Diagnosis of Pheo / Para...

Biochemical diagnosis of pheochromocytomas and paragangliomas: A guide for patients in 2012

Graeme Eisenhofer PhD, Professor & Chief, Division of Clinical Neurochemistry, Institute of Clinical Chemistry & Laboratory Medicine and Department of Medicine, University Hospital Dresden, Dresden, Germany

What are pheochromocytomas and paragangliomas? Pheochromocytomas are tumors that arise from the adrenal glands, which sit atop each kidney. Paragangliomas are tumors that arise from similar cells, but at non-adrenal gland locations; they most commonly occur in the abdomen, but can also develop in the chest, head and neck. Both types of tumors are usually suspected because of effects produced by secretions of certain hormones, called catecholamines. Secretion of catecholamines by the tumors causes increases in blood pressure and a wide range of symptoms (e.g., headaches, sweatiness, palpitations, anxiety, panic attacks).

What are catecholamines? There are three catecholamines, the most well known of which is adrenaline (epinephrine) formed almost entirely in the adrenal glands. The other catecholamines include noradrenaline (norepinephrine) and dopamine. Noradrenaline is secreted mainly by nerves that go to the blood vessels, heart and other organs. Noradrenaline is also formed in the adrenal glands where it is converted to adrenaline. Dopamine is the building block catecholamine from which noradrenaline is formed.

Which catecholamines are formed by what tumors? The three catecholamines are formed within tumors in different amounts depending on tumor type. About a half of all pheochromocytomas produce both adrenaline and noradrenaline in various proportions, while the other half produce only noradrenaline. In contrast, most paragangliomas secrete only noradrenaline, but others produce both dopamine and noradrenaline in varying amounts, including some that produce only dopamine. Other paragangliomas, particularly those of the head and neck, produce no catecholamines.

Which catecholamines cause what effects? It is only the tumors that produce adrenaline, noradrenaline or both catecholamines that cause increases in blood pressure and symptoms such as headaches, sweatiness, and palpitations. Tumors that produce only dopamine, as well those that produce no catecholamines, do not cause high blood pressure or the usual symptoms. However, some tumors that produce adrenaline or noradrenaline do not secrete the hormones in large enough amounts to produce increases in blood pressure or symptoms of catecholamine excess. Such tumors and tumors that produce only dopamine are usually found for other reasons.

What biochemical tests are used for diagnosis of pheochromocytomas and paragangliomas? Biochemical tests used for diagnosis of pheochromocytomas and paragangliomas include measurements in blood or urine of the catecholamines and various breakdown products (metabolites) of the catecholamines. The metabolites most commonly measured include normetanephrine, the breakdown product of noradrenaline, and metanephrine, the breakdown product of adrenaline. These two metabolites, normetanephrine and metanephrine, are commonly referred to in the plural form as the “metanephrines”. It is incorrect to refer to these metabolites in their single form as “normetanephrines” and “metanephrines”. In addition to normetanephrine and metanephrine, some laboratories also measure methoxytyramine, which is the breakdown product of dopamine. Another biochemical test still offered by some laboratories involves measurements in urine of vanillymandelic acid (commonly referred to as VMA), which represents the final major breakdown product of both noradrenalne and adrenaline. Measurements of plasma chromogranin A (CgA) are another test sometimes used to diagnose pheochromocytomas and paragangliomas. Chromogranin A is not a catecholamine or a catecholamine metabolite, but is secreted by the same cells that secrete catecholamines.

What biochemical tests are recommended for diagnosis of pheochromocytomas and paragangliomas? It is recommended that initial testing for pheochromocytomas and paragangliomas always include measurements of either or both urine or blood levels of metanephrines.

Why are measurements of catecholamines by themselves not recommended for biochemical diagnosis? The catecholamines formed in nerves, adrenal glands or in pheochromocytomas and paragangliomas are packaged and concentrated within cells in very tiny bag-like structures known as storage vesicles. The catecholamine contents of these storage vesicles are normally secreted by nerves to control blood pressure and the beating of the heart or they are secreted by the adrenal glands in response to stress. Pheochromocytomas and paragangliomas also can secrete catecholamines, but these secretions are not controlled and some tumors do not always secrete catecholamines. In the latter situation catecholamines in urine and blood will be normal and their measurements will fail to show up a tumor. This is called a false-negative test result, which is not good for patients with the tumors since the tumors will remain undetected. These tumors nevertheless are still highly dangerous, since secretion of catecholamines by the tumors can be provoked under certain situations (e.g., childbirth, accidents, surgery, certain medications), resulting in dangerous increases in blood pressure or even death.

