The Pheochromocytoma/Paraganglioma syndrome: an overview on mechanisms, diagnosis and management

ABSTRACT Pheochromocytomas/paragangliomas (PPGL) are rare, metastatic, and potentially fatal neuroendocrine tumors, often neglected because they present symptoms similar to other prevailing clinical conditions such panic syndrome, thyrotoxicosis, anxiety, hypoglycemia, etc., delaying diagnosis and treatment. The rate of diagnosis of PPGL has been increasing with the improvement in the measurement of catecholamine metabolites and the expanding availability of imaging procedures. Its essential genetic nature has been extensively investigated, comprising more than 20 genes currently related to PPGL and more new genes will probably be revealed. This overview will shed some light on the clinical, laboratory, topographical, genetic diagnosis, and management of PPGL.

PPGL is a serious, potentially metastatic, and fatal disease that often goes unnoticed by unexperienced doctors. Approximately 85-90% of PPGL are localized in the adrenals and 10-15% are extra-adrenal, being called paragangliomas (PGL); the latter may be found from the base of the skull to the testicles but are mostly found within the abdomen (4)(5)(6)(7).
In this mini-review article we survey on clinical, laboratory, topographical, genetic, and therapeutic aspects of PPGL, a condition that has been showing an increase in incidence with the improvement of methods to measure catecholamine metabolites and imaging techniques.

EPIDEMIOLOGY
The prevalence of PPGL among the hypertensive population is 1:500-1,000, but 75% of the cases are diagnosed postmortem, and in 55% of them PPGL directly contributed to death. In autopsy studies, the prevalence of PPGL ranges from 250 to 1,300 cases per million. Thus, clinical suspicion of PPGL still draws little attention (5,6,8).
The incidence of PPGL has been increasing over time, despite a fall in the number of necropsies, and this is due to the increase demand in the number of imaging exams and improved methods for measuring catecholamine metabolites (6).

CLINICAL PICTURE AND INVESTIGATION
The symptomatology of patients with PPGL is variable. Systemic arterial hypertension (SAH) is the most frequent clinical manifestation of the disease, being present in 90% of cases. However, paroxysms (headache, palpitation, and sweating) are the most characteristic findings, resulting from release of catecholamines by the tumor and consequent stimulation of adrenergic receptors. They are often accompanied by increased blood pressure, tremor, pallor, chest or abdominal pain, and less commonly, facial flushing. Paroxysms do not occur in all patients. In some series, one or more components of the classical triad were present in more than 90% of patients. (4,(7)(8)(9)(10)(11) The frequency of paroxysms is unpredictable and varies from 30 times a day to a single episode every 2-3 months. Near 75% of patients have one or more spells per week. Duration ranges from a few minutes (usually 15 to 60 min.) to days. They may arise spontaneously or be precipitated by activities that compress the tumor or elicit an increase in catecholamine secretion, such as exercises, pressure on the abdomen, urination, defecation, the act of smoking, and drugs like beta-blockers, anesthetic agents, radiologic contrasts, glucagon, metoclopramide, and tricyclic antidepressants (1)(2)(3)(6)(7)(8)(9)(10)(11)(12)(13).
There are clinical scores based on signs and symptoms that have high diagnostic predictability. Among the signs and symptoms are hyperhidrosis, palpitation, pallor, tremor, nausea, heart rate >85 bpm plus body mass index (BMI) (14). SAH may be paroxysmal, but more commonly are persistent (in ~60% of cases). It tends to be severe and/or refractory to antihypertensive medications and present with ample fluctuations. Sudden elevation of blood pressure (associated or not with other symptoms) may occur during abdominal manipulation, labor, intubation, anesthetic induction, surgery, or other invasive procedures. Norepinephrine (NE)-secreting tumors are usually associated with constant SAH, whereas those that secrete substantial amounts of epinephrine (E) in addition to NE are associated with episodic SAH. Conversely, when tumors secrete solely E, they provoke hypotension instead of hypertension; in this situation, the clinical feature may be of a cardiogenic shock. Orthostatic hypotension may be present in 40% of patients (12)(13)(14).
Cardiac abnormalities such as left ventricular hypertrophy occur quite commonly in patients with SAH, and myocarditis or dilated cardiomyopathy may result from circulating excess catecholamines. Palpitations and arrhythmias are common and occasionally fatal (12,15).
Pre-diabetes is present in 50% of cases and diabetes mellitus (DM) in 10-20%. They are secondary to suppression of insulin secretion and increased hepatic glucose output, induced by excess catecholamines. Hypercalcemia may also occur due to concomitance of hyperparathyroidism or tumor production of PTH-related protein (PTHrp).
Atypical manifestations such as ACTHdependent Cushing's syndrome, acute abdomen, cardiovascular (shock, myocarditis, cardiac arrhythmias, acute pulmonary edema, heart failure, Takotsubo syndrome) and neurological events (altered mental status, seizures, stroke, and focal neurological manifestations), weight loss, fever of indeterminate origin, aqueous diarrhea, or constipation simulating pseudo-obstruction and paralytic ileus may also be found. Fever of mild to severe intensity (reaching up to 41 0 C) is not uncommon and has been attributed to IL-6 secretion (11)(12)(13).

