Immune Complexes in Urine and Serum of Patients with Bladder Cancer

OD22-5347/84/l3l3-0L163$02,00/0

VoL 131, l\1:m:'C;h

THE JOURNAL OP UROLOGY

Printed in U.S.A.

Copyright © 1984 by The Williams & VVi!kins Co.

IIVl:MUNE COIVIPLEXES IN URINE AND SERUM OF PATIENTS WITH BLADDER CANCER R JOSEPH BABAIAN, DEBORAH A WATSON

AND

JOE M. JONES

From the Departments of Uro/,ogy and Patho/,ogy, The University of Arkansas for Medical Sciences, Little Rock, Arkansas

ABSTRACT

The urine and/or serum of 43 patients with bladder cancer was tested for the presence of immune complexes, Immune complexes were present more frequently in the urine (38 per cent) than in the serum (23 per cent). Urinary immune complexes were elevated in 10 per cent of the patients with no tumor (group 1), 25 per cent with superficial neoplasms (group 2) and 89 per cent with infiltrating or metastatic disease (group 3), which compared favorably to elevated serum immune complexes in 20, 25 and 33 per cent of the patients, respectively. The incidence of urinary immune complexes was statistically significant when compared in patients with and without active tumors (p less than 0,03), and when group 3 patients were compared to those in group l or 2 (p less than 0.01 and p less than 0.03, respectively), Unlike semm immune complexes urinary immune complexes appeared to correlate with the presence and stage of bladder cancer. The immunogenic nature of bladder cancer has been demonstrated by several investigators. 1- 3 One of the components of the immune response believed to be activated in patients with transitional cell carcinoma of the bladder is the production of antibodies, 4 ' 5 A portion of the antibodies is presumed to be directed against tumor-associated antigens occurring in these patients. The presence of immune complexes has been reported recently in the sernm of a small percentage of patients with bladder cancer. 6 Because urine comes in direct contact with bladder neoplasms and frequently has been known to contain immunoglobulins, 7 • 8 antigens 9 • 10 and exfoliated tumor cells 11 we examined urine as well as serum for the presence of immune complexes. MATERIALS AND METHODS

From December 1981 to September 1982 voided urine and/ or serum was collected from 5 women and 38 men, including 5 patients who underwent multiple evaluations for bladder cancer. Urine and serum were collected in 25 cases, while serum only was collected in 22 and urine only was collected in 1. Patient age ranged from 44 to 92 years, with a median age of 63.5 years. AH patients underwent cystoscopy, bladder biopsy and/or transurethral resection of a bladder neoplasm. Of 28 µ,au,:;ucc 10 had stage 0, 6 stage A, 6 stage 4 stage C and 2 stage D bladder cancer. There was no difference in t:ransfusi.on among patients without tumors, or with superficial or infiltrating bladder cancer. All samples were collected before and on the day of cystoscopy. were conducted and the data were tabulated without knowledge of the characteristics and disease status of the patients. The serum was stored at -70C until tested. Each urine sample was centrifuged at 1,000 times gravity. The supernatant was concentrated 50-fold at 4C overnight by pressure dialysis against phosphate buffered saline, pH 7.0, followed by Minicon Bl5 concentration. The dialyzed concentrated urine was centrifuged at 1,000 times gravity and the supernatant was stored at -70C until tested. The protein content of the urine was determined using the Lowry method and bovine albumin standards. Concentrated urine samples from 31 patients were subjected to polyacrylamide gel electrophoresis (5 per cent gel, Accepted for publication August 12, 1983. Supported by National Cancer Institute Grant RCDA K0400630 and American Cancer Society Grant IM-293.

