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Original Studies |
Clinical Neurocardiology Section and Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (G.E., T.-T.H., S.-T.L., A.V., D.S.G.); Urologic Oncology Branch, National Cancer Institute (M.M.W., W.M.L.); and Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development (S.R.B., K.P.), National Institutes of Health, Bethesda, Maryland 20892; Department of Clinical Pathophysiology, University of Florence (M.M.), Florence, Italy; and Department of Internal Medicine, St. Radboud University Hospital (J.W.M.L.), Nijmegen, The Netherlands
Address all correspondence and requests for reprints to: Dr. Graeme Eisenhofer, Building 10, Room 6N252, National Institutes of Health, 10 Center Drive, MSC 1620, Bethesda, Maryland 20892-1620. E-mail: ge{at}box-g.nih.gov
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In some pheochromocytomas catecholamine secretion appears to be continuous, whereas in others, particularly epinephrine-secreting tumors, secretion is episodic (2, 5). Such differences may account for why some patients with pheochromocytoma present with sustained hypertension, whereas others present with paroxysmal hypertension and attacks of sweating, tachycardia, or anxiety. In some patients with pheochromocytoma, particularly those in whom the tumor is discovered during periodic screening for hereditary pheochromocytoma or as an incidentaloma during imaging studies for other medical conditions, the tumor may not produce signs or symptoms (6, 7, 8, 9, 10). In this setting, plasma and urinary catecholamines often are normal, indicating little secretion of the amines by the tumor.
We recently reported that measurements of plasma concentrations of normetanephrine and metanephrine, the O-methylated metabolites of norepinephrine and epinephrine, provide a particularly sensitive test for detecting pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome or multiple endocrine neoplasia type 2 (MEN 2) (11). These multisystem neoplastic disorders are inherited in an autosomal dominant fashion and account for most currently identified hereditary pheochromocytomas. The continuous nature of production of metanephrines within tumor tissue, production that is independent of catecholamine secretion, revealed that pheochromocytomas from patients with MEN 2 produce metanephrine, whereas those from VHL patients produce almost exclusively normetanephrine (11). These findings suggest that pheochromocytomas in the VHL syndrome are characterized by a noradrenergic biochemical phenotype, whereas those in patients with MEN 2 are characterized by an adrenergic phenotype.
Although previous studies indicated that many patients with MEN 2 have epinephrine-secreting pheochromocytomas (9, 12, 13, 14), the basis for this is not established, and these studies did not compare tumor phenotypes in patients with MEN 2 and VHL syndrome. This study examined the hypothesis that pheochromocytomas in MEN 2 and VHL patients exhibit specific noradrenergic vs. adrenergic biochemical phenotypes that reflect mutation-dependent differential expression of genes regulating catecholamine synthesis. More specifically this study aimed to establish whether differences in the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and of phenylethanolamine N-methyltransferase (PNMT), the enzyme that converts norepinephrine to epinephrine, might account for different biochemical phenotypes and clinical presentations of pheochromocytomas in patients with MEN 2 and VHL syndrome.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The patient database for this report includes 49 patients with histologically proven adrenal pheochromocytomas, 30 (12 women and 18 men) associated with VHL syndrome and 19 (9 women and 10 men) with MEN 2 (18 with MEN 2a and 1 with MEN 2b). Three VHL patients and 1 MEN 2 patient had pheochromocytomas removed on 2 separate occasions, between 1–4 yr apart, giving a total of 53 cases of adrenal pheochromocytoma. At the time of tumor resection, VHL patients had a mean (±SD) age of 29 ± 14 yr (range, 8–63 yr) and MEN 2 patients were aged 36 ± 10 yr (range, 22–53 yr). The diagnosis of VHL syndrome or MEN 2 was confirmed by identification of germline mutations of the VHL tumor suppressor gene or the RET protooncogene in all patients. The absence or presence of symptoms attributable to pheochromocytoma (e.g. headache, diaphoresis, and palpitations) or of hypertension, and whether hypertension was sustained or paroxysmal, were assessed by review of patient records. Studies were approved by the appropriate institutional review boards, and all patients gave informed consent to participate.
Collection of blood and tissue samples
Blood samples were obtained from all patients using an indwelling iv catheter inserted into a forearm vein, with patients supine for at least 20 min before blood collection. Samples of blood were transferred into tubes containing heparin as anticoagulant and immediately placed on ice until centrifuged (4 C) to separate the plasma. Plasma samples were stored at -80 C until assayed.
