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Urinary Proteins as Biological Markers: Bladder Cancer Diagnosis Versus Urinary Tract Infection
0022-534 7/80/1246-0802$02.00/0 Vol. 124, December Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1980 by The Williams & Wilkins Co.
URINARY PROTEINS AS BIOLOGICAL MARKERS: BLADDER CANCER DIAGNOSIS VERSUS URINARY TRACT INFECTION PATRICIA O'BRIEN, JAMES J. GOZZO*
AND
ANTHONY P. MONACO
From the College of Pharmacy and Allied Health Professions, Northeastern University, and Cancer Research Institute, New England Deaconess Hospital and Department of Surgery, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Urine specimens from normal individuals, and from patients with bladder cancer, bladder papillomas and urinary tract infections were assayed for the presence of bladder tumor-related antigens. Ten-fold concentrated urine specimens were reacted in Ouchterlony gel diffusion against various anti-human monospecific antisera. With these antisera urine specimens from normal individuals were distinguished from those from patients with bladder carcinoma as well as bladder papilloma. However, the urine samples from individuals with urinary tract infections showed significant reactivity with many of the monospecific antisera as did specimens from patients with bladder cancer and bladder papilloma. Thus, investigations involved in the assay of bladder cancer biological markers should take the proteinuria associated with urinary tract infection into consideration. The potential importance of detecting tumor-specific components for early diagnosis and treatment of bladder neoplasms is stressed. An assay that detects urine-associated tumor components for the early diagnosis of bladder cancer would be highly desirable to screen and monitor individuals in high risk populations. Various biological markers present in urine have been associated with bladder cancer. McGregor and associates reported an increased excretion of the amino acid isoleucine in urine specimens from patients with bladder cancer compared to those from normal individuals. 1 Posey and Morgan have demonstrated increased activity of the enzymes arylsulfatase A and B, alkaline phosphatase and lactate dehydrogenase in the urine of patients with bladder carcinoma. 2 Urine specimens from patients with bladder cancer also have been associated with increased ,8-glucuronidase activity. 3 Wajsman and associates demonstrated a 90 per cent bladder cancer detection rate when correlating urine cytology and the presence of urinary fibrinogen products. 4 Increased urinary fibrinogen degradation products in patients with advanced carcinoma of the bladder also have been reported by Ohtaki. 5 Hall6 and Fraser7 and their associates indicated that the urine of patients with bladder cancer contained increased amounts of carcinoembryonic antigen. Johansson and Kistner detected increased quantities of albumin, transferrin and immunoglobulins in the urine of patients with uroepithelial tumors when compared to normal controls. 8 Previous Ouchterlony gel diffusion studies from our laboratory have indicated that, when compared to samples of normal urine, specimens from patients with bladder cancer have increased levels of a-2-macroglobulin, haptoglobin, fibrinogen, IgG, IgA, transferrin and orosomucoid. 9 All urine specimens analyzed in previous studies were from individuals that were negative for urinary tract infection by routine bacteriological culture. The objectives of our study were to extend previous data relating to the proteinuria associated with bladder cancer and to include data on the proteinuria associated with urine from individuals with non-cancerous associated urinary tract infection.
