32 Pheochromocytoma, paraganglioma, neuroblastoma

Lothar Thomas

Pheochromocytoma and paraganglioma are neuroendocrine tumors with differentiation of chromaffine cells originated from neural crests.

Pheochromocytomas are tumors arising from the chromaffin cells of the adrenal medulla. Paragangliomas are extra-adrenal tumors of the sympathetic or parasympathetic nervous system and arise from ganglia along the sympathetic and parasympathetic chain. Due to the close relationship with neural tissue, composite lesions of pheochromocytoma and paraganglioma are known both in the adrenal and extra adrenal locations /1/.

Sympathetic paraganglioma (neuroblastoma) are derived from post ganglionic neurons of the sympathetic nervous system which produces catecholamines such as norepinephrine, epinephrine and dopamine. The great majority of neuroblastoma produces high levels of vanillylmandelic acid (MA) and homovanillic acid (HVA) /3/.

Parasympathetic paraganglioma, typicaly occuring in the head and neck, do not produce catecholamines and are very rare conditions with a prevalence of only 2 to 8 new cases permillion persons per year.

Pheochromocytoma and paraganglioma secreting excessive amounts of catecholamines (epinephrine, norepinephrine and dopamine), cause severe morbidity and/or lethal complications (e.g., cerebrovascular and cardiovascular) from effects of excess circulating catecholamines and from hypertension /2/. Pheochromocytoma usually secrete norepinephrine and epinephrine.

32.1 Metanephrines

Catecholamines (e.g., epinephrine, norepinephrine and dopamine) are metabolized within chromaffin cells to metanephrines, the respective O-methylated metabolites (e.g., norepinephrine to normetanephrine, epinephrine to metanephrine and dopamine to methoxytyramine, respectively). Because pheochromocytoma and paragangliomas (PGLs) do not always secrete catecholamines continuously, measurements of these analytes in plasma and urine often fail to reveal the presence of the tumor. In contrast pheochromocytoma (Pheo) and PGL continuously metabolize catecholamines to the O-methylated metabolites by a process that is independent of variations in catecholamine release /4/.

The measurement of plasma free normetanephrine and free metanephrine, represent the most sensitive tests to diagnose catecholamine production of Pheo and PGL. Measurements of fractionated metanephrines (i.e., normetanephrine and metanephrine measured separately) in plasma or urine provide superior diagnostic sensitivity and specificity in comparison to the measurement of catecholamines /5/.

32.1.1 Indication

Symptomatic patients /6/:

• Hypertension (sustained hypertension in 50–60% of cases)
• Paroxysmal hypertension (30%), orthostatic hypertension (10–50%)
• Headache (60–90%), palpitations (50–70%), sweating (55–75%), pallor (40–45%), hyperglycemia (40%).

Patients with adrenal incidentaloma

Individuals who have an hereditary risk for developing a Pheo or PGL.

32.1.2 Method of determination

Plasma free normetanephrine and free metanephrine are quantified by liquid chromatography with electrochemical detection, isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS) or immunoassay. In plasma free normetanephrine and free metanephrine are predominantly present.

For discrimination of different hereditary forms of Pheo the determination of methoxytyramine is used besides the measurement of free normetanephrine and free metanephrine /7/.

Chromogranin A is a marker with high diagnostic sensitivity but low specificity for the detection of Pheo. Refer to Section 14.5.2 – Chromogranin A.

The determination of urinary metanephrine and normetanephrine differs from plasma determination in that the mainly conjugated metanephrines and normetanephrines are deconjugated prior to determination and subsequently measured as free metanephrine and normetanephrine.

Liquid chromatography with electrochemical detection

Principle: a column with cation exchange resign and ammoniacal methanol elution are used to extract metanephrines from the plasma. The eluate is dried, dissolved in the mobile phase of the reversed phase column, and then injected into the liquid chromatography column for separation. Normetanephrine and metanephrine are detected using an electrochemical detector /8/.

LC-MS/MS

Principle: at first, interfering plasma proteins are removed from the sample using isopropanol precipitation, cation exchange cartridges, or other commercially available extraction agents. In the next step, the metanephrines are separated using liquid chromatography and detected and quantified using mass spectrometry /8, 9/.

Immunoassay

For the determination of plasma free metanephrines, interfering proteins are first removed using acid precipitation and metanephrines and normetanephrines are then acylated to their acyl derivatives. The acylated metanephrines and normetanephrines compete with a fixed number of solid phase bound antibody-binding sites (rabbit anti-metanephrine or rabbit anti-normetanephrine). When the system has reached equilibrium, free antigen antibody complexes are removed by washing. The acylated metanephrine and normetanephrine that are bound to solid-phase as immune complexes are detected using tetramethylbenzidine labeled antibodies directed against rabbit IgG. The amount of antibody bound to the solid phase metanephrine and normetanephrine is inversely proportional to the concentration of metanephrine and normetanephrine in the sample /10/.

32.1.3 Specimen

Heparin plasma: 2 mL

24-hour collection of urine in adults or random specimen in children. Refer to Section 32.1.6 – Comments and problems.

32.1.4 Reference interval

Refer to:

• Tab. 32.1-1 – Reference intervals for plasma catecholamines
• Tab. 32.1-2 – Reference intervals for catecholamines in urine
• Tab. 32.1-3 – Reference intervals for metanephrines and normetanephrines in random urine of children.