INVESTIGATION
Candidate subjects for a PPGL screening are: 1) young hypertensive patients under 30 years of age; 2) hypertensive patients refractory to treatment with 3 classes of antihypertensive drugs in effective doses; 3) hypertensive patients with paroxysms (headache, palpitation and sweating), seizures, unexplained shock, mucous neuromas, orthostatic hypotension, weight loss, presence of type I neurofibromatosis, family history of PPGL, medullary thyroid carcinoma, von Hippel-Lindau syndrome and familial PGL syndrome; 4) adrenal incidentalomas, especially in cases where pre-contrast attenuation values on computed tomography (CT) are ≥10 HU (Hounsfield units) and contrast washout <60%; 5) marked blood pressure lability; 6) episodes of shock or severe blood pressure responses during anesthesia induction, surgeries, invasive procedures, labor and use of β-blockers; 7) Takotsubo syndrome; 8) new-onset diabetes mellitus in a young lean hypertensive patient (12,14).
Hereditary PPGL are classified according to their transcription signature and are divided into three clusters as shown in Table-1. Next (and in Table-2) we describe briefly the main syndromic features that are associated with specific PPGL syndromes: von Hippel-Lindau (VHL) Syndrome PPGL occurs in 10 to 30% of patients with VHL. The VHL syndrome is classified as: type 1, in which PPGL does not manifest, and type 2, which is subdivided into 3 subtypes: 2A (encompassing PPGL plus retinal and CNS hemangioblastomas, and low risk for renal carcinoma), 2B (PPGL plus retinal and CNS hemangioblastomas and kidney and pancreatic tumors), and 2C (PPGL only).
PV occur in the VHL gene, which is a tumor suppressor located on chromosome 3p25, responsible for regulating hypoxia-induced genes by ubiquitination and subsequent degradation of HIF2α. VHL disease has a penetration >90% at 65 years of age and missense PV are likely associated with the development of PPGL, whereas truncated or large variants are associated with the presence of hemangioblastomas and renal cell carcinoma (17)(18)(19)(20)(21)(22).

Paragangliomas
PV of succinate dehydrogenase (SDH) subunits D, B, C, A, and A2F are associated with PGL. These subunits are related to signals responsive to oxygen level so that PV in the respective genes would lead to a chronic state of hypoxia and, therefore, cell proliferation. PGL are classified as follows: PGL1: results from PV in SDHD, located on chromosome 11q23, with a maternal imprint mechanism, which results in the PV almost always being transmitted by the father and a PV frequency of 3 to 5%, penetrance of 31 to 50% and frequency of metastases less than 5%; these PGL are usually located in the head, neck, and adrenals bilaterally, and may or may not be functioning. In 75% of cases, the disease manifests around the age of 40 years. Renal carcinomas are found in 8% and pituitary adenomas have been reported in a few cases. PGL2: results from PV of the SDHA2F gene. Initially described in 2009, this PV is rarely found in PGL. Located on chromosome 11q13 and, as in cases that present PV in SDHD, transmission is also by maternal imprint and almost always results from paternal transmission. PGL usually appear around 22 years of age and are often multifocal, although non metastatic.
PGL3: results from PV of the SDHC gene, located on chromosome 1q21, with autosomal dominant transmission, and PV frequency below 0.1%, unknown penetrance and indeterminate frequency of metastases; tumors in PGL3 localize in the head and neck and are not functioning.
PGL4: results from PV in the SDHB gene, located on chromosome 1p36.3, with autosomal dominant inheritance, and frequency PV ranging from 2 to 7%, penetrance of 50 to 70% and frequency of metastases from 34 to 70%; these PGL are usually located in the thorax, abdomen and adrenal bilaterally and are always functioning. Renal carcinomas occur in 14% and GIST in 2% of cases.
PGL5: results from PV of the SDHA gene that rarely cause PGL; corresponds to 3% of cases and has low penetrance. GIST and pituitary adenomas may be present. (10-13, 23, 24).