0.2 M. tris-glycine buffer, pH 8.8) and stained with Coomassie blue. Serum from 8 patients with systemic lupus erythematosus and urine from 5 noncancer patients with culture-proved urinary tract infections were obtained and analyzed for immune complexes. Retrospective analysis revealed that urine cultures had been done in 8 patients with and 8 without urinary immune complexes. Immune complexes. Samples of serum (25 µI.) and concentrated urine (100 µl.) were assayed for immune complexes by the method of Barkas. 12 Briefly, samples were diluted to 400 µI. with phosphate buffered saline containing 0.01 M. ethylenediaminetetraacetic acid and 0.1 per cent sodium azide, mixed with 2 ml. 7.5 per cent polyethylene glycol in 0.05 M. barbitol buffer, pH 8.6, and incubated at 4C overnight. Samples then were centrifuged at 5,000 times gravity and the pellets were dissolved in 425 µL phosphate buffered saline. Aliquots of 200 µl. then were mixed with 100 µl. protein A positive staphylococci in 5 per cent fetal calf serum, incubated for 1 hour at room temperature and washed twice with 0.5 ml. phosphate buffered saline containing 1 mg. bovine albumin per ml. Then, 50 ng. (10 tiCi,/ µ.g.) 125iodine protein A were added, The pellets 'JVere incubated for 30 minutes at room temperature and washed twice with 0.5 mL phosphate buffered saline containing 1 mg. bovine albumin per ml. The rllrlio:adivi1;v of the pellet then was measured. Each sample was tested in quadruplicate. The radioactivity of the test samples was plotted on a standard curve obtained from assays of heat aggregated (64C for 30 minutes) human IgG mixed with 25 samples of immune complex negative normal human serum or 100 µl. samples of concentrated immune complex negative human urine from a single donor. When tested in this assay 3 samples of normal serum and concentrated normal urine obtained from healthy subjects showed negligible values. A sample was considered positive if the average radioactivity minus 1 standard deviation exceeded the average normal control (without aggregated human IgG) plus 1 standard deviation. Positive values were calculated as µg. immune complexes per ml. undiluted serum and ng. immune complexes per ml. unconcentrated urine. Before testing all serum samples were absorbed with 0.1 volume of formalin-fixed packed protein A negative Staphylococcus aureus cells to avoid complications from the presence of antibody directed against staphylococci. All data were analyzed for statistical significance by Student's t test or the chi-square method.

463

464

BABAIAN, WATSON AND JONES RESULTS

Histologic interpretations of 15 patients with cystoscopically abnormal mucosal findings revealed no demonstrable malignancy (group 1). There was no significant difference in the parameters measured between patients with (7) and without (8) a history of bladder cancer in this group. The remaining 28 patients had histologically proved bladder cancer (26 transitional and 2 squamous cell). Group 2 consisted of patients with superficial disease (stages O and A) and group 3 consisted of those with infiltrating or metastatic disease (stages B to D). The Marshall modification of the Jewett-Strong staging system and the World Health Organization grading criteria were used.1a, 14 The amount of immune complexes relative to aggregated human lgG in serum and urine for the 3 groups is shown in figure 1. The frequency and level of serum immune complexes were not significantly different between patients with or without proved malignancy. There also was no correlation between the level of serum and urine immune complexes. Despite the higher concentration of serum immune complexes in positive samples, urine samples from patients with active neoplasms were positive more frequently than were serum samples. Unlike 10,000

• = serum IC = urine IC

0

= ·;::

0

C:

::,

e .......

Cl C:

0

e ...::,.... .,..

e ~

I.DOD

..3,

0

c:::i

::c

serum immune complexes, the occurrence of urinary immune complexes correlated with the stage of the neoplasm (see table). The average level of urinary immune complexes in group 3 was significantly higher than in group 1 (p <0.05). The following controls indicated that immune complexes were being detected selectively: 1) serum from the 3 normal healthy controls used as standards exhibited negligible activity while the serum panel from 8 patients with systemic lupus erythematosus exhibited immune complexes values of 201 ± 40 ,ug./ml. and 2) the activity of positive urine and serum samples could be removed by absorption with protein A positive staphylococci but not by absorption with protein A negative staphylococci. The immunoglobulin contained in the detected immune complexes is likely to be lgG. With human immunoglobulins protein A binds selectively to lgG 1, lgG2 and IgG4 but it also exhibits some activity for other classes and subclasses of immunoglobulins. 15 The average proteinuria level was significantly higher in patients with urine that was positive rather than negative for immune complexes (p <0.02). Within the positive group there was no correlation between proteinuria and the amount of urinary immune complexes. There also was no significant difference in proteinuria when patients with (groups 2 and 3) and without (group 1) malignancy were compared. Urine samples from 31 patients were examined by polyacrylamine gel electrophoresis. Of these patients 9 (7 with and 2 without bladder cancer) had protein bands that did not appear to correspond to bands detected in normal serum or urine including 5 who had a band with a migration of 0.23 relative t~ albumin. The urine of 4 of these patients was positive for immune complexes, while 1 was not tested. All urine samples found to have immune complexes also exhibited a protein band with a mobility of serum lgG, while only 20 per cent (4 of 20) of the samples without immune complexes demonstrated this band. Urinary immune complexes were detected in only 3 of the 5 noncancer controls with urinary tract infection. Of the 8 patients with cancer positive urine cultures were noted in 6 with and 1 without detectable urinary immune complexes.