32.1.5 Clinical assessment

Serious morbidity and mortality rates are associated with Pheo and PGL which are related to the effects of catecholamines on various organs, especially those of the cardiovascular system.

32.1.5.1 Incidence, signs and symptoms of pheochromocytoma and paraganglioma

Pheo are tumors that arise from the chromaffine cells of the adrenal glands. Adrenal Pheo account for 80–85% of these tumors. About 5–7% of Pheo are adrenal incidentalomas and about 10% of Pheo are malignant.

PGLs, the extra-adrenal counterparts of Pheo, arise from ganglia along the sympathetic and parasympathetic chain. PGLs have a prevalence of about 15% that of the adrenal tumors. Approximately 30% of these tumors are known to be hereditary. Malignancy occurs in 30–40% of PGLs. The most common lesion in PGL syndrome is gastrointestinal stromal tumor.

The prevalence of Pheo and PGL is about 1 in 2,500 to 6,500 individuals and the incidence in hypertensive patients is about 0.3–0.5%. The mean age at diagnosis is 43 years. Approximately 10–20% of Pheo or PGL are diagnosed in children /11/.

Catecholamine secretion from Pheo and PGL is often episodic, causing headache, perspiration, palpitations, and hypertension. If not recognized and treated Pheo and PGL can lead to stroke, arrhythmia, myocardial infarction, and death.

PGLs have a prevalence of approximately 15% that of the adrenal tumors and can occur in different parts of the body. Populations at increased risk for Pheo and PGL are those with germ line mutations. More than 20 genetic (mostly germ line, but some are sporadic) mutations are associated with the pathogenesis of 35% of Pheo and PGL. An additional 15% of tumors are associated with somatic mutations in these same genes. In children this rate is even higher /12/, with 69% of pediatric Pheo and PGL cases and 87.5% of metastatic Pheo and PGL patients who developed their first tumor in childhood linked to underlying germ line mutations /13/. The major genetic mutations in PGL are listed to the year of discovery are /14/: NF1, RET, VHL, SDHC, SDHD, SDHB, EGLN1, KIF1B, SDHAF2, IDH, SDHA, TMEM 127, MAX, BAP1, EPAS1, FH, MDH2 und ATRX.

The presence and characteristics of tumors in hereditary syndromes associated with Pheo and PGL are shown in:

• Tab. 32.1-4 – Hereditary syndromes associated with pheochromocytoma and paraganglioma
• Tab. 32.1-5 – Proportion of pheochromocytoma associated syndromes.

32.1.5.2 Biochemical investigation

Pheo and PGL almost all produce, store, metabolize, and secrete catecholamines or their metabolites. Epinephrine and the breakdown product metanephrine are secreted from tissue of the adrenal medulla and normetanephrine the break down product of norepinephrine is primarily secreted by PGLs.

32.1.5.2.1 Diagnostic biochemical results

Plasma free metanephrine and free normetanephrine are the catecholamines with the highest diagnostic sensitivity for diagnosing Phe and PGL. Concentrations of one or both of these that are greater than 4 times the upper reference interval value [greater than 2.19 nmol/L (400 ng/L) for normetanephrine and greater than 1.20 nmol/L (236 ng/L) for metanephrine] have a diagnostic sensitivity of 100% for the presence of a Pheo and PGL. These tumors can be excluded on the basis of normal values and further investigations are only necessary if indicated by clinical or imaging findings /5/. Plasma is preferred over urine as a specimen because free metanephrines in plasma have a higher diagnostic specificity than fractionated metanephrines in urine Conjugated metanephrines in urine are also produced in other organs.

Refer to:

• Fig. 32.1-1 – Biochemical diagnosis of pheochromocytoma and paraganglioma
• Tab. 32.1-6 – Diagnostics of pheochromocytoma and associated syndromes.

32.1.5.2.2 Threshold biochemical results

Approximately 20–30% of patients with Pheo and PGL, in particular those who have hereditary syndromes or incidentalomas of the adrenal medulla, present values that are equivocal for plasma metanephrines below 4 times above the upper reference limit. These patients are usually normotensive and asymptomatic /15/. In such cases, it must be possible to distinguish between a false positive and a true positive result. Isolated cases of sporadic Pheo and PGL also occur in which clinical symptoms are present but metanephrines are normal. These are usually small tumors measuring less than 1 cm in diameter that are detected only because the patient has a known hereditary predisposition.

Due to the low prevalence of Pheo and PGL combined with the large number of investigations requested, the number of false positive results significantly exceeds the number of true positives. As a consequence, borderline results are often not pursued further. Studies /16/ have shown that, in up to 72% of cases where initial investigation shows borderline increases in free normetanephrines, the results are not followed up with repeat testing. For example, out of 10 patients with an adrenal mass and borderline biomarkers, only three underwent repeat testing.

Borderline increases in plasma free metanephrine and free normetanephrine are commonly caused by medications such as tricyclic antidepressants, phenoxybenzamine, and β-receptor blockers. In one study /15/, these drugs were responsible for 41% of the false positive results for plasma normetanephrine and 45% of the false positive results for urinary norepinephrine. Sympathoadrenal activation (e.g., before blood sampling) is another cause of false positive results.

