Paraganglioma of the urinary bladder: Immunohistochemical, ultrastructural, and DNA flow cytometric studies

Paraganglioma of the Urinary Bladder: Immunohistochemical, Ultrastructural, and DNA Flow Cytometric Studies DAVID J. GRIGNON, MD,* JAE Y. RO, MD, BRUCE MACKAY, NELSON G. ORD6r;lEZ, MD, ADEL EL-NAGGAR, MD, THOMAS J, MOLINA, MD, DAVID T. SHUM, MB, AND ALBERT0 G. AYALA, MD Primary paraganglioma arises infrequently in the urinary bladder. We present the clinicopathologic, immunohistochemical, ultrastructural, and DNA flow cytometric findings in three cases (one man and two women). Ages at diagnosis were 19,35, and 45 years. One female presented with paroxysmal headaches and hypertension that followed urination; the remaining two patients presented with hematuria. Immunohistochemical studies revealed positive reactivity for chromogranin (three patients), met-enkephalin (three), leuenkephalin (three), vasoactive intestinal polypeptide (two), serotonin (one), and S-100 protein (one; sustentacular cells only). Neurosecretory granules were identified in all cases; in the patient with hypertension, the granules were small with eccentric cores similar to those of adrenal pheochromocytomas. A nondiploid DNA flow cytometric pattern was present in all three patients, an aneuploid pattern was present in two, and a tetraploid pattern was present in one. After diagnosis, one patient was alive without progression at 7 years, one died of an uncertain cause at 5 years, and one suffered multiple recurrences over a 24-year period before developing metastatic disease. While the presence of aneuploidy has been shown to be a predictor of malignant behavior in adrenal pheochromocytomas, our study illustrates that DNA ploidy cannot be used as a diagnostic criterion for malignancy in urinary bladder paraganglioma. HUM PATHOL 22:1162-1169. Copyright 0 1991 by W.B. Saunders Company

Paragangliomas (pheochromocytomas) are rare tumors in the urinary bladder, accounting for only approximately 0.06% of all bladder neoplasms’ and 6% of extra-adrenal paragangliomas.’ In the urinary bladder, approximately half the patients have a typical triad of hypertension (often paroxysmal), hematuria, and micturitional attacks. Previous ultrastructural and immunohistochemical studies have noted the similarities between these tumors and other extra-adrenal paragangliomas.’ Approximately 14% of urinary bladder paragangliomas are said to be malignant; however, no good pathologic criteria exist to separate these tumors from

Frcm the Dcpartmrnt of Pathology. Thr University of Texas M. D. .4nde1-son (:anccr (:entrr. Houston, TX; t’athology Consultants. Beaumont, TX and Victoria Hospital, I,ondon. Onrario. Canada. Accepted fol- publication .Janua~y 29. 199 1, *I-‘rr.rrntnddrf,sr: Ikpxtment of Patholo~, Victoria Hospital, 375 South St. London. Ontario, (Canada N6A 4Ct.5. Dr Grignon was support4 by a MrEarhct-n Award of the
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the benign neoplasms.‘,” Recent studies have suggested that DNA ploidy, as determined by flow cytometry, provides useful prognostic data for pheochromocytomas of the adrenal medulla.5 The purpose of this report is to present the clinicopathologic, immunohistochemical, ultrastructural, and DNA flow cytometric findings in three cases of urinary bladder paraganglioma. CASE REPORTS Case No. 1 A 45-year-old woman presented to her family physician with complaints of occasional pounding headaches that were worsened after urinating, sometimes to the point of causing her to faint. Hematuria was also present. At cystoscopy, nrultiple submucosal nodules, measuring up to 2 cm, were noted and biopsied. Immediately following biopsy. an acute hypertensive crisis (blood pressure, ?10/140 mm Hg) developed. The patient underwent partial cystectomy and remains well with no evidence of recurrent disease 7 years later. Case No. 2 A 35-year-old man presented with hematuria. Clinical information is scant, and it is not known whether he was hypcrtensive or had symptoms related to micturition. A L’.5-cn1 polypoid mass was found at cystoscopy and a partial cystrctonry was performed. The patient remained well for 5 years with no evidence of local recurrence. He then developed multiple pulmonaI_): nodules, and a primary bronchogenic carcinoma was clinically diagnosed. The patient subsequently diecl and an autopsy was not performed.

