INTRODUCTION

Pheochromocytomas and abdominal paragangliomas are catecholamine-producing tumors arising from sympathetic paraganglia located within and outside, respectively, of the adrenal medulla (1, 2). Less than 10% of sporadic pheochromocytomas and 15–35% of abdominal paragangliomas are malignant (1). The hereditary forms of the disease, which constitute approximately 10% of cases, are usually benign (3). Clinical and histopathological distinction between benign and malignant lesions is difficult, and reliable diagnostic and prognostic markers are lacking (2, 4). Today the only indisputable proof of malignancy is the presence of metastasis at the time of diagnosis or during follow-up (5). Because metastases may occur up to decades after primary surgery (6), lifelong follow-up of all patients has been advocated (7, 8). Hence, other classification systems and new markers of malignancy are required to identify the patients who are likely to follow a benign course and select patients requiring closer follow-up and an individualized management program.

Telomerase is an enzyme complex that stabilizes the chromosomes by preventing the normal shortening of telomeric structures with each cell division. It is present in germ-line tissues and stem cells but is not expressed in most normal somatic cells. Activation of telomerase is considered a crucial step for malignant cells to become immortal. Human telomerase reverse transcriptase (hTERT), the catalytic part of the complex, is the major determinant for activation of telomerase (9). Telomerase activity (TA) has been found to be a sensitive marker of malignancy in almost all cancer types investigated, including most endocrine tumors (9, 10, 11). However, little is known about its potential diagnostic and prognostic implications in pheochromocytomas and abdominal paragangliomas.

The Ki-67 antigen is a nuclear protein that is abundantly expressed in G1 through S phase of the cell cycle but is rapidly degraded after mitosis (12). Ki-67 expression can be detected with the monoclonal antibody MIB-1 and is used as a marker of proliferative activity. It has been suggested as a predictor of malignancy in many cancer types, including pheochromocytoma (13, 14, 15, 16, 17).

Matrix metalloproteinases (MMP) are a group of proteases that play important roles in the processes of tumor invasion and metastasis (18). Extracellular matrix metalloproteinase inducer (EMMPRIN) is expressed on the tumor cell's surface and enhances the fibroblast synthesis of MMP-2, which is involved in matrix remodeling and cell migration (19, 20). Overexpression of various MMPs, particularly MMP-2, is correlated with poor prognosis in many cancer types including adrenocortical cancer, breast cancer, and thyroid malignancies (18, 21, 22, 23) but has until now been studied only to a very limited extent in pheochromocytomas (16).

The aim of this study was to evaluate hTERT expression, telomerase activity, proliferative activity, and mRNA expression of EMMPRIN and MMP-2, as prognostic markers of malignancy, in pheochromocytomas and abdominal paragangliomas.

MATERIALS AND METHODS

Patients and Tumor Samples

Twenty-one pheochromocytoma and 13 abdominal paraganglioma samples were obtained from 32 patients undergoing surgery between 1985 and 1997 at the Karolinska Hospital. All patients were subsequently followed until death or until June 2001. The hospital's ethics committee approved use of the tumor tissue for this study.

All tumor samples were histopathologically reevaluated by one of the authors (AH). The classification of the tumors was based on their localization within or outside the adrenal glands and their morphological pattern. By immunohistochemistry, all tumors were shown to be positive for neuroendocrine markers such as chromogranin A and synaptophysin (2). Malignancy was defined according to the criteria published by the Armed Forces Institute of Pathology (AFIP), that is, presence of metastasis and/or extensive local invasion (2). Three samples of normal adrenal medulla from patients operated on for renal carcinoma were included as controls.

