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Original Studies |
Department of Pathology, Haartman Institute, University of Helsinki (K.S., J.A., R.V., A.I.K., P.H., J.L.), and HUCH Laboratory Diagnostics (K.S., J.A., R.V., A.I.K., P.H., J.L.) and Departments of Internal Medicine (V.I.) and Surgery (C.H.), Helsinki University Central Hospital, FIN-00014 Helsinki; and Department of Pediatrics, Kuopio University Hospital (R.V., J.L.), FIN-70211 Kuopio, Finland
Address all correspondence and requests for reprints to: Dr. Kaisa Salmenkivi, Haartman Institute, Department of Pathology, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail address: kaisa.salmenkivi{at}helsinki.fi
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Abstract |
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-subunit and either a ßA- or a
ßB-subunit. Activins are composed of ß-subunits only. Immunohistochemically inhibin/activin ßB-subunit was strongly positive in the normal adrenal medulla, but the cortex was negative. A striking difference was found in inhibin/activin ßB expression between benign and malignant pheochromocytomas. The majority of benign adrenal tumors (27 of 30) showed strong or moderate immunoreactivity, whereas all seven malignant tumors were negative or only weakly positive for inhibin/activin ßB-subunit. The percentage of positively staining cells varied greatly in extraadrenal pheochromocytomas and in those benign tumors that showed over 5 mitoses/10 high power fields, necrosis, or capsular or vascular invasion, here called borderline tumors. Inhibin/activin ßB messenger ribonucleic acid was also found in pheochromocytomas. However, no significant differences in messenger ribonucleic acid levels were found in various types of tumors. Weak immunohistochemical positivity for inhibin/activin ßA-subunit was detected in the adrenal cortex, but the medulla and most of the pheochromocytomas were negative.
Our data show that inhibin/activin ßB-subunit is expressed in normal adrenal medullary cells. Strong staining is found in most benign adrenal pheochromocytomas, whereas malignant tumors are almost negative. This suggests that loss of inhibin/activin ßB-subunit expression in pheochromocytomas may be used as an indicator of malignant potential.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibins and activins are glycoproteins of the transforming growth
factor-ß superfamily. Inhibins consist of an -subunit linked to
either a ßA (inhibin A) or a ßB (inhibin B) subunit, whereas
activins are heterodimers of ßA and ßB or homodimers of either one
(5). Inhibin/activin -, ßA-, and ßB-subunits are
expressed in gonadal tissues (5) and in a wide range of
extragonadal sites, including pituitary, bone marrow, kidney, spinal
cord, brain, adrenal, and placenta (6, 7, 8). In the adrenal
gland, strong immunoreactivity for inhibin -subunit is found mainly
in the inner zones of the cortex, whereas zona glomerulosa and medulla
are immunohistochemically negative (9, 10, 11). Diffuse
immunostaining of all three cortical zones has been reported for both
ßA- and ßB-subunits (9, 12), but no positivity was
found in the medulla (12). We have previously shown that
most adrenocortical tumors are positive for inhibin -subunit,
whereas pheochromocytomas are negative (10). However,
little is known about the expression profile of ßA- and ßB-subunits
in pheochromocytomas. To study further the role of inhibins and
activins in adrenals, particularly in the medulla and
chromaffin-derived tumors, we examined the expression of
inhibin/activin ßA- and ßB-subunits in normal adrenal glands and in
a large number of pheochromocytomas. The potential value of ßA- and
ßB-subunit expression in the estimation of the malignancy of
pheochromocytomas was considered.
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Materials and Methods |
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Tissue specimens were obtained at operations performed at
the Department of Surgery, Helsinki University Central Hospital, from
1985–1999. Normal adrenal glands were from patients undergoing
nephrectomy for renal tumor. Tissue specimens were dissected, and
visible medullary parts were carefully separated within 0.5 h if
used for ribonucleic acid (RNA) analysis. Tissues were fixed in
buffered 10% formalin. Samples were routinely processed for light
microscopic study and stained with hematoxylin-eosin. Sections were
histologically rereviewed by two pathologists (K.S. and P.H.)
independently. There was no discrepancy between the two pathologists in
classifying the tumors. Tissue material consisted of 7 normal adrenal
glands, 83 pheochromocytomas, and 5 metastases. The clinical data are
summarized in Table 1
. The patients’
ages ranged from 17–82 yr, and there were 46 females and 34 males.