Case No. 3 A 1%year-old woman presented with hematuria; no hypertension was reported. A tumor was found in the bladder and was treated by transurethral resection. Local recurrences developing 1, 9, 12, 16, and 18 years after the initial surgery were also managed by trdnsurethrdl resection. Twenty-four years after the initial ‘presentation, a right-sided pelvic mass developed, and computed tomography demonstrated paraaortic lymphadenopathy and tumor involvement of the right bladder wall with extension into perivesical adipose tissue. ‘“‘Imetaiodobenzylguadinine (MIBG) scintigraphy demonstrated uptake in and around the bladder. At cystoscopy. four cherryred nodular lesions, ranging from 0.5 to 1 .O ~11 in size. were seen. Laparotomy revealed a mass on the right side of the bladder extending around the internal iliac blood vess&, peritoneal seeding, and enlarged para-aortic lymph nodes. A 3-month course of chemotherapy with Adriamycin (doxorubicin; Adria I.aboratories, Columbus. OH) and dacarbazinr

PARAGANGLIOMA

FIGURE 1. x100.)

Photomicrograph

OF THE URINARY BLADDER (Grignon et al)

of tumor from case no. 1 illustrating typical “zellballen”

(1X1(:, Miles I’h;~t~t~~a~eutic-als.West Haven, CT) W;IS given. The patient remained alive with stable disease 9 months after c.essation of c-hrmothet-spy.

MATERIALS

AND METHODS

Light Miicroscopy FW ILISCS no. 1 and 2, paraffin-embedded, formalin-fixed tissue from the partial cystectomy specimens were available. In addition, wet tissue, fixed in 10% neutral-buffered formalin, was available for case no. ?. Materials available from case no. 3 included paraffin-embedded, formalin-fixed tissue from the recurrences at 16 and 18 years and biopsy specimens from the bladder and para-aortic lymph nodes at the most recent admission. Cellular feat.ures observed by light microscopy were evaluated from hematoxylin-eosin-stained sections and included the presence or absence of typical small nests (“zellballen”). nuclear pleomorphism, and mitotic activity. Nuclear pleomorphism was judged to be present if nuclei were seen that measured at least three times the size of adjacent nuclei; pleomorphism was considered mild if it involved less than 10% of cells, model-ate if it involved 10% to 30% of cells. and severe if it involved more than 30% of cells. Mitotic rates were evaluated by counting 40 consecutive high-power fields (HPFs; X 10 ocular. X40 objective) and were expressed as the number of mitoses per- 10 HPFs.

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separated

by vascular septa. (Magnification

lmmunohistochemistry Immunohistochemical studies were carried out using the avidin-biotin peroxidase complex (ABC) method of Hsu et al.” Antibodies against the following antigens were used: chromog-ranin (mouse monoclonal; Boehringer Mannheim Biochemicals. Indianapolis, IN; dilution, 1:40). neurofilaments (mouse monoclonal; Boehringer Mannheim; dilution, 1:50), met-enkephalin (rabbit polyclonal; Incstar. Indianapolis, IN; dilution, 1: 1,000). leu-enkephalin (rabbit polyclonal; Incstar; dilution 1: 1 ,OOO), vasoactive intestinal polypeptide (VIP) (rahl:SOO), serotonin (rat monobit polyclonal; Incstar; dilution, clonal; Bioproducts for Science, Inc, Indianapolis, IN; dilution, 1: 1,500). somatostatin (rabbit polyclonal: Dako (:orp. Santa Barbara, CA; dilution, 1:3), calcitonin (rabbit polyclonal; Immunonuclear Corp. Stillwater, MN; dilution, 1 :!!OO), S-l 00 protein (rabbit polyclonal; Dako; dilution, 1:700), and cytokeratin (mouse monoclonal [AEI/AIS]; !Bochringer Mannheim; dilution, 1:400).

Electron Microscopy In case no. 1, tissue for electron microscopy was recovered from the paraffin blocks. In case no. 2. for-malin-fixed tissue was transferred to 4% glutaraldehyde and processed in the usual manner. In case no. 3, fresh tissue from the bladder and para-aortic lymph node biopsies at the most recent admission was fixed in 4% glutaraldehyde.

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FIGURE 2.

Photomicrograph

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1991)

of tumor from case no. 3 (1988) showing enlarged pleomorphic

DNA Flow Cytometry In each case, DNA flow cytometry was performed on from one to three paraffin-embedded blocks of formalin-fixed tissue using a modification of the method of Hedley et al7 as previously descrihed.8 Analysis was performed on an Epics Profile flow cytometer (Coulter Corp, Hialeah, FL). Single-parameter histograms were used in evaluating the tumors. The first distinct peak was assumed to represent the GO/Cl diploid peak. DNA nondiploidy was defined by the presence of a distinct second peak to the right of the first GO/ Gl peak. The half-peak coefficient of variation was calculated for all GO/G1 peaks, as was the DNA index for all nondiploid populations. RESULTS

nucleus. (Magnification

x400.)

nodes at the most recent admission showed moderate pleomorphism and 5 mitoses/lO HPFs (Fig 2). lmmunohistochemistry All cases showed strong reactivity for chromogranin in the majority of cells (Fig 3); in addition, all cases showed at least some reactivity for both met-enkephalin and leu-enkephalin. Reactivity for VIP was seen in two cases and was particularly intense in case no. 2. Serotonin reactivity was present in only case no. 3, but was diffuse and strong. Reactivity for S-100 protein was only found in case no. 3 and was limited to sustentacular cells (Fig 4). No reactivity was present for cytokeratin, somatostatin, calcitonin, or neurofilaments in any of the cases.

