Brief communication
Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urin&-ybladder I. Levin, B. Klein, S. Segal, A. Eyal, J. Gopas, J. Kaneti, M. Saadon, 0. Kuperman. Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urinary bladder. Tissue Antigens 1992: 39: 19-22: ,
Israel Levin', Baruch Kleinz, Shraga Segal', Arnona Eyal', Jacob Gopas5, Jacob Kaneti', M. Saadon3 and Oded Kuperman' Departments ol 'Urology, ?mmunology and Microbiology, 'Institute of Pathology and 'Institute of Oncology, The Soroka Medical Center and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva and 'Department of Oncology, Beilinson Medical Center, Petah Tiqva and Sackler School of Medicine, Tel Aviv University, Israel.
Key words: bladder cancer - HLA class I Received 10 July 1990, revised, accepted for publication 30 July 1990
The major histocompatibility complex (MHC) is one of the most important factors affecting the membrane-associated molecules which play a pivotal role in the interaction of a cell with its humoral and cellular microenvironment (1). The activity of cytotoxic T cells is, in fact, MHC class I antigenrestricted (2). In many different mouse tumors, a deviant and unbalanced expression of glycoproteins encoded by different loci of the MHC has been described (1). Also in several human tumors a reduced expression of class I antigens has been reported (3-6). Since MHC glycoproteins are of importance in regulation of target cell recognition and elimination by both specific and non-specific killer cells, it was the aim of this study to investigate the expression of MHC class I molecules on normal bladder cells and on malignant cells of patients with transitional cell carcinoma (TCC) of the bladder. Forty-two biopsies were obtained from patients with TCC, 28 biopsies from normal bladder tissue of TCC patients and an additional 22 biopsies were This work was supported by the Israel Cancer Assocation, Grant N17187. Helsinki Committee permission was given for this study.
obtained from patients treated surgically for reasons other than malignancy who served as controls. To prevent antigenic modulation due to thermal damage to the urothelial tissue, biopsies were excised with biopsy forceps during cystoscopy without using a cautery loop. The biopsies were snap-frozen in liquid nitrogen immediately after resection and stored at - 70°C until used. 4 pm sections, cut with a cryocut, were used for immunofluorescence studies. Histopathological diagnosis was established on paraffin sections. An additional frozen section from each tissue was evaluated by the same pathologist before and after the specimen was examined by immunofluorescence. The following murine mAb were used: a) Cappel anti-HLA-ABC, catalog No. 010 1-2027. b) Dakopatts anti HLA-ABC (W6/32), catalog No. M-736. c) American type culture collection antiHLA-ABC (MB 40.5), catalog No. HB 116. The polyclonal antibodies (pAb) used more rabbit antihuman B-2M (Dakopatts A-072), which served as a primary antibody. As secondary antibody we used fluorescein-conjugated rabbit antimouse immunoglobulins (Dakopatts F-26 1) for the detection of HLA-ABC, and fluorescein-conjugated swine antirabbit immunoglobulin (Dakopatts F-
Levin et a].