Samples of tumor tissue were obtained at surgery from 18 VHL patients and 12 MEN 2 patients within 1 h of surgical removal. Tumors were placed on ice immediately after removal, extraneous tissue was removed, dimensions of tumors were recorded, and small samples (50–400 mg) were dissected away from surrounding tissue, placed on dry ice or in liquid nitrogen, and then stored at -80 C.
Plasma and tissue catecholamines and metanephrines
Plasma and tissue concentrations of catecholamines (norepinephrine, epinephrine and dopamine) were quantified by liquid chromatography with electrochemical detection. Samples of tissue were weighed and homogenized in at least 5 vol 0.4 mol/L perchloric acid containing 0.5 mmol/L ethylenediamine tetraacetate. Homogenized samples were centrifuged (1500 x g for 15 min at 4 C), and supernatants collected and stored at -80 C until assayed. Concentrations of catecholamines were determined after extraction from plasma or perchloric acid tissue supernatants using alumina adsorption as described previously (15).
Plasma concentrations of metanephrines (normetanephrine and metanephrine) were determined using a different liquid chromatography procedure after extraction onto solid phase ion exchange columns (16).
Intraassay coefficients of variation were 1.9% for norepinephrine, 3.0% for epinephrine, 4.2% for normetanephrine, and 3.3% for metanephrine. Interassay coefficients of variation were 3.2% for norepinephrine, 9.9% for epinephrine, 7.1% for normetanephrine, and 5.1% for metanephrine.
Tissue TH activity
The activity of TH in samples of pheochromocytoma tumor tissue was determined from measurements of the formation dihydroxyphenylalanine from tyrosine according to a previously described assay (17). In brief, an appropriately diluted sample of each tissue preparation was incubated with 0.1 mol/L acetate buffer (pH 6.0), 200 µmol/L L-tyrosine, 1.25 mmol/L m-hydroxybenzylhydrazine, 1 mmol/L D,L-6-methyltetra-hydropterine dihydrochloride, 19.5 x 103 U/mL catalase, 3.8 U/mL dihydropteridine reductase, 1 mmol/L NADPH, and 0.4 mol/L glycerol to a total volume of 200 µL. The mixture was incubated at 37 C for 10 min, the incubation was terminated by adding 100 µL 0.4 mol/L perchloric acid, and dihydroxyphenylanine was quantified by liquid chromatography with electrochemical detection. TH activity (picomoles per min/mg wet wt tissue) was calculated using the formula: TH activity = (DOPAsample - DOPAblank)/(weight of tissue x time of incubation).
Western blot analysis of TH and PNMT
Cytosolic proteins were prepared according to the procedure of
Andrews and Faller (18). Samples of pheochromocytoma
tissue (
15 mg) were homogenized using a Dounce homogenizer (Kontes
Co., Vineland, NJ) in 0.5 mL 10 mmol/L HEPES-KOH buffer (pH 7.9)
containing 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5
mmol/L dithiothreitol, 0.2 mmol/L phenylmethylsulfonylfluoride, and
protease inhibitors. Cytosolic fractions were separated from pelleted
debris by centrifugation at 17,000 x g for 10 s
(4 C).
Cytosolic proteins (20 µg) were then electrophoresed on a 12% SDS-polyacrylamide gel for PNMT and on a 10% SDS-polyacrylamide gel for TH and transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA) using a transblot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). After transfer, polyvinylidene fluoride membranes were incubated in blocking buffer [50 mmol/L Tris (pH 7.4), 0.9% NaCl, 0.05% Tween-20, and 10% dry milk] overnight at 4 C. Membranes were then washed three times for 10 min each time with Tris-buffered saline [50 mmol/L Tris (pH 7.4) and 0.9% NaCl] containing 0.05% Tween-20, and then incubated with either rabbit anti-PNMT polyclonal antibody (1:1000 dilution; Chemicon, Temecula, CA) or mouse anti-TH monoclonal antibody (1:4000 dilution; Calbiochem, San Diego, CA) for 1 h at room temperature.
Membranes were washed again three times for 10 min each time with Tris-buffered saline containing 0.05% Tween-20 and then incubated for 1 h with horseradish peroxidase-conjugated antirabbit IgG or antimouse IgG at a 1:20,000 dilution for both antibodies. Membranes were again washed three times for 10 min each time with Tris-buffered saline containing 0.05% Tween-20. PNMT and TH bands were visualized using the enhanced chemiluminescence method.