Refer to Tab. 32.1-7 – Diagnostic sensitivity and specificity of laboratory tests for diagnosis of pheochromocytoma and paraganglioma.

32.1.5.2.3 Clonidine suppression test

The clonidine suppression test is recommended in the event of borderline increases in free normetanephrine and free metanephrine.

Clonidine suppresses the release of norepinephrine at sympathetic nerve terminals by activating α₂-adrenoceptors in the brain /15/. Suppression of the plasma normetanephrine concentration 3 h after oral administration of clonidine indicates increased sympathetic activity while a steady concentration indicates autonomic secretion. The clonidine test cannot be used for tumors secreting only metanephrine, because almost 99% of metanephrine is derived from the adrenal gland.

Criteria /15/:

• Normal response to clonidine is a decline in plasma normetanephrine to within the reference interval or a decline to less than 50% of baseline values considered normal
• Lack of decrease or a small decrease of elevated plasma levels of normetanephrine confirm Pheo and PGL with high specificity.

In one study /15/ the following normetanephrine concentrations were determined:

• In patients with a questionable result, 2.98 ± 1.69 nmol/L before clonidine and 1.03 ± 0.79 nmol/L after clonidine
• In patients with Pheo 11.14 ± 9.85 nmol/L before clonidine and 10.71 ± 11.29 nmol/L after clonidine.

The clonidine test has a diagnostic sensitivity of 97% and a specificity of 100% /17/. The procedure for performing the clonidine test is outlined in Tab. 32.1.8 – Clonidine test.

32.1.5.3 Diagnosis of hereditary catecholamine secreting tumors

Underlying mutations in susceptibility genes are associated with approximately 35% of Pheo and PGL. Besides biochemical investigations genetic testing is important to differentiate underlying similarities in various forms of hereditary Pheo and PGL.

32.1.5.3.1 Biochemical investigations

Different patterns have been shown to be associated with different mutations. In a study /7/:

• Patients with MEN 2 and neurofibromatosis type 1 (NF1) could be discriminated from those with VHL, SDHB, and SDHD mutations on the basis of increased concentrations of metanephrines (indicating increased epinephrine production)
• Patients with VHL mutations invariably showed increases in normetanephrine (indicating increased norepinephrine production). Additional increases in 3-methoxytyramine (indicating increased dopamine production) characterized 70% of patients with mutations in SDHB and SDHD
• Patients with NF1 and MEN 2 could be discriminated from those with VHL, SDHB, and SDHD gene mutations in 99% of cases by the combination of normetanephrine and metanephrine
• Elevated 3-methoxytyramine discriminated patients with SDHB and SDHD mutations from those with VHL mutations in an additional 78% of cases.

The determination normetanephrine, metanephrine, and 3-methoxytyramine in plasma and urine is helpful for classifying patients in the absence of clear clinical signs of hereditary Pheo and PGL and can serve as a basis for further differentiation based on molecular genetics. For example, differentiation based on the RET oncogene is only necessary for tumors that predominantly secrete metanephrine. The pattern of free metanephrine, free normetanephrine, and 3-methoxytyramine elevation indicates mutations in the VHL, SDHS, and SDHB genes

In patients with malignant disease secondary to a Pheo and PGL, almost 50% have SDHB mutations and two-thirds of patients with SDHB related PGL or Pheo will develop metastatic disease. Approximately 70–80% of patients respond to chemotherapy with vincristine, cyclophosphamide, and dacarbazine /18/.

Association with other diseases

Patients with MEN 2B have an approximately 50% risk of developing Pheo and a 100% risk of developing medullary thyroid cancer (MTC). Refer to Section 28.12 – Calcitonin. Pheo can develop in patients with familial MTC, generally at a later stage than the MTC. Patients with MEN 2A develop multi focal, bilateral MTC and have an approximately 50% risk of developing Pheo. Patients with MEN 2A also have a 20–35% risk of developing hyperparathyroidism.

32.1.5.3.2 Molecular genetic investigations

Around 30% of catecholamine producing tumors have a genetic basis and are mainly caused by germ line mutations.

Genetic testing should be considered if a patient has one of the following /17/:

• PGL
• Bilateral Pheo
• Unilateral Pheo and family history of Pheo and PGL
• Unilateral Pheo onset at young age (< 45 years)
• Other clinical findings suggestive of one of the associated syndromic disorders.

An asymptomatic person known to be at risk for disease on the basis of family history of Pheo and PGL should have genetic testing only if an affected family member has a known mutation.

To chose a proper genetic test, the biochemical profile of catecholamine secretion, age of the patient, localization of the primary tumor, and previous family history must be carefully evaluated and included in the genetic algorithm /19/.

Family members with a known mutation must be investigated periodically for the presence of Pheo and PGL. Because not all genetic causes of these tumors are known, all first degree relatives of patients with Pheo and PGL should undergo biochemical screening (e.g., determination of metanephrines in plasma). Asymptomatic individuals at known risk of Pheo and PGL should undergo genetic testing only if the family member with Pheo and PGL has a known mutation /19/.