205) for the detection of B-2M. The staining procedure, consisting of a double-stage technique, was used in all cases (7). Briefly, sections were incubated with primary antibodies diluted 1:lO for 30 minutes at room temperature, washed three times with saline (0.9% NaCI) and then incubated in the presence of the fluorescein-conjugated reagent. Following three additional washings, the biopsies were counter-stained with Evans blue 1% for 5 min, washed three times with saline and mounted with buffered glycerol. The expression of the tested antigens was determined by using a fluorescence microscope with an ultraviolet light source (Bausch and Lomb). The staining of HLA class I antigens on peripheral blood lymphocytes was tested in several patients in which B-2M was expressed on their tumor cells without concomitant detection of HLA class I antigens using the same immunofluorescence procedure. This served as specificity control. In such cases, the lymphocytes were found to express class I antigens, confirming that the failure to stain these patients’ tumor cells for HLA class I antigens does not result from the inability of the anti-HLA-ABC antibodies to detect the relevant class I antigens. The staining was a quantitative phenomenon and the sensitivity of detection was identical for B-2M and HLA class I antigens as tested with peripheral blood lymphocytes and normal bladder specimens. Most of the normal urothelial tissue expresses practically undetectable levels of MHC-encoded antigens, subject to the limitations and specific sensitivity of our analysis. Only 4 out of 22 biopsies originating in the normal bladder were stained with HLA class I antigens (18%) and only 5 out of 22 showed reactivity to B-2M (22%). In every case, regardless of whether the urothelium was stained positively or negatively for the expression of glycoproteins, stromal elements in the same biopsy specimen were always stained positively, indicating that normal urothelial cells indeed express a practically undetectable level of the glycoproteins in comparison to other normal cells belonging to the connective tissue. The staining was evident on cell membrane as well as stromal elements. These results are in disparity to those that were obtained with TCC cells in which it was found that 40 out of 42 biopsies (95%) were stained for B2M, especially the cell membrane (Fig. 1, panel A), and 25 out of 42 (60%) showed a clear staining of HLA class I heavy chain also mainly observed on the cell membrane (Fig. 1, panel B). Interestingly, bladder tissue biopsies from TCC patients who were diagnosed histopathologically as normal showed higher proportions of staining for both B2M (57%) and HLA class I heavy chain (28%) than normal control urothelium. Moreover, in 20
Figwc. I. Expression of HLA class I antigens and B-2M on TCC cells. Panel A: 6-2M staining in TCC. Staining mainly seen on the cell membrane (see arrows). Panel B: HLA-ABC staining in TCC. Staining is evident on the cell membrane (see arrows).
every case in which positive staining of MHC class I antigens and B-2M was found on normal urothelium from TCC patients there was also a positive staining on the counterpart malignant tissue. The reverse situation was not found, namely staining of these antigens on TCC tissue was not necessarily accompanied by a concomitant expression on the normal tissue of the same patients (Table 1). In the present study we found that a high percentage of malignant urothelial tissues were stained with anti-HLA class I antibodies. Moreover, 95% of the same malignant tissue showed reactivity to the B-2M molecule. A puzzling observation is that, on a relatively high proportion of malignant TCC cells, HLA class I heavy chains are not detected despite enhanced reactivity of B-2M. This suggests at least two explanations. First, that no class I HLA heavy chain is produced but B-2M nevertheless is expressed on the cell surface. This is unlikely since it is generally accepted that synthesis of class
Brief communications Table 1 Expression of MHCencoded antigens on normal bladder and TCC of the same patients Normal Bladder of TCC Patients
TCC Patients
1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
HLA Class I
B-2M
++ +++ ++++ ++ +++
+++ ++++ ++++ +++ ++++ ++++
-
+
++ + + ++ +
+
+
++ ++++ + ++
+++
++++ -++++ +++ + +t+
HLA Class I
B-2M
+++ +++
+++ +++ ++ +++ + ++ ++++ + + + -
-
++++ ++++ + + ++ ++ +++
+++ + +++
56% (14125)
92% (24125)
60% (15/25)
-
-
+
++ -
-
* The expression of these antigens was graded between (+) to (+ + + +) according to the intensity of the fluorescence.
I and B-2M chain must be concomitant although B-2M is bound to an unrelated component in order to be expressed on the cell membrane. A second more intriguing possibility is that the malignant cells produce class I molecules which bind B-2M but are not detected by the monoclonal antibodies used. These class I heavy chains might be aberrant and express other than the classical HLA class I antigenic determinants and are unrecognizable by our antibodies. The possibility also exists that these cells express a B-2M binding Tla/Qa-like class I human equivalent as part of a malignant process of differentiation (8). It is interesting to note that apparently histologically non-neoplastic urothelial tissue in TCC patients was also stained with MHC class I antigens and, in particular, B-2M. This interesting phenomenon may be explained by at least two different approaches: 1) It is known that interferons and other cytokines (i.e. TNF) may cause the enhanced expression of both class I: and class I1 MHC glycoproteins (9). Therefore, one may assume that an inflammatory process mounted by the host against the possibly antigenically deviant premalignant