The clinical data are detailed for each case in Table 1. Twenty-one patients were classified as having benign tumors (Group A), including one patient with multiple, well-circumscribed, noninvasive paraganglioma in the urinary bladder, in whom a further local tumor was detected after 42 months. Six of the patients with benign tumors had a familial form of pheochromocytoma. The 11 primary tumors classified as malignant based on AFIP criteria could be divided into three groups: Group BI, comprising three tumors from patients with no evidence of metastasis or local recurrence during follow-up (two of these patients were followed for 68 and 78 mo, respectively, whereas the third patient died 11 mo after operation from a nonrelated cancer); Group BII, comprising three tumors from patients who developed metastasis and/or local recurrence at 36, 41, and 87 months respectively, from the time of initial surgery; and Group BIII, consisting of five tumors from patients with known metastases at the time of diagnosis. From two patients with malignant paraganglioma, both the primary tumor and metastatic tissue, removed at 40- and 49-month intervals, respectively, were analyzed (Group C).

TABLE 1 - Clinical Data for the 32 Patients with Pheochromocytoma or Abdominal Paraganglioma.

Full table

Fresh-frozen tumor material was used for analyses of hTERT expression and telomerase activity. Routinely formalin-fixed, paraffin-embedded, 5-m sections of the same tumors were used for immunohistochemical analysis of Ki-67/MIB-1 immunoreactivity and mRNA in situ hybridization for EMMPRIN and MMP-2 expression. Fresh tumor samples were snap-frozen in liquid nitrogen immediately after surgical removal and then stored at -70° C. To ensure that the tumor samples were suitable for TA and hTERT analyses, representative sections were cut from all specimens and subjected to histopathological examination, which confirmed that the proportion of tumor cells was 70% in all cases.

RNA Extraction and RT-PCR

Total cellular RNA was extracted using the ULTRASPEC-II RNA kit (Biotecx Lab., Houston, TX). cDNA synthesis and polymerase chain reaction (PCR) for hTERT mRNA was performed essentially as described elsewhere (24). In brief, PCR was carried out using cDNA derived from 100 ng of total RNA in 25-L reactions containing 1 PCR buffer, 2.5 mm MgCl₂, 0.2 mm of each dNTP, 0.1 m of each primer, and 0.625 U Taq DNA polymerase. The following thermocycling conditions were applied: an initial denaturation at 94° C for 3 minutes was followed by 35 step cycles of denaturation at 94° C for 45 seconds, annealing at 60° C for 60 seconds, and elongation at 72° C for 90 seconds, with a final extension at 72° C for 5 minutes. Amplification of ₂-microglobulin (M) was used as a control for RNA presence and quality. Primer sequences for ₂-M and hTERT were as described (24). PCR products were resolved in 2% agarose gels, stained with ethidium bromide, visualized under UV light and photographed. RNA derived from leukemic HL-60 cells and H₂O were included in each PCR run as positive and negative controls, respectively.

Telomerase Activity Assay

A commercial telomerase PCR enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics Scandinavia AB, Stockholm, Sweden), based on the telomeric repeat amplification protocol (TRAP assay), was used to determine TA in all the samples according to the manufacturer's protocol, using 28 PCR cycles (25). The optical density (OD) level was determined to 0.09–0.11 for the clearly negative control samples: three normal adrenal medulla specimens, differentiated DMSO-treated HL-60 cells, and cultured normal human fibroblasts. As positive control, HL-60 leukemic cells, giving an OD level of 1.1, were used. Tumor samples with an OD value of >0.18 (2 that of the negative control) were scored as positive for telomerase activity.

Immunohistochemistry

Immunohistochemical analyses of the proportion of Ki-67/MIB-1 immunoreactive cells were performed using the Avidin-Biotin-Complex method (Vectastain; Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Paraffin sections 5 m thick were incubated overnight at +8° C with monoclonal antibodies against Ki-67 (clone MIB-1, diluted 1:150; Immunotech, Marseille, France). To intensify the immunoreaction, the sections were heated in citrate buffer, pH 6.0, during two 5-minute periods in a microwave oven at 300W, before incubation with the antibody. Known positive samples of breast cancer tissue were included as positive controls. As negative controls, the antibodies were replaced by albumin in the incubation step for one tissue section.