<

.s ... £: ;;; "' ....

DISCUSSION

0

8 0

. ...

100

-·..

...:.

Group 1

&ooo Group 2

0

Group 3

Patient Groups Fm. 1. Urine a1;1d sel'l!-m imrr_mne comt?lex levels relative to aggregated human IgG m patients with and without superficial and infiltrating bladder cancer.

The presence of immune complexes has been demonstrated in a number of inflammatory and malignant diseases. The significance and function of the immune complexes are not understood clearly, particularly in relation to malignant disease. In some studies with other types of malignancies the presence or quantity of immune complexes in the serum was found to correlate with prognosis or tumor burden while in others it did not. 16-18 We found that serum immune ~omplexes did not correlate with the presence or stage of bladder cancer which agreed with the previously reported 15 per cent incidenc~ of serum immune complexes without apparent correlation to disease stage in patients with transitional cell carcinoma. 19 Based on reported observations related to the content of

Comparison of frequencies of immune complexes Total Determinations/No. Pos.

Group: 1 2 3

2 and3 Grade: I

II III

Group 2

Group 1 Serum

Urine

Serum

Urine

15/3 20/4 12/4 32/8

10/1 8/2 8/7 16/9

Not significant Not significant Not significant

Not significant p <0.01* p <0.03*

5/0 12/4 12/4

0 5/3 9/6

Not significant Not significant Not significant

Not available Not significant p <0.03*

* Significant difference using chi-square test.

Serum

Urine

Not significant

Not significant

Not significant

p<0.03*

465

!lb Fm. 2. Polyacrylamide gel eledropho.resis of urine (arwws) from patients who exhibited novel protein bands, which are absent from normal human serum (NHS) or normal human urine (NHU). Channels l to 6 and 9 are from patients with cancer, while channels 7 and 8 are from patients without tumors at time of mine collection. Urinary immune complexes were elevated in patients represented by channels 4 to 6 and 8 (7 was not tested). Hb, human hemoglobin. lgG, human IgG.

human urine and despite the absence of reports with examination of urine for irD.mune cuu1µ1i'-'·'°',.""' per Since there is no con-elation between urinary and serum immune complexes, and between the presence of m,,.u1,1Jccui.cy and levels of proteinuria it seems m,,u,,.v,,;y that immune COill])IE,XE,S merely have leaked from the serum. Immune coma mechanism similar plexes could be shed from a neoplasm to that described by Johannson and Kistner 19 or u1.HAJ.,u1v 1,R,uu1ins teleased into the urine could bind to ant1~;en,2 present. The detected immune -.,u,.u 1;,,o,2.,c,s may be an a.ggregr,te sum of reactions to the tumor and responses the such as infection and tissue destruction. 'Nhi1e more work 1s needed to isolate and to characterize these i::.THT1uns ~.,,,.,.,~,,.uu there does not to be a S1g;mnc:ar.rr r•B1ll.t1Clmi!1Ip between infection and Although important, the questions of components of the immune '1,VHAJf-'''"'''o detected in the differ from those in the serum or whether those au,u,;c,,·w are derived from or exhibit specificity for bladder cancer were not examined by this study. Further work i.n this area seems justified and is ongoing in our laboratory. REFERENCES 1. Bubenik, J., Perlmann, P., Helmstein, K. and Moberger, G.: Cel-

lular and humoral immune responses to human urinary bladder carcinomas. Int. J. Cancer, 5: 310, 1970. 2. Takasugi, M., Mickey, M. R. and Terasaki, P. I.: Reactivity of lymphocytes from normal persons on cultured tumor cells. Cancer Res., 33: 2898, 1973.

