedure for blood sampling (42).
In a large multicenter study it was found that plasma free metanephrines and urinary fractionated metanephrines had the best diagnostic sensitivity while urinary total vanillylmandelic acid and urinary total metanephrines had the best specificity. The sensitivity and specificity was highest for plasma free metanephrines at equivalent levels using receiver operating characteristic curves in comparison to other tests (43).
Blood samples collected for plasma-free metanephrines should be collected in the morning after a minimum of 15 min rest in the supine position in a patient who has fasted the night before, which is like the recommendations for plasma catecholamines (44).
However it is important that patients avoid antidepressants (selective norepinephrine reuptake inhibitors, selective serotonin reuptake inhibitors, monoamine oxidase inhibitors and most importantly tricyclic antidepressants), caffeine and cigarette smoking which may falsely elevate metanephrines. Although alpha and beta-blockers may mask catecholamine related symptoms and signs they may falsely elevate plasma-free normetanephrine levels as well.
Phenoxybenzamine is associated with elevation in norepinephrine and normetanephrine and can lead to high false positive rates. Thus a patient should not be treated with the drug until the biochemical testing is complete. Substitutes for controlling hypertension in the interval period are selective alpha1 blockers and calcium channel blockers (45). Acetaminophen should also be avoided before blood sampling as it has been found to interfere with high-performance liquid chromatographic assays (46, 47).
Increases in the plasma concentrations of normetanephrine and metanephrine four times above the upper reference limit are almost non-existing in patients without pheochromocytoma but occur in 70-80% of patients with the tumor (48). Measurements of plasma-free normetanephrine and metanephrine provide a sensitive test for the diagnosis of pheochromocytoma but may fail to detect tumors that produce predominantly dopamine. Therefore additionally measuring plasma-free methoxytyramine (the O-methylated metabolite of dopamine) may be useful in patients with paragangliomas for the identification of tumors that produce predominantly dopamine (49). In a recent study Eisenhofer et al concluded that plasma methoxytyramine (>0.2 nmol/L) is a novel biomarker for metastatic pheochromocytomas and paragangliomas. Such patients have increased likelihood of SDHB positivity, tumor size (>5 cm) and extra-adrenal location of their tumor all predictors for higher likelihood of malignancy (50).
In patients with levels in the intermediate range that is above normal and below four times normal a clonidine suppression test is done to confirm the diagnosis. The clonidine suppression test was first introduced by Bravo in 1981 to address the problem of managing these patients with inconclusive biochemistries (51). The increase in plasma catecholamines takes place through activation of the sympathetic nervous system in normal individuals however in pheochromocytoma patients the increase results from diffusion of excess catecholamines synthesized by the tumor into the circulation bypassing normal storage and release mechanisms (52). Since clonidine decreases resting plasma catecholamines by inhibition of centrally mediated stimulatory adrenergic influences it would not be expected to suppress catecholamine release in pheochromocytoma (53). This is the basis behind the clonidine suppression test. It is performed with administration of 0.3 mg/70 kg of oral clonidine and plasma values are measured 3 hours later. Before performing the clonidine suppression test it is important to rule out the intake of any medications such as beta-blockers, thiazide diuretics and tricyclic antidepressants by the patient that might cause false positive results (54, 55).
Two criteria that were used to indicate a normal (negative) clonidine test is a fall in plasma norepinephrine to within the normal range or a fall in plasma norepinephrine to less than 50% of baseline values. More recently plasma normetanephrine levels were measured 3 hours post clonidine and they remained elevated in 96% of patients with pheochromocytoma compared with only 67% for norepinephrine. A fall in plasma normetanephrine to a normal range or to less than 40% of baseline values implies a normal clonidine suppression test. Thus, measuring normetanephrine responses to clonidine enabled one to exclude pheochromocytomas more reliably as it confirmed 40% more pheochromocytomas than did norepinephrine level measurement (45). In a recent study that evaluated the sensitivity and specificity of available criteria that were using catecholamine levels they found that the best sensitivity (93%) and specificity (95%) was with plasma noradrenaline + adrenaline > 2.96 nmol/l at 3h post clonidine or a baseline plasma adrenaline plus noradrenaline > 11.82 nmol/l (56). A newer study at the same hospital comparing plasma-free metanephrine plus normetanephrine (measured using ELISA) with plasma catecholamines (measured using HPLC) in the investigation of suspected pheochromocytoma showed that both were equally effective during CST. Hence use of the faster easier and equally effective biochemical test of measuring metanephrines using ELISA for the diagnosis of pheochromocytoma should be preferred during the clonidine suppression test (57). Chromogranin A is an acidic protein that is stored with catecholamines in secretory granules and co-released with them. It is not specific for pheochromocytomas but elevated levels are seen with many neuroendocrine tumors. Chromogranin A has a sensitivity of 83-89% for identifying pheochromocytomas and maybe elevated in both non-secretory and secretory tumors. High plasma levels indicate malignancy and correlate with tumor mass. Their levels may also be used to gauge tumor response and relapse (58).