Neurofibromatosis (NF)
PPGL may be associated with type 1 NF, whose diagnosis is clinical and generally does not pose diagnostic problems. The NF-1 gene localize on chromosome 17q11.2 and is responsible for encoding a protein called neurofibromine; its inheritance is autosomal dominant. In NF-1, PV inactivate the gene and occur in 1 to 5% of the cases, when PPGL is not accompanied by hypertension and in up to 50% of those with hypertension. PPGL associated to PV in NF-1 is similar to sporadic ones, occurring in older patients; less frequently they are bilateral and extra-adrenal. PPGL was present in 3 to 13% of individuals who underwent autopsy (10-13, 23, 24-26).

Multiple Endocrine Neoplasia (MEN)
In MEN 2A (medullary thyroid carcinoma [MTC], PPGL, and primary hyperparathyroidism) and 2B (MTC, PPGL, and mucous neuromas/intestinal ganglioneuromas and marfanoid habit), PPGL may be present in 50% of cases. PV in the RET proto-oncogene (Rearranged During Transfection, localized on chromosome 10q11.2) is of missense germline. This gene encodes a tyrosinekinase receptor that is expressed in various tissues derived from the neural crest, including the CNS and peripheral nervous system, and neuroendocrine tissues.

TMEM127
The TMEM127 gene, described by Dahia et al. in 2010, is positioned on chromosome 2q11; it is a tumor suppressor that, like the NF-1 gene, promotes gene inactivation (20). In a cohort of 103 samples, PV was present in 30% of cases and in 3% of apparently sporadic PPGL (23,24).

Laboratory Diagnosis
Laboratory diagnosis of PPGL is usually accomplished by measuring blood and urine metanephrines. The current gold standard is a plasma metanephrine (MN) measurement that achieves a sensitivity of 99% for sporadic and hereditary functioning PPGL and a specificity of 99% for hereditary (and 89% for sporadic), superior to any combination of tests. Normal plasma MN virtually excludes functioning PPGL. Preferably, plasma MN and/or urinary MN should be the tests of choice for the diagnosis of PPGL.
Chromogranin A (ChrA), an acid glycopeptide co-secreted by PPGL, can be measured during laboratory investigation; it has a diagnostic sensitivity of 83-86% and specificity of 76-98%. ChrA is not influenced by an-tihypertensive drugs and exhibits an increase in positive predictive value (PPV) when combined with plasma MN. ChrA may be elevated in cervical PGL that do not have elevated plasma and/or urinary MN, thus functioning as a tumor marker in this situation. However, ChrA may be increased in the following conditions: renal failure (creatinine clearance <80mL/min), use of proton pump inhibitors, liver failure, and atrophic gastritis. Also, ChrA has low specificity since other neuroendocrine tumors (NET) can also produce it.
In Figure-1, we described a laboratory flowchart for the diagnosis of functioning PPGL.
Use of MRI for the diagnosis of PPGL has the following advantages: (1) high sensitivity (93-100%) in detecting adrenal disease, (2) presence of a "hypersignal" in T2 sequence compared to the liver, in at least 75% of PPGL, (3) better sensitivity to localize intracardiac PGL, (4) possibility of visualization and confirmation of bone metastases suggested by mIBG scintigraphy, and (5) can be performed in pregnant women (second trimester on) (without contrast) and in children and carriers of germline variants, since there is no exposure to ionizing radiation. In Figure-2, we described the MRI with sporadic pheochromocytoma on the left adrenal with some typical features.
CT has a sensitivity of 93-100%, but low specificity (70%). Sensitivity is lower for small adrenal PPGL and for adrenal medullary hyperplasia. It is also less sensitive in the detection of PGL, small metastases and early recurrence of tumors in the adrenal surgical bed. CT is currently recommended as the first choice for topographic diagnosis of PPGL (11,25,30,34,(36)(37)(38).