Secretion of either metanephrines or normetanephrines was elevated in
all 51 adrenal pheochromocytoma patients in whom data were
available.
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). Two patients
had bilateral adrenal tumors, and 1 patient had a bilateral
paraganglioma in both carotid bifurcations. The mean follow-up time was
73.3 months (range, 16–131) for the adrenal tumors and 47.6 months
(range, 22–86) for the extraadrenal ones.
Tumors without metastases but having any of the histologically
suspicious features, i.e. more than 5 mitoses/10 high power
fields, confluent tumor necrosis, or vascular or capsular invasion
(4), were evaluated as a separate group. This group
included 29 pheochromocytomas, and we called them borderline tumors.
Twenty-three of these were located in the adrenal glands, and 6 were
extraadrenal (Table 1). The follow-up times of these patients were 88.2
months (range, 24–144) and 71.2 months (range, 0–183),
respectively.
Seven tumors (8.4%) were considered malignant, having histologically
or radiologically proven metastases, and one had extensive invasion to
a spinal vertebral body. All three malignant adrenal tumors were
located in the right adrenal. Four malignant tumors were extraadrenal:
two located in the retroperitoneum, one in the aortic bifurcation, and
one in right upper abdomen. All primary tumors had histologically
suspicious features. Four patients had metastases at the time of
surgery, whereas in two cases the metastases were diagnosed 55 and 91
months after the primary operation. The follow-up data of the patients
with malignant disease are summarized in Table 2. As metastases may occur years after
primary operation, actual clinical and survival data were checked by
the end of November 2000. The survival data and the cause of death were
obtained from the Population Registry of Finland.
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Five-micron sections were cut from paraffin-embedded
blocks, deparaffinized in xylene, and rehydrated in a series of graded
alcohols. The sections were pretreated in a microwave oven in 10 mmol/L
citrate buffer, pH 6.0, at 600 watts for 20 min. Endogenous peroxidase
activity was blocked in 0.5%
H2O2 for 30 min. Primary
monoclonal antibodies for both inhibin/activin ßA (MCA950S) and ßB
(MCA1661; both from Serotec Ltd., Oxford, UK) subunits
were applied overnight at 1:50 dilution. The detections were performed
using the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s
instructions, and sections were lightly counterstained with
hematoxylin. To exclude the effect of possible endogenous biotin on
immunohistochemical staining, biotin blocking (avidin-biotin blocking
kit, Vector Laboratories, Inc.) was performed in at least
one sample of each diagnostic group before the addition of the primary
antibody. Granular red-brown cytoplasmic staining was considered
positive. Omission of primary antibodies and staining with nonimmunized
mouse IgG2 and (in the case of ßA-subunit) with the primary antibody
preabsorbed with a 7-fold molar excess of recombinant human activin A,
were used as negative controls. Recombinant activin B was not available
for preabsorption. Sections of normal ovary were used as positive
controls for both inhibin/activin ßA- and ßB-subunits. The
percentage of positively stained cells was analyzed from one slide per
tumor. Five representative high power fields were chosen, and the
number of positive staining cells was calculated. The final result was
estimated from the whole slide. The following categories were used:
negative; weak, 1–10%; moderate, 11–50%; and strong, over 51%. The
results were scored by two independent pathologists (K.S. and J.A.),
who were blinded to the subtype of the tumors. The agreement between
the pathologists was very good in all groups. The 
values were
0.873, 0.901, and 1.000 in benign, borderline, and malignant groups,
respectively.
