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Increased Expression of Cyclooxygenase-2 in Malignant Pheochromocytomas

Department of Pathology, Haartman Institute, University of Helsinki and HUCH Laboratory Diagnostics (K.S., A.R., J.A., P.H.), and Department of Surgery (C.H.), Helsinki University Central Hospital, FIN-00014 Helsinki; and Molecular and Cancer Biology Program, Biomedicum Helsinki, University of Helsinki (A.R.), FIN-00014 Helsinki, Finland

Address all correspondence and requests for reprints to: Kaisa Salmenkivi, M.D., Haartman Institute, Department of Pathology, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: kaisa.salmenkivi{at}helsinki.fi

Abstract

Pheochromocytomas are rare tumors of the adrenal medulla or the paraganglion system. There are no histological or chemical markers available that define the malignant behavior of these tumors; so far only the discovery of metastases reveals malignancy. Cyclooxygenase (Cox) is the key enzyme in conversion of arachidonic acid to PGs, and two isoforms, Cox-1 and Cox-2, have been identified. Cox-2 has been associated with carcinogenesis, and it is overexpressed in many human malignancies. We have now investigated the expression of Cox-2 in normal adrenal gland, in 92 primary pheochromocytomas and in six metastases using immunohistochemistry and Northern blot and Western blot analyses. Cox-2 protein was expressed in the adrenal cortex, whereas the medulla was negative as detected by immunohistochemistry. Interestingly, all malignant pheochromocytomas (n = 8), regardless of the primary location of the tumor, showed moderate or strong Cox-2 immunoreactivity, whereas 75% of the benign adrenal tumors (n = 36) showed no or only weak immunopositivity. The staining was negative or weak in 79% of the adrenal tumors that showed histologically suspicious features (n = 24), but had not metastasized. Most of the pheochromocytoma samples studied also expressed low levels of Cox-2 mRNA.

Our data show that normal adrenal medulla does not express Cox-2 immunohistochemically. However, strong Cox-2 protein expression was found in malignant pheochromocytomas, whereas most benign tumors expressed Cox-2 only weakly. To our knowledge, this is the first report on Cox-2 expression in pheochromocytomas and enhanced expression in malignant pheochromocytomas. These findings suggest that negative or weak Cox-2 expression in pheochromocytomas favors benign diagnosis.

PHEOCHROMOCYTOMAS ARE RELATIVELY uncommon tumors arising from the human sympathoadrenal system, mostly from chromaffin cells of the adrenal medulla. Extra-adrenal pheochromocytomas, also referred to as paragangliomas, are derived from the paraganglion system. Distinguishing between benign and malignant pheochromocytomas is extremely difficult, and the diagnosis of malignant disease is based on the evidence of extensive local invasion or metastasis to sites where chromaffin tissue is not normally present (1). The proportion of malignant tumors out of all pheochromocytomas has been reported to range from 2.4% (2) to 14% (3). The tumor is malignant more frequently if the primary tumor is located extra-adrenally, has coarse nodularity, confluent tumor necrosis, or absence of hyaline globules (4). Pheochromocytomas metastasize via hematogenous or lymphatic pathways, and the most common metastatic sites are lymph nodes, bone, lung, and liver (5).

Cyclooxygenase (Cox) is the key enzyme in the conversion of arachidonic acid to PGs. Two Cox genes have been characterized, Cox-1 and Cox-2, and they share over 60% homology at amino acid level (6). Cox-1 is constitutively expressed in normal human tissues and its expression is not usually regulated, whereas Cox-2 is usually absent in most normal cells but can be highly induced in response to cell activation by hormones, proinflammatory cytokines, growth factors, and tumor promoters (6, 7). Thus, the role of Cox-2 has been connected to inflammation, reproduction, and carcinogenesis (8, 9). High Cox-2 expression is found in gastrointestinal tumors (10, 11, 12, 13, 14), but increased expression has also been described in several other human malignancies, including lung carcinoma (15), hepatocellular carcinoma (16), pancreatic carcinoma (17), squamous cell carcinoma of the head and neck (18), prostate adenocarcinoma (19), retinoblastoma (20), and urinary bladder carcinoma (21).

The purpose of this study was to investigate the expression of Cox-2 in normal adrenal gland and in our large collection of benign and malignant pheochromocytomas.