tissue may result in the secretion of such products which, in turn, may induce the enhanced expression of MHC antigens. 2) The expression of MHC glycoproteins was found to be under the control of both negative and positive regulatory molecules which are, in certain instances, differentiation-dependent (10) and, therefore, the possibility exists that at the very initial premalignant stages, even before characteristic neoplastic changes have taken place, the premalignant urothelium is activated to express B-2M concomitantly with or followed by the expression of either classical or deviant (embryonic) class I heavy chains. If, indeed, the expression of MHC molecules is induced at a very early premalignant stage long before the appearance of an established TCC, then the present findings may be of significant clinical importance because, in addition to providing a possible insight as to the etiology and pathophysiology of TCC, it may serve as a basis for early diagnosis by screening of populations at risk. Although infection could induce the expression of HLA class I in the present study, there was no evidence of inflammatory infiltrate in the specimens studied. In support of the possible importance of recognizing premalignant stages are the studies which show that antigenic changes take place in premalignant cells, such as the colon I1 1 ) and skin (12). Our present results are in contrast to the results reported by Walton et al. (13), who observed a diminished expression of B-2M in TCC specimens which correlated inversely with the degree of malignancy. This apparent discrepancy may be explained by the fact that “normal” material in the study by Walton may have been derived from patients who suffered from urinary tract infections in the past, which may have led to an in sitri immune response. In conclusion, our results suggest that TCC tumor cells are characterized by an enhanced reactivity to MHC glycoproteins commencing possibly at the very earliest stages of malignant transformation. The suggestion that the apparent histologically normal urothelial cells which express high levels of B-2M in TCC patients are actually premalignant cells may explain the relatively high frequency of recurrent disease in TCC patients as a result of failure to surgically excise premalignant tissue possessing normal histological characteristics.
References 1 . Goodenow R, Vogel J. Linsk R. Histocompatibility antigens on murine tumors. Science 1985: 230: 777-83. 2. Benacerraff B. Role of MHC gene products in immune regulation. Science 1981: 212: 1229-38. 3. Mombeug F, Muller P, Moldenhauer G, Hammerling G.
21
Levin et al. Loss of HLA-ABC in colorectal carcinoma is related to the degree of differentiation. J Immunogenet 1986: 13: 195-9. 4. Eyal A, Levin I, Segal S, Levi I, Klein B, Kuperman 0. Variation of HLA-ABC cells surface antigens expression on adenocarcinoma of the colon in correlation to the degree of differentiation. Nat Immrin Cell Growth Reg 1990: 9 3 1-7. 5. Klein B, Klein T,Kunichezky M, et al. The expression of HLA class I antigen in germ cell testicular cancer GCTC. Am J Cfin Puthol 1990 93: 202-7. 6. Klein B, Klein T,Figer A, et al. The expression of HLA class I and class I antigens gastric carcinoma. Tumor Biol (in press). 7. Poulter L, Chilosi M, Seymour G, Hobbs S, Janossy J. Immunofluorescence membrane staining and cytochemistry, applied in combination for analyzing cell interaction in situ. In: Polak J, Van-Noorder S, eds:Immunohistochemistry today. Edinburgh: Churchill-Livingstone, 1983: 233-48. 8. Gazit E, Gotheif Y, Gil R, et al. Alloantibodies to DNAactivated lymphocytes detect human Qa-like antigens. J bnmunol 1984 132: 165-9. 9. Wallach D, Fellows M,-Revel M. Preferential effect of gamma interferon on the synthesis of HLA antigens and their mRNAs in human cells. Nature 1982: 299: 833-6. I
22
10. Ehrlich R, Aguirre JE, Singer DS. Identification of negative and positive regulatory elements associated with a class I major histocompatibility complex gene. Mol Cell Bioll988: 8: 695-703. 11. Umpelby HC, Heineman D, Symes MO, Williamson RCM. Expression of histocompatibility, antigens and characterization of mononuclear cell infiltrates in normal and neoplastic colorectal tissues of humans. J Natl Cancer Inst 1985: 7 4 1161-5. 12. Turbitt U, Mackie RM. Loss of B-2 microglobulin from the cell surface in cutaneous malignant and prernalignant lesions. Br J Dermatof 1981: 104: 507-13. 13. Walton G, McCue P, Graham S. Beta-2-microglobulins as a differentiation marker in bladder cancer. J Urol 1986: 136: 1197-200. Address: Dr. Israel Levin Department of Urology Soroka Medical Center P.O.Box 151 Beer Sheva Israel Telephone: Israel-57-660394
Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urin&-ybladder I. Levin, B. Klein, S. Segal, A. Eyal, J. Gopas, J. Kaneti, M. Saadon, 0. Kuperman. Expression of HLA class I-encoded cell surface antigens in transitional cell carcinoma of the urinary bladder. Tissue Antigens 1992: 39: 19-22: ,
Israel Levin', Baruch Kleinz, Shraga Segal', Arnona Eyal', Jacob Gopas5, Jacob Kaneti', M. Saadon3 and Oded Kuperman' Departments ol 'Urology, ?mmunology and Microbiology, 'Institute of Pathology and 'Institute of Oncology, The Soroka Medical Center and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva and 'Department of Oncology, Beilinson Medical Center, Petah Tiqva and Sackler School of Medicine, Tel Aviv University, Israel.