MATERIALS AND METHODS
Urine collection and initial processing. We collected 12hour samples from individuals with proved untreated bladder cancer and bladder papillomas who had no associated bacterial infection as determined by standard bacteriological culture techniques. Histological grading of all tumors was according to the classification defined by Koss. 10 Infected urine (~100,000 colony count) was obtained from patients who were known to be free of uroepithelial cancers, and was collected as single voided specimens before antibiotic treatment. Twelve to 24hour normal urine specimens were collected from laboratory personnel and from individuals accepted as renal transplant donors. Only normal donors without a history of urinary inflammation were accepted. All urine specimens (bladder cancer, bladder papilloma, infected and normal) were maintained on ice during collection. Samples were centrifuged at 3,000 times gravity for 20 minutes to remove any sediment, dialyzed overnight against saline at 4C and concentrated 10 times using B15 minicon units. t Commercial antisera. We used commercially available goat anti-human IgG, IgM, ,8-2-macroglobulin, haptoglobin, IgD, Clq, C3, C4, rabbit anti-human fibrinogen, transferrin, antirabbit orosomucoid, a-2-macroglobulin, plasminogen, IgA, a-1antitrypsin, ceruloplasmin, immature placenta, mature placenta, C-reactive protein, ferritin and carcinoembryonic antigen. Each commercial antiserum then was reacted by immunoelectrophoresis against whole normal human plasma to determine its degree of specificity. Ouchterlony gel diffusion. A total of 9 ml. 1 per cent agarose was layered onto a 16 X 4 cm. polyester backing consisting of 4 Ouchterlony gel diffusion plates. Each plate consisted of 1 well in the center surrounded by 6 wells (5 mm. apart) arranged in a circle 5 mm. from the center well. The outer wells were filled with 10 times concentrated urine specimens (15 µl.) and were reacted with various undiluted antisera (15 µl.), which were placed in the center well. The plates were reacted overnight in a humid chamber followed by incubation at 4C for an additional 24 hours. The plates then were washed in saline with continuous mixing for 7 days at 4C followed by a 1-day wash with distilled water. Plates then were air dried, stained with 1.0 per cent Amido Black lOB protein stain, destained in 1 per cent acetic
Accepted for publication March 21, 1980. Supported by the National Cancer Institute Grant Number CA2088804 through the National Bladder Cancer Project, Worcester, Massachusetts. * Requests for reprints: Department of Immunology, Cancer Research Institute, New England Deaconess Hospital, 194 Pilgrim Rd., Boston, Massachusetts 02215.
t Amicon Corporation, Lexington, Massachusetts. 802
803
URINARY PROTEINS AS BIOLOGICAL MARKERS
Various monospecific antisera reacted in gel diffusion against 10 times concentrated urine samples. Values listed are per cent of urine showing a precipitation band Urine Specimens Antisera
IgG IgA lgM IgD Transferrin Orosomucoid Haptoglobin Fibrinogen a-1-Antitrypsin a-2-Macroglobulin C3 Clq C4 Ceru!oplasmin Immature placenta Mature placenta Carcinoembryonic antigen C-reactive protein Ferritin Plasminogen ,8-2-Microglobulin
Bladder Papilloma
Normal
Bladder Ca
Infected
No.
%Pos.
No.
%Pos.
No.
%Pos.
No.
%Pos.
25 25 26 26 26 23 26 26 26 26 26 26 26 4 3 3 22
16 4 0 0 8 13 0 0 4 0 0 0 0 0 0 0 0
18 18 19
19
19 33 33 33 28 33 33 33 33 14 14 14 14 7 5 19
79 53 0 0 70 71 42 30 42 33 29 14. 7 29 29 0 0
24 24 24 24 24 24 24 24 24 24 24 24 24 23 23 17 24
88 63 29 0 67 75 29 25 58
19 3 2 2 18
34 28 0 0 53 35 11 0 21 11 0 0 0 0 0 0 0
3 3 4 26
0 0 0 0
2 2 3 18
0 0 0 0
6 5 14 33
0 0 0 9
23 23 17 24
0 0 12 38
19 19
17 19 19 19
19 19 19
16
8 0 8 7 4 12 1
levels oflgG, IgA, transferrin, orosomucoid, haptoglobin, fibrinogen and a-1-antitrypsin. These same proteins also were found in urine from patients with bladder neoplasms. If we or others were to use assays more sensitive than Ouchterlony gel diffusion, such as radioimmunoassay, it is reasonable to expect that there would be few differences when comparing proteinuria in patients with bladder cancer to that in patients with urinary tract infection. Preliminary results using the method of microcomplement fixation confirm this assumption. The probable causes of the proteinuria observed may include serum or plasma leakage into the urine caused by tumor vascularization and inflammation, immunological or acute phase responses by the host to the tumor or infection and a direct product of the destructive process of the tumor or infection. Therefore, these studies urge caution when using methods that measure normal plasma, tissue or inflammatory components in urine of patients with bladder cancer for the diagnosis of bladder cancer. They suggest further the importance of using and producing an antiserum that detects specific tumor-associated antigens. REFERENCES 1. McGregor, R. F., Johnson, D. E., Sharon, M. S., Crawford, R.,
2.
acid and methanol, and mounted on 2 X 2 photographic slides for projection to facilitate reading. RESULTS
3. 4.