The proliferative activity, given as the percentage of Ki-67/MIB-1 immunoreactive cells, was calculated with the aid of an 10 10–square ocular grid. To estimate the cell density of each tumor section, the total number of cells (positive and negative) was counted in one row of the grid (10 squares) and multiplied by 10. Then the total number of immunoreactive cell nuclei was counted within the whole grid, and the procedure was repeated at 10 different locations in the areas with the highest proliferative activity. In this way, a minimum of 2400 cells was counted in all tumor samples. The calculations were done by two independent observers.

mRNA In Situ Hybridization

Full-length cDNA of EMMPRIN, as well as MMP-2, subcloned into Bluescript transcription vectors, was generously supplied by Huiming Guo (19) and Gregory I. Goldberg (26). The clones were linearized by appropriate restriction enzymes for in vitro transcription using Riboprobe System components from Promega (Madison, WI) and ³⁵S-UTP (10 mCi/mL, Amersham Pharmacia Biotech Inc, Piscataway, NJ) to form antisense- and sense-labeled RNA probes. The probes, EMMPRIN (1.6 kb) and MMP-2 (1.2 kb), were then purified by ultrafiltration (Microcon 100; Amicon, Inc., Beverly, MA) and generated at a specific activity of 2.1–3.1 10⁶ cpm/L and 1.4–2.9 10⁶ cpm/L, respectively. Sense RNA probes were used for all preparations as negative controls. As positive controls, hybridization to adrenocortical cancer tissue was performed, and -actin probe hybridization verified tissue mRNA presence.

The hybridization procedures used in this study were essentially as previously described (22, 23). After labeling, the probes were applied to paraffin-embedded tissue sections of 5 m and hybridized at 55° C overnight. The slides were then washed, with the most stringent step being 0.1 SSC for 15 minutes at 60° C, and treated with RNAse. After being dipped in autoradiographic emulsion, the slides were exposed at 4° C for 14 days, developed, and counterstained with hematoxylin and eosin. The slides were evaluated semiquantitatively by two independent observers (22).

Statistical Analyses

Statistical analyses were performed using StatView 4.5 software for Windows (Abacus Concepts, Inc., Berkeley, CA). Probabilities of <.05 were accepted as significant. Diagnostic accuracy was defined as the percentage of benign tumors showing no expression and of malignant tumors with positive expression per the total number of cases examined; sensitivity as the percentage of malignant tumors with positive expression per the total number of malignant tumors; specificity as the percentage of benign tumors showing no expression per the total number of benign tumors; positive predictive value as the percentage of malignant tumors with positive expression per the number of both benign and malignant tumors with positive expression; and negative predictive value as the percentage of benign tumors showing no expression per the number of both benign and malignant tumors showing no expression. In these calculations, the three tumors classified as malignant on AFIP criteria but without proven metastasis and/or local recurrence (Group BI) were excluded because of the uncertain clinical outcome of these tumors.

RESULTS

HTERT Gene Expression and TA

In total, 11 of the 34 tumor samples were positive for hTERT (one only weakly positive), and 6 samples were positive for TA (Table 2; Fig. 1). Seven of 11 (64%) primary tumors classified as malignant were positive for hTERT expression. Of the five tumors with known metastases at the time of diagnosis, four were hTERT positive, as well as two of the three tumors with metastases or local recurrence during follow-up (75% of the unequivocally malignant tumors). In comparison, 2/21 (10%) benign tumors were positive for hTERT expression. Telomerase activity (TA) was detectable in three malignant and one benign primary tumors (all positive for hTERT expression). Both samples from metastatic tissue were positive for hTERT expression and TA. hTERT was expressed in 4 of 21 pheochromocytomas and 5 of 11 paragangliomas. All three samples of normal adrenal medulla were negative for hTERT expression and TA.

FIGURE 1.