Key words: bladder cancer - HLA class I Received 10 July 1990, revised, accepted for publication 30 July 1990
The major histocompatibility complex (MHC) is one of the most important factors affecting the membrane-associated molecules which play a pivotal role in the interaction of a cell with its humoral and cellular microenvironment (1). The activity of cytotoxic T cells is, in fact, MHC class I antigenrestricted (2). In many different mouse tumors, a deviant and unbalanced expression of glycoproteins encoded by different loci of the MHC has been described (1). Also in several human tumors a reduced expression of class I antigens has been reported (3-6). Since MHC glycoproteins are of importance in regulation of target cell recognition and elimination by both specific and non-specific killer cells, it was the aim of this study to investigate the expression of MHC class I molecules on normal bladder cells and on malignant cells of patients with transitional cell carcinoma (TCC) of the bladder. Forty-two biopsies were obtained from patients with TCC, 28 biopsies from normal bladder tissue of TCC patients and an additional 22 biopsies were This work was supported by the Israel Cancer Assocation, Grant N17187. Helsinki Committee permission was given for this study.
obtained from patients treated surgically for reasons other than malignancy who served as controls. To prevent antigenic modulation due to thermal damage to the urothelial tissue, biopsies were excised with biopsy forceps during cystoscopy without using a cautery loop. The biopsies were snap-frozen in liquid nitrogen immediately after resection and stored at - 70°C until used. 4 pm sections, cut with a cryocut, were used for immunofluorescence studies. Histopathological diagnosis was established on paraffin sections. An additional frozen section from each tissue was evaluated by the same pathologist before and after the specimen was examined by immunofluorescence. The following murine mAb were used: a) Cappel anti-HLA-ABC, catalog No. 010 1-2027. b) Dakopatts anti HLA-ABC (W6/32), catalog No. M-736. c) American type culture collection antiHLA-ABC (MB 40.5), catalog No. HB 116. The polyclonal antibodies (pAb) used more rabbit antihuman B-2M (Dakopatts A-072), which served as a primary antibody. As secondary antibody we used fluorescein-conjugated rabbit antimouse immunoglobulins (Dakopatts F-26 1) for the detection of HLA-ABC, and fluorescein-conjugated swine antirabbit immunoglobulin (Dakopatts F-
Levin et a].