Reactivity of urine samples in Ouchterlony gel diffusion against monospecific antisera. Urine from individuals with bladder cancer showed a high reactivity rate against antisera to IgG (79 per cent), IgA (53 per cent), transferrin (70 per cent), orosomucoid (71 per cent), haptoglobin (42 per cent), fibrinogen (30 per cent), a-1-antitrypsin (42 per cent) and a-2-macroglobulin (33 per cent) (see table). Urine specimens from individuals with bladder papillomas were more frequently reactive with antisera to transferrin (53 per cent), IgG (34 per cent), IgA (28 per cent) and orosomucoid (35 per cent). Urine specimens from individuals with urinary tact infections were most frequently reactive with antisera to IgG (88 per cent), IgA (63 per cent), transferrin (67 per cent), orosomucoid (75 per cent), a-1-antitrypsin (58 per cent), haptoglobin (29 per cent), fibrinogen (25 r,
The occurrence of increased quantities of normal plasma or tissue-related components in the urine of patients with bladder cancer has been reported previously. 9 Previous data from our laboratory support the potential usefulness of an antiserum panel against these components for the immunological diagnosis of bladder cancer. 9 The relationship ofproteinuria to bladder cancer also has been studied by a number of other investigators.2· 4-B A major limitation in assessing proteins accurately is that patients with urinary tract infection also have levels of these same components. This study shows clearly that specimens from patients with urinary tract infection have detectable
5. 6.
7. 8.
9. 10.
Brown, B. and Johnston, D.: Urinary amino acid excretion. Comparison of normal individuals and patients with bladder cancer. Urology, 9: 538, 1977. Posey, L. E. and Morgan, L. R.: Urine enzyme activities in patients with transitional cell carcinoma of the bladder. Clin. Chim. Acta, 74: 7, 1977. Tanaka, K.: Beta-glucuronidase in patients with bladder carcinoma. Acta Urol. Jap., 23: 557, 1977. Wajsman, Z., Merrin, C. E., Chu, T. M., Moore, R.H. and Murphy, G. P.: Evaluation of biological markers in bladder cancer. J. Urol., 114: 879, 1975. Ohtaki, M.: Serum and urinary fibrin/fibrinogen degradation products (FDP) in patients with urological malignancies. Jap. J. Urol., 68: 1172, 1977. Hall, R.R., Lawrence, D. J. R., Darcy, D., Stevens, U., James, R., Roberts, S. and Neville, A. M.: Carcinoembryonic antigen in the urine of patients with urothelial carcinoma. Brit. Med. J., 3: 609, 1972. Fraser, R. A., Ravry, M. J., Segura, J. W. and Go, V. L. W.: Clinical evaluation of urinary and serum carcinoembryonic antigen in bladder cancer. J. Urol., 114: 226, 1975. 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. Gozzo, J. J., Gottschalk, R., O'Brien, P., Cronin, W. and Monaco, A. P.: Use of heterogeneous and monospecific antisera for the diagnosis of bladder cancer. J. Urol., 118: 748, 1977. Koss, L. G.: Tumors of the urinary bladder. In: Atlas of Tumor Pathology. Washington, D. C.: Armed Forces Institute of Pathology, 2nd series, fasc. 11, pp. 19-28, 1975. EDITORIAL COMMENT
This study demonstrates some of the difficulties encountered in screening urine for substances that may be associated with the development of bladder cancer if these substances are non-specific reflections of an inflammatory response. Moreover, interpretation of such a screening program depends upon the appropriateness of control groups that have been used to assess differences from a population of patients with bladder cancer. Also, before this type of screening program can be applied to the individual patient, either a quantum step must be made in the sensitivity and specificity of these particular tests, since they are associated with a particular neoplastic process, or more specific tests must be developed. Michael J. Droller Brady Urological Institute The Johns Hopkins Hospital Baltimore, Maryland
THE JOURNAL OF UROLOGY
Copyright© 1980 by The Williams & Wilkins Co.