Materials and Methods

Histopathology and clinical data

Tissue specimens were obtained at operations performed at Helsinki University Central Hospital, from 1976–2001. Normal adrenal glands were from five patients undergoing nephrectomy for a renal tumor. Tissue specimens were dissected, and visible medullary parts were carefully separated within 0.5 h if used for mRNA analysis. Tissues were fixed in 10% buffered formalin. Samples were routinely processed for light microscopic study and stained with hematoxylin-eosin. All samples were histologically rereviewed by two pathologists (K.S. and P.H.) independently. There was no discrepancy between the two pathologists in classifying the tumors. The tissue material consisted of five normal adrenal glands, 92 pheochromocytomas, and six metastases. The clinical data are summarized in Tables 1 and 2. Secretion of either metanephrines or normetanephrines was increased in 54 adrenal pheochromocytoma patients for whom data were available.

The pheochromocytomas that were considered clinically and histologically benign included 36 adrenal and 18 extra-adrenal tumors (Table 1). The mean follow-up time was 68.1 months (range, 8–281) for the adrenal tumors and 42.4 months (range, 0–82) for the extra-adrenal ones.

Tumors without metastases, but having any of the histologically suspicious features [i.e. over five mitoses per 10 high-power fields, confluent tumor necrosis, vascular or capsular invasion (4)], were evaluated as a separate group. Twenty-four of these were located in the adrenal glands, and six were extra-adrenal (Table 1). The follow-up times of these patients were 94.1 months (range, 24–281) and 58.2 months (range, 0–116), respectively.

Eight patients (9.4%) were considered to have malignant pheochromocytoma, seven patients with histologically or radiologically proven metastases, and one patient with extensive tumor invasion to a spinal vertebral body. All three adrenal malignant pheochromocytomas were located in the right gland (Table 1). Five malignant tumors were extra-adrenal, two located in the retroperitoneum, one in the aortic bifurcation, one in the carotid bifurcation, and one in the right upper abdomen. All primary tumors displayed histologically suspicious features. Five 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 metastases were located in the cervical and para-aortal lymph nodes, liver, bones, and lungs. One patient died during the operation, and one died after 11 months because of metastasized pheochromocytoma. Two patients are alive with residual or metastasized disease 65 and 90 months after operation. Four patients have no evidence of disease. The follow-up data are summarized in Table 2. Because metastases may occur years after primary operation, the 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.

Immunohistochemistry

Four-micrometer 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 for 4 x 5 min in 700 watts in 0.01 M Na-citrate buffer (pH 6.0) for antigen retrieval. The slides were immersed in 0.6% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity and in blocking solution (1.5:100 normal horse serum in PBS) for 15 min to block unspecific binding sites. Cox-2-specific antihuman monoconal antibody (160112; Cayman Chemical Co., Ann Arbor, MI) was applied overnight at a dilution of 1:200 in PBS containing 0.1% sodium azide and 0.5% BSA at room temperature. Then, the sections were treated with biotinylated horse antimouse immunoglobulin (1.200; Vector Laboratories, Inc., Burlingame, CA), and antibody-binding sites were visualized by avidin-biotin peroxidase complex (Vectastain ABComplex; Vector Laboratories, Inc.) and 3- amino-9-ethylcarbazole (Sigma, St. Louis, MO). The counterstaining was performed with Mayer’s hematoxylin. Specificity of the antibody was determined by preadsorption of the primary antibody with human Cox-2 control peptide (10 µg/ml; Cayman Chemical) for 1 h at room temperature before the staining. 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. The percentage of positive staining 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, 1–19%; moderate, 20–49%; and strong, over 50% (14). The results were scored by two independent pathologists (K.S. and P.H.) who were blinded to the subtype of the tumors. The agreement between the pathologists was good in the benign ( value 0.736) and the borderline ( value 0.760) group. In the malignant group the agreement was very good ( value 1.000).

mRNA analysis

Total RNA was isolated from one normal medullary tissue and from one normal cortex, three benign adrenal, three borderline adrenal, three malignant pheochromocytomas, and two metastases. RNA isolation was performed as described previously (22). Human Cox-2 and ß-actin cDNAs were labeled using [-³²P]deoxy-CTP (NEN Life Science Products, Boston, MA) and Prime-a-Gene kit (Promega Corp., Madison, WI) (23). Probes were purified with nick columns (Pharmacia, Uppsala, Sweden) and used at 1 x 10⁶ cpm/ml. Hybridizations were performed at 60 C for 16 h in ExpressHyb Hybridization solution (CLONTECH Laboratories, Inc., Palo Alto, CA). Membranes were rinsed several times and washed three times at 50 C for 15 min each time, and two times for 20 min at 50 C in 0.1x SSC and 0.1% SDS. The relative intensities of the autoradiographic signals were quantified by densitometric scanning.