32.1.6 Comments and problems

Preanalytics

The most common cause of false positive results is inappropriate blood sampling conditions associated with sympatho-adrenal activation, particularly sampling performed with the patient in the seated rather than supine position /9/. For 12–14 hours before blood sampling, patients must abstain from coffee and other sources of caffeine and refrain from smoking or engaging in strenuous physical activity. Drugs such as tricyclic antidepressants, acetaminophen, phenoxybenzamine, β-receptor blockers, and diuretics should be discontinued 5 days prior to blood sampling.

Blood sampling

Blood samples should be obtained with the patient resting in a supine position, 30 minutes after the insertion of an intravenous catheter. Collect 5–10 mL of blood using a heparin containing collection tube. The sample must be transported immediately to the laboratory and centrifuged without delay. Alternatively, blood can be stored at 4 °C for up to 6 hours prior to centrifugation /20/.

Urine collection or spot urine samples

Fractionated metanephrines: samples do not need to be acidified to prevent degradation if they are analyzed within one week /20/.

Method of determination

HPLC with electrochemical detection and LC-MS/MS yield comparable results for free normetanephrine and metanephrine in plasma. Immunoassays also yield comparable normetanephrine concentrations but lower metanephrine values. Immunoassays are not recommended as a first choice for measurements of plasma free metanephrines /21/.

Conversion factors

To convert nmol/L into ng/L, multiply by:

• 183 for normetanephrine
• 169 for norepinephrine
• 197 for metanephrine
• 183 for epinephrine.

Stability

Urinary catecholamines: acidified to a pH of 2–3 and at 4–8 °C stable for up to 2 weeks.

Urinary fractionated metanephrines: preservation of samples is not necessary if samples are assayed or frozen within 1 week /20/.

Plasma free normetanephrines and free metanephrines: using HPLC and electrochemical detection, the samples can be stored for 3 days at 4 °C or for longer periods at –20 °C /21/. For the determination using LC-MS/MS samples can be stored 24 h at room temperature and up to 7 days at 10 °C or 4 °C /22/.

32.1.7 Biochemistry and physiology

Functional Pheo and PGL produce catecholamines, which include the following biogenic amines:

• Dopamine, norepinephrine, and epinephrine
• Their metabolic products 3-methoxytyramine, normetanephrine, metanephrine, homovanillic acid, and vanillylmandelic acid.

Catecholamine production

Catecholamines are synthesized in the adrenal medulla, the brain, and the sympathetic nerve terminals. Epinephrine is produced mainly in the adrenal medulla and norepinephrine is produced mainly by sympathetic nerve terminals /15, 23/. However, up to 91% of the metanephrine and 23% of the normetanephrine released into the plasma as a result of the metabolism of norepinephrine to normetanephrine and of epinephrine to metanephrine originates in the adrenal medulla /23/.

Catecholamine metabolism

Dopamine, epinephrine, and norepinephrine are metabolized via the following pathways (Fig. 32.1- 2 – Catecholamine metabolism):

• Catechol-O-methyltransferase (COMT) catalyzes the methylation of the OH group in position C3 of the benzene ring to produce 3-methoxytyramine from dopamine, normetanephrine from norepinephrine, and metanephrine from epinephrine.
• Deamination by mitochondrial monoamine oxidase. This results in the production of homovanillic acid from 3-methoxytyramine and vanillylmandelic acid from normetaphrine and metanephrine.

Following their release into the circulation, normetanephrine, metanephrine, and 3-methoxytyramine are converted into sulfate esters and glucuronides, with the result that both free and conjugated forms are present in the blood and urine. Only about 3% of the normetanephrine and metanephrine in the urine exists in free form; the remainder is conjugated. In the case of Pheo and PGL, however, there is a higher proportion of free epinephrine and norepinephrine and a disproportionate increase in normetanephrine.

Catecholamines and clinical assessment

In the biochemical diagnosis of Pheo and PGL false positive results remain a problem /10/:

• Patients with Pheo and PGL usually have larger relative increases in metanephrine and normetanephrine than of epinephrine and norepinephrine, whereas patients with false positive results due to sympathoadrenal activation usually have larger increases in epinephrines and norepinephrines. The differences are partly due to the amounts of free metanephrine and free normetanephrine formed continuously within the Pheo and PGL and released into the circulation.
• Another factor is the large contribution of the adrenal medulla to normal circulating levels of metanephrine and normetanephrine, a contribution that is again independent of epinephrine and norepinephrine release. Thus, during sympathoadrenal activation, increase in plasma free metanephrine is negligible and increase in normetanephrine is smaller than those of the respective parent amines. This explains why a patient with elevated plasma normetanephrine or metanephrine, but normal or slightly elevated norepinephrine and epinephrine is more likely to have Pheo and PGL than a patient with highly elevated norepinephrine or epinephrine and slightly elevated normetanephrine and metanephrine.

Most of the norepinephrine released is taken up into the nerve terminals and granules in native form before it reaches the effector cells. The high rate of false positive findings when measuring free normetanephrine in the context of chronic tricyclic antidepressant use is thought to be due to inhibition of norepinephrine re uptake /15/.

Phenoxybenzamine is a nonspecific α-adrenoceptor blocker commonly used to treat patients with Pheo. It presumably increases catecholamine release by attenuating the feedback inhibition of norepinephrine and epinephrine release /15/.