URINARY PROTEINS AS BIOLOGICAL MARKERS: BLADDER CANCER DIAGNOSIS VERSUS URINARY TRACT INFECTION PATRICIA O'BRIEN, JAMES J. GOZZO*
AND
ANTHONY P. MONACO
From the College of Pharmacy and Allied Health Professions, Northeastern University, and Cancer Research Institute, New England Deaconess Hospital and Department of Surgery, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Urine specimens from normal individuals, and from patients with bladder cancer, bladder papillomas and urinary tract infections were assayed for the presence of bladder tumor-related antigens. Ten-fold concentrated urine specimens were reacted in Ouchterlony gel diffusion against various anti-human monospecific antisera. With these antisera urine specimens from normal individuals were distinguished from those from patients with bladder carcinoma as well as bladder papilloma. However, the urine samples from individuals with urinary tract infections showed significant reactivity with many of the monospecific antisera as did specimens from patients with bladder cancer and bladder papilloma. Thus, investigations involved in the assay of bladder cancer biological markers should take the proteinuria associated with urinary tract infection into consideration. The potential importance of detecting tumor-specific components for early diagnosis and treatment of bladder neoplasms is stressed. An assay that detects urine-associated tumor components for the early diagnosis of bladder cancer would be highly desirable to screen and monitor individuals in high risk populations. Various biological markers present in urine have been associated with bladder cancer. McGregor and associates reported an increased excretion of the amino acid isoleucine in urine specimens from patients with bladder cancer compared to those from normal individuals. 1 Posey and Morgan have demonstrated increased activity of the enzymes arylsulfatase A and B, alkaline phosphatase and lactate dehydrogenase in the urine of patients with bladder carcinoma. 2 Urine specimens from patients with bladder cancer also have been associated with increased ,8-glucuronidase activity. 3 Wajsman and associates demonstrated a 90 per cent bladder cancer detection rate when correlating urine cytology and the presence of urinary fibrinogen products. 4 Increased urinary fibrinogen degradation products in patients with advanced carcinoma of the bladder also have been reported by Ohtaki. 5 Hall6 and Fraser7 and their associates indicated that the urine of patients with bladder cancer contained increased amounts of carcinoembryonic antigen. Johansson and Kistner detected increased quantities of albumin, transferrin and immunoglobulins in the urine of patients with uroepithelial tumors when compared to normal controls. 8 Previous Ouchterlony gel diffusion studies from our laboratory have indicated that, when compared to samples of normal urine, specimens from patients with bladder cancer have increased levels of a-2-macroglobulin, haptoglobin, fibrinogen, IgG, IgA, transferrin and orosomucoid. 9 All urine specimens analyzed in previous studies were from individuals that were negative for urinary tract infection by routine bacteriological culture. The objectives of our study were to extend previous data relating to the proteinuria associated with bladder cancer and to include data on the proteinuria associated with urine from individuals with non-cancerous associated urinary tract infection.
