Oncology 1990;47:37-42
< 1990 S. Karger A G . Basel 0030 2414 90 047] 0037 $ 2.75 0
Tissue Polypeptide Antigen (TPA) in Urinary Bladder Cancer Cytology: A Follow-Up Study S. Senatoreab, L. Zi:ziab, C. Blcisib, G. Alfierib, G. Saccani Jottic, M. Gabriellic, S. Luccarelliá a Morbid Anatomy ‘Di Summa' Hospital. Brindisi; b Gruppo di Studi in Oncología del Territorio Jonico-Salentino. Brindisi; c Institute of Pathology. Parma University School of Medecine, Parma; d Department of Urology. 'Di Summa' Hospital. Brindisi, Italy
Key Words. Tissue polypeptide antigen • Urinary bladder cancer cytology ■Immunocytochemistry • Follow-up
Introduction Tissue polypeptide antigen (TPA) is a tumor-related protein originally isolated from extracts of pooled tumors [1]. Antigenically.it is a cytoplasmic constituent of epithelial tissue with well-defined biochemical pro perties [2]. It can be considered an epithelial tissue mar ker of normal nonsquamous epithelia and derived neo plasms [3]. TPA is considered as both a differentiation marker [4] and/or proliferation marker [5]. TPA distribution in body fluids from cancerous patients has been widely investigated with significant results in tumor diagnosis [6. 7], monitoring [8], and follow-up [9], In particular, elevated levels of TPA have been found in serum and urine samples of a wide variety of cancer patients and thus the potential use of TPA as an index for the presence of tumor has been suggested [10]. Recently, both TPA distribution and staining pat terns in normal human tissues and in derived neo plasms have been studied [3, 10-13], Although the
epithelial distribution and biochemical characteristics of TPA show some similarity with intermediate fila ment proteins [14, 15], i.e. low molecular weight cytokcratins [3], recent data has demonstrated that ac tive and clinically relevant TPA is distinct from cytokeratin [5]. Both these findings and the peculiar patterns of the expression of TPA in cells, which appear to be related with urinary bladder cancer (UBC) grading [12], sug gest that the detection of TPA in cells in the urine of UBC patients, could act as a valuable parameter for follow-up controls to increase the diagnostic accuracy of cytology alone. At present, urine cytology in both bladder cancer detection and follow-up has given vari able results [16, 17], Although morphological criteria are highly diagnostic for anaplastic urothelial cells [18-20], they are of little use in the diagnosis for low-grade lesions characterized by well-differentiated tumor cells [17, 20], Moreover, several nonneoplastic conditions can express atypical/dysplastic cells [ 16, 21, 22] which decrease cytologic accuracy [16].
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Abstract. Tissue polypeptide antigen (TPA) has been detected by immunocytochemical assay on urine cytological samples from 28 asymptomatic patients, who previously had a histological diagnosis of papillary transitional cell carcinoma (PTCT) of the urinary bladder (UB), in order to evaluate its role in follow-up controls. TPA staining intensity (SI) in urothelial cells was evaluated to improve the diagnostic accuracy of cytology. Differentiated tumor cells were strongly stained for TPA, heavier than normal urothelial cells. Undifferentiated neoplastic cells were less stained for TPA with a wide range of SI. TPA detection revealed positive cytology in 21 (75%) of the considered cases. The accuracy of our cytological findings compared with both routine examinations and subsequent histopathological diagnosis was of 95.2%. Follow-up urinary cytology limits could be reduced by TPA searching in differentiated tumor cells, deriving from low-grade neoplasms.