The main catecholamines produced by Pheo and PGL are epinephrine and norepinephrine, although some tumors also produce large quantities of dopamine. Epinephrine and norepinephrine are released intermittently and have a short half life. Therefore, the concentration of these catecholamines in plasma or in a random specimen of urine fluctuates significantly.

The diagnostic superiority of free normetanephrine and metanephrine in plasma over other tests include /24/:

• Tumor cells of Pheo and PGL have a high level of catechol-O-methyltransferase (COMT) which leads to immediate metabolism. Therefore, continuously normetaphrine, metanephrine, and 3-methoxytyramine are released in the circulation. This is not the case for norepinephrine and epinephrine, which are released intermittently from vesicles.
• The half life of normetanephrine, metanephrine, and 3-methoxytyramine is longer than that of norepinephrine and epinephrine.

References

1. Lam AK. Update on paragangliomas and pheochromocytomas. Turk Patoloji Derg 2015; 31 (suppl): 105–12.

2. Phillips RA. Pheochromocytoma. J Clin Hypertens 2002; 4: 62–72.

3. Brodeur G, Pritchard J, Berthold F, Carlsen NLT, Castel V, Castleberry RP, et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993; 11: 1466–77.

4. Eisenhofer G, Keiser H, Friberg P, Mezey E, Hyunh TT, Hiremagular B, et al. Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase with tumors. J Clin Endocrinol Metab 1998; 83: 2175–85.

5. Lenders JW, Pacak K, Walther MM, Lineham WM, Manelli M, Fiberg P, et al. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA 2002; 287: 1427–34.

6. Lenders JWM, Eisenhofer G, Menelli M, Pacak K. Pheochromocytoma.. The Lancet 2005; 366: 665–74.

7. Eisenhofer G, Lenders JWM, Timmers H, Mannnelli M, Grebe SK, Hofbauer LC, et al. Measurements of plasma methoxytyramine, normetanephrine, and metanephrine as discriminators of different hereditary forms of pheochromocytoma. Clin Chem 2011; 57: 411–20.

8. Lenders JWM, Eisenhofer G, Armando I, Keiser HR, Goldstein DS, Kopin IJ. Determination of metanephrines in plasma by liquid chromatography with electrochemical detection. Clin Chem 1993; 39: 97–103.

9. Eisenhofer G, Peitzsch M. Laboratory evaluation of phechromocytoma and paraganglioma. Clin Chem 2014; 60: 1486–99.

10. Lenz T, Zorner J, Kirchmaier C, Pillitteri D, Badenhoop K, Bartel C, et al. Multicenter study on the diagnostic value of a new RIA for the detection of free plasma metanephrines in the work-up for pheochromocytoma. Ann NY Acad Sci 2006; 1073: 358–73.

11. Martucci VL, Pacak K. Pheochromocytoma and paraganglioma: diagnosis, genetics, management, and treatment. Curr Probl Cancer 2014; 38; 7–41.

12. Cascon A, Inglada-Perez L, Comino-Mendez I, De Cubas AA, Leton R, Moro J. Genetics of pheochromocytoma and paraganglioma in Spanish pediatric patients. Endocr Relat Cancer 2013; 20: L1–L6.

13. King KS, Prodanov T, Kantorovic V, Fojo T, Hewitt JK, Zacharin M, et al. Metastatic pheochromocytoma/paraganglioma related to primary tumor development in childhood or adolescence: significant link to SBHD mutations. J Clin Oncol 2011; 29: 4137–42.

14. Mercado-Asis LB, Wolf KI, Jochmanova I, Taieb D. Pheochromocytoma: a genetic and diagnostic update. Endocr Pract. 2018, 24: 78–90.

15. Eisenhofer G, Goldstein DS, Walther CM, Friberg P, Lenders JWM, Reiser HR, Pacak K. Biochemical diagnosis of pheochromocytoma: how to distinguish true- from false-positive test results. J Clin Endocrinol Metab 2003; 88: 2656–66.

16. Anas SS, Vasikaran SD. An audit of management of patients with borderline increased plasma-free metanephrines. Ann Clin Biochem 2010; 47: 554–8.

17. Estay MP, Diamandis EP. Pheochromocytoma Clin Chem 2013; 59: 466–72.

18. Eisenhofer G, Pacak K, Mahler ER, Young WF, de Krijger R. Pheochromocytoma. Clin Chem 2013; 59: 466–72.

19. Chen H, Sippel RS, O’Dorisio S, Vinik AI, Llooyd RV, Pacak K. The North American neuroendocrine tumor society consensus guideline for the diagnosis and management of neuroendocrine tumors. Pancreas 2010; 39: 775–83.

20. Willemsen J, Ross AH, Lenders JWM, Sweep FCGJ. Stability of urinary fractionated metanephrines and catecholamines during collection, shipment, and storage of samples. Clin Chem 2007; 53: 268–72.

21. Pillai D, Callen S. Pilot quality assurance programme for plasma metanephrines. Ann clin Biochem 2010: 47: 137–42.

22. De Jong WHA, Graham KS, van der Molen JC, Links TP, Morris MR, Ross HA, et al. Plasma free metanephrine measurement using automated online solid-phase extraction HPLC-tandem mass spectrometry. Clin Chem 2007; 53: 1684–93.