Agarose gel electrophoresis of RT-PCR products obtained at the screening for hTERT expression in pheochromocytomas and abdominal paragangliomas. Representative benign tumors (Cases 3 and 9) without detectable hTERT mRNA are shown together with two strongly positive malignant tumors (Cases 32 and 30). The positive control for hTERT constitutes of the leukemia cell-line HL-60, and amplification of -microglobulin serves as a positive control of mRNA presence and quality.

Full figure and legend (33K)

TABLE 2 - Results from Analysis of hTERT Expression, Telomerase Activity (TA), Ki-67 Immunoreactivity and mRNA Expression of EMMPRIN and MMP-2.

Full table

MIB-1/Ki-67 Immunoreactivity

All of the benign tumors showed <1% proliferative activity, as measured by Ki-67/MIB-1 staining (Table 2; Fig. 2). Three of 11 (27%) primary tumors classified as malignant showed significantly increased proliferative activity (16–29% positive staining). All 3 were from the 8 patients with proven metastases or invasive local recurrence (38%). Both samples of metastatic tissue showed increased proliferative activity at the same level as that of the primary tumors. All cases with elevated proliferative activity were diagnosed as malignant paraganglioma. None of the three samples of normal adrenal medulla showed an increased proliferative activity.

FIGURE 2.

Immunohistochemical staining with MIB-1 antibodies directed against the proliferation associated Ki-67 antigen in a benign pheochromocytoma (top, Case 9) and a malignant paraganglioma (bottom, Case 33). The MIB-1 index is <1% in the benign tumor (arrow indicates a single positive cell) and is 29% in the malignant tumor.

Full figure and legend (63K)

In the three patients with tumors classified as malignant who have not developed metastasis and/or local recurrence (Group BI), no tumors showed elevation of either proliferative or telomerase activity, whereas one tumor was positive for hTERT gene expression. In all three patients who developed metastases and/or invasive local recurrence during follow-up (Group BII), the tumors were positive for either hTERT expression or Ki-67/MIB-1 immunoreactivity. In one patient with malignant metastatic disease, the tumor was negative for all markers analyzed.

Metalloproteinase Expression

mRNA expression of the metalloproteinase EMMPRIN was detected in 29/31 tumors analyzed (Table 1; Figs. 3, 4). In 7 tumors, expression was detected in single cells (+); in 10, in a moderate number of cells (++); and in 12, in the vast majority of cells (+++). mRNA expression of the metalloproteinase MMP-2 was detected in 20/31 tumors analyzed. In 5 tumors, expression was detected in single cells (+); in 8, in a moderate number of cells (++); and in 7, in the vast majority of cells (+++). The EMMPRIN expression was seen in both stroma and tumor cells, whereas the expression of MMP-2 was most frequently found in stroma cells.

FIGURE 3.

Example of mRNA in situ hybridization analyses showing expression of EMMPRIN in the vast majority of cells (+++; top) in Case 26. Hybridization with sense probe was used as a negative control (bottom). The hybridization signals appear as white spots in dark-field microscopy (left) and as black spots in light-field microscopy (right).

Full figure and legend (65K)

FIGURE 4.

Summary of expression pattern of the metalloproteinases EMMPRIN and MMP-2. - = no expression detected; + = expression detected in a single cell; ++ = expression detected in a moderate amount of cells; +++ = expression detected in the vast majority of cells.

Full figure and legend (43K)

Predictive Value

There was a significant difference in hTERT expression between benign and unequivocally malignant tumors (Group BII and BIII; P < .002; ² analysis, P < .03; Fisher's exact test). However, TA was not a significant discriminator with the number of samples included. Proliferative activity did also significantly discriminate between benign and malignant tumors (P < .02; ², P < .05; Fisher's exact test). There was no significant difference in the level of expression of MMP-2 between benign and malignant tumors. Clearly malignant tumors did show a significantly higher level of EMMPRIN expression than benign tumors (P < .03; ANOVA), but also approximately half of the benign tumors showed a high expression. The accuracy, sensitivity, specificity, negative and positive predictive values for hTERT gene expression, Ki-67/MIB-1 expression, and EMMPRIN are listed in Table 3.