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Senatore/Zizzi/Blasi/Alfieri/Saccani Jotti/Gabrielli/Luccarelli
Material and Methods Urine cytological samples were obtained from 28 asymptomatic UBC patients, aged from 58 to 79 years old. All patients were male with previous histological diagnosis of papillary transitional cell tumor (PTCT), graded according to the WHO classification [23] on biopsy material obtained by TUR. With regard to the accuracy of the technique. 4 urine samples were collected on 4 different days every 3 months for 2 years and examined for cells [24. 25], TPA cell immunoreaction and bacterial presence. No urinary-tract infections were detected. The urinary samples obtained from 5 healty volunteers were used as controls. Morning urinary samples were added to an equal amount of 70% ethanol, centrifuged after 1 h and the sediment was reccntrifuged at 1,500 rpm for 10 min. using a Shandon Southern Cytospin. Cell concentration, routine HE and TPA immunocytochcmical evaluations were performed for each sample. The WHO classification for UBC was applied [23] on cytologic material. Those patients who had positive cytology were submitted for cystoscopy and urinary bladder mapping. Materials Sera used for immunocytochcmical evaluations were as follows: normal swine serum (Dako, lot. No. 043); rabbit anti TPA (AB Sangtec Medical, lot. No. 5011 Cl 105; swine serum Ig to rabbit Ig (Dako. lot. No. 101 B); PAP complexes (Dako. lot. No. 083). Immunocytochemistry Prior to immunocylochemical staining with a PAP method [26], endogenous peroxidase activity was blocked with methanol for 15 min. TPA antiserum was used undiluted, swine Ig to rabbit Igat 1:50 and soluble PAP complexes at 1:100. A solution of 3-amino-9cthylcarbazolc (AEC), 0.02%, and N-N-dimethyl-formamide. 5%, in acetate buffer (0.1 M , pH 5.2) to which 0.02% hydrogen peroxide was then added. Counterstaining with Mayer's hemalum stain was performed, normal samples were considered as positive controls. Negative controls were obtained by omitting the primary antiserum and substituting with nonimmune serum. TPA staining intensity (SI) was evaluated. Negative, weak, strong and heavy immunorcactions coded from 0 to 3, were con sidered as different staining degress. SI grade I, i.e. weak, was considered as that expressed from urothelial cells with the classic cytological criteria of normality [22] observed in urine cytology of unaffected subjects.
Results Neoplastic urothelial cells during routine observa tions were found in 13 (46%) of the considered cases with differing degrees of atypia. In particular, well differentiated tumor cells were detected in 8 cases, poorly differentiated cells in 5 cases (table 1). Urothelial cells in all control cases were normal. TPA Staining Normal urothelial cells showed a weak TPA im munoreaction (SI grade 1). Within individual cells, TPA staining was granular, with heavier perinuclear or peripheral location (fig. 1). On the basis of both morphology and immunoreactivity, normal cells were easily distinguished from neoplastic cells. In par ticular, TPA staining patterns, indicating different neoplastic differentiation stages, appear to be an avail able and distinctive diagnostic tool for the detection of the degree of tumor cell atypia. Well differentiated neoplastic cells were heavily stained (SI grade 2-3) (fig. 2). Both peripheral honey comb-like and cytoplasmic intervacuolar immuno staining were observed. A globular cap-like staining has been observed around the nucleus. Although staining distribution in these cells was similar to that of normal urothelial cells, their SI was clearly different (table 2) (fig. 3). Undifferentiated neoplastic cells were less stained for TPA with a wide range of SI, from marked to weak or negative (table 2; fig. 3). TPApositive reactivity decreased consistently in anaplastic cells (fig. 4, 5). Both uneven staining patterns and SI were observed in these cells. In our study, tumor cell detection by ordinary cy tology and TPA immunocytochemical evaluation gave different results (fig. 6). In the latter, neoplastic Table 1. Cytologic degree of anaplasia Tumor cell grading
Go G, Gj Gj
Cases n
%
15 2 6 5
53.5 7.1 21.4 17.9
Go = No evidence of anaplasia; G i = well-differentiated tumor cells; G 2 = medium-differentiated tumor cells; G 3 = poorly differentiated tumor cells.
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In this study, urine cytological samples obtained by follow-up examinations of asymptomatic patients were assayed for TPA by immunocytochemistry. These patients previously underwent transurethral re section (TUR) with UBC diagnosed histologically. Both TPA immunostaining and morphological pat terns in urothelial cells were correlated to clarify their diagnostic value in cases in which individual cells were not sufficiently abnormal to give an unequivocal diag nosis of malignancy, as in residual or recurrent tumor diagnosis.
39
TPA in Urinary Bladder Cancer Cytology
Fig. I. a TPA in normal urothelial cells showing a cap-like staining around the nucleus, b TPA staining with peripheral location.
Fig. 2. TPA staining patterns in well-differentiated neoplastic cells.