RNA analysis
Total RNA was isolated from four carefully dissected samples of normal adrenal medullary tissue and from nine benign, three borderline, and six malignant pheochromocytomas. RNA isolation and Northern blotting were performed as described previously (13). The inhibin/activin ßB-subunit messenger RNA (mRNA) was detected by Northern blots hybridized with two radiolabeled oligonucleotides complementary to different regions of human inhibin ßB mRNA. The sequences were 5'-GTG GAA GGA GGA GGC AGA GCC GGG GAC CCC-3' and 5'-GGG CAC GTC CCG CTT GAC GAT GTT GTA CTC-3', complementary to nucleotides 864–893 and 1002–1031 of the ßB-subunit mRNA, respectively (GenBank accession no. M13437) (14). The mouse ribosomal 28S RNA complementary DNA was used as a loading control (15). The oligonucleotides and 28S complementary DNA were labeled as previously described (13). The relative intensities of the autoradiographic signals were quantified by densitometric scanning.
Statistical analysis
The statistic was used to examine the interobserver
variation in the scoring of the staining. Statistical analysis of the
RNA and immunohistochemical results was performed using the
Mann-Whitney test. The correlation between ßB-subunit
immunoreactivity and RNA expression was analyzed by Spearman’s test.
The level of significance was chosen as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strong positive inhibin/activin ßB-subunit immunostaining was
found in the normal adrenal medulla, whereas the cortex was negative
(Fig. 1). The distribution and intensity
of the staining were similar in all normal adrenal glands (n = 7).
In contrast, weak diffuse immunopositivity for inhibin/activin
ßA-subunit was seen in the cortical tissue, mainly in the inner
zones, leaving medullary cells negative.
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). Two thirds of the benign
adrenal tumors (20 of 30) showed strong reactivity for the ßB-subunit
(Table 3). Seven tumors were moderately
positive, and 1 was weakly positive. Only 2 tumors were completely
negative. The profile of the ßB-subunit immunoreactivity was slightly
different in benign extraadrenal pheochromocytomas (n = 17). Seven
tumors were negative; 3 had weak, 5 had moderate, and only 2 had strong
positivity for the ßB-subunit. Twenty-three benign tumors were
stained with the ßA-subunit specific antibody: all adrenal ones
(n = 13) were negative, whereas 3 of 10 extraadrenal tumors were
weakly positive (data not shown).
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). Thirteen tumors exhibited strong, 4 moderate,
and 4 weak immunopositivity for the ßB-subunit. Only 2 borderline
adrenal tumors were negative. Extraadrenal borderline pheochromocytomas
(n = 6) expressed less ßB-subunit than the adrenal cases: 4
tumors were negative, 1 was moderately positive, and 1 was strongly
positive. Thirteen tumors classified as borderline were stained for the
ßA-subunit; 11 were negative, and 2 showed occasional positively
staining cells.
Malignant pheochromocytomas (n = 7), regardless of their
primary location, showed very little, if any, immunohistochemical
staining for the ßB-subunit (Fig. 1). One adrenal tumor was
completely negative, and all other cases had occasional
ßB-subunit-positive tumor cells (Table 3). One of the five metastases
expressed moderate immunopositivity, whereas the rest were negative or
only weakly positive for the ßB-subunit (Table 2).
Immunohistochemistry with the ßA-subunit-specific antibody revealed
six negative tumors among the seven malignant pheochromocytomas. In one
malignant tumor occasional ßA-subunit positive cells were seen.
Statistically, a significant difference was found in immunohistochemical inhibin/activin ßB-subunit expression between benign and malignant adrenal tumors (P = 0.006) as well as between borderline and malignant adrenal tumors (P = 0.027). There were no statistical differences between any other groups. No correlation between inhibin/activin ßA- and ßB-subunit immunoreactivity was found.