TABLE 3 - Predictive Values of hTERT, Ki-67, and EMMPRIN Expression in Clearly Malignant and Benign Pheochromocytomas and Abdominal Paragangliomas.

Full table

DISCUSSION

Unlike the case with many other tumor types, the distinction between benign and malignant pheochromocytomas and abdominal paragangliomas is often difficult. There are no reliable distinguishing clinical parameters, except that paragangliomas are more often malignant than pheochromocytomas (1, 6, 8, 27, 28). Histopathological features usually associated with malignancy, such as nuclear atypia, pleomorphism, vascular invasion, and mitotic activity, may be seen in benign as well as malignant lesions (2, 4). Therefore, it has been suggested that the presence of distant metastasis would be the only criterion for malignancy in this tumor type (5). An alternative classification is the one published by the AFIP, in which extensive local invasion is recognized as a criterion for malignancy in addition to the presence of metastasis (2).

Is lifelong follow-up necessary for all patients with catecholamine-producing tumors? The reported recurrence rate of clearly benign tumors varies between 0 and 8%, in part depending on how malignancy is defined (7, 8, 29, 30). In a recent retrospective review of 85 consecutive patients with pheochromocytomas and abdominal paragangliomas who underwent surgery at the Karolinska Hospital between 1976 and 1999, none of the 71 patients with tumors initially classified as benign using AFIP criteria had recurrence after a median follow-up time of 144 months (range, 7–287; 28). However, some tumors classified as malignant on the basis of extensive local invasion have not recurred after a long period of follow-up. Markers that might provide additional prognostic information to guide individualized follow-up and treatment would be valuable.

To date there have been a number of attempts to develop useful prognostic markers without great success. Conflicting results have been reported for DNA flow cytometry (13, 31, 32), overexpression of the proto-oncogene products c-erb B-2 and bcl-2 (a marker of apoptosis; 16, 33, 34), mutations in the tumor suppressor gene p53 and its product (14, 33, 35), elevated plasma neuropeptide Y levels (36, 37), elevated urinary dopamine levels reflecting an immature secretion pattern (27), and absence of S-100 sustentacular cells (16, 17). Immunohistochemical staining for the cell adhesion molecule E-cadherin (14), the oncogene product of HER-2/neu (14), PCNA (a marker of proliferative activity; 13), somatic mutations in the RET proto-oncogene (17), and angiogenesis assessed by high microvascular count (15) seem to have no potential diagnostic utility in this tumor type. On the other hand, topoisomerase alpha II, a cell cycle–related protein that correlates with Ki-67 expression (14), and loss of inhibin/activin betaB-subunit expression may prove to be of diagnostic value but need further evaluation (38).

TA previously has been assessed in a small number of pheochromocytoma and paraganglioma samples and in normal adrenal medulla (39, 40, 41). In a study by Kubota et al. (39) using the AFIP criteria for malignancy, TA was detected in 3/3 malignant but not in 16 benign pheochromocytoma nor in normal adrenal medulla. We found that TA was more frequently elevated in malignant pheochromocytomas and abdominal paragangliomas than in benign ones, but TA could not be used to accurately discriminate between malignant and benign tumors. However, TA was negative in all 3 tumors classified as malignant that did not metastasize during follow-up (Group BI). On the other hand, hTERT expression was significantly associated with malignancy in this study. Of the 8 primary tumors with metastases at the time of diagnosis or during follow-up, hTERT was positive in six. The one hTERT-positive tumor with no recurrence was followed for only 12 months because of death from intercurrent disease. As for its negative predictive value, hTERT was negative in 4 tumors classified as malignant. One of these had metastasized at the time of diagnosis and was thus clearly falsely negative. The remaining three did not metastasize, with follow-up periods of 68 and 79 months for two and with an invasive local recurrence at 87 months in the third patient. This suggests that as for TA, hTERT-negative tumors, classified as malignant on AFIP criteria, may follow a less aggressive course with later or absent recurrence.