Quantitative PCR analysis of TH and PNMT
Ribonucleic acid (RNA) was extracted from pheochromocytoma tissue using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). Traces of DNA were removed by digestion with deoxyribonuclease-free ribonuclease (Gene Hunter, Nashville, TN). Total RNA (1 µg) was reversibly transcribed to complementary DNA (cDNA) using random hexamers together with the Superscript Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc.). Real Time Quantitative PCR (TaqMan PCR), using a 7700 Sequence Detector (Perkin-Elmer Corp./PE Applied Biosystems, Foster City, CA), was used for quantification of PNMT or TH messenger RNA (mRNA) as described previously (19). The amounts of PNMT and TH mRNA were determined by amplification of the cDNA target using the following primers and TaqMan probes designed from the human PNMT or TH gene sequences by the Primer Express program from Perkin-Elmer Corp./PE Applied Biosystems: PNMT forward primer, 5'-GCA GCC ACT TTG AGG ACA TCA-3'; PNMT reverse primer, 5'-GGC TGT ACA TGC TCC AGT TGA A-3'; PNMT TaqMan probe, 5' (FAM)-CAG ATT TCC TGG AGG TCA ACC GCC A-(TAMRA) 3'; TH forward primer, 5'-CGG ATG AGG AAA TTG AGA AGC T-3'; TH reverse primer, 5'-TCT GCT TAC ACA GCC CGA ACT-5'; and TH TaqMan probe, 5' (FAM)-CCA CGC TGT CAT GGT TCA CGG TG-(TAMRA) 3'.
To normalize quantification of PNMT or TH mRNA for differences in the amount of total RNA added to each cDNA reaction, 18S ribosomal RNA served as a housekeeping gene, which was detected using the TaqMan Ribosomal RNA Control Reagents (Perkin-Elmer Corp./PE Applied Biosystems). To minimize random errors, PCR amplification of PNMT or TH genes and 18S ribosomal RNA was carried out in the same tube. Reaction mixtures contained 5 µL cDNA product as template, 1 x TaqMan Universal PCR Master Mix, 3 µmol/L for each PNMT or TH forward and reverse primer, 2 µmol/L for the PNMT or TH TaqMan probe, 10 µmol/L for each 18S forward and reverse primer, 40 µmol/L for the 18S TaqMan probe, and water to a final volume of 50 µL. The following temperature parameters were cycled 50 times: 15 s at 95 C and 1 min at 60 C. Input RNA amounts were calculated manually using the Comparative CT method for both target genes and 18S. The amount of PNMT or TH mRNA was normalized by division by the amount of 18S RNA in each sample.
Electron microscopy
Pheochromocytoma tissue was fixed for 3 h in 2% formaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.3. Tissue slices were postfixed for 90 min (2% OsO4 in 0.1 mol/L cacodylate buffer, pH 7.3), dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined and photographed at 80 kV in a Phillips CM10 electron microscope (Phillips Electronic Instruments, Mahway, NJ).
Statistics
The distributions of plasma and tumor tissue concentrations of
catecholamines and plasma concentrations of metanephrines in patients
with pheochromocytoma were highly skewed. Normal distributions were
obtained after logarithmic transformation of the data. Mean values for
these variables are therefore provided as geometric means.
Corresponding SEs were established from the normalized
data. All other results for normally distributed data are expressed as
the arithmetic mean ± SEM. Where data showed
nonnormal distributions, statistical tests of significance were carried
out on normalized data. These tests included paired t tests
and ANOVAs with post-hoc tests carried out using the
Scheffé F test. 
2 analysis was used to
examine differences in presenting signs and symptoms. Differences among
relationships between plasma concentrations of catecholamines or
between tumor size or catecholamine content and the presence or absence
of symptoms or hypertension were examined by multiple linear regression
analysis.
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Results |
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Pheochromocytomas from VHL patients displayed a distinctly and
consistently noradrenergic phenotype, with norepinephrine
concentrations representing 98.0 ± 0.4% and epinephrine
concentrations only 1.5 ± 0.4% of the total catecholamine
content (Fig. 1A
). In contrast,
epinephrine accounted for 47.5 ± 6.3% and norepinephrine for
52.3 ± 6.3% of the total catecholamine content of
pheochromocytoma tumor tissue in patients with MEN 2. Dopamine was a
minor component, amounting to less than 0.5% of the total
catecholamine content of tumors from VHL and MEN 2 patients.
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). However, patients with MEN 2 also
had 2.3-fold higher tumor tissue concentrations of norepinephrine
(P = 0.019) and 3.7-fold higher concentrations of
dopamine (P < 0.001) than VHL patients. Plasma catecholamines and metanephrines
Plasma concentrations of norepinephrine were 2-fold higher
(P = 0.009) in VHL patients than in MEN 2 patients with
pheochromocytoma (Fig. 1B). In contrast, plasma concentrations of
epinephrine were 5-fold higher (P < 0.001) in MEN 2
patients than in VHL patients with pheochromocytoma.
