28 K in Squamous Metaplasia of the Bladder in Patients with Spinal Cord Injury

28 K in squamous metaplasia of the bladder in patients with spinal cord injury

J Z Montgomerie MB ChB FRACP,l H J Holshuh DVM? A J Keyser PhD? C J Bennett MD,4 D G Schick BSI 2 1 Department of Infectious Diseases, Comparative Medical and Veterinary Services, 4Urology Service and Departments of Medicine and Surgery, Rancho Los Amigos Medical Center, 7601 E Imperial Highway, Downey, California 90242; 3Departments of Chemical Pathology and Surgery, University of Southern California School of Medicine, Los Angeles, California 90033, USA.

Antibody to 28 K was used to examine sections of bladder biopsies obtained by cystoscopy from 14 patients with spinal cord injury (SCI). Most of the biopsies were obtained from patients with indwelling catheters during the investigation for possible malignancy. Sections of bladder were stained by the streptavidin procedure. The 28 K in the normal transitional epithelium of the bladder was in the superficial cells (umbrella cells). All the biopsies from patients with indwelling urethral catheters showed areas of squamous metaplasia usually associated with evidence of chronic inflammation. Cystitis cystica glandularis was also seen in one patient. Staining was most marked in the areas of squamous metaplasia with intracellular granular staining. The basal layers were not well stained. With marked squamous metaplasia, there was a superficial hyperkeratotic layer that stained variably and often did not stain at all. Staining was less marked in areas of hyperplasia, regenerating urothelium, and cystitis cystica glandularis. These findings raise the possibility that the presence of 28 K glycoprotein in the tissues or released into the urine may be used as an indicator of squamous metaplasia and chronic inflammation of the bladder.

Keywords: spinal cord injury; urinary bladder metaplasia; urinary bladder malignancy; 28 K glycoproteins.