205) for the detection of B-2M. The staining procedure, consisting of a double-stage technique, was used in all cases (7). Briefly, sections were incubated with primary antibodies diluted 1:lO for 30 minutes at room temperature, washed three times with saline (0.9% NaCI) and then incubated in the presence of the fluorescein-conjugated reagent. Following three additional washings, the biopsies were counter-stained with Evans blue 1% for 5 min, washed three times with saline and mounted with buffered glycerol. The expression of the tested antigens was determined by using a fluorescence microscope with an ultraviolet light source (Bausch and Lomb). The staining of HLA class I antigens on peripheral blood lymphocytes was tested in several patients in which B-2M was expressed on their tumor cells without concomitant detection of HLA class I antigens using the same immunofluorescence procedure. This served as specificity control. In such cases, the lymphocytes were found to express class I antigens, confirming that the failure to stain these patients’ tumor cells for HLA class I antigens does not result from the inability of the anti-HLA-ABC antibodies to detect the relevant class I antigens. The staining was a quantitative phenomenon and the sensitivity of detection was identical for B-2M and HLA class I antigens as tested with peripheral blood lymphocytes and normal bladder specimens. Most of the normal urothelial tissue expresses practically undetectable levels of MHC-encoded antigens, subject to the limitations and specific sensitivity of our analysis. Only 4 out of 22 biopsies originating in the normal bladder were stained with HLA class I antigens (18%) and only 5 out of 22 showed reactivity to B-2M (22%). In every case, regardless of whether the urothelium was stained positively or negatively for the expression of glycoproteins, stromal elements in the same biopsy specimen were always stained positively, indicating that normal urothelial cells indeed express a practically undetectable level of the glycoproteins in comparison to other normal cells belonging to the connective tissue. The staining was evident on cell membrane as well as stromal elements. These results are in disparity to those that were obtained with TCC cells in which it was found that 40 out of 42 biopsies (95%) were stained for B2M, especially the cell membrane (Fig. 1, panel A), and 25 out of 42 (60%) showed a clear staining of HLA class I heavy chain also mainly observed on the cell membrane (Fig. 1, panel B). Interestingly, bladder tissue biopsies from TCC patients who were diagnosed histopathologically as normal showed higher proportions of staining for both B2M (57%) and HLA class I heavy chain (28%) than normal control urothelium. Moreover, in 20
Figwc. I. Expression of HLA class I antigens and B-2M on TCC cells. Panel A: 6-2M staining in TCC. Staining mainly seen on the cell membrane (see arrows). Panel B: HLA-ABC staining in TCC. Staining is evident on the cell membrane (see arrows).
every case in which positive staining of MHC class I antigens and B-2M was found on normal urothelium from TCC patients there was also a positive staining on the counterpart malignant tissue. The reverse situation was not found, namely staining of these antigens on TCC tissue was not necessarily accompanied by a concomitant expression on the normal tissue of the same patients (Table 1). In the present study we found that a high percentage of malignant urothelial tissues were stained with anti-HLA class I antibodies. Moreover, 95% of the same malignant tissue showed reactivity to the B-2M molecule. A puzzling observation is that, on a relatively high proportion of malignant TCC cells, HLA class I heavy chains are not detected despite enhanced reactivity of B-2M. This suggests at least two explanations. First, that no class I HLA heavy chain is produced but B-2M nevertheless is expressed on the cell surface. This is unlikely since it is generally accepted that synthesis of class
Brief communications Table 1 Expression of MHCencoded antigens on normal bladder and TCC of the same patients Normal Bladder of TCC Patients
TCC Patients
1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
HLA Class I
B-2M
++ +++ ++++ ++ +++
+++ ++++ ++++ +++ ++++ ++++
-
+
++ + + ++ +
+
+
++ ++++ + ++
+++
++++ -++++ +++ + +t+
HLA Class I
B-2M
+++ +++
+++ +++ ++ +++ + ++ ++++ + + + -
-
++++ ++++ + + ++ ++ +++
+++ + +++
56% (14125)
92% (24125)
60% (15/25)
-
-
+
++ -
-
* The expression of these antigens was graded between (+) to (+ + + +) according to the intensity of the fluorescence.