Unlike the pattern for norepinephrine, plasma concentrations of
normetanephrine did not differ among VHL and MEN 2 patients with
pheochromocytoma (Fig. 1C). Plasma concentrations of metanephrine,
however, were 16-fold higher (P < 0.001) in MEN 2
patients than in VHL patients with pheochromocytoma.
The predominant features distinguishing the biochemical diagnostic
presentation of the two hereditary pheochromocytoma syndromes were
higher plasma concentrations of metanephrine and epinephrine in MEN 2
patients than in VHL patients (Fig. 2).
However, half of all MEN 2 patients with pheochromocytoma had normal
plasma epinephrine concentrations, whereas all had elevated plasma
concentrations of metanephrine. Very few patients with VHL disease had
elevations of either plasma epinephrine (3%) or metanephrine (9%). In
the three VHL patients who had elevated plasma concentrations of
metanephrine, the increases above the upper reference limit of normal
were slight (<20%). Thus, whereas there was considerable overlap in
plasma concentrations of epinephrine among VHL and MEN 2 patients,
there was no overlap in plasma concentrations of metanephrine.
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Quantitative TaqMan PCR revealed that PNMT mRNA was expressed in
pheochromocytoma tumor tissue from VHL patients at 22% the level of
expression in tumor tissue from patients with MEN 2 (P
< 0.001; Fig. 3A). Similarly, Western
blot analysis showed lower levels of expression of TH protein in
pheochromocytoma tissue from VHL patients than from MEN 2 patients
(Fig. 3B). Moreover, levels of TH enzyme activity in tumor tissue from
VHL patients were 19% (P < 0.002) those observed in
tissue from patients with MEN 2 (Fig. 3C). Levels of TH mRNA correlated
positively with TH enzyme activity (r = 0.62; P =
0.011) and total catecholamine contents of tumors (r = 0.69;
P = 0.006).
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Quantitative TaqMan PCR revealed that PNMT mRNA was expressed in
pheochromocytoma tumor tissue from VHL patients at less than 2%
(P < 0.001) the level of expression in tumor tissue
from patients with MEN-2 (Fig. 4A).
Similarly, Western blot analysis showed consistent expression of PNMT
protein in pheochromocytoma tissue from MEN-2 patients and a general
lack of expression in VHL patients (Fig. 4B).
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Electron microscopic analysis revealed distinct ultrastructural
differences between pheochromocytoma tumor cells from VHL and MEN 2
patients (Fig. 5). Chromaffin tumor cells
from patients with MEN 2 shared many of the characteristics of normal
adrenal medullary chromaffin cells, whereas tumor cells from VHL
patients did not. The cytoplasm of MEN 2 tumor cells was filled with
two types of secretory granules in similar amounts: 1) epinephrine-
containing large, round or elongated, medium density granules with
a particulate substructure; and 2) small norepinephrine-containing,
electron-dense granules (Fig 5, A and C). In MEN 2 tumor cells the two
types of secretory granules were evenly distributed throughout the
cytoplasm, and in some cells there were enlarged round mitochondria. In
contrast, pheochromocytoma tumor cells from VHL patients contained
fewer granules than did MEN 2 tumor cells, and most vesicles exhibited
a dense core with a large lucent halo typical of
norepinephrine-containing granules (Fig 5, B and D). Pheochromocytoma
tumor cells from VHL patients showed an increased amount of rough
endoplasmic reticulum, and the secretory granules were most frequently
lined up for exocytosis along cell membranes.
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Plasma concentrations of total metanephrines (combined sum of
plasma normetanephrine and metanephrine) and total catecholamines
(combined sum of plasma norepinephrine and epinephrine) were positively
correlated (P < 0.001) with tumor size (Fig. 6). The relationships were stronger for
metanephrines (r = 0.87) than for catecholamines (r = 0.69)
and differed among VHL and MEN 2 patients. Relative to tumor size,
patients with MEN 2 had higher (P = 0.010) plasma
concentrations of total metanephrines, but lower (P <
0.001) plasma concentrations of total catecholamines than did VHL
patients.
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).
Relationships between total tumor catecholamine content and plasma
total metanephrines were similar for VHL and MEN 2 patients, whereas
relative to tumor catecholamine content, VHL patients had higher
(P = 0.019) plasma concentrations of catecholamines
than did MEN 2 patients.