Once the diagnosis of pheochromocytoma/paraganglioma is made with a positive biochemistry it is necessary to locate the tumor for surgical resection. Anatomical imaging is most widely used in the initial evaluation of patients as it is offers the advantage of low cost, almost universal availability, and less skill for use (3). CT scan can be used to localize adrenal tumors > 1 cm and extra adrenal tumors > 2 cm (59). MRI with or without gadolinium enhancement is superior for the detection of extra-adrenal tumors. (60) Inspite of their excellent sensitivity, CT and MRI lack the specificity required to confirm a mass as a pheochromocytoma and thus functional imaging modalities are used which have a higher specificity for detection (3).
Whole body scanning using MIBG labeled with radioiodine (MIBG scintigraphy) has a higher specificity and helps to overcome the limitations of anatomical imaging (61). MIBG scanning may be carried out with either 123I or 131I-MIBG scanning with 123I offers a number of advantages over 131I like much better sensitivity (62, 63), additional utility for imaging by SPECT and shorter half-life hence higher doses can be used (63). A recent study however found that low-dose diagnostic 123I-MIBG whole-body scans at 6h and 24h detect less lesions compared to 3 days post treatment 131I-MIBG whole-body scan in malignant pheochromocytoma and paraganglioma (64). Positron Emission Tomography is another functional modality used due to its superior spatial resolution and low radiation exposure. 18F-DA PET is more sensitive overall than 123I-MIBG scintigraphy or somatostatin receptor scintigraphy (SRS) with 111In-pentetreotide on a per patient basis. Hence it should be used in the evaluation of pheochromocytomas if available and if unavailable 123I-MIBG scintigraphy should be used for non-metastatic or adrenal pheochromocytomas and SRS should be used for metastatic pheochromocytomas (65). Since pheochromocytomas and paragangliomas at different sites take up these biomarkers for imaging differently the sensitivity of various functional imaging studies varies for tumors according to site. Adrenal tumors should be imaged with either 123I-MIBG scintigraphy, 18F-DA PET or 18F-DOPA PET to detect/exclude multifocal or metastatic disease. Adrenal tumors associated with a VHL gene mutation are best imaged by 18F-DA PET. In patients with extra-adrenal tumors imaging with MIBG is often not very accurate and imaging with 18F-DA PET or 18F-DOPA PET is a better approach. 18F-DOPA PET is particularly useful to detect head and neck paragangliomas. In patients with metastatic pheochromocytomas and paragangliomas 123I-MIBG scintigraphy has a limited role unless it is being used to determine whether or not the patient is eligible for 131I-MIBG treatment. It is found that 18FDG PET is superior in patients with metastatic SDHB paragangliomas and the 18F-DOPA PET performs poorly. This radiopharmaceutical FDG is non-specific and taken up by rapidly growing tumors hence it can be used in patients with metastatic pheochromocytoma that is becoming undifferentiated to take up more specific agents. Provided that somatostatin receptors are not lost during dedifferentiation somatostatin analogues like 111In-penetreotide, 68Ga-DOTATOC and 68Ga-DOTANOC maybe useful (3, 66, 67).
Genetic testing is important in patients who are <45 years, with multiple tumors, extra-adrenal tumors, family history, metastatic tumors and increased dopamine secretion. Decision for the sequence in which to test the genes is based on clinical presentation and investigations. About 40-50% patients with malignant pheochromocytoma will have mutations in SDHB (~35 %), VHL (~5%) and SDHD (~1%) and hence should be offered testing for at least these 3 genes. The importance of genetic testing lies in that it helps predict prognosis for the patient, risk for the family and helps guide therapy (39).