3. Catalana, W. J.: Commentary on the immunobiolog-y of bladder cancer. J. Urol., 118: 2, 1977. 4. Hakala~ T. R.J Castro A. E., Elliot, A. Y. andFraley E. E.: Humeral cytotoxicity in human transitional cell carcinoma. J. UroL, 111: 382, 1974. 5. Troye, M., Perlmann, P., Pape, G. R, Spiegelberg, H. L., Naslund, L and Gidliif, A.: The use of Fab fragments of anti-human immunoglobulin as analytic tools for establishing the involvement of immunoglobulin in the spontaneous cytotoxicity to cultured tumors cells by lymphocytes from patients with bladder carcinoma and from healthy donors. J. ImmunoL, 119: 1061, 1977. 6. Pesce, A. J., Phillips, T. M., Ooi, B. S., Evans, A., Shank, R. A., HI and Lewis, M. G.: Immune complexes in transitional cell carcinoma. J. UroL, 123: 486, 1980. 7. Johansson, B., Kistner, S. and Norberg, R.: Proteinuria in patients with urinary tract tumours. Scand. J. Urol. Nephrol., 5: 229, 1

1

1971.

8. O'Brien, P., Gozzo, J., Cronin, W. and Monaco, A.: Qualitative of proteinuria associated with bladder cancer. Invest. 17: 28, 1979. 9. O'Brien, P. and rvfonaco, A. P.: Detecin urine from patients with 1980. J.O. and Morton, D. L.: Tumor-associated autologous sera in urine of patients with 29: 18, 1980. 11. Rifej C. C., FarTOVJ~ Utz, D. Co: Urine cytology of transitional cell neoplasms. Urol. Clin. N. Arner., 6: 599, 1979. 12. Barkas, T.: A simple, rapid and sensitive assay for immune complexes using Staphylococcus aureus immunoadsorbent. J. Clin. Lab. ImmunoL, 5: 59, 1981. 13. Marshall, V. F.: The relation of the preoperative estimate of the pathologic demonstration of the extent of vesical neoplasms. J. Urol., 68: 714, 1952. 14. Mostofi, F. K., Sobin, L. H. and Torlini, H.: Histological Typing of Urinary Bladder Tumours. International Histological Classification of Tumours, No. 10. Geneva: World Health Organization, 1973. 15. Langone, J. J.: Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci. Adv. Immunol., 32: 157, 1982. 16. Theofilopoulos, A. N., Andrews, B. S., Urist, M. M., Morton, D. L. and Dixon, F. J.: The nature of immune complexes in human cancer sera. J. Immunol., 119: 657, 1977.

466

BABAIAN, WATSON AND JONES

17. Horvath, M., Fekete, B. and Rahoty, P.: Investigation of circulating immune complexes in patients with breast cancer. Oncology, 39: 20, 1982. 18. Heier, H. E., Landaas, T. 0. and Marton, P. F.: Circulating immune complexes and prognosis in human malignant lymphoma, a prospective study. Int. J. Cancer, 23: 292, 1979. 19. Johannson, B. and Kistner, S.: Proteinuria in patients with uroepithelial tumours with special regard to tumour size, clinical staging and grade of malignancy. Scand. J. Urol. Nephrol., 9: 45, 1975.

EDITORIAL COMMENT The authors demonstrate that nearly 90 per cent of the patients with infiltrating or metastatic bladder cancer have detectable immune

complexes in the urine compared to 25 per cent of the patients with superficial bladder cancer arid 10 per cent of the healthy controls. In contrast, the presence of circulating immune complexes in the serum was only slightly more prevalent among patients with infiltrating or metastatic bladder cancer (33 per cent) than among those with superficial bladder cancer (25 per cent) or healthy controls (20 per cent). The authors have not yet performed studies to determine the nature of these immune complexes. The presence of immune complexes in cancer patients is of theoretical importance because immune complexes can inhibit host immunologic mechanisms. The antigen portion of the immune complex can bind to cytotoxic antibodies or lymphocytes pre-empting their cytotoxic potential, while the antibody portion of the complexes can bind to tumor-associated antigens protecting tumor cells from cytotoxic attack. Further studies will be necessary to determine the clinical significance of the observations presented. W. J. C.