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Many of the patients with pheochromocytoma, particularly the VHL
patients, were normotensive and asymptomatic (Table 1). Only 18% of VHL patients with
pheochromocytoma presented with hypertension, which was usually
persistent. In contrast, hypertension was present in 40% of patients
with MEN 2 and was usually paroxysmal. Thus, paroxysmal hypertension
was considerably more common (P = 0.005) in MEN 2
patients than in VHL patients.
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). Moreover, MEN 2 patients reported that these
symptoms occurred as distinct attacks or episodes over three times more
frequently (P = 0.006) than VHL patients. The most
commonly reported symptoms were headache, diaphoresis, palpitations,
and anxiety. Other less common symptoms were tiredness or fatigue
(n = 8), dizziness or faintness (n = 5), facial flushing
(n = 4), nausea with or without vomiting (n = 3),
constipation (n = 3), and tremulousness (n = 1). Apart from
constipation, which was only reported in three VHL patients, none of
these symptoms appeared to distinguish VHL from MEN 2 patients. Relationships of signs and symptoms to tumor size and plasma catecholamines
Tumor size was a significant determinant of hypertension
(P = 0.005) and the presence of symptoms of
pheochromocytoma (P = 0.002) for the combined data from
VHL and MEN 2 patients. However, the influence of tumor size on the
presence of hypertension and symptoms tended to be stronger in MEN 2
patients than in VHL patients, so that all MEN 2 patients with tumors
larger than 5 cm in average diameter presented with hypertension and
symptoms compared with only 50% of VHL patients (Fig. 8A).
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). Thus, relative to plasma
concentrations of total catecholamines, patients with MEN 2 had a much
higher frequency of hypertension (P = 0.015) and
symptoms of pheochromocytoma (P = 0.010) than did VHL
patients. |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Pheochromocytomas in patients with MEN 2 express PNMT and thus display an adrenergic biochemical phenotype, whereas those in VHL patients express negligible PNMT and therefore show a distinctly noradrenergic biochemical phenotype. Consequently, MEN 2 patients with pheochromocytoma all show elevations in plasma metanephrine, the metabolite of epinephrine, whereas VHL patients typically show elevations only in normetanephrine, the metabolite of norepinephrine. Moreover, due to greater expression of TH, and thus higher rates of catecholamine biosynthesis, pheochromocytomas in MEN 2 patients contain considerably larger amounts of catecholamines than those in VHL patients. Consequently, MEN 2 patients show larger elevations of plasma metanephrines than VHL patients and have a higher frequency of hypertension and symptoms, particularly of a paroxysmal nature. Paradoxically, however, basal plasma levels of total catecholamines are lower in MEN 2 patients with pheochromocytoma than in VHL patients, indicating that pheochromocytomas from VHL and MEN 2 patients differ in their propensity for continuous release of catecholamines.
Although numerous studies have reported plasma and urinary catecholamines in VHL patients with pheochromocytoma (8, 20, 21), none appears to have noted, as reported here, the almost exclusive production of norepinephrine in this form of hereditary pheochromocytoma. Also, although several studies have documented that MEN 2 patients with pheochromocytoma often have elevated plasma or urinary levels of epinephrine (9, 12, 13, 14), none has established that epinephrine production represents a consistent finding among different kindreds and in large numbers of patients with MEN 2 and pheochromocytoma.
Previous failure to recognize the clear-cut differences in the adrenergic and noradrenergic biochemical phenotypes of pheochromocytomas in MEN 2 and VHL syndrome may stem from two factors. Differences in plasma concentrations of normetanephrine and metanephrine reflect underlying differences in tumor catecholamine phenotype better than do differences in plasma or urinary norepinephrine and epinephrine, and advances in molecular genetic diagnosis now allow unambiguous identification of the underlying mutation in the two disorders, whereas previously this was based largely on clinical presentation.
The superiority of assays of plasma free metanephrines over catecholamines for identifying adrenergic or noradrenergic biochemical phenotypes in pheochromocytoma is illustrated by the considerable overlap in plasma concentrations of epinephrine and the absence of overlap in plasma concentrations of metanephrine among patients with the two hereditary syndromes. Many patients with MEN 2 had normal plasma concentrations of epinephrine, and many VHL patients had normal levels of norepinephrine. In contrast, all patients with MEN 2 had elevations of plasma metanephrine, often accompanied by an increase in normetanephrine, and almost all patients with VHL syndrome had elevations in normetanephrine with little increase in plasma metanephrine. Thus, compared with plasma metanephrines, plasma catecholamines fail to adequately indicate both the presence and the underlying neurochemical phenotype of a pheochromocytoma.