In individuals who meet the criteria for genetic testing but for whom clinical indicators, biochemical indicators and imaging do not indicate which genes to test, immunohistochemistry seems to offer an opportunity to narrow the mutations that should be tested for.
In an individual with SDHB positive immunohistochemistry staining one should test for VHL, RET or NF1 mutations. In those with negative staining implying absence of SDHB protein expression one should test for SDHB, SDHC or SDHD mutations. SDHB immunohistochemistry has a sensitivity of about 100% and a specificity of about 84% in a prospective series to detect the presence of an SDH mutation (68, 69). SDHA immunohistochemistry has recently been shown to reveal the presence of SDHA mutations in about 3% of patients who are affected by apparently sporadic tumors (70).
There are no histological features that are certain for malignancy and the presence of lymph node involvement or distant metastases is the only widely accepted criteria for malignancy. About 10% of pheochromocytomas are malignant at the time of initial surgery or at follow up. The clinical value of the Pheochromocytoma of the Adrenal Gland Scaled Score (PASS score) based on invasion, diffuse growth, focal or confluent necrosis, high cellularity or tumor spindling has not been confirmed. Tumors that have increased likelihood of malignancy are usually larger in size, extra-adrenal, and secrete more dopamine. Also an index of tumor differentiation the epinephrine to epinephrine plus norepinephrine ratio, is lower in malignant than benign tumors (71, 72).
To achieve optimum management of a patient with pheochromocytoma a multi-disciplinary approach is required with involvement from a number of specialists an endocrinologist, a surgeon, an anesthesiologist and an oncologist at the very least along with ancillary staff. The ultimate goal of therapy is tumor removal. In benign tumors usually this is not such a difficult task and once the tumor is removed the patient can have a normal symptom free life. However the task that is difficult is to know at the time of initial treatment which patients have benign tumors versus patients with malignant tumors. Unfortunately it is not possible to predict the long-term outcome of a tumor initially and hence patients with seemingly benign tumors also need follow up to detect recurrence or malignancy.
The mainstay of medical therapy is achieving adequate blood pressure control pre and intra-operatively to avert a hypertensive crisis. Postoperative hypotension maybe a risk and hence the patients need to be monitored closely as well.
Depending on the patient's symptomatology appropriate medical therapy must be started. Most patients will have hypertension and will need an antihypertensive medication. An alpha-blocker is the initial drug of choice and phenoxybenzamine (irreversible non-competitive alpha blocker) is used most often. It is started in a dose of 10 mg twice a day, and then titrated upwards (increments of 10-20 mg every 2-3 days) until adequate blood pressure control is achieved (total dose usually 1 mg/kg daily) unless postural hypotension develops earlier. Other agents used are the competitive, shorter acting, selective alpha 1 agents such as prazosin, doxazosin and terazosin (73). They can cause postural hypotension after the first dose and hence must be used with caution. A combined alpha plus beta blocker labetalol may also be used in a dose of 200-600 mg daily though it has a stronger beta antagonistic activity than alpha (4-6:1) and this means that a sufficient antihypertensive dose will certainly lead to bradycardia (74).
Some patients may have clinical symptoms of beta-receptor stimulation such as palpitations, anxiety, and chest pain and may need a beta-blocker such as propranolol, atenolol or metoprolol. A beta-blocking agent should never be used in absence of an alpha-blocking agent as that could precipitate a hypertensive crisis (75). Metyrosine a competitive inhibitor of tyrosine hydroxylase, the rate-limiting step in catecholamine synthesis decreases production of catecholamines thus making pre and intra operative blood pressure control easier (76). Metyrosine is administered in an initial dose of 250 mg orally every 6-8 hours and the dosage is then titrated in increments of 250-500 mg to achieve adequate blood pressure control up to a maximum dose of 1.5-4 g/day. It crosses the blood brain barrier and hence has sedative and depressant effects on the brain. Therefore this drug is used only in patients with severe hypertension uncontrolled with other medications. Calcium channel blockers have also been used to control blood pressure before and during surgery (77).