Why are measurements of metanephrines recommended for diagnosis? As outlined above the catecholamines formed in nerves, adrenal cells and pheochromocytoma or paraganglioma tumor cells are packaged in a highly concentrated form within tiny bag-like structures called storage vesicles. These storage vesicles are slightly porous and constantly leak catecholamines into the fluid inner contents (cytoplasm) of the cells. High concentrations of those catecholamines inside this cellular fluid is not good for the cells. The cells therefore have ways of dealing with this by breaking down the catecholamines into metabolites. It is because of this that most initial metabolic breakdown of catecholamines occurs in the same cells in which the catecholamines are formed. Metabolic breakdown also occurs after secretion from the cells, but the initial metabolic breakdown of catecholamines after release by cells is relatively minor compared to the metabolism within the cells. Importantly, the breakdown of catecholamines within pheochromocytoma and paranganglioma cells occurs by a process that produces the metanephrines. These metabolites of the catecholamines are therefore formed continuously within the tumors cells. Since this process is independent of catecholamine release, measurements of the metanephrines provide advantages for diagnosis compared to catecholamines. As outlined above the catecholamines may not always be secreted by tumors. In such situations measurements of catecholamines will not show that a pheochromocytoma or paraganglioma is present, whereas the metanephrines formed continuously with tumors cells will be elevated and thus show the presence of a tumor.

What about urine vanillymandelic acid - VMA? Catecholamines undergo many sequential steps during their metabolic breakdown. Formation of metanephrines is just one of three initial of the sequential steps that lead to formation of VMA, which is the final metabolic breakdown product of both noradrenaline and adrenaline. VMA is formed almost totally in the liver, in part from metanephrines extracted from blood by liver cells, but mainly from different breakdown products formed in the nerves that control blood pressure and heart function. Therefore, VMA does not provide a good test for diagnosis of pheochromocytomas and paragangliomas and is no longer recommended for this purpose.

What about chromogranin A - CgA? CgA is secreted like the catecholamines from the same storage bags (vesicles) that contain the catecholamines. Therefore, like the catecholamines blood levels of CgA can also be normal when tumors are not secreting the contents of the vesicles. Nevertheless measurements of CgA can be useful under certain circumstances, some of which are outlined later.

How do measurements of metanephrines in urine and blood differ? The metanephrines measured in urine are in fact largely different metabolites from the metanephrines measured in plasma. The metanephrines released into the bloodstream from catecholamine-producing tumors are further metabolized in other parts of the body by addition of a sulfate group. This makes the metabolites more easy to excrete into urine. Nevertheless, measurements of metanephrines in urine are nearly as good measurements in blood for diagnosis. Some clinicians prefer the urine test and others the blood test. The blood test has advantages in certain situations, but is not always available and at some places may not be as reliable as the urine test. In the United States measurements of metanephrines are now usually carried out by good modern methods involving mass spectrometry. It can be useful to find out what measurement methods are used. If measurements are by high pressure liquid chromatography or involve mass spectrometry then they are likely to be reliable.

How should patients be positioned for measurements of metanephrines in blood? Unfortunately, not all centers adequately prepare their patients for blood measurements of metanephrines. Ideally blood samples for measurements of metanephries should be taken after patients have rested in the lying (supine) position for at least 20 minutes. This is because any upright posture causes the nerves in the body to release catecholamines in order to tighten blood vessels. This tightening of blood vessels keeps blood flowing to the brain. If this automatic response did not happen blood pressure would fall and there would be loss of consciousness due to lack of blood to the brain (a faint). Therefore, when measuring catecholamines as well as metabolites of catecholamines it is useful to take blood when normal secretions of catecholamines are at their lowest possible (i.e., with patients resting comfortably and under minimal stress in the lying position). This reduces the “noise” from normal sources of metanephrines, which might otherwise lead to elevated blood concentrations not from a tumor but due to activated release from nerves and adrenals (a false-positive result).