Fig. 3. SI related to cytologic degree of anaplasia. TPA staining patterns. Go = No evidence of anaplasia; Gi = well-differentiated tumor cells: G 2 - medium-differentiated tumor cells; G j = poorly differentiated tumor cells.
Fig. 4. TPA-positive immunoreaction in anaplastic cell.
Fig. 5. TPA quite negative staining in anaplastic cell.
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4
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Senatore Zizzi Blasi Alfieri Saccani Jotti/Gabrielli/Luccarelli
Table 2. TPA findings related to cytologic degree of anaplasia Tumor cell grading
Stained cases, %
Stainig intensity
Well-stained cells < 25%
100 100 100 100
Go G, G,
Gj
+ 1 + 2- + 3 + 2 -+ 3 + 1 —1-2
< 50%
Cases > 50%
+ + + +
11
%
7 7 9 5
25 25 32.1 17.9
Fig. 6. Cytologic accuracy in both routine (—) and TPA immunocytochcmical evaluations ( —). Tumor grading as in figure 3.
urothelial cells were found in 21 (75%) of the considered cases. Morpho-functional abnormalities were assessed in 16 cases exhibiting differentiated tumor cells and in 5 with poorly differentiated cells (table 2). In those cases showing different cytologic degrees of anaplasia and immunoreactivity in the urinary samples, the one with the high grade was considered. The accuracy of our morpho-functional evaluations, compared with histopathological diag nosis on renewed biopsies at subsequent follow-up examinations, was of 95.2%, i.e. 20 positive cases.
Discussion Conventional urine cytology is often inadequate for distinguishing between cells derived from benign bladder papillomas [18, 22, 24, 27, 28] and those from well-differentiated UBC [16]. In particular, it was ob served that urine cytology was less often positive in first grade PTCT than in second or third grades [29],
In these conditions, several routine cytological evalua tions of urinary samples from each patient need to be requested to provide a suitable diagnosis in the man agement and follow-up of UBC patients [24, 25], In our follow-up study, both cytological and immunocytochemical evaluations were performed to increase the diagnostic accuracy of cytology alone. Previous studies demonstrated that TPA im munoreactivity in PTCT was different from that of normal urothelium and was related to the tumor grad ing [12]. TPA findings indicated that tumor grading and stage of invasiveness were unrelated [12]. TPA, a tissue-related [2, 3], differentiation/proliferation marker [4, 5], was employed as a highly available diagnostic tool in UBC [12]. Our TPA findings in urine cytology seem to indicate different patterns of tumor cell biology, i.e. differentiation and/or proliferative patterns. In addition, TPA is abundant in cells undergoing mitosis, while interphase cells contain less TPA [14], On this basis, tumor cell TPA expres sion seems to be a valuable functional index of the cell status [12]. Both TPA staining patterns and intensity may represent a diagnostic parameter indicating the morpho-functional tumor growth rate [14, 30]. Such an immunoreaction clearly indicates different tumor cell populations. In our experience, there is no definite relationship between cytological and TPA immunocytochemical findings (table 1,2; fig. 6). Welldifferentiated tumor cells were easily distinguished from normal cells by a heavier TPA immunoreaction. Such a finding could be due to a modified cell cycle, with a reduction of the interphase period and an increase of DNA replication and mitosis rate. These TPA immunostaining patterns are in accordance with serum TPA values in bladder cancer evolution [31]. Occasionally, these staining patterns exhibited by neo plastic cells could be observed in apparently normal
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Tumor grading as in table 1.