MATERIALS AND METHODS
Urine collection and initial processing. We collected 12hour samples from individuals with proved untreated bladder cancer and bladder papillomas who had no associated bacterial infection as determined by standard bacteriological culture techniques. Histological grading of all tumors was according to the classification defined by Koss. 10 Infected urine (~100,000 colony count) was obtained from patients who were known to be free of uroepithelial cancers, and was collected as single voided specimens before antibiotic treatment. Twelve to 24hour normal urine specimens were collected from laboratory personnel and from individuals accepted as renal transplant donors. Only normal donors without a history of urinary inflammation were accepted. All urine specimens (bladder cancer, bladder papilloma, infected and normal) were maintained on ice during collection. Samples were centrifuged at 3,000 times gravity for 20 minutes to remove any sediment, dialyzed overnight against saline at 4C and concentrated 10 times using B15 minicon units. t Commercial antisera. We used commercially available goat anti-human IgG, IgM, ,8-2-macroglobulin, haptoglobin, IgD, Clq, C3, C4, rabbit anti-human fibrinogen, transferrin, antirabbit orosomucoid, a-2-macroglobulin, plasminogen, IgA, a-1antitrypsin, ceruloplasmin, immature placenta, mature placenta, C-reactive protein, ferritin and carcinoembryonic antigen. Each commercial antiserum then was reacted by immunoelectrophoresis against whole normal human plasma to determine its degree of specificity. Ouchterlony gel diffusion. A total of 9 ml. 1 per cent agarose was layered onto a 16 X 4 cm. polyester backing consisting of 4 Ouchterlony gel diffusion plates. Each plate consisted of 1 well in the center surrounded by 6 wells (5 mm. apart) arranged in a circle 5 mm. from the center well. The outer wells were filled with 10 times concentrated urine specimens (15 µl.) and were reacted with various undiluted antisera (15 µl.), which were placed in the center well. The plates were reacted overnight in a humid chamber followed by incubation at 4C for an additional 24 hours. The plates then were washed in saline with continuous mixing for 7 days at 4C followed by a 1-day wash with distilled water. Plates then were air dried, stained with 1.0 per cent Amido Black lOB protein stain, destained in 1 per cent acetic
Accepted for publication March 21, 1980. Supported by the National Cancer Institute Grant Number CA2088804 through the National Bladder Cancer Project, Worcester, Massachusetts. * Requests for reprints: Department of Immunology, Cancer Research Institute, New England Deaconess Hospital, 194 Pilgrim Rd., Boston, Massachusetts 02215.
t Amicon Corporation, Lexington, Massachusetts. 802
803
URINARY PROTEINS AS BIOLOGICAL MARKERS
Various monospecific antisera reacted in gel diffusion against 10 times concentrated urine samples. Values listed are per cent of urine showing a precipitation band Urine Specimens Antisera
IgG IgA lgM IgD Transferrin Orosomucoid Haptoglobin Fibrinogen a-1-Antitrypsin a-2-Macroglobulin C3 Clq C4 Ceru!oplasmin Immature placenta Mature placenta Carcinoembryonic antigen C-reactive protein Ferritin Plasminogen ,8-2-Microglobulin
Bladder Papilloma
Normal
Bladder Ca
Infected
No.
%Pos.
No.
%Pos.
No.
%Pos.
No.
%Pos.
25 25 26 26 26 23 26 26 26 26 26 26 26 4 3 3 22
16 4 0 0 8 13 0 0 4 0 0 0 0 0 0 0 0
18 18 19
19
19 33 33 33 28 33 33 33 33 14 14 14 14 7 5 19
79 53 0 0 70 71 42 30 42 33 29 14. 7 29 29 0 0
24 24 24 24 24 24 24 24 24 24 24 24 24 23 23 17 24
88 63 29 0 67 75 29 25 58
19 3 2 2 18
34 28 0 0 53 35 11 0 21 11 0 0 0 0 0 0 0
3 3 4 26
0 0 0 0
2 2 3 18
0 0 0 0
6 5 14 33
0 0 0 9
23 23 17 24
0 0 12 38
19 19
17 19 19 19
19 19 19
16
8 0 8 7 4 12 1
levels oflgG, IgA, transferrin, orosomucoid, haptoglobin, fibrinogen and a-1-antitrypsin. These same proteins also were found in urine from patients with bladder neoplasms. If we or others were to use assays more sensitive than Ouchterlony gel diffusion, such as radioimmunoassay, it is reasonable to expect that there would be few differences when comparing proteinuria in patients with bladder cancer to that in patients with urinary tract infection. Preliminary results using the method of microcomplement fixation confirm this assumption. The probable causes of the proteinuria observed may include serum or plasma leakage into the urine caused by tumor vascularization and inflammation, immunological or acute phase responses by the host to the tumor or infection and a direct product of the destructive process of the tumor or infection. Therefore, these studies urge caution when using methods that measure normal plasma, tissue or inflammatory components in urine of patients with bladder cancer for the diagnosis of bladder cancer. They suggest further the importance of using and producing an antiserum that detects specific tumor-associated antigens. REFERENCES 1. McGregor, R. F., Johnson, D. E., Sharon, M. S., Crawford, R.,
2.
acid and methanol, and mounted on 2 X 2 photographic slides for projection to facilitate reading. RESULTS
3. 4.