Four cases showed positive hTERT gene expression but no elevation of TA. Alternatively spliced hTERT transcripts or unbalanced levels of expression and/or posttranscriptional modifications of telomerase subunits could account for enzyme inactivity and thus explain those discrepancies. hTERT expression may also represent an earlier event than reactivation of telomerase enzyme in tumorigenesis. If contaminating RNAses remain in the protein extracts, this can lead to degradation of the telomerase protein-RNA complex and subsequently give false-negative results on the TA assay. Furthermore, contaminating hemoglobulin from red blood cells may function as PCR inhibitors. Therefore, determination of hTERT expression may be a more sensitive method for detecting telomerase activation in pheochromocytomas and abdominal paragangliomas than the enzyme activity assay.

The Ki-67 antigen is expressed in the proliferative phases of the cell cycle. Because the monoclonal antibody MIB-1 recognizes a formalin-resistant epitope of Ki-67, it can be used in routinely fixed, paraffin-embedded tissue (42). Ki-67/MIB-1 immunostaining has become clinically relevant for other endocrine tumor types in situations where discrimination between benign and malignant tumors is challenging (17). For example, in the investigation of pituitary tumors and their potential aggressive/invasive behavior, a cutoff level of proliferative activity has even been adapted in a recent World Health Organization classification of endocrine tumors (5). A few previous studies of MIB-1 staining in pheochromocytoma have shown promising results (13, 14, 15, 16), including a large series of 110 cases reported by van der Harst et al. (17) in which 18/36 metastasizing, malignant pheochromocytomas, as compared with none of the benign, exhibited a proliferative activity greater than 2.5%. In this study, we found increased proliferative activity in 3 of 11 primary malignant tumors (defined by AFIP criteria) and 2 metastatic specimens but not in any of the benign tumors. All three MIB-1–positive tumors were among the 8 malignant tumors with proven metastasis and/or local recurrence. This association was similar to our TA and hTERT expression results but was more sensitive for benign tumors and nonrecurrent malignant tumors. The Ki-67 antigen is very sensitive to fixation and prolonged storage, which could explain failure of detecting elevated Ki-67/MIB-1 expression levels in some malignant tumors (15). Also, because pheochromocytomas are generally slow-growing tumors, a substantial proportion of malignant cells might not be in a proliferating cell cycle phase at the time of tumor removal.

The value of EMMPRIN and MMP-2 as predictive markers was limited. In this study, MMP-2 was expressed in the majority of tumor samples examined without correlation to malignancy. This is in agreement with a previous immunohistochemical study of collagenase IV and cathepsin B and D (all substances capable of degrading extracellular matrix) that indicates that metalloproteinases are important in the tumorigenesis of pheochromocytoma and paraganglioma but do not predict malignancy (16). EMMPRIN was able to distinguish significantly between benign and malignant tumors but was present in all malignant tumors except one, whether or not recurrence occurred during the period of follow-up.

In conclusion, telomerase activation as measured by hTERT expression, increased proliferation rate, and EMMPRIN expression are all significant discriminators between malignant and benign pheochromocytomas and abdominal paragangliomas. Tumors defined on AFIP criteria as benign generally do not recur. Absence of Ki-67/MIB-1 immunoreactivity and hTERT expression is useful as supportive evidence for a benign course in this group and distinguishes those malignant tumors on AFIP criteria that are less likely to follow an aggressive course. The combined use of Ki-67/MIB-1 and hTERT may become a valuable diagnostic addition for pheochromocytomas and abdominal paragangliomas.

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Acknowledgements

We thank Prof. Bertil Hamberger for insightful comments on the manuscript and Lisa Åhnfalk for valuable technical assistance. This study was financially supported by grants from the Swedish Cancer Foundation, the Swedish Medical Research Council, the Cancer Society in Stockholm, the Torsten and Ragnar Söderberg Foundations, the Stockholm County Council and 'Förenade Liv' Mutual Group Life Insurance Company, Stockholm, Sweden.