Introduction Methods In a previous study of human urothelium, Specimens were obtained from the patho we identified a component of the umbrella logy laboratory at Rancho Los Amigos cells of normal human urinary bladder Medical Center (RLAMC). These were epithelium that was also present in squam routinely processed biopsies of the bladder ous metaplastic cells in the bladder. 1 West from 14 patients with spinal cord injury who ern immunoblotting and tissue immuno had undergone cystoscopy. The main indic chemistry identified a 28 Kd molecule which ation for cystoscopy and biopsy had been to was present within the surface cells of the examine the bladder for evidence of neopla urothelium (umbrella cells) of the human sia in persons who had used long term bladder. Antiserum to 28 Kd also stained indwelling catheterization. Three patients areas of squamous metaplasia in the blad with SCI who had developed hematuria and der. In the present study we examined the did not have indwelling catheters were also distribution of staining in biopsies of the included. Biopsies had been obtained from bladder of patients with longstanding spinal any abnormal areas. In addition, biopsies cord injury. were taken from the right and left bladder 106 Montgomerie et al Paraplegia 31 (1993) 105-110 walls, the dome of the bladder, and the sis were observed in almost all of the trigone in most patients. All biopsies had biopsies. Squamous metaplasia of the blad been fixed in formalin and embedded in der was found in one or more biopsies of all paraffin blocks. patients that had had catheters in place The methods for the isolation of 28 K (range 9-36 years) and stained + to + + + bladder surface component and the produc in all instances. The staining of squamous tion of antibody were described previously. 1 metaplasia (Fig 1) was usually throughout Briefly, hyperimmune antiserum, obtained the thickness of the epithelium but often the from rabbits immunized with partially puri basal cells did not stain. Superficial keratin fied scrapings from the luminal surface of stained variably and often remained un human urinary bladders, was absorbed with stained (Fig 2). Cystitis cystica glandularis IgG-agarose and human type 0 blood. This was present in multiple biopsies of the resulted in specificity to the 28 K antigen. bladder of one patient with squamous meta Absorbed antibody in TRIS buffered saline plasia being present only at the urethra. pH 7.5 was applied to the sections for 2 Areas of cystitis cystica glandularis did not hours at 37°C. Slides were washed and stain. incubated with biotinylated antibody against One patient showed a significant degree rabbit IgG (link antibody) for 30 minutes at of epithelial dysplasia, basal hyperplasia and 37°C. Slides were then washed, incubated disorderly maturation of the urothelium. with streptavidin conjugated horseradish This included mitotic figures in occasional peroxidase for 30 minutes, and washed midzonal cells displaying early keratiniza again. The substrate, 3-amino-9-ethylcarba tion as well as lobular downgrowth of the zole (AEC), was used to visualize bound proliferating epithelium. There was signific antibody and the slides counterstained with ant anisokaryosis but no evidence of pene Mayer's hematoxylin. Dilutions of the ab tration of the urothelial basement mem sorbed antibody were selected after titration brane or other features needed to confirm of the antibody using the index case of carcinoma-in-situ. Two malignancies stu squamous cell carcinoma (RIV). Negative died offered contrasting patterns. The trans controls included tissue sections with sub itional cell carcinoma (TCC) grade 1 was stitution of the primary antibody with non reactive, while a case of squamous cell normal rabbit serum. carcinoma was intensely stained, the dis Areas of squamous metaplasia, keratin tribution of the 28 K antigen appearing to be ization, cystitis cystica glandularis, hyper directly related to the degree to which areas plasia, epithelial regeneration and normal within the malignancy were differentiated. urothelium were identified in each biopsy. One of three patients cystoscoped for For the purpose of this study, squamous hematuria but without indwelling urethral metaplasia was defined as urothelium in catheter had squamous metaplasia - one which the first few outer layers of the other patient had TCC grade 1. urothelial cells and nuclei become increas Areas of hyperplastic epithelium and re ingly flattened. The layers of cells from generating urothelium stained poorly. basal cells to surface cells are nucleated, Hyperplastic epithelium, while generally partially nucleated and finally non nucle staining poorly, showed immunoreactivity ated. The degree of staining with the 28 K in the cells closer to the basal layer. Areas of antibody was recorded in each of these areas normal urothelium were found in one or and graded 0 to + + +. The presence of more biopsies of 5 patients. The staining of inflammation was also recorded. these areas was comparable to that seen in a normal subject with staining localized to the luminal side of the cell (unpublished stu Results dies). The results of the study are shown in Table I. Acute and chronic inflammatory cells (including neutrophils, lymphocytes, occa Discussion sional mast cells and monocytes) with fibro- In this study we found squamous metaplasia Duration of Reaction w! Antibody to 28 K "" ;;- catheter (yrs) Sex/age SM NEp Other Bladder site Other "E- ......

Patients with indweIling catheters � ...... '0 '0 9 M 55 ++ Urethra Bladder calculi � R waIl ...... + + C cyst 0 v. + 0 C cyst L waIl I ...... 0 C cyst + reg ep Dome ...... 0 28 M 51 + Trigone Bladder calculi + R wall + Dome 17 F 33 ++ R waIl Bladder calculi +++ L waIl + Dome 11 F 36 ++ ++ Dysplasia Trigone Bladder calculi + ++ Dysplasia R waIl + ++ Dysplasia L waIl ++ ++ Dysplasia Dome I/) ..t:l :;;: 13 F 54 ++ Trigone � � ++ + R wall \:) :;:: ++ L wall '" + Dome '"� M 41 + Urethra Urethra fistula .§ + Trigone suprapubic catheter '"is' ++ L wall ;:;- ++ R wall 5- 36 F 56 +++ Not specified '";;. +++ Not specified <::l" is' 34 M 49 + Trigone i::l.. + L wall �.... + + R wall 5- + Dome 1/) 33 F 71 + Trigone Q ++ + Hyperplasia R wall ...... ++ L wall 0 -.I + Posterior wall I-' o 00 Table I (cant) � Duration of Reaction w/Antibody to 28 K � catheter (yrs) Sex/age SM NEp Other Bladder site Other aQ <::>

'"3 Patients with indwelling catheters .... (i;. 21 F 65 ++ SCC Not specified Squamous cell carcinoma � 12.. 24 F 45 + R wall ++ L wall + Dome + Posterior wall 10 M 54 + Trigone + R wall + Hyperplasia Dome

Patients without catheters

M 34 0 TCC Trigone 17 years quadriplegic (C5) ++ R wall Hematuria ++ L wall ++ reg ep Posterior wall M 38 +++ Not specified 22 years paraplegic (T7) Bladder calculi F 44 + reg ep Not specified 16 years paraplegic (L2) Hematuria Alkaline encrusting cystitis Corynebacterium sp �.... {5 SM = squamous metaplasia � NEp = normal epithelium is· = w C cyst cystitis cystica glandularis ...

