Acta histochem. (lena) 96, 303 - 308 (1994) Gustav Fischer Verlag Jena . Stuttgart· New York

Acla hislocheMica

Unusual immunostaining pattern of chromogranin in normal urothe­lium and in transitional cell neoplasms

Kien T. Mail, D. Garth Perkinsl, William Parks2, Peter Rippstein2 and lame Farmer2

1 Anatomical Pathology, Laboratory Medicine, The Ottawa Civic Hospital and Department of Pathology and Laboratory Medicine, University of Ottawa, 1053 Carling Ave., Ottawa, Ontario, K1Y4E9 and 2 Anatomical Pathology, Laboratory Medicine, The Ottawa Civic Hospital and Department of Pathology, Ottawa, Ontario, Canada

Accepted 3 April 1994

Summary

We report an unusual pattern of immunostaining for chromogranin A in 24 transitional cell carcinomas (TCC). Positive immunostaining was seen with chromogranin A antisera in 10 of 16 urinary bladder TCC, neither of 2 prostatic TCC, 3 of 3 ureteral TCC and 2 of 3 metastatic TCC. This staining was demonstrated within the cytoplasm of transi­tional cells at all levels of the neoplastic epithelium, including cells near the basement membrane and at the free surface (umbrella cells). In 5 of 7 cases there was also im­more activity of the non-neoplastic urothelium. Immunostaining with neuron-specific enolase and synaptophysin was negative in all of these cases and no neuro-secretory granules were identified in 7 cases examined by electron microscopy. This pattern of im­munostaining is probably due to reactivity with chromogranins or certain chromogranin­like proteins in the transitional cells, particularly in the umbrella cells.

Key words: chromogranin - neuroendocrine cells - urothelial neoplasm

Introduction

Chromogranins are a family of acidic glycoproteins, present in normal and neoplastic neuroendocrine tissues as first described in 1965 (Banks and Helle, 1965). Their function is not well known (Eiden, 1987); however, antibodies to chromogranins, particularly to chromogranin A (CgA) and chromogranin B, are widely used to identify neuroendocrine differention within tumours by immunohistochemical methods (Lloyd et aI., 1988).

In the investigation of neuroendocrine differentiation in urinary bladder tumours, we encountered a peculiar positive immunostaining pattern for CgA in benign urothelium and transitional cell carcinomas (TCC). This led us to the present study which examines neoplastic and non-neoplastic urothelium of the genitourinary tract utilizing 3 different sources of antisera for CgA.

Correspondence to: K. T. Mai

304 Kien T. Mai et al.

Materials and Methods

Surgical Pathology files of the Ottawa Civic Hospital were searched for cases of cystectomy, biopsy (blad­der, ureter or prostate) and metastases with TCC accessioned from January 1992 to April 1993. Carcinoid tumour of lung, pancreatic tissue, and colonic tissue were used for positive and negative controls. For each case, 1 block representative of the area of interest was submitted for hematoxylin phloxine and saffron stain and Grimelius stain for argyrophil granules.

For Immunohistochemical studies, the peroxidase-antiperoxidase method was applied using monoclonal chromogranin A (CgA) from 3 sources (Boehringer Mannheim, 1 : 1000, Hybritech Incor­porated perchased 1987; 1: 1 200, and Hybritech Incorporated perchased 1992; 1: 1 000), polyclonal neuron specific enolase (NSE; Dako; 1 : 1 000) and monoclonal synaptophysin (Boehringer Mannheim; 1: 50).

For electron microscopy, either formaldehyde-fixed wet tissue or paraffin-embedded material was utilized. The formaldehyde-fixed tissue was washed, fixed with 1.6070 glutaraldehyde and then processed routinely, and examined with a Hitachi 7 100 electron microscope. For the paraffin embedded tissue, the area of interest was obtained, deparaffinized with xylene, washed with alcohol and then processed as described above.

Results

Twenty-four (24) surgical cases were retrieved. They consisted of 16 bladder biopsies showing Tee (3 carcinoma in situ, 5 non-invasive papillary Tee grade 1, 3 non-invasive papillary Tee grade 2, 5 invasive TeC), 2 transurethral resections of the prostate for Tee, 3 ureteronephrectomy specimens for non-invasive Tee of ureter and renal pelvis, and 3 specimens of metastatic Tee (1 in the retroperitoneum, 2 in the lung). In addition 3 bladder biopsies, in cases without previous history of Tee, showing non­specific chronic inflammation were used as control. Positive egA immunostaining was observed in 10 of 16 Tee of the bladder, neither of 2 Tee of the prostate, 3 of 3 Tee of the ureter and the renal pelvis, 2 of 3 metastatic Tee (Table 1).

The non-invasive tumours showed weak to strong focal (up to 70070 of cell popula­tion) immunostaining in cells throughout all levels of the urothelium, including cells near the basement membrane as well as at the free surface (Fig. 1 A). The invasive tumours showed weak and focal (10 to 20% of cells) immunostaining (Fig. 1 B). Non­neoplastic urothelium was present in 7 cases of Tee and showed immunoreactivity in 5. In 3 cases of chronic inflammation, immunostaining was observed in 1. Staining of the non-neoplastic urothelium was weak and more focal as compared to non-invasive neoplastic tissue. Immunostaining of the umbrella cells was strong in certain cases (Fig. 1 C).