Light Microscopy All cases demonstrated the classic histologic features of paragangliomas, with cells arranged in zellballen, separated by vascular septa (Fig 1). Individual cells were characterized by abundant eosinophilic cytoplasm and central nuclei. All cases showed some degree of nuclear pleomorphism and mitotic activity. In case no. 3, specimens obtained 6 and 8 years prior to development of overt malignant behavior showed minimal pleomorphism and 0 mitoses/lO HPFs. In contrast, material obtained from both the bladder and para-aortic lymph 1164

Electron Microscopy Ultrastructural findings in cases no. 1 and 2 were virtually identical but differed from those in case no. 3. In cases no. 1 and 2, the cells had regular, round-tooval nuclei with dispersed chromatin and small nucleoli. Occasional, poorly formed junctions were seen, but desmosomes were not found. The cytoplasm contained scattered mitochondria and abundant membranebound, dense core granules. These granules were characterized by the eccentric location of the dense cores

PARAGANGLIOMA

FIGURE 3. lmmunoperoxidase stain for chromogranin tumor cells. (Magnification x400.)

OF THE URINARY BLADDER (Grignon et al)

in case no. 3 demonstrating

(Fig 5). In case no. 3, the tumor cells had more abundant cytoplasm with prominent cell processes, many containing microtubules. Cell junctions were more numerous and better formed, hut again, no desmosomes were present. The neurosecretory granules were fewer in number and showed a considerable degree of variation in size and shape. Electron-dense cores were not seen. DNA Flow Cytometry The DNA histograms in cases no. 1 and 2 demonstrated the presence of aneuploidy with DNA indices of 2.33 and 1.31, respectively. In case no. 3, the tissue from the resections performed 6 and 8 years prior to the onset of malignancy was diploid. In contrast, the most recent bladder biopsy specimens showed a tetraploid pattaern. with 22.8% of cells in the &+M region (DNA indlex, 2.0!1) (Fig 6). DISCUSSION In 1953. Zimmerman et al” reported the first case ot‘ pheocl”-o~~,oc~toma arising in the urinary bladder. Since then. well over 100 cases have been reported in the English literature.“.‘.“‘.” Zimmernran et al” believed such t u’11ors arose from paraganglionic tissue

intense cytoplasmic

reactivity in virtually all the

present within the wall of the urinary bladder; the presence of paraganglionic tissue within the bladder wall has been demonstrated recently in three cases, with immunohistocl~emical confirmation.” The tumors may occur over a wide age range, from childhood to old age, and are found roughly equally in males and females.4~“’ Clinically, patients often present with symptoms related to catecholamine excess (headache, tachycardia, dizziness, fainting, and hypertension) and, in many, the episodes are associated with micturition. Hematuria is relatively common, and episodes of paroxysmal hypertension with painless hematuria in association with micturition are practically pathognomanic.’ In most cases, diagnosis may be confirmed by measurement of catecholamines and their metabolites in serum and urine. In addition, ““I-MIBC; scintigraphy has been shown to confirm localization of the lesion within the bladder in these cases.“’ In most cases, the clinical course is benign. Davaris et al’ collected 86 welldocumented cases from the literature and li,und lymph node metastases to have occurred in 12! cases (13.8%) and distant metastases in only two cases (2.3%). Such metastases can develop years after the initial diagnosis and treatment.“.” Our three cases occurred in two women and one man aged 19. 45, and 35 years. Hematuria was present

HUMAN PATHOLOGY

FIGURE 4. lmmunoperoxidase (Magnification x400.)