Adequate precautions must be taken and careful monitoring of the patient's blood pressure and heart rate are crucial for the safety of the patient especially during induction, intubation and during peritoneal incision, tumor handling and devascularization as this can lead to large variations in blood pressure and heart rate. Infusions of short acting vasodilators or anti-arrhythmic agents should be prepared in advance so that they are ready to administer as soon as required (71).
Laparoscopic adrenalectomy is a safe and effective approach in most patients with a benign pheochromocytoma less than 6 cm and offers significant post-operative benefits. Patients with large adrenal tumors (larger than 6 cm), evidence of venous involvement, or invasion into surrounding tissue should be approached cautiously (78, 79). A recent study where 23 patients were operated via laparoscopic adrenalectomy and another 23 patients via posterior retroperitoneoscopic adrenalectomy, they concluded that they are both safe and effective approaches in patients with pheochromocytomas. In their experience, retroperitoneoscopic approach results in decreased operative time, blood loss, and postoperative length of stay compared with laparoscopic adrenalectomy. Hence posterior retroperitoneoscopic adrenalectomy is now becoming the preferred approach for patients with PHEO (80). It is a technique that is safely performed for a variety of adrenal lesions and is ideal for patients who have undergone earlier abdominal surgery, and is feasible in obese patients (81). Patients with hereditary adrenal pheochromocytomas may undergo adrenal cortical sparing surgery, which is an alternative approach that aims to balance tumor removal with preservation of adrenocortical function. These patients were followed up for a mean of 36 months and the patients had normal catecholamine levels and remained tumor free by imaging (82).
Frequent blood pressure monitoring is very important postoperatively. Patients are prone to develop hypotension due to acute withdrawal of catecholamines after resection of the tumor. Volume replacement is the treatment of choice and the volume of fluid required is often large about 0.5-1.5 times the patients total blood volume during the first 24-48 hours after surgery and lower volumes thereafter (125ml/hour). The patient may also develop hypertension from pain, volume overload, autonomic instability, essential hypertension or residual tumor.
The plasma or urinary metanephrine concentration should be checked 10 days after surgery, as catecholamine levels may remain high for the first week after tumor resection until all the extra-tumoral pools of catecholamines have emptied (71).
In malignant disease, surgical debulking of the recurrence or metastases is the primary management. In patients not amenable to surgery or in those that require additional therapy post surgery a number of palliative options are available and discussed below.
131I-MIBG therapy is indicated in patients with malignant inoperable 123I-MIBG positive pheochromocytomas or paragangliomas. Its contraindications are mainly related to radiation safety and toxicity. Thyroid blockade with potassium iodide is necessary to protect the gland from radiation. The main side effects of this therapy are vomiting and rise in blood pressure (early side effects) and myelotoxicity and hypothyroidism inspite of blockage (late side effects). The routine initial dose in treatment of pheochromocytomas and paragangliomas is about 15-18.5 GBq (405-500 mCi). Doses greater than that show a significant increase in hematological toxicity without an additional improvement in response rate (83). A retrospective review of 116 patients showed symptomatic relief in 76%, tumor response in 30%, and biochemical response in 45% (84). A phase II clinical trial of high dose 131I-MIBG therapy (492-1,160 mCi) in 50 patients showed a 64% 5-year survival rate. Since treatment with such high doses of 131I-MIBG is myeloablative, stem cells from the peripheral blood must be obtained before treatment (85).
In metastatic pheochromocytomas that are rapidly progressing chemotherapy is the preferred treatment option. A combination of cyclophosphamide, vincristine and dacarbazine showed a 57% complete or partial radiologic response while 79% had some biochemical response (86). The NIH updated this study with 4 more patients (total 18 patients) and followed them for 22 years. 55% patients had radiologic response to treatment and 72% had a biochemical response. The authors concluded that CVD therapy is not indicated in every patient with metastatic disease, and should be used for patients with symptoms where tumor shrinkage might be beneficial and could lead to possible surgical resection. In this study they found that based upon long-term follow-up, there is no statistically significant difference in overall survival between patients whose tumors responded to CVD compared to those whose tumors did not respond (87). In another study done in 2011 33% of patients with metastatic pheochromocytoma/sympathetic paraganglioma experienced improvements in tumor size or hypertension (dose reduction/antihypertensive discontinuation) after chemotherapy. They also observed a trend toward longer survival in the responders than in the nonresponders to chemotherapy. Cyclophosphamide and dacarbazine combined with vincristine-based and/or doxorubicin-based chemotherapy was the regimen received by these patients (88). CVD therapy can cause a hypertensive crisis due to catecholamine release during treatment and hence patients should be blocked beforehand (89).