The importance of molecular genetic diagnosis for unambiguously identifying a mutation of either the RET protooncogene or the VHL tumor suppressor gene is illustrated by several reports that VHL families with pheochromocytoma were diagnosed erroneously, based on clinical presentation, as having MEN 2 or familial pheochromocytoma (22, 23, 24, 25). As different kindreds can present with different phenotypes, it can sometimes be difficult to identify the specific hereditary syndrome, based on clinical presentation alone. In particular, some VHL families have mainly pheochromocytoma, with occult or delayed manifestations of the syndrome in the central nervous system, eye, or other organs (25, 26, 27). As MEN 2 features a high penetrance of medullary thyroid cancer (28), it is generally less of a problem to identify this syndrome on clinical grounds. Nevertheless, in one kindred of the present series, there was no history of either medullary thyroid cancer or parathyroid disease, and increased plasma concentrations of metanephrine provided the only initial clinical evidence that the underlying mutation was of the RET gene rather than of the VHL gene. The above examples illustrate how differences in plasma levels of normetanephrine and metanephrine can provide a supplementary guide to clinical presentation in deciding which gene to test to unambiguously identify a particular germline mutation.
The low frequency of hypertension or symptoms found during screening of VHL and MEN 2 patients with pheochromocytoma agrees with other reports (6, 7, 8, 9, 10), reflecting in part early detection of small tumors that produce insufficient amounts of catecholamines to produce the typical clinical manifestations of the tumor. Comparisons with patients with sporadic pheochromocytoma suggest that the small tumors detected in MEN 2 families may be more often functional than those detected in VHL families (9, 10). This suggestion is supported by the present findings of a higher frequency of symptoms and hypertension in MEN 2 than in VHL patients with pheochromocytoma.
Epinephrine has more potent 
- and
ß2-adrenoceptormediated vascular and
metabolic effects than norepinephrine (29, 30). Thus,
differences in expression of PNMT and production of epinephrine
probably contribute to the higher frequency of signs and symptoms of
pheochromocytomas in MEN 2 than in VHL patients. The greater expression
of TH in pheochromocytomas from MEN 2 than VHL patients is in agreement
with previous findings of higher TH activity in tumors from MEN 2
patients than in those from other patients (31). The
resulting higher rates of catecholamine synthesis and releasable tissue
stores of catecholamines in pheochromocytomas from MEN 2 than from VHL
patients probably also contribute to the higher frequency of signs and
symptoms of pheochromocytoma in MEN 2 than in VHL patients.
Despite higher tumor tissue contents of catecholamines and production of metanephrines, the lower plasma concentrations of total catecholamines in MEN 2 than in VHL patients indicate that pheochromocytomas in MEN 2 patients do not secrete catecholamines as readily or as continuously as those in VHL patients. Possibly this difference may reflect the morphological findings that secretory vesicles in VHL tumor cells are concentrated around cell membranes in apparent readiness for release, whereas those in MEN 2 tumor cells are distributed evenly throughout the cytoplasm. The possibility that this might result in a more continuous pattern of catecholamine release from tumors in VHL patients compared with a more episodic pattern in MEN 2 patients is consistent with previous findings that patients with epinephrine-secreting tumors present more often with paroxysmal signs and symptoms than patients with predominantly norepinephrine-secreting tumors (2, 5). Greater down-regulation of adrenoceptors resulting from continuously higher plasma concentrations of catecholamines in VHL patients compared with the more intermittently elevated concentrations in MEN 2 patients might also contribute to differences in the clinical presentation of pheochromocytoma among the two groups.
Apart from an influence of early tumor detection, low tissue levels of catecholamines in pheochromocytomas from VHL patients and low rates of basal catecholamine secretion from tumors in MEN 2 patients may be other factors responsible for the low frequency of hypertension and symptoms in hereditary pheochromocytoma. Low rates of basal catecholamine secretion combined with high rates of catecholamine biosynthesis and consequently metabolism to metanephrines also explain the present and previous finding that MEN 2 patients with pheochromocytoma have proportionally larger increases in urinary or plasma metanephrines than in catecholamines (12, 32).