RNA analysis
Northern blot analysis revealed a predominant 4.8-kb
inhibin/activin ßB-subunit transcript in all pheochromocytomas as
well as in the normal adrenal medullary samples (Fig. 2). Additional lower molecular weight
transcripts were seen in the samples with the highest ßB-subunit
expression. The relative ßB-subunit mRNA levels varied considerably
in different tumors and in normal adrenal medullary samples. However,
among the samples analyzed, there was no statistical difference in the
ßB-subunit mRNA levels among the different tumor groups (nine benign,
three borderline, and six malignant tumors).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Distinction between malignant and benign pheochromocytomas is very difficult, and it has been generally accepted that only metastasized tumors are malignant. Some pheochromocytomas are associated with multiple endocrine neoplasia type 2 and von Hippel-Lindau disease. In these syndromes, malignant pheochromocytomas are rare (16). Multiple endocrine neoplasia type 2-associated oncogenic RET mutations do not associate with aggressive tumor behavior in sporadic pheochromocytomas (17). The prognosis of pheochromocytomas is unpredictable on the basis of clinical, biochemical, or histopathological characteristics (18). No definite immunohistochemical markers have been found for malignant pheochromocytomas, even though p53 mutations are believed to have a role in the development of multiple and malignant pheochromocytomas (19). In our series, all seven malignant pheochromocytomas had only weak or no immunoreactivity for the ßB-subunit. In contrast, most benign tumors showed strong or moderate immunopositivity. It seems that pheochromocytomas arising from chromaffin cells lose their potential to express inhibin/activin ßB-subunit when they become malignant.
Although the diagnosis of a malignant pheochromocytoma requires evidence of metastases, Linnoila et al. reported that malignancy often correlates with extraadrenal location and with histological features, including coarse nodularity, confluent necrosis, and absence of hyaline globules (4). Therefore, we decided to evaluate the inhibin/activin expression separately in a group of tumors with these histological features and called them borderline tumors. The staining pattern for inhibin/activin ßB-subunit in both borderline and benign extraadrenal tumors was heterogeneous. Immunohistochemically, 4 of 6 borderline extraadrenal tumors were totally negative for the ßB-subunit. If loss of ßB expression in pheochromocytomas is considered a sign of malignancy, some of these negatively staining extraadrenal borderline tumors may truly have had malignant potential. On the other hand, it seems that in the case of extraadrenal pheochromocytomas, the lack of ßB-subunit expression is not a reliable sign of malignancy, as 7 of 17 benign pheochromocytomas were negative for the ßB-subunit.
As metastases of a malignant pheochromocytoma may not be detected until several years after primary operation, a long follow-up is needed to exclude malignancy in patients originally considered to have a benign tumor. Most of our patients had a long follow-up, but in 30 patients the follow-up was less than 5 yr. None of the patients with a tumor classified as benign, including borderline tumors, as checked by clinical and survival data, had shown any sign of metastases by the end of November 2000. This fact does not naturally exclude a malignant potential of the primary tumor. Part of the borderline tumors showed immunohistochemical properties similar to those of their malignant counterparts. These tumors might have been malignant in nature, but without metastases at the time of operation and thus were treated curatively by surgery.
RNA analysis revealed that the inhibin/activin ßB-subunit mRNA is indeed expressed in normal adrenal medulla and in various pheochromocytomas. However, compared with immunohistochemical data, no clear differences in ßB- subunit mRNA levels were found among the different pheochromocytoma types, suggesting posttranscriptional regulation of the ßB-subunit. On the other hand, only a small number of samples was available for RNA analysis.
In summary, we found both inhibin/activin ßA- and ßB-subunit expression in the normal adrenal gland. The ßA-subunit was located in the cortex and the ßB-subunit in the medulla. Benign adrenal pheochromocytomas were mostly positive for the ßB-subunit, whereas malignant ones had weak or no expression of it. We therefore believe that a clear ßB-subunit expression in pheochromocytomas favors a benign nature.
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Acknowledgments |
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Footnotes |
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Received July 12, 2000.
Revised December 28, 2000.
Accepted January 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ßA and ßB
subunits in various tissues predicts diverse functions. Proc Natl Acad
Sci USA. 85:247–251. in adrenocortical tumours reflects the
hormonal status of the neoplasm. J Endocrinol. 165:223–229.[Abstract]
inhibin. J
Clin Pathol. 51:114–116.[Abstract]
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