131
I-mIBG scintigraphy has diagnostic sensitivity and specificity of 77-90% and 95-100%, respectively. When 123 I is used instead, sensitivity reaches higher values: 83-100%, without loss of specificity. Its use should be considered in cases of adrenal Pheo that are suggestive of benignity. False negative results occur in 15% (approximately 60% of PGL are not avid for mIBG), and false positives can also occur, since 50% of normal adrenals have physiological uptake. The following are indications for pre-surgery mIBG: diagnostic confirmation, inconclusive biochemical results, familial disease, extra-adrenal tumors, and the possibility of treatment with therapeutic mIBG in metastatic PPGL. Post-surgical mIBG are indicated to search for disease recurrence and metastases (1, 5, 7-9). 18 FDG PET-CT is recommended for aggressive metastatic PPGL, lesions greater than 8 cm and those with PV in the SDHB gene. Sensitivity ranges from 74-100%. In Figure- Ga PET-CT DOTATATE, DOTATOC or DOTANOC have high sensitivity and specificity for neuroendocrine tumors as well as for tumor dedifferentiation; its recommendations parallel those of 18 FDG PET-CT (11,25,32,34). The histological concept of malignancy in PPGL is rather complex, since histological features of malignancy can be identified in "benign" PPGL, and histological absence of malignancy may be found in "malignant" tumors. Thus, malignancy is defined when there
The most used histological classification to aid in establishing malignancy potential is the PASS score (Pheochromocytoma of the Adrenal Gland Scaled Score) which considers the following items (Table-3): Non-metastatic PPGL have a score ≤3 and those potentially more aggressive ≥4 points. To date, there is no stratification model that combines histological and genetic data.
Tumor immunohistochemistry for the succinate dehydrogenases, especially the investigation of SDHB is indicated, as the loss of its expression suggests a germline PV in the SDHB gene and implies greater aggressiveness; this analysis is part of COOPS (Composite Pheochromocytoma/Paraganglioma Prognostic Score) system, in which necrosis (focal or confluent), loss of S100 expression, vascular invasion, loss of SDHB expression and size greater than 7 cm are evaluated. Scores greater ≥3 have a higher risk of metastasis (12,35).
The 8 th edition of the AJCC (American Joint Committee on Cancer) staging system includes a special chapter for PPGL, but not for parasympathetic PGL, as metastatic behavior is less than 5%. Pheo smaller than 5 cm in their longest axis and without vascular invasion are classified as T1; those ≥5 cm or sympathetic PGL of any size and without extra-adrenal invasion are classified as T2. PPGL of any size with invasion of surrounding tissues such as liver, pancreas, spleen and kidneys are classified as T3. Regarding lymph node involvement: Nx (without knowledge of involvement), N0 (without involvement of lymph nodes) and N1 (with involvement of regional lymph nodes). Regarding distant metastases, M0 (no distant metastases), M1a (distant metastases to bone only), M1b (distant metastases to distant lymph nodes/liver or lung) and M1c (distant metastases to bone and multiple other organs).

Clinical treatment
Treatment of PPGL is surgical whenever possible since there is a possibility of reversal of SAH. In addition, complications of an untreated PPGL can be fatal and there is a chance of metastases in 15-17% of cases.
Preoperative clinical therapy for a minimum of 7-30 days (15 days, on average) is man- datory, aiming to prevent an intraoperative hypertensive crises and cardiac arrhythmias, and to avoid hypotension after tumor removal. The best drugs for this purpose are α-blockers, such as prazosin, doxazosin, and terazosin; phenoxybenzamine has been less accepted in Brazil, as it has a longer biological half-life (and should be withdrawn 48h preoperatively, leaving the patient a period of 2 days rather unprotected) and may produce reflex tachycardia after its withdrawal. Prazosin and doxazosin are the most widely used drugs, in doses ranging from 1 to 16-20 mg per day. On average, 12 mg prazosin and 10 mg of doxazosin warrant good blood pressure control and prevention of paroxysms. Additionally, calcium channel blockers (amlodipine, diltiazen, verapamil and nifedipine) and angiotensin-converting enzyme (ACE) inhibitors may also be used. The use of β-blockers should be kept for when tachycardia and tachyarrhythmias are present, but always after effective control of hypertension with α-blockade; on average, β-blockers may be used after 3 days of the introduction of α-blockade.
α-Methyl-paratyrosine blocks the synthesis of catecholamines by inhibiting tyrosine hydroxylase, a key enzyme in the hormonal synthesis process; it can reduce catecholamine excretion by 35-80%. In general, it is recommended to treat SAH in patients with unresectable tumors or in those with metastases and in the preoperative period when there is no effective control with α-adrenergic blockers. Initial dose is 250mg 4x per day, a dose that can be adjusted every 3-4 days according to blood pressure response and possible side effects (sedation, psychiatric disorders, extrapyramidal symptoms, urolithiasis). The largest recommended dose is 4g/day (2,8,12,15,32).