The findings of this study that pheochromocytomas in MEN 2 and VHL patients are characterized by distinctly different clinical, biochemical, and morphological features raise the question of how RET and VHL gene mutations result in differences in TH and PNMT expression. As the expressions of TH and particularly PNMT are controlled by actions of steroids on glucocorticoid receptors (33, 34, 35, 36), differences in the expression of these receptors or in local production of steroids might account for the differences in catecholamine production. Additionally, several other transcription factors known to regulate TH and PNMT gene expression (36, 37, 38, 39, 40) might be involved in linking RET and VHL gene mutations to differences in catecholamine synthesis. The above possibilities are supported by recent findings that some of these transcription factors are differentially expressed in predominantly norepinephrine-producing compared with epinephrine-producing pheochromocytomas (41).
As the adrenal medulla is comprised of subpopulations of noradrenergic and adrenergic chromaffin cells (42, 43, 44), the different pheochromocytoma tumor cell phenotypes in MEN 2 and VHL patients may simply reflect mutation-dependent development of tumors from different types of chromaffin cells. The present and future work aimed at establishing the mechanisms that link germline and somatic mutations of genes to expression of specific pheochromocytoma tumor cell phenotypes should lead to improved understanding of the molecular basis of tumorigenesis, variations in the rate of disease progression, tendency to recurrence, metastatic potential, and development of novel treatments.
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Acknowledgments |
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Received August 25, 2000.
Revised January 8, 2001.
Accepted January 14, 2001.
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References |
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L. Green-Golan, C. Yates, B. Drinkard, C. VanRyzin, G. Eisenhofer, M. Weise, and D. P. Merke Patients with Classic Congenital Adrenal Hyperplasia Have Decreased Epinephrine Reserve and Defective Glycemic Control during Prolonged Moderate-Intensity Exercise J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3019 - 3024. [Abstract] [Full Text] [PDF]
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F. M Brouwers, S. Glasker, A. F Nave, A. O Vortmeyer, I. Lubensky, S. Huang, M. S Abu-Asab, G. Eisenhofer, R. J Weil, D. M Park, et al. Proteomic profiling of von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2 pheochromocytomas reveals different expression of chromogranin B Endocr. Relat. Cancer, June 1, 2007; 14(2): 463 - 471. [Abstract] [Full Text] [PDF]
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C. Jimenez, G. Cote, A. Arnold, and R. F. Gagel Should Patients with Apparently Sporadic Pheochromocytomas or Paragangliomas be Screened for Hereditary Syndromes? J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 2851 - 2858. [Abstract] [Full Text] [PDF]
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E. R Woodward and E. R Maher Von Hippel-Lindau disease and endocrine tumour susceptibility. Endocr. Relat. Cancer, June 1, 2006; 13(2): 415 - 425. [Abstract] [Full Text] [PDF]
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 T.-T. Huynh, K. Pacak, F. M Brouwers, M. S Abu-Asab, R. A Worrell, M. M Walther, A. G Elkahloun, D. S Goldstein, S. Cleary, and G. Eisenhofer Different expression of catecholamine transporters in phaeochromocytomas from patients with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2 Eur. J. Endocrinol., October 1, 2005; 153(4): 551 - 563. [Abstract] [Full Text] [PDF]
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 S. J. Marx and W. F. Simonds Hereditary Hormone Excess: Genes, Molecular Pathways, and Syndromes Endocr. Rev., August 1, 2005; 26(5): 615 - 661. [Abstract] [Full Text] [PDF]
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M. S. Lora, S. G. Waguespack, J. F. Moley, and E. C. Walvoord Adrenal Ganglioneuromas in Children with Multiple Endocrine Neoplasia Type 2: A Report of Two Cases J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4383 - 4387. [Abstract] [Full Text] [PDF]
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T. W. A. Vogel, F. M. Brouwers, I. A. Lubensky, A. O. Vortmeyer, R. J. Weil, M. M. Walther, E. H. Oldfield, W. M. Linehan, K. Pacak, and Z. Zhuang Differential Expression of Erythropoietin and Its Receptor in von Hippel-Lindau-Associated and Multiple Endocrine Neoplasia Type 2-Associated Pheochromocytomas J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3747 - 3751. [Abstract] [Full Text] [PDF]