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stain for S-100 protein in case no. 3 illustrating

in all three patients, and the classic history of paroxysmal hypertension associated with micturition was present in one. Measurements of catecholamines and metabolites were available only for the last admission in case no. 3 and were normal at that time. A ‘“‘I-MIBG scan performed in that patient confirmed localization to the vesical tumor. Two patients were treated by partial cystectomy: one patient remained alive and well after 7 years and the other died of an apparent bronchogenic carcinoma after 5 years. A paraganglioma in the third patient was managed initially by transurethral resection, as were subsequent local recurrences over an l&year period. Frank malignant behavior developed 24 years after the initial resection, with extensive local invasion and peritoneal and lymph node metastases. Pathologic predictors of behavior for these neoplasms are not well defined.“.” Necrosis, vascular invasion, and the presence of mitoses have been associated with a more aggressive clinical course in extra-adrenal paragangliomas in general,‘” but studies have not specifically looked at these features in urinary bladder tumors. In our cases, no association was seen between histologic features and malignancy; it is of interest to note, however, that in the case which ultimately showed malignant behavior, tissue specimens from two earlier occurrences had shown no mitotic activity, while tissue 1166

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intense reactivity

limited to sustentacular

cells.

specimens obtained at the time the aggressive course developed showed a mitotic count of 5/10 HPFs. Kliewer et alI7 indicated that the presence of SLIStentacular cells, identified by either immunohistochemistry or electron microscopy, seemed to indicate a benign clinical course. In our “malignant” case, numerous sustentacular cells were present, a feature also noted by Korat et all8 in one of three malignant extra-adrenal paragangliomas (none from the urinary bladder). In 1971, Lewis”’ studied 15 adrenal pheochromocytomas by quantitative Feulgen cytophotometry and reported that tumors with a high percentage of hyperdiploid cells behaved in a malignant manner in contrast to those with predominantly diploid nuclei. More recently, DNA flow cytometric studies have yielded conflicting results.“.“’ In the largest study to date, Hosaka et al5 found that the presence of tetraploidy/polyploidy or aneuploidy was a significant predictor of malignant behavior in adrenal pheochromocytoma. In the current series, an aneuploid DNA pattern was obtained in two cases; in one of these cases, the patient was alive and well at 7 years after diagnosis and in the other the patient died of an apparently unrelated cause at 5 years after diagnosis. In the third case, a diploid pattern was seen in two resections done 6 and 8 years prior to the discovery of the tumor, demonstrat-

PARAGANGLIOMA

OF THE URINARY BLADDER (Grignon et al)

FIGURE 5. (Top) Electron micrograph of tumor from case no. 1 illustrating abundant dense core granules in the cytoplasm, rE?gular oval nuclei, and absence of desmosomes. (Bottom) Higher magnification illustrating neurosecretory granules with eccentric (jense cores.

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FIGURE 6. (Left) Single-parameter DNA histogram from tumor in case no. 3 (1982) illustrating a diploid DNA pattern. (Right) Singleparameter DNA histogram from tumor in 1988 now having a tetraploid DNA pattern, with 22.8% of the cells failing in the G2+M region (DNA index, 2.09).

ing malignant behavior. Tissue resected at the time metastases developed showed a tetraploid DNA pattern. Although our data are based on a small series, it appears that DNA ploidy is not a useful predictor of malignancy in urinary bladder paragangliomas. Immunohistochemical studies have been reported for at least six cases of urinary bladder paragangliomas.?l-‘~ All cases studied have shown reactivity for neuron-specific enolase; other neuroendocrine markers that have been looked for include chromogranin (two of three cases), leu-enkephalin (one of one), enkephalin (none of two), neuropeptide (one of two), serotonin (four of four), calcitonin (two of three), somatostatin (three of three), gastrin (two of three), and glucagon (two of three). S-100 protein reactivity has been found in four of four cases studied; in these studies, positivity was not restricted to sustentacular cells. All cases tested were nonreactive for cytokeratin, carcinoembryonic antigen, and epithelial membrane antigen. Our results are, for the most part, in keeping with these studies; one exception, however, was the presence of S-l 00 protein positivity in only one of our cases and its limitation to sustentacular cells. This result more closely reflects the experience with this antibody and paragangliomas as discussed in the literature in general.” Ultrastructural findings have been described in at least five cases of urinary bladder paraganglioma4*22-‘4 and have for the most part been similar to findings observed in paragangliomas arising in other sites.24 All of these studies have reported the presence of dense core granules. In two of our cases, these granules were typical of adrenal catecholamine-secreting tumors with eccentric dense cores. In the third case, the granules showed considerable pleomorphism, a feature that has been associated by some investigators with a more aggressive clinical course. All tumors in our series showed at least a few cell junctions, but desmosomes were not found. 1168

In summary, we have presented the clinicopathologic, immunohistochemical, ultrastructural, and DNA flow cytometric features of three urinary bladder paragangliomas, one of which demonstrated malignant behavior. None of the features we studied, however, appeared to be of predictive value for these rare neoplasms. Acknowledgnmt.

excellent

secretarial

The

authors thank Mona Poitras for her

assistance.

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OF THE URINARY BLADDER (Grignon et al)

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