Western blot analysis

For Western blotting samples from three benign adrenal, five borderline, and two malignant pheochromocytomas as well as one metastasis were homogenized in radioimmunoprecipitation assay buffer [150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 50 mM Tris (pH 8.0)] supplemented with Complete mini protease inhibitor cocktail tablets (ROCHE Diagnostics GmbH, Mannheim, Germany). Proteins (100 µg) were transferred electrophoretically to Hybond-C membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK), and nonspesific binding was blocked by TBS-NP40–5% low-fat dry milk solution overnight at 4 C. For immunodetection the membranes were incubated with the monoclonal Cox-2 antibody (1:1000) for 1 h. Then, the membranes were washed three times in TBS-NP40, and incubated with sheep antimouse antibodies conjugated to horseradish peroxidase (1:2000; ECL Western blotting analysis system; Amersham Pharmacia Biotech) for 1 h.

Statistical analysis

Statistics was used to examine the interobserver variation in the scoring of the staining. Statistical significance in immunohistochemisty was calculated with the Mann-Whitney U test. The level of significance was chosen as P less than 0.05.

Results

The cortex of the normal adrenal glands (n = 5) was positive for Cox-2 as detected by immunohistochemistry using the monoclonal antibody (Fig. 1). The distribution and intensity of the staining were similar in all normal adrenal glands. In contrast, no Cox-2 immunoreactivity was evident in the medulla of the normal adrenals.

View larger version (99K): [in this window] [in a new window]   Figure 1. Expression of Cox-2 in normal adrenal gland, in benign, and in malignant pheochromocytoma. The top panel shows normal adrenal tissue, where all cortical zones express strong immunoreactivity while medulla is negative (magnification, x70). A benign pheochromocytoma in the middle panel shows a single positive tumor cell (magnification, x300). The bottom panel shows a malignant pheochromocytoma with strong immunopositivity for Cox-2 (magnification, x300). Immunohistochemical staining with the Cox-2 antibody was performed as described in Materials and Methods.

All malignant pheochromocytomas, regardless of their primary location, showed strong (6 of 8, 75%) or moderate (2 of 8, 25%) Cox-2 immunoreactivity (Table 3 and Fig. 1). We also studied the expression of Cox-2 in six metastatic cases. The immunoreactivity was weak in the metastases from all three adrenal tumors, whereas metastases from the extra-adrenal tumors exhibited either moderate or strong immunoreactivity.

Statistically a significant difference was found in immunohistochemical Cox-2 expression between benign and malignant adrenal tumors (P = 0.014) as well as between borderline and malignant adrenal tumors (P = 0.012). There were no statistical differences between any other groups.

The immunohistochemical expression was confirmed by Western blotting (see Fig. 2). Samples from three benign adrenal, four borderline adrenal, one borderline extra-adrenal, and two malignant pheochromocytomas as well as from one metastasis were available for analysis. A 70-kDa band corresponding to Cox-2 protein was detected in each tumor.

View larger version (64K): [in this window] [in a new window]   Figure 2. One borderline and two malignant pheochromocytomas as well as one metastasis were analyzed by using Western blotting and the monoclonal Cox-2 antibody. An approximately 70-kDa band corresponding to Cox-2 protein was detected in all samples studied.

View larger version (39K): [in this window] [in a new window]   Figure 3. A representative Northern blot showing expression of Cox-2 mRNA in normal adrenal cortex, normal medulla, benign adrenal pheochromocytoma, two malignant pheochromocytomas, and two metastases. Total RNA was extracted from frozen tissues, and the Northern blot was prepared with 20 µg RNA for each lane. The filter was sequentially hybridized with 32P-labeled Cox-2 and ß-actin cDNA probes.