= � TCC transitional cell carcinoma f-' '-D SCC = squamous cell carcinoma '-D reg ep = regenerating epithelium � f-' o = changes not present in biopsy Vl I = 0-+ ++ degree of reaction with antibody ..... f-' o Paraplegia 31 (1993) 105-110 Squamous metaplasia in the bladder in SCI 109

sies with squamous metaplasia only in the urethra. This incidence of squamous metaplasia in these patients with spinal cord injury is higher than previous studies. Broeker et al2 studied bladder biopsies of 50 patients with indwelling catheters for> 10 years, and 31 patients with external urinary catheter drainage for> 15 years. Squamous metapla sia occurred in 11 patients (28%) with indwelling catheters and 4 patients (8%) with external catheter drainage. Squamous metaplasia may occur with any form of bladder management. Polsky3 et al used cytology and bladder biopsy from 36 pa tients to demonstrate an 86% incidence of squamous metaplasia of the bladder among patients with chronic infection, and a 64% incidence in patients with bladder diversion. The reason for the higher incidence in the present study is not clear. Biopsies obtained from multiple sites may have been import ant, although in most patients squamous metaplasia was seen in all biopsies. Squamous metaplasia of the urinary blad der has been associated with chronic inflam matory processes, especially those seen in Figure 1 Squamous metaplasia of the human conjunction with longstanding indwelling urinary bladder showing reaction with antibody catheters, infection and lithiasis. Squamous to 28 K. metaplasia was found in the biopsy of one of the patients without a catheter. This patient of the bladder in all patients with indwelling also had bladder calculi. We did not study catheters in whom the catheter had been patients who had been catheterized for <9 present for 9-36 years. One patient had years. The presence of squamous metaplasia cystitis cystica glandularis in multiple biop- may be suggestive of a variety of potentially

Figure 2 Squamous metaplasia of the bladder stained with antibody to 28 K showing marked staining of the midzone, less staining of the basal layer and little or no staining of the superficial keratin. 110 Montgomerie et al Paraplegia 31 (1993) 105-110 serious problems that may develop within stammg of the 28 K suggested that it was the bladder. The patient with significant being produced in the more superficial urothelial dysplasia was demonstrating early layers of the urothelium. Staining was most development of a staining pattern seen in marked in the umbrella cells and its pres squamous cell carcinoma. There is con ence suggested that it may be being se tinuing debate as to whether the condition creted.1 The absence of 28 K in cystitis may be considered precancerous. Many cystica and cystitis glandularis might suggest factor" are apparently capable of stimulating that this substance is not part of 'mucin' . urothclial squamous metaplasia including 28 K may be a useful marker for squam potent carcinogens such as nitrosamines ous metaplasia. The consequences of (which may be produced in the urine of SCI squamous metaplasia are not well under patients), physical agents such as catheters, stood. This change may represent only a or even the installation of substances as reaction to foreign bodies and other irrit benign as sterile water. 4 The progression to ants, but there is also concern that the neoplasia appears to depend on the nature change is precancerous. This marker has of the agent involved and the duration of the potential as a noninvasive means of invest exposure. igating squamous metaplasia. In previous studies the distribution of the

References

1 Montgomerie JZ, Keyser AJ, Holshuh HJ. Schick DG (1992) 28 K antigen in the urothelium. JUral 147: 1388-1390. 2 Broecker BH, Klein FA. Hackler RH (1981) Cancer of the bladder in spinal cord injury patients. JUral 139: 428-432. 3 Polsky MS, Weber CH Jr, Williams JE III, Nikolewski RF, Barr MT, Ball TP Jr (1976) Chronically infected and postdiversionary bladders. Cytologic and histopathologic study. Urology 7(5): 531. 4 Murphy WM, Blatnik AF, Shelton TB, Soloway MS (1986) Carcinogenesis in mammalian urothelium: Changes induced by non-carcinogenic substances and chronic indwelling catheters. JUral 135: 840.