I and B-2M chain must be concomitant although B-2M is bound to an unrelated component in order to be expressed on the cell membrane. A second more intriguing possibility is that the malignant cells produce class I molecules which bind B-2M but are not detected by the monoclonal antibodies used. These class I heavy chains might be aberrant and express other than the classical HLA class I antigenic determinants and are unrecognizable by our antibodies. The possibility also exists that these cells express a B-2M binding Tla/Qa-like class I human equivalent as part of a malignant process of differentiation (8). It is interesting to note that apparently histologically non-neoplastic urothelial tissue in TCC patients was also stained with MHC class I antigens and, in particular, B-2M. This interesting phenomenon may be explained by at least two different approaches: 1) It is known that interferons and other cytokines (i.e. TNF) may cause the enhanced expression of both class I: and class I1 MHC glycoproteins (9). Therefore, one may assume that an inflammatory process mounted by the host against the possibly antigenically deviant premalignant
tissue may result in the secretion of such products which, in turn, may induce the enhanced expression of MHC antigens. 2) The expression of MHC glycoproteins was found to be under the control of both negative and positive regulatory molecules which are, in certain instances, differentiation-dependent (10) and, therefore, the possibility exists that at the very initial premalignant stages, even before characteristic neoplastic changes have taken place, the premalignant urothelium is activated to express B-2M concomitantly with or followed by the expression of either classical or deviant (embryonic) class I heavy chains. If, indeed, the expression of MHC molecules is induced at a very early premalignant stage long before the appearance of an established TCC, then the present findings may be of significant clinical importance because, in addition to providing a possible insight as to the etiology and pathophysiology of TCC, it may serve as a basis for early diagnosis by screening of populations at risk. Although infection could induce the expression of HLA class I in the present study, there was no evidence of inflammatory infiltrate in the specimens studied. In support of the possible importance of recognizing premalignant stages are the studies which show that antigenic changes take place in premalignant cells, such as the colon I1 1 ) and skin (12). Our present results are in contrast to the results reported by Walton et al. (13), who observed a diminished expression of B-2M in TCC specimens which correlated inversely with the degree of malignancy. This apparent discrepancy may be explained by the fact that “normal” material in the study by Walton may have been derived from patients who suffered from urinary tract infections in the past, which may have led to an in sitri immune response. In conclusion, our results suggest that TCC tumor cells are characterized by an enhanced reactivity to MHC glycoproteins commencing possibly at the very earliest stages of malignant transformation. The suggestion that the apparent histologically normal urothelial cells which express high levels of B-2M in TCC patients are actually premalignant cells may explain the relatively high frequency of recurrent disease in TCC patients as a result of failure to surgically excise premalignant tissue possessing normal histological characteristics.
References 1 . Goodenow R, Vogel J. Linsk R. Histocompatibility antigens on murine tumors. Science 1985: 230: 777-83. 2. Benacerraff B. Role of MHC gene products in immune regulation. Science 1981: 212: 1229-38. 3. Mombeug F, Muller P, Moldenhauer G, Hammerling G.
21
Levin et al. Loss of HLA-ABC in colorectal carcinoma is related to the degree of differentiation. J Immunogenet 1986: 13: 195-9. 4. Eyal A, Levin I, Segal S, Levi I, Klein B, Kuperman 0. Variation of HLA-ABC cells surface antigens expression on adenocarcinoma of the colon in correlation to the degree of differentiation. Nat Immrin Cell Growth Reg 1990: 9 3 1-7. 5. Klein B, Klein T,Kunichezky M, et al. The expression of HLA class I antigen in germ cell testicular cancer GCTC. Am J Cfin Puthol 1990 93: 202-7. 6. Klein B, Klein T,Figer A, et al. The expression of HLA class I and class I antigens gastric carcinoma. Tumor Biol (in press). 7. Poulter L, Chilosi M, Seymour G, Hobbs S, Janossy J. Immunofluorescence membrane staining and cytochemistry, applied in combination for analyzing cell interaction in situ. In: Polak J, Van-Noorder S, eds:Immunohistochemistry today. Edinburgh: Churchill-Livingstone, 1983: 233-48. 8. Gazit E, Gotheif Y, Gil R, et al. Alloantibodies to DNAactivated lymphocytes detect human Qa-like antigens. J bnmunol 1984 132: 165-9. 9. Wallach D, Fellows M,-Revel M. Preferential effect of gamma interferon on the synthesis of HLA antigens and their mRNAs in human cells. Nature 1982: 299: 833-6. I
22
10. Ehrlich R, Aguirre JE, Singer DS. Identification of negative and positive regulatory elements associated with a class I major histocompatibility complex gene. Mol Cell Bioll988: 8: 695-703. 11. Umpelby HC, Heineman D, Symes MO, Williamson RCM. Expression of histocompatibility, antigens and characterization of mononuclear cell infiltrates in normal and neoplastic colorectal tissues of humans. J Natl Cancer Inst 1985: 7 4 1161-5. 12. Turbitt U, Mackie RM. Loss of B-2 microglobulin from the cell surface in cutaneous malignant and prernalignant lesions. Br J Dermatof 1981: 104: 507-13. 13. Walton G, McCue P, Graham S. Beta-2-microglobulins as a differentiation marker in bladder cancer. J Urol 1986: 136: 1197-200. Address: Dr. Israel Levin Department of Urology Soroka Medical Center P.O.Box 151 Beer Sheva Israel Telephone: Israel-57-660394