23. Eisenhofer G, Rundqvist B, Aneman A, Friberg P, Dakak N, Kopin IJ, et al. Regional release and removal of catecholamines and extraneural metabolism to metanephrines. J Clin Endocrinol Metab 1995; 80: 3009–17.

24. Peaston RT, Weinkove C. Measurement of catecholamines and their metabolites. Ann Clin Biochem 2004; 41: 17–38.

25. Eisenhofer G, Lenders JWM, Goldstein DS, Mannelli M, Csako G, Walther MM, et al. Pheochromocytoma catecholamine phenotypes and prediction of tumor size and location by use of plasma free metanephrines. Clin Chem 2005; 51: 735–44.

26. Griffin A, O’Shea P, FitzGerald R, O’Connor G, Tormey W. Establishment of a paedriatric reference interval for the measurement of urinary total fractionated metanephrines. Ann Clin Biochem 2011; 48: 41–4.

27. Peaston RT, Ball S. Biochemical detection of phaeochromocytoma: why are we continuing to ignore the evidence? Ann Clin Biochem 2008; 45: 6–10.

28. Neumann HPH, Young Jr WF, Eng C. Pheochromocytoma and paraganglioma. N Engl J Med 2019; 381: 552–65.

32.2 Neuroblastoma

Neuroblastoma is the most common extracranial solid tumor of childhood and the third most common pediatric malignancy. Neuroblastoma arise from neural crest cells of the adrenal gland and sympathetic chain, accounts for up to 15% of all cancer fatalities and represents about 97% of all neuroblastíc tumors of childhood. Overall, about 46% of neuroblastomas arise from the adrenal gland, 18% arise from an extra-adrenal abdominal location, 14% arise from the posterior mediastinum or thorax, the reminder arise from the neck, pelvis, and other locations /1, 2/.

The manifestation of neuroblastoma is variable depending on the location and the absence or presence of paraneoplastic syndromes. Clinical signs and symptoms include distension, respiratory distress, hypertension, bone pain, and neurologic symptomes due to cord compression. Patients may also present with paraneoplastic syndromes /1/:

• watery diarrhea (Verner-Morrison syndrome)
• miosis, ptosis, and anhydrosis (Horner syndrome)
• jerking limbs and rapid eye movements (opsoclonus myoclonus ataxia syndrome)
• Phox2B mutation-associated disorders such as congenital central hypoventilation
• Hirschsprung disease; a condition that affects the colon and causes problems with passing stool.

Neuroblastoma are of aggressive nature and high likelihood of metastatic disease. The prognosis of neuroblastoma varies with age.Children who are less than 1 year old at diagnosis of neuroblastoma have a higher 5-year survival rate with those who receive the diagnosis later /1/.

The diagnosis of neuroblastoma is made on the basis of imaging, chemical characteristics and immunohistochemical stains. The diagnosis is confirmed using:

• immunohistochemical stains for biologic markers such as neuron specific enolase (NSE), S-100 protein, and chromogranin
• determination of catecholamine metabolites. The great majority of neuroblastoma produce higher than normal excretions of catecholamines. Dopamin is metabolized to homovanillic acid (HVA) and norepinephrin is metabolized to 3-methoxy-4-hydroxymandelic acid (vanillylmandelic acid, VMA).

The presence of elevated excretions of HVA and VMA‚ with the aforementioned investigations increases the probability of having a peripheral neuroblastic tumor /3/.

32.3 Homovanillic acid (HVA), Vanillylmandelic acid (VMA)

32.3.1 Indication

Suspicion of embryonic tumor of the peripheral sympathetic nervous system.

32.3.2 Method of determination

High performance liquid chromatography with electrochemical detection /4/. This method represents the gold method for the determination of VMA and HVA.

Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) /5/.

32.3.3 Specimen

24 hours urine. The sample is preserved in a bottle containing 6 N HCl (5 mL) /6/.

Random urine adjusted to pH 1–3 with HCl /7/ (with or without determination of creatinine in urine /8/).

32.3.4 Reference interval

Refer to Ref. /8/, /9/, /10/, /11/ and Tab. 32.3-1 – Reference intervals for VMA, HVA and dopamine.

32.3.5 Clinical assessment

Neuroblastomas are embryonal tumors of the autonomic nervous system that arise from partially uncommitted precursor cells of the neural crest.

Incidence, signs and symptoms of neuroblastoma

Genetic testing for mutations in ALK and PHOX2B should be considered whenever a patient has a family history of neuroblastoma or has an other clinical conditions that are strongly suggestive of highly penetrant transmissible mutation, such as bilateral tumors of the adrenal glands /12/.

Biochemical investigations

VMA spot urine test results in a low detection rate and a high false positive rate. Using quantitative high-performance liquid chromatography for screening at 6 months of age is recommended for testing of VMA and HVA in urine. The false positive rate is 2.4%. It is important to measure HVA and VMA when the biopsy of a large tumor shows Ganglioneuroma (Schwannian stroma-dominant) or ganglioneuroblastoma intermixed (Schwannian stroma-rich).

Screening

The vast majority of tumors detectable by the screening in infants are biologically favorable neuroblastoma (MYCN non amplified and favorable histology), and a considerable number of them seem to have a potential of spontaneous regression. Screening unnecessarily identifies neuroblastoma patients whose tumor will not be detectable clinically. In biologically unfavorable tumors (MYCN amplified and unfavorable histology) mass screening results in increased detection of neuroblastic tumors, though no chance in overall survival /13, 14/.