Many other experimental and targeted therapies have been developed and are being tried for the management of malignant pheochromocytomas and paragangliomas. Some of them are sunitinib – a receptor tyrosine kinase inhibitor, imatinib mesylate – a tyrosine kinase inhibitor, thalidomide, everolimus, temozolomide and trastuzumab. A number of new therapeutic targets are being considered such as mTOR inhibitors, HIF inhibitors, Prolyl hydroxylase activators ERBB2 inhibitors and heat shock protein 90 inhibitors (89). With so many newer drugs and targets the future for these malignant tumors looks hopeful and prognosis for patients will most likely improve over time.
It cannot be used as a primary mode of therapy and does not prevent local recurrence, though it is mainly beneficial for relief from symptoms and pain associated with bone and lymph node metastasis (89).
Ablation can be used to control disease locally to alleviate symptoms like pain and hypertension related to the tumor and to prolong a patient's functionality. Bone, chest wall, and retroperitoneal lesions are treated with either radiofrequency (RF) ablation or cryoablation, and liver tumors are treated with either RF ablation or ethanol ablation (RF ablation was deemed unsafe for lesions adjacent to the central bile ducts). Amongst the patients available for follow up imaging that was done at a mean of 3.7 months 56% of these patients had no evidence of recurrence (90).
When recurrences are small with an accessible vascular supply surgical removal maybe preceded or replaced with therapeutic embolization. Preoperative embolization of the tumor can minimize blood loss and facilitate subsequent resection. Transcatheter arterial chemo-embolization (TACE) is particularly useful in hepatic metastases and can lead to significant reduction of the tumor mass (number and size of the lesions) in the liver without any major side effects. In one report they used mitomycin C-Lipiodol emulsion followed by small pieces of gelatin sponge. An increase in blood pressure after every TACE could be controlled by sufficient pre-administration of an alpha-blocker (91). A more recent case report states that they used Epirubicin-Lipiodol emulsion and multiple gelatin particles for chemo-embolization of the liver (92). Another case report of a patient with recurrence of the metastases post transarterial bland embolization Drug-Eluting Bead Transarterial Chemo-embolization (DEB-TACE) of Hepatic Metastases with doxorubicin was used in the patient with follow up CT showing complete devascularization of all three hypervascular lesions (93).
The somatostatin receptors are expressed in pheochromocytomas, particularly subtype 2A and 3 (94). This therapy utilizes radiolabeled 111In, 90Y, or 177Lu-labelled somatostatin analogues and is of use in patients with unresectable metastatic disease.
Symptomatic improvement may occur with all radiolabeled somatostatin analogues though tumor size reduction is achieved with 90Y and 177Lu. High tumor uptake on somatostatin receptor scintigraphy and limited amount of liver metastases predict higher likelihood of tumor remission. Serious side-effects like myelodysplastic syndrome or renal failure are rare. The median duration of the therapy response for [90Y-DOTA(0),Tyr(3)]octreotide is 30 months and for [177Lu-DOTA(0),Tyr(3)]octreotate it is more than 36 months. This treatment is being used for neuroendocrine tumors and is now gaining popularity for pheochromocytomas as well (95).
The long-term survival of patients after successful removal of a benign pheochromocytoma is basically the same as normal individuals adjusted for age due to advances in surgery and a multi-disciplinary approach in the management of these patients (96). Patients should be followed up lifelong with annual check-ups of blood pressure measurement and urine or plasma metanephrine levels. In the long term follow-up of 613 patients with pheochromocytoma/paraganglioma, the tumor was metastatic at some time 11.4% patients out of which about half had metastases detected at the time of initial presentation and about half during follow up of an apparently benign tumor. (71).
This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.
Conflict of interest
The authors declare no conflict of interest.
Manger WM, Gifford RW, Jr., Hoffman BB. Pheochromocytoma: a clinical and experimental overview. Curr Probl Cancer. 1985;9(5):1-89