SURGICAL TREATMENT
Only experienced surgeons and anesthesiologists should be responsible for the PPGL surgical procedure. The laparoscopic approach is preferred for tumor access, except for cases of suspected metastases and tumor size larger than 7 cm, conditions in which the classic open access is mandatory. Ideally, the entire immediate postoperative (post-op) period should be done in an intensive care unit (ICU), because even with adequate preparation there is a risk of arrhythmias and blood pressure instability, with the possibility of hypertensive crises and hypotension in the post-op period. There is still also a risk of hypoglycemia in the post-op, and installation of a 10% IV glucose solution is recommended for a period of 48h, with capillary glucose controls. The patient may remain hypertensive for a period of 2 weeks, after which a new 24h-plasma and/ or urinary MN measurement is recommended.
For PPGL patients with metastases, the target is to achieve tumor reduction and to control hypertension. Large PPGL can be reduced through surgery to obtain symptom relief and control of blood pressure levels; however, rarely will this surgery be curative, as there are often distant metastases, especially in bones (70%). Exceptionally, when metastases are restricted to the liver but are not surgically removable, transplantation will be an option. Tumor reduction can also be achieved by other interventional techniques such as transcatheter selective embolization or chemoembolization.
Thermal perfusion of the liver with cytotoxic drugs is used in some centers in cases of hepatic metastases.

Treatment with 131 I-mIBG
The use of radiolabeled mIBG in metastatic PPGL therapy should be considered, as mIBG may cross the cell membrane and be stored in cytoplasmic granules via VMS transporters (VMAT1 and 2). Since 1984, several patients with PPGL have been treated using different therapeutic protocols. Such patients are selected by demonstrating significant uptake of the radioisotope during a scintigraphy with 123 I/ 131 I mIBG.
The only impediment to this treatment is the total dose of radiation delivered to vital organs, such as bone marrow. Approximately 60% of metastases are avid for 131 I-mIBG. Recently, quantitative determination of VMAT 1 and 2 expression in surgical specimens proved useful in selecting patients suitable for treatment with 131 I-mIBG (12,36,37).
In general, patients with limited disease have an increased chance of tumor response. Similarly, soft tissue metastases respond better than bone metastases. Hormonal and symptomatic responses to 131 I-mIBG are independent of tumor size response (36)(37)(38).

Treatment with radioactive somatostatin analogues
Due to the expression of somatostatin receptors in metastatic PPGL, the use of radiopharmaceuticals (RP) based on somatostatin analogues has been tested.
Patients who will benefit from treatment are those who have an increased tumor uptake on scintigraphy (currently 68 Ga PET-CT with DOT-ATATE, DOTATOC or DOTANOC).
Stabilization or decrease in hormonal secretion and tumor growth have been reported in 20-25% of cases. Main side effects include leukopenia and thrombocytopenia.
Treatment with unlabeled octreotide is generally unsuccessful and only in some patients a transient response was observed because they express a low density of subtype 2 somatostatin receptors (SST2) (7,11,12).

Chemotherapy
Chemotherapy (QT) is an option when the tumor is inoperable and/or there is extensive residual disease. The combination of cyclophosphamide, vincristine and dacarbazine (CVD) may provide partial remission and transient symptomatic relief in up to 50% of patients with metastatic PPGL, although short-lived (1,7,9,11,12,36,37).
Other QT options are etoposide and cisplatin, anthracycline plus CVD and arabinoside cytokine. Some authors suggest a combination of lomustine and 5-fluorouracil or capecitabine for tumors with slow progression, whereas for rapidly progressive tumors, the best option would be the association of etoposide with a drug based and platinum (7,9,11,12,36,37).

New and emerging therapies
New antineoplastic therapies are being tested in patients with metastatic PPGL. The combination of temozolomide and thalidomide provided biochemical and radiological responses in 40 and 33% of the cases, respectively; however, lymphopenia accompanied by opportunistic infections occurred in most patients.

Follow-up
Patients with PPGL should undergo annual reevaluations by measuring urinary or plasma MN and chromogranin A. Follow-up is for life. When there is clinical and laboratory recurrence, with no radiological evidence, a new full body scintigraphy should be performed with 123 I/ 131 I-mIBG, or 68 Ga PET-CT with DOTATE, DOT-ATOC or DOTANOC or 18 FDG-PET-CT (11)(12)(13).