The diagnostic evaluation of neuroblastoma includes the determination of urinary VMA, HVA, and serotonine less frequently, of norepinephrine, epinephrine, normetanephrine, and metanephrine. It is recommended that the cutoff for increased levels of serum or urine catecholamines or their metabolites be retained at 3.0 standard deviation above the mean per milligram creatinine for age when making a diagnosis of neuroblastoma in a patient. The rate of neuroblastoma patients with positive VMA and HVA or serotonine at diagnosis varies with the stage of disease. Serum ferritin and lactate dehydrogenase may be useful prognostic markers for neuroblastoma at diagnosis, but they lack sensitivity and specificity to monitor disease activity. Serum NSE and chromogranin A are more specific, but they are not as sensitive /9/.

The diagnostic sensitivity of tests for measuring VMA, HVA, and serotonine excretion depends on the disease stage.

VMA and HVA are only sensitive enough to detect tumors from stage III on; as a result, earlier stages of disease and small tumors may be missed. The renal excretion of VMA and HVA is increased only when tumors weighing more than 5 g are present.

Measurement of VMA excretion is better than of dopamine secretion for diagnosing neuroblastoma. In a study /15/ of 249 children, 20 of whom were confirmed cases of neuroblastoma, the ratios of VMA to creatinine and dopamine to creatinine were calculated. From receiver operator characteristic curves (ROC curves), the VMA/creatinine ratio was found to have an area under the curve of 0.96 (with a diagnostic sensitivity of 95% and a specificity of 86%) compared with only 0.72 for the dopamine/creatinine ratio.

Not only the disease stage but also the age of the patient must be considered. For instance, the number of children with stage I/II neuroblastoma and high biomarker secretion is higher if the screening examinations are performed at a younger age. The diagnostic sensitivity shown in Tab. 32.3-2 – MA, HVA, ferritin, and NSE in the diagnosis of neuroblastoma have a corresponding specificity of 99% /16/. The number of elevated values requiring further testing has been found to be 2.36% /17/.

A small percentage of patients with neuroblastoma do not secrete VMA or HVA or secrete elevated HVA in isolation. However, the VMA and HVA level is only one piece of information to rule in or rule out neuroblastoma, and may be used in the context with clinical findings, symptoms and abdominal mass /3/.

Neuroblastomas are heterogeneous with regard to both their genotype and their clinical presentation /15/. The main genetic alterations found in certain neuroblastomas are:

• Hyperdiploidy or near triploidy; the majority of neuroblastomas show a modal diploid karyotype
• Loss of heterozygosity (LOH) of the distal end of the short arm of chromosome 1 (from 1p36.1 to 1p36.3) and of the long arm of chromosome 14 (14q)
• Amplification of the MYCN proto oncogene
• Expression of the receptor tyrosine kinases (RTKs).

On the basis of cytogenetic and molecular findings (karyotype, MYCN amplification, RTK expression) as well as the DNA content of the neuroblastoma cells, three genetically different neuroblastoma subtypes, each with a different prognosis, are presumed to exist.

Classification of neuroblastoma

According to genetic and histopathological analysis neuroblastomas are classified into 3 stages based on the three most common and prognostically important genetic alterations (MYCN amplification, 1p deletion, and 17q gain) /13/:

• Type 1; these tumors show none of the three significant genetic alterations and have a good prognosis
• Type 2; these tumors show 17q gain only or 17q gain together with 1p deletion. These are progressive tumors with a poor prognosis.
• Type 3; these tumors show all three significant genetic alterations, progress rapidly, and compared to type 2, occur in younger children and are associated with shorter survival times /15/.

The classification of neuroblastomas according to their differing responses to therapy and their prognosis is shown in Tab. 32.3-3 – Biologic and clinical subtypes of neuroblastoma. Patients with neuroblastomas type 2 and 3 are usually older than 1 year. Tumors type 2 initially respond well to treatment. In around half of patients, however, rapid disease progression occurs within 6–12 months, accounting for a 5 year survival rate of only 40–50%. Patients with neuroblastoma type 3 (MYCN proto oncogene amplification, stage 3/4, age 1–5 years) have the poorest prognosis.

Other prognostic indicators for neuroblastoma include tumor cell factors and results of laboratory investigations (Tab. 32.3-4 – Neuroblastoma: prognostic factors) /19/.

32.3.6 Comments and problems

24 h urine collections are difficult to obtain in pediatrics and are often incomplete. For these reasons spot urine is routinely used since their interchangeability with 24 h collections is possible /20/.

HVA and VMA daily excretion increases linearly with age with no difference between the two sexes while creatinine excretion increases in proportion with growing muscle mass. For these reasons age-related reference intervals and decision limits for VMA and HVA have to take into account for these variations.

3-methoxy-4-hydroxy phenyl glycol and homogentisinic acid (2,5-dihydroxy phenyl acetic acid) are expected to interfere in the chromatography and at the electrochemical detector /2/.