cells. In these cases, we suggest that only neoplastic cells having both morphofunctional variable pa rameters be considered. In this study, cytological and immunocytochemical diagnostic conclusions were similar in poorly-differen tiated tumor cells. The particular TPA distribution observed in clearly anaplastic urothelial cells was probably related to their complex neoplastic cell changes. Moreover, both the complex mechanism of TPA release from tumor cells [32] and its absence after cell death [33] must be considered. In these cells, im paired methabolic pathways, e.g. protein synthesis, can be involved [12]. These staining patterns, which could reflect an increase in the interphase period in anaplastic cells, indicate different stages of neoplastic cell disruption. Both an increased production or an impaired se cretion could explain the TPA ‘engulfed' picture in well-differentiated tumor cells wich is highly diagnos tic for low-grade neoplasms. Neoplastic cell auton omy, antigen masking and other functional disruption in the cell, e.g. those regarding tumor cell cycle, could explain the TPA-‘depleted' picture in anaplastic cells, diagnostic for high-grade tumors. TPA detection in urine cytology aids in the follow-up of UBC patients, focusing on the actual functional status of the neo plastic urothelial cells [12, 30, 33] derived from un classified tumors. On the basis of our results, the limitations of conventional urinary cytology [16, 20] could be successfully reduced by assaying for TPA in well-differentiated tumors. Samples collected at follow-up, as well as those obtained from high-risk patients for UBC, should be assayed for TPA to ac quire more information concerning the biology and behavior of the tumor for a better management of cancer patients. Acknowledgments This work was supported in part by a grant from the Reggio Emilia Section of the Italian League Against Cancer and in part by the Gruppo di Studi in Oncologia; the authors wish to thank Mr. G. Capano and Mr. P. Libetta for assistance in the preparation of the manuscript.
References I Bjorklund, B.; Lundblad. G.; Bjorklund. V.: Antigenicity of pooled human malignant and normal tissue by cytoimmunological technique. II. Nature of tumor antigen. Int. Archs Allergy appl. Immun. 12: 241-261 (1958).
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2 Bjorklund. B.: Serological analysis of human cancer antigen. Proe. 6th int. Congr. Microbiol., Rome 1953, vol. 2, p. 344 (Int. Microbiol. Association, Rome 1953). 3 Nathrath. W.B.J.; Ileidenkummcr, P.; Bjorklund. V.: Bjorklund. B.: Distribution of tissue polypeptide antigen (TPA) in normal human tissue: immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissue. J. Hislochem. Cytochem. 33: 99-109 (1985). 4 Boyse, E.A.: Old. L.J.: Some aspects of normal and abnormal cell surface genetics. Annu. Rev. Genet. 3: 269 (1969). 5 Bjorklund, B.; Bjorklund, V.: The enigma of a human Tumor marker: TPA revisited. Third Int. Conf. on "Human Tumor Markers. Biology and Clinical Applications', Naples 1986, p. 24, (Int. Academy of Tumour Marker Oncology. Naples. 1986). 6 Mommsen, S.; Aagard, J.; Sell, A.: Presenting symptoms, treat ment delay and survival in bladder cancer. Scand. J. Urol. Nephrol. 17: 163-167 (1983). 7 Oehr. P.: Adolphs. H.-D.; Altmann. R.: Clinical use of TPA in Cancer of the urinary bladder using CEA for comparison; In Peeters Protides of the biological Fluids, pp. 483-486 (Pcrgamon Press. Oxford. 1984). 8 Liithgens, M.; Schlegel. G.: Combined use of carcinocmbryonic antigen and tissue polypeptide antigen in oncologic therapy and surveillance. Cancer Detect. Prevent. 6: 51-59 (1983). 9 Kumar, S.; Wilson. P.; Brenchley, P.; et al.: Frequent elevation of tissue polypeptide antigen in the sera of workers exposed to bladder carcinogens. Int. J. Cancer 22: 542-545 (1978). 10 ShefT, C.; Lin. C.W.; Fujime, M.; Prout. G.R.: Localization of tissue polypeptide antigen in urological tumors by immuno histochemical techniques. (Abstract). 78 Annu. Meet. Am. Urol. Ass.. Las Vegas, 1983. pp. 330 (American Urological Associa tion, Baltimore 1983). 11 Bjorklund, V.; Bjorklund, B.: Localization of synthesis of TPA in normal and malignant human tissues by immunohistological techniques. Protides Biol. Fluids 27: 229-232 (1979). 12 Scnatorc. S.: Attolini. A.; Luccarclli. S.; Candita. F.: Trabucco. M.: Tissue polypeptide antigen in normal and neoplastic uri nary bladder. Preliminary reports. Oncology 44: 118-123 (1987). 13 Oehr. P.; Vogel. J.: Staining for TPA and CFA as and AID in search for primary tumors and micrometastases; in Peeters. Protides of the biological flu d, vol. 32: pp. 727-730 (Pergamon Press, Oxford 1984). 14 Bjorklund. V.; Bjorklund, B.: Differential immunocytochemical localization of tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA) in breast cancer. Cancer Detect. Prevent. 6: 193-198 (1983). 15 Bjorklund. V.; Bjorklund. B.: Immunohistochemistry of TPA: notes on the methodology; in Peeters, Protides of the biological fluid, pp. 341 346 (Pergamon Press. Oxford 1984). 16 Fl-Boldainy, M.N.: Cytology of bladder carcinoma. J. Urol. 124: 20-22 (1980). 17 Umiker. W.: Accuracy of cytologic diagnosis of cancer of the urinary tract. Acta Cytol. 8: 186-193 (1964). 18 Esposti, P. L.; Moberger, G.; Zajicck. J.: The cytologic diagnosis of transitional cell tumours of the urinary bladder and its his tologic basis. A study of 567 cases of urinary-tract disorder including 170 untreated and 182 irradiated bladder tumors. Acta Cytol. 14: 145-155 (1970).