At many centers blood samples are taken with patients in the seated position, which is often much more convenient for the staff taking the blood samples. Such sampling can be okay, but only as long as the doctor is aware of the increased likelihood of a positive result. If a blood test in the sitting position returns a positive test result then there is usually a need to repeat the sampling at a later date with the patient lying for at least 20 minutes before the blood sample is taken. If on the other hand the blood test in the sitting position returns a negative test result (i.e., normal) then there is no need to repeat the test.

What other precautions are necessary before blood samples are taken for metanephrines? Other precautions that may be required depend on the center and measurement methods that are used. Usually patients will be instructed to refrain from smoking and drinking caffeinated beverages after wakening and preceding the blood draw. At some centers patients may also be asked to fast overnight up until the blood draw, which is particularly important when measurements include methoxytyramine. At other centers there may instructions to avoid certain medications (e.g., acetaminophen, Tylenol) over a certain time period, this also dependent on the center and measurement method.

What is required for urine measurements? Urine measurements commonly require complete collection of urine over a twenty-four hour period so that results can be expressed in terms of amount of substance excreted per day. The precise instructions for collections may vary from center to center, but usually patients are instructed to start collecting their urine in the morning after discarding their first morning’s urine (i.e., when they first arise after awakening) as usual into a toilet. Thereafter all urines must be collected into a specified urine container up until the next morning’s first urine, which is also collected into the container. Patients should also avoid excessive exercise, which can increase release of catecholamines from nerves and adrenal glands and cause false-positive results for measurements of catecholamine metabolites. There may also be medications that should not be taken depending on the center and particular measurement method used. Once all the urine is collected it should be returned to the clinic or to a specified laboratory as soon as possible. The test is reasonably easy for clinical staff to implement, but does usually require two trips for patients, one to pick up the empty urine container and another to drop off the full container. At some centers there may be additional instructions to follow on the use of preservatives, such as concentrated hydrochloric acid. It is very important that all urine produced over the course of a day is collected into specified containers.

What determines a positive or negative biochemical test result? Determination of whether a biochemical test result is positive (which suggests a tumor may be present) or negative (which suggests a tumor is unlikely) is dependent on the reference intervals or cut-offs for the particular test. Typically these cut-offs are determined using a reference population of at least 120 volunteers without the disease. Cut-offs are most usually determined from the 2.5% and 97.5% percentiles or 95% confidence intervals of a population mean. In both cases it can be expected that 2.5% of all patients without disease would have values above the upper limits for the reference intervals and another 2.5% below the lower limits. Results that are positive (for metanephrines this is above these limits) therefore do not always define the presence of a tumor, but they do increase the likelihood that a tumor is present.

For diagnosis of pheochromocytoma using blood levels of metanephrines it is important that reference intervals are determined to ensure that a result above the upper limits (i.e., a positive result) occurs in almost all patients with the tumors. This then provides confidence that a normal result reliably excludes disease. Unfortunately not all laboratories have established their upper reference intervals using blood samples taken after resting for at least 20 minutes in the lying position. Such reference intervals determined from samples taken in seated subjects are usually too high for a negative test result to reliably exclude disease. The situation is somewhat further complicated by the fact that measurements of metanephrines involve both normetanephrine and metanephrine, with normal results for both required to exclude disease. Increasing levels of normetanephrine with advancing age represent another complication. Thus, while an upper cut-off of 0.9 nmol/L (165 pg/mL) might be okay for patients over 50 years these limits are too high for anyone under 40 years, and particularly children in whom upper limits for blood levels of normetanephrine should be closer to 0.55 nmol/L (100 pg/mL). 