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 G. Eisenhofer, J. W.M. Lenders, D. S. Goldstein, M. Mannelli, G. Csako, M. M. Walther, F. M. Brouwers, and K. Pacak Pheochromocytoma Catecholamine Phenotypes and Prediction of Tumor Size and Location by Use of Plasma Free Metanephrines Clin. Chem., April 1, 2005; 51(4): 735 - 744. [Abstract] [Full Text] [PDF]
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W H van Houtum, E P M Corssmit, P B Douwes Dekker, J C Jansen, A G L van der Mey, A H J T Brocker-Vriends, P E M Taschner, M Losekoot, M Frolich, M P M Stokkel, et al. Increased prevalence of catecholamine excess and phaeochromocytomas in a well-defined Dutch population with SDHD-linked head and neck paragangliomas Eur. J. Endocrinol., January 1, 2005; 152(1): 87 - 94. [Abstract] [Full Text] [PDF]
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G Eisenhofer, T-T Huynh, K Pacak, F M Brouwers, M M Walther, W M Linehan, P J Munson, M Mannelli, D S Goldstein, and A G Elkahloun Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome Endocr. Relat. Cancer, December 1, 2004; 11(4): 897 - 911. [Abstract] [Full Text] [PDF]
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G. Eisenhofer, S. R Bornstein, F. M Brouwers, N.-K. V Cheung, P. L Dahia, R. R de Krijger, T. J Giordano, L. A Greene, D. S Goldstein, H. Lehnert, et al. Malignant pheochromocytoma: current status and initiatives for future progress Endocr. Relat. Cancer, September 1, 2004; 11(3): 423 - 436. [Abstract] [Full Text] [PDF]
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G. Mansmann, J. Lau, E. Balk, M. Rothberg, Y. Miyachi, and S. R. Bornstein The Clinically Inapparent Adrenal Mass: Update in Diagnosis and Management Endocr. Rev., April 1, 2004; 25(2): 309 - 340. [Abstract] [Full Text] [PDF]
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 J. Bryant, J. Farmer, L. J. Kessler, R. R. Townsend, and K. L. Nathanson Pheochromocytoma: The Expanding Genetic Differential Diagnosis J Natl Cancer Inst, August 20, 2003; 95(16): 1196 - 1204. [Abstract] [Full Text] [PDF]
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 D. S. Goldstein, G. Eisenhofer, and I. J. Kopin Sources and Significance of Plasma Levels of Catechols and Their Metabolites in Humans J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 800 - 811. [Abstract] [Full Text] [PDF]
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W. G. Meijer, S. C.V.M. Copray, H. Hollema, I. P. Kema, N. Zwart, I. Mantingh-Otter, T. P. Links, P. H.B. Willemse, and E. G.E. de Vries Catecholamine-Synthesizing Enzymes in Carcinoid Tumors and Pheochromocytomas Clin. Chem., April 1, 2003; 49(4): 586 - 593. [Abstract] [Full Text] [PDF]
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 A. V. Kuznetsova, J. Meller, P. O. Schnell, J. A. Nash, M. L. Ignacak, Y. Sanchez, J. W. Conaway, R. C. Conaway, and M. F. Czyzyk-Krzeska von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination PNAS, March 4, 2003; 100(5): 2706 - 2711. [Abstract] [Full Text] [PDF]
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C. A. Koch, K. Pacak, and G. P. Chrousos The Molecular Pathogenesis of Hereditary and Sporadic Adrenocortical and Adrenomedullary Tumors J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5367 - 5384. [Abstract] [Full Text] [PDF]
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M. Weise, D. P. Merke, K. Pacak, M. M. Walther, and G. Eisenhofer Utility of Plasma Free Metanephrines for Detecting Childhood Pheochromocytoma J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 1955 - 1960. [Abstract] [Full Text] [PDF]
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 A. L. Bauer, W. R. Paulding, J. B. Striet, P. O. Schnell, and M. F. Czyzyk-Krzeska Endogenous von Hippel-Lindau Tumor Suppressor Protein Regulates Catecholaminergic Phenotype in PC12 Cells Cancer Res., March 1, 2002; 62(6): 1682 - 1687. [Abstract] [Full Text] [PDF]
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S. J. Marx and L. K. Nieman Aggressive Pituitary Tumors in MEN1: Do They Refute the Two-Hit Model of Tumorigenesis? J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 453 - 456. [Full Text] [PDF]
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) and
epinephrine (
) in pheochromocytoma tumor tissue (A) or plasma (B)
and concentrations of normetanephrine (
) compared with MEN 2 patients (•). Relationships
for VHL patients are shown by dashed regression
lines, and those for MEN 2 patients are shown by
solid regression lines. Plasma concentrations of total
metanephrines represent the sum of plasma concentrations of
normetanephrine and metanephrine. Plasma concentrations of total
catecholamines represent the sum of plasma concentrations of
norepinephrine and epinephrine. Multiple regression analysis
revealed that relative to tumor size, patients with MEN 2 have higher
(P = 0.010) plasma concentrations of total
metanephrines and lower (P < 0.001) plasma
concentrations of total catecholamines than VHL patients.
and