This study demonstrates the absence of Cox-2 expression in normal adrenal medullary cells and its expression immunohistochemically in pheochromocytomas, particularly increased expression in malignant pheochromocytomas. Cox-2 has been associated with carcinogenesis (8, 9), and it is overexpressed in gastrointestinal carcinomas (10, 11, 12, 13, 14), as well as in other human malignancies (15, 16, 17, 18, 19, 20, 21). To our knowledge, there are no previous reports on Cox-2 expression in pheochromocytomas.

Cox-2 has been most thoroughly studied in colorectal cancers. Its expression is increased in 85–90% of human colorectal adenocarcinomas (24). In colorectal cancers, Cox-2 has been found in epithelial tumor cells, inflammatory cells, vascular endothelium, and fibroblasts (14), whereas in sporadic colorectal adenomas Cox-2 is expressed predominantly by interstitial macrophages (25). Sheehan et al. (14) demonstrated that Cox-2 overexpression in colorectal cancers correlates with more advanced Dukes’ stage, larger tumor size, and is particularly evident in tumors with lymph node involvement. Cox-2-expressing cells have also been shown to be more invasive than nonexpressing human colon cancer cells (26). Furthermore, Ristimäki et al. (21) reported that Cox-2 immunoreactivity is most prominent in invading cells of transitional cell bladder carcinoma. In this study, normal adrenal medulla showed no Cox-2 expression by immunohistochemistry. Most of the benign adrenal pheochromocytomas had no or only weak immunopositivity, whereas strongly enhanced immunostaining was seen in malignant pheochromocytomas. These results are, thus, concordant with previous studies.

Cox-2 overexpression has been reported to enhance hematogenous metastasis of human colorectal cancer (27) and lymphatic invasion and metastasis of human gastric carcinoma (28). We also studied metastases from six malignant pheochromocytomas. Interestingly, four metastases expressed weaker immunopositivity than their primary tumors, while in two cases immunopositivity remained strong. In these two cases the patients have not developed new metastases. However, the number of cases studied is too small for definite conclusions to be drawn.

The distinction between malignant and benign pheochromocytomas is difficult, and it is generally accepted that only tumors with metastases or local invasion into adjacent tissues are malignant. Linnoila et al. (4) reported that the malignancy of pheochromocytomas correlates more often with extra-adrenal location, greater tumor weight and histological features, including confluent tumor necrosis, vascular or capsular invasion, coarse nodularity, and absence of intracytoplasmic hyaline globules. We, therefore, evaluated the Cox-2 expression separately in extra-adrenal pheochromocytomas as well as in a group of pheochromocytomas that showed capsular or vascular invasion, confluent tumor necrosis, or over five mitoses per 10 high-power fields, here called borderline tumors. Five borderline adrenal tumors and most extra-adrenal tumors (83%) showed strong or moderate Cox-2 expression, like the tumors that were malignant. It is, therefore, possible that part of the tumors evaluated as borderline may in fact have had malignant potential, but were without metastases at the time of operation, and were thus treated curatively by surgery. On the other hand, the strong Cox-2 expression in extra-adrenal pheochromocytomas may be a consequence of several matters. Firstly, extra-adrenally located tumors tend to behave more aggressively (4). Secondly, extra-adrenal tumors lack the hormonal environment of the adrenal gland, which may also influence the Cox-2 expression.

Normal adrenal medulla was negative by immunohistochemistry, but showed low Cox-2 mRNA expression. This may be due to the fact that adrenal medulla is macroscopically difficult to separate from cortex, and clusters of cortical cells are sometimes situated within the medulla. Low levels of Cox-2 mRNA were detected in most of the pheochromocytomas studied, although one tumor in each diagnostic group did not show any transcript.

In summary, we found strong or moderate Cox-2 immunopositivity in all malignant pheocromocytomas, whereas most benign tumors stained weakly or negatively for Cox-2. Most pheochromocytoma samples studied also expressed low levels of Cox-2 mRNA. We, therefore, suggest that Cox-2 might have a role in malignant the transformation of pheochromocytomas.

Acknowledgments

We thank Kaija Antila, Merja Haukka, Elina Laitinen, and Sari Nieminen for excellent technical assistance.

Footnotes

This work was supported by Helsinki University Central Hospital Research Contracts TYH 0208 (to C.H.), TYH 0235 (to A.R.), and TYH 9107 (to P.H.).

Abbreviation: Cox, Cyclooxygenase.

Received February 22, 2001.

Accepted July 30, 2001.

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