Reactivity of urine samples in Ouchterlony gel diffusion against monospecific antisera. Urine from individuals with bladder cancer showed a high reactivity rate against antisera to IgG (79 per cent), IgA (53 per cent), transferrin (70 per cent), orosomucoid (71 per cent), haptoglobin (42 per cent), fibrinogen (30 per cent), a-1-antitrypsin (42 per cent) and a-2-macroglobulin (33 per cent) (see table). Urine specimens from individuals with bladder papillomas were more frequently reactive with antisera to transferrin (53 per cent), IgG (34 per cent), IgA (28 per cent) and orosomucoid (35 per cent). Urine specimens from individuals with urinary tact infections were most frequently reactive with antisera to IgG (88 per cent), IgA (63 per cent), transferrin (67 per cent), orosomucoid (75 per cent), a-1-antitrypsin (58 per cent), haptoglobin (29 per cent), fibrinogen (25 r,
The occurrence of increased quantities of normal plasma or tissue-related components in the urine of patients with bladder cancer has been reported previously. 9 Previous data from our laboratory support the potential usefulness of an antiserum panel against these components for the immunological diagnosis of bladder cancer. 9 The relationship ofproteinuria to bladder cancer also has been studied by a number of other investigators.2· 4-B A major limitation in assessing proteins accurately is that patients with urinary tract infection also have levels of these same components. This study shows clearly that specimens from patients with urinary tract infection have detectable
5. 6.
7. 8.
9. 10.
Brown, B. and Johnston, D.: Urinary amino acid excretion. Comparison of normal individuals and patients with bladder cancer. Urology, 9: 538, 1977. Posey, L. E. and Morgan, L. R.: Urine enzyme activities in patients with transitional cell carcinoma of the bladder. Clin. Chim. Acta, 74: 7, 1977. Tanaka, K.: Beta-glucuronidase in patients with bladder carcinoma. Acta Urol. Jap., 23: 557, 1977. Wajsman, Z., Merrin, C. E., Chu, T. M., Moore, R.H. and Murphy, G. P.: Evaluation of biological markers in bladder cancer. J. Urol., 114: 879, 1975. Ohtaki, M.: Serum and urinary fibrin/fibrinogen degradation products (FDP) in patients with urological malignancies. Jap. J. Urol., 68: 1172, 1977. Hall, R.R., Lawrence, D. J. R., Darcy, D., Stevens, U., James, R., Roberts, S. and Neville, A. M.: Carcinoembryonic antigen in the urine of patients with urothelial carcinoma. Brit. Med. J., 3: 609, 1972. Fraser, R. A., Ravry, M. J., Segura, J. W. and Go, V. L. W.: Clinical evaluation of urinary and serum carcinoembryonic antigen in bladder cancer. J. Urol., 114: 226, 1975. 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. Gozzo, J. J., Gottschalk, R., O'Brien, P., Cronin, W. and Monaco, A. P.: Use of heterogeneous and monospecific antisera for the diagnosis of bladder cancer. J. Urol., 118: 748, 1977. Koss, L. G.: Tumors of the urinary bladder. In: Atlas of Tumor Pathology. Washington, D. C.: Armed Forces Institute of Pathology, 2nd series, fasc. 11, pp. 19-28, 1975. EDITORIAL COMMENT
This study demonstrates some of the difficulties encountered in screening urine for substances that may be associated with the development of bladder cancer if these substances are non-specific reflections of an inflammatory response. Moreover, interpretation of such a screening program depends upon the appropriateness of control groups that have been used to assess differences from a population of patients with bladder cancer. Also, before this type of screening program can be applied to the individual patient, either a quantum step must be made in the sensitivity and specificity of these particular tests, since they are associated with a particular neoplastic process, or more specific tests must be developed. Michael J. Droller Brady Urological Institute The Johns Hopkins Hospital Baltimore, Maryland