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TPA in Urinary Bladder Cancer Cytology
19 Reichdorn-Kjenncrud. S.; Hocg, K..: The value of urine cytology in the diagnosis of recurrent bladder tumors: a preliminary report. Acta Cytol. 16: 269 272 (1972). 20 Lewis. R.W.; Jackson. A.C.. Jr.; Murphy. W.M.; Leblanc. G.A.: Meehan. W.L.: Cytology in the diagnosis and follow-up of transitional cell carcinoma of the urothelium: a review with a case series. J. Urol. 116: 43 46 (1976). 21 De Voogt. H.J.; Rathcrt, P.; Beyer-Boon, M.E.: Urinary cytol ogy- PP- 55 61 (Springer. New York 1977) 22 Holmquist. N.D.: Diagnostic cytology of the urinary tract. Monogr. clin. Cytol., vol. 6 (Kargcr, Basel 1977). 23 Mostofi. F. K.: Histologic typing of urinary bladder tumors. Int. Histological Classification of Tumors. Wld Hlth Org.. No. 10 (1973). 24 Foot. N.C.; Papanicolaou. G.N.; Holmquist. N.D.; Scybolt. J. F.: Exfoliative cytology of urinary sediments: review of 2.829 cases. Cancer //. 127 137 (1958). 25 Cullen. T.H.; Popham, R.R.; Voss, H.J.: An evaluation of routine cytological examination of the urine. Br. J. Urol. 39: 615 (1967). 26 Sternbcrger, L.A.: Immunocytochemistry. 2nd ed. (John Wiley & Sons. Chichester 1979). 27 Johnson. W.D.: Cytopathological correlations in tumours of the urinary bladder. Cancer 17: 867 880 (1964). 28 Umikcr. W.; Lapides. J.; Soureene. R.: Exfoliative cytology of papillomas and intraepithelial carcinomas of the urinary blad der. Acta Cytol. 6: 255-266 (1962).
Senatore/Zizzi/Blasi, Alfieri/Saccani Jotti/Gabrielli/Luccarelli
29 National Bladder Cancer Collaborative Group A: Cytology and histopathology of bladder cancer cases in a prospective lon gitudinal studi. Cancer Res. 37: 2911 2915 (1977). 30 Bjorklund. B.: Tumor products reflecting growth activity; in Stoll, Cancer treatment: end point evaluation, pp. 251 278 (John Wiley & Sons, Chichester 1983). 31 Holyoke, E.D.: Chu. T.M.: Tissue polypeptide antigen; in Herberman. Mclntire. Immunodiagnosis of cancer, pp. 513-521 (Marcel Dekkcr. New York 1979). 32 Oehr. P.; Schult. B.; Altehoefer, C.: Mechanism of TPA - Re lease from tumor cells and the significance for measurement of TPA values. Third Int. Conf. on 'Human Tumor Markers. Biology and Clinical Applications'. Naples 1986. p. 164 (Int. Academy of Tumour Marker Oncology, Naples, 1986). 33 Vogel, J.; Maiscy. R.: Oehr. P.; Adolphs. H.D.: Comparison of tissue polypeptide antigen (TPA) with carcinoembryonic anti gen (CEA) in human normal and urinary bladder cancer tissue and plasma. 2nd Int. Conf. on Human Tumor Markers. Vienna, 1984. Cancer Detect. Prevent. 6: 615 (1983).