References

1. Hazard FK, Shimada H. The role of the clinical laboratory in the diagnosis of neuroblastoma. JALM 2020; 254–6.

2. Swift CC, Eklund MJ, Kraveka JM, Alazraki AL. Updates in diagnosis, management, and treatment of neuroblastoma. Radio Graphics 2018; 38 (2): doi: 10.1148/rg.2018170132.

3. Maris JM. Recent advances in Neuroblastoma. N Engl J Med 2010; 362: 2202–11.

4. Moleman P, Borstrok JJM. Determination of urinary vanillylmandelic acid by liquid chromatography with electrochemical detection. Clin Chem 1983; 29: 878–81.

5. Sadlikova K, Dugaw K, Benjamin D, Jack RM. Analysis of vanillylmandelic acid and homovanillic acid acid by UPLC-MS/MS in serum for diagnostic testing for neuroblastoma Clin Chim Acta 2013; 424: 253–7.

6. Regianin LJ, McGill AC, Pinheiro CM, Brunetto AL. Vanillylmandelic acid and homovanillic acid levels in patients with neural crest tumor: 24-hour urine collection versus randon sample. Pediatric hematology and Oncology 1997; 14: 259–65.

7. Pussard E, Neveux M, Guigueno N. Reference intervals for urinary catecholamines and metabolites from birth to adulthood. Clin Biochem 2009, 42. 536–9.

8. Barco S, Gennai J, Reggiardo G, Galleni B, Barbagallo L, Maffia A, et al. Urinary homovanillic acid and vanillylmandelic acid in the diagnosis of neuroblastoma: report from the Italian cooperative group for neuroblastoma. Clin Biochem 2014; 47: 848–52.

9. Brodeur G, Pritchard J, Berthold F, Carlsen NLT, Castel V, Castleberry RP, et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993; 11: 1466–77.

10. Griffin A, O’Shea P, FitzGerald R, O’Connor G, Tormey W. Establishment of a paedriatric reference interval for the measurement of urinary total fractionated metanephrines. Ann Clin Biochem 2011; 48: 41–4.

11. Aydin GB, Kutluk MT, Yalcin B, Varan A, Akyuz C, Buyukpamukcu M. The prognostic significance of vanillylmandelic acid in neuroblastoma. Pediatr Hematology Oncology 2010; 27: 435–48.

12. Brodeur GM, Seeger RC, Barrett A, Berthold F, Castleberry RB, D’Angio G, et al. International criteria for diagnosis, staging response to treatment in patients with neuroblastoma. J Clin Oncol 1988; 6: 1874–81.

13. Sawada T, Sugimoto T, Kawakatsu H, Matsumura T, Matsuda Y. Mass screening for neuroblastoma in Japan. Pediatr Hematol Oncol 1991; 8: 93–109.

14. Murphy SB, Cohn SL, Craft AW, Woods WG Sawada T, Castleberry RP, et al. Do children benefit from mass screening for neuroblastoma? Consensus statement from the American Cancer Society workshop on neuroblastoma screening. Lancet 1991; 337: 344–6.

15. Sies CW, Florkowski CM, Sullivan M, Mackay R, George PM. Urinary VMA, dopamine and the likelihood of neuroblastoma: a preferred way of reporting laboratory results? Ann Clin Biochem 2006; 43: 300–5.

16. Carlsen NLT. Neuroblastoma: epidemiology and pattern of regression. Am J Pediatr Hematol Oncol 1992; 14: 103–10.

17. Berthold F, Brandeis WE, Lampert F. Neuroblastoma: diagnostic advances and therapeutic results in 370 patients. Monogr Paediat 1986; 18: 206–23.

18. Brodeur GM. Molecular basis for heterogeneity in human neuroblastomas. Eur J Cancer 1995; 31A: 505–10.

19. Lastowska M, Cullinane C, Variend S, O’Neill SS, Mazzocco K, Roberts P, Nicholson J, et al. Comprehensive genetic and histopathologic study reveals three types of neuroblastoma tumors. J Clin Oncol 2001; 19: 3080–90.

20. Kushner BH. Neuroblastoma: a disease requiring a multitude of imaging studies. J Nuclear Med 2004; 45: 1172–88.

21. Mundschenk J, Lehnert H. Malignant pheochromocytoma review. Exp Clin Endocrinol Diabetes 1998; 106: 373–6.

22. Eisenhofer G, Goldstein DS, Walter CM, Friberg P, Lenders JWM, Keiser HR, Pacak K. Biochemical diagnosis of pheochromocytoma:how to distinguish true- from false-positive test results. J Clin Endocrinol Metab 2003; 88: 2676–66.

23. Cryer PE. Pheochromocytoma. West J Med 1992; 156: 399–407.

24. Willemsen JJ, Ross HA, Lenders JWM, Sweep FCGJ. Stability of urinary fractionated metanephrines and catecholamines during collection, shipment and storage of samples. Clin Chem 2007; 53: 268–72.

25. Eisenhofer G, Keiser H, Friberg P, Mezey E. Plasma metanephrines are markers of pheochomocytoma produced by catechol-o-methyltransferase within tumors. J Clin Endocrinol Metab 1998; 83: 2175–85.

26. Davidson DF. Simultaneous assay for 4-hydroxy-3-methoxy-mandelic acid, 5-hydroxy indolacetic acid and homovanillic acid by isocratic HPLC with electrochemical detection. Ann Clin Biochem 1989; 26: 137–43.