Table 1. Positive immunostaining of chromogranin in different groups of TCC

Type of Tumour Number of Cases in Study Number of Cases with Positive Immunostaining for CgA

Tee in situ 3 TCe grade 1 5 4 Tee grade 2 3 3 Invasive TCC 5 2 TCC/prostate 2 1 Tee/ureter, pelvis 3 3 Tee/secondary 3 2 Total 24 16

Chromogranin in normal urothe1ium and transitional cell neoplasms 305

Immunoperoxidase stains for NSE and synaptophysin, and the Grimelius stains were negative in all cases.

Paraffin-embedded tissue from the areas corresponding to strong CgA immunostain­ing of 3 cases (1 TCC in situ, 1 TCC grade 1, 1 TCC grade 2) and formaldehyde-fixed wet tissue of another 4 biopsy cases with strong diffuse CgA immunostaining (2 TCC grade 1, I TCC grade 2, 1 TCC grade I of ureter) were examined by electron microscopy. The state of preservation of the tissue was poor with paraffin embedded tissue and ade­quate with formaldehyde-fixed tissue; however, organelles such as mitochondria and lysosomes were still visualized in the paraffin embedded tissue. There were no neurosecretory granules identified in the neoplastic or non-neoplastic urothelium.

Discussion

Neuroendocrine differentiation is a common phenomenon in tumours of many different body sites such as lung and colon, and the prostate as more recently described (Abenoza, 1986). In addition to small cell carcinoma, neuroendocrine differentiation has been reported in adenocarcinoma (de Matteis, 1992) and inverted papillomas (Summers et ai., 1991) of the urinary bladder. This has been demonstrated with immunostaining utilizing a battery of antisera including NSE, CgA and specific neuroendocrine hormones, as well as by the ultrastructural identification of neurosecretory-type dense core granules. CgA immunostaining is usually considered to be a good screening method due to its commer­cial availability and its relative specificity (Angletti, 1986).

In this study, a large proportion of TCC (67070) showed positive immunostaining for CgA. The pattern of staining seen, however, was distinct from that of neuroendocrine tumours in which there is diffuse cytoplasmic staining of cells near the basement mem­brane. Furthermore, the negative immunostaining with NSE and synaptophysin, the negative Grime1us agyrophil staining, and the absence of neurosecretory granules at the electron microscopic level are all arguments against true neuroendocrine differentiation within the study cases. Possible reasons for this peculiar staining pattern with CgA in the neoplastic and the non-neoplastic urothelium include: 1) staining artefact produced by cautery induced changes. This is unlikely as positive staining was also seen in the cystec­tomy and nephroureterectomy specimens and metastatic lesions; 2) false positive staining due to non-specific antisera. This is largely excluded by using CgA antisera from 3 dif­ferent sources; 3) reactivity of CgA antisera with CgA or CgA-like proteins present in the tissue studied. We believe that the last possibility is the case. Chromogranin antiserum has been reported to stain positively bovine salivary ductal cells (Angletti, 1986) and alveolar type II cells in animals (Kalima and Grimelius, 1991). In mice aged 5 to 6 weeks, CgA immunoreactivity has been detected in submandibular glands with the im­munolocalization and double immunoelectrophoresis techniques (Letic-Gavrilovic and Abe, 1990; Letic-Gavrilovic et al 1990). In toads, CgA has been identified in secretory granules of epithelial cells of the urinary bladder (Davis et aI., [990). Therefore, there ist increasing evidence that chromogranins or chromogranin-like proteins are present not only in neuroendocrine cells but also in non-endocrine cells (Winkler and Fischer-Col­brie, 1992).

In summary, chromogranin antisera showed positive reactivity in a large proportion of the cases of TCC and non-neoplastic urothelium studied. This unusual immunostain­ing was distinct from the pattern of staining seen in true neuroendocrine cells. The significance of this positive immunostaining is unknown. Caution should be exercised in the classification of urethelial tumours showing positive immunostaining with CgA. False classification of a tumour as a neuroendocrine lesion can be avoided by using a panel which includes at least NSE in addition to CgA.

306 Kien T. Mai et al.

Fig.1A

Fig.1B

Chromogranin in normal urothelium and transitional cell neoplasms 307

Fig.IC

Fig. 1. Immunostaining for CgA in neoplastic and benign urothelium: A) TCC in grade 2 shows strong positive cytoplasmic reaction (arrow); B) Invasive TCC in the retroperitoneal space shows weak positive cytoplasmic reaction (arrow head); C) Non-neoplastic urothelium shows strong cytoplasmic positive reac­tion of superficial cells and weak positive reaction of basal cells. Original magnification x 334,4.

Acknowledgments

The authors thank Ms B. Burton for secretarial assistance and Dr V. W. Walley for reviewing the manuscript.

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