Are normal test results for plasma or urinary free metanephrines sufficient to rule out a catecholamine-producing? This depends on the circumstances under which a catecholamine-producing tumor is suspected. Patients may undergo biochemical testing for pheochromocytoma or paraganglioma for a range of different reasons. The most common reason is because of a presentation of high blood pressure or spiking increases in blood pressure, along with other suspicious symptoms (e.g., headaches, sweatiness, palpitations) that alert the clinician to a possible catecholamine-producing tumor. Presence of high blood pressure despite use of multiple blood pressure medicines provides another justification for clinicians to test for catecholamine-producing tumors. Under both these circumstances biochemical testing is relatively straightforward and can simply include measurements of plasma or urinary metanephrines or both. Provided reference intervals are appropriate and there is no laboratory error, then normal results for measurements of both normetanephine and metanephrine reliably exclude the tumor and no further testing should usually be required. If on the other hand test results are positive then this may or may not reflect a tumor and further testing is invariably required to refute or better confirm the diagnosis.

Are there circumstances that require an individualized approach for biochemical testing? Biochemical testing for catecholamine-producing tumors may be carried out even in the absence of increased blood pressure or symptoms of catecholamine excess. Such circumstances include follow-up of patients who have had a pheochromocytoma or paranganglioma surgically removed, but in whom there is always a risk of recurrent or even malignant disease. Another circumstance involves patients who undergo imaging studies, such as computed tomography (CT) or magnetic resonance imaging (MRI), for reasons unrelated to any suspicion of a catecholamine-producing tumor and in whom an unusual mass is found in the adrenal gland or elsewhere. In such patients it is important to rule out that the mass is not a catecholamine-producing tumor. Another increasingly important circumstance involves the patient and family members identified with gene mutations that predispose to development of catecholamine-producing tumors. Although measurements of plasma or urinary metanephrines under these circumstances remain important, sometimes these measurements are insufficient alone for reliable diagnosis or exclusion of the tumor. All the above circumstances can benefit from an individualized approach to biochemical testing.  

What individualized approaches are required for biochemical testing in patients with germ-line mutations of tumor-susceptibility genes? Today it is widely recommended that patients with identified mutations of tumor-susceptibility genes undergo routine screening for pheochromocytomas or paragangliomas regardless of the presence or absence high blood pressure or symptoms of catecholamine excess. The form that this testing takes depends on the mutation, but it is generally recommended that the testing be carried out once every year or two years. The one hereditary syndrome where testing may depend on other evidence or may be carried less frequently is neurofibromatosis type 1. Pheochromocytomas in this syndrome occur in less than 2% of cases.

Pheochromocytomas and paragangliomas occurring as a consequence of various mutations of the different tumor susceptibility genes have different clinical presentations depending on the affected gene. These differences include the type of catecholamine produced which impacts choice of biochemical testing strategies and interpretation of test results. Many of the hereditary causes of pheochromocytomas and paragangliomas carry additional risk for other types tumors, which must also be considered when patients visit for routine check-ups. The individualized approach required for such patients is therefore best carried out at specialist centers.

A good example of a required individualized approach involves patients with mutations of the gene for succinate dehydrogenase sbunit B (SDHB), who are at particular risk for malignant disease. Tumors in these patients often produce noradrenaline and dopamine, with some only producing dopamine. Testing in these patients therefore should include both measurements of normetanephrine and methoxytyramine, the latter only available at a few centers. Also occasional tumors in these patients can be so immature that they do not produce any catecholamines. Measurements of chromogranin A can therefore also be useful, while additional imaging including of the head and neck is also called for to detect non-functional paragangliomas.

Where should a patient have their biochemical testing carried out? For testing in common circumstances involving high blood pressure and symptoms suggestive of a catecholamine-producing tumor, testing can be carried out by any qualified clinician with an up-to-date knowledge of endocrine tumors and access to a good laboratory for biochemical measurements of blood or urine for metanephrines. For other patients, such as those requiring individualized attention as outlined above, testing is best done at specialist centers with staff experienced in hereditary causes of pheochromocytomas and paragangliomas, as well as how to best follow-up affected patients and others with a previous history of the tumors. Referral to specialist centers in cases of positive test results can also facilitate more efficient follow-up testing in order to better confirm and then locate tumors. Treatment of patients and particularly their pre- to post-surgical care is best undertaken by staff experienced with all recommended approaches, such as laparascopic surgical removal and adrenal sparing surgery where this may be indicated.


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