Dr. Silvano Scnatorc Morbid Anatomy ‘Di Summa' Hospital USE BR/4 1-72100 Brindisi (Italy)
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42
< 1990 S. Karger A G . Basel 0030 2414 90 047] 0037 $ 2.75 0
Tissue Polypeptide Antigen (TPA) in Urinary Bladder Cancer Cytology: A Follow-Up Study S. Senatoreab, L. Zi:ziab, C. Blcisib, G. Alfierib, G. Saccani Jottic, M. Gabriellic, S. Luccarelliá a Morbid Anatomy ‘Di Summa' Hospital. Brindisi; b Gruppo di Studi in Oncología del Territorio Jonico-Salentino. Brindisi; c Institute of Pathology. Parma University School of Medecine, Parma; d Department of Urology. 'Di Summa' Hospital. Brindisi, Italy
Key Words. Tissue polypeptide antigen • Urinary bladder cancer cytology ■Immunocytochemistry • Follow-up
Introduction Tissue polypeptide antigen (TPA) is a tumor-related protein originally isolated from extracts of pooled tumors [1]. Antigenically.it is a cytoplasmic constituent of epithelial tissue with well-defined biochemical pro perties [2]. It can be considered an epithelial tissue mar ker of normal nonsquamous epithelia and derived neo plasms [3]. TPA is considered as both a differentiation marker [4] and/or proliferation marker [5]. TPA distribution in body fluids from cancerous patients has been widely investigated with significant results in tumor diagnosis [6. 7], monitoring [8], and follow-up [9], In particular, elevated levels of TPA have been found in serum and urine samples of a wide variety of cancer patients and thus the potential use of TPA as an index for the presence of tumor has been suggested [10]. Recently, both TPA distribution and staining pat terns in normal human tissues and in derived neo plasms have been studied [3, 10-13], Although the
epithelial distribution and biochemical characteristics of TPA show some similarity with intermediate fila ment proteins [14, 15], i.e. low molecular weight cytokcratins [3], recent data has demonstrated that ac tive and clinically relevant TPA is distinct from cytokeratin [5]. Both these findings and the peculiar patterns of the expression of TPA in cells, which appear to be related with urinary bladder cancer (UBC) grading [12], sug gest that the detection of TPA in cells in the urine of UBC patients, could act as a valuable parameter for follow-up controls to increase the diagnostic accuracy of cytology alone. At present, urine cytology in both bladder cancer detection and follow-up has given vari able results [16, 17], Although morphological criteria are highly diagnostic for anaplastic urothelial cells [18-20], they are of little use in the diagnosis for low-grade lesions characterized by well-differentiated tumor cells [17, 20], Moreover, several nonneoplastic conditions can express atypical/dysplastic cells [ 16, 21, 22] which decrease cytologic accuracy [16].
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Abstract. Tissue polypeptide antigen (TPA) has been detected by immunocytochemical assay on urine cytological samples from 28 asymptomatic patients, who previously had a histological diagnosis of papillary transitional cell carcinoma (PTCT) of the urinary bladder (UB), in order to evaluate its role in follow-up controls. TPA staining intensity (SI) in urothelial cells was evaluated to improve the diagnostic accuracy of cytology. Differentiated tumor cells were strongly stained for TPA, heavier than normal urothelial cells. Undifferentiated neoplastic cells were less stained for TPA with a wide range of SI. TPA detection revealed positive cytology in 21 (75%) of the considered cases. The accuracy of our cytological findings compared with both routine examinations and subsequent histopathological diagnosis was of 95.2%. Follow-up urinary cytology limits could be reduced by TPA searching in differentiated tumor cells, deriving from low-grade neoplasms.
38
Senatore/Zizzi/Blasi/Alfieri/Saccani Jotti/Gabrielli/Luccarelli
Material and Methods Urine cytological samples were obtained from 28 asymptomatic UBC patients, aged from 58 to 79 years old. All patients were male with previous histological diagnosis of papillary transitional cell tumor (PTCT), graded according to the WHO classification [23] on biopsy material obtained by TUR. With regard to the accuracy of the technique. 4 urine samples were collected on 4 different days every 3 months for 2 years and examined for cells [24. 25], TPA cell immunoreaction and bacterial presence. No urinary-tract infections were detected. The urinary samples obtained from 5 healty volunteers were used as controls. Morning urinary samples were added to an equal amount of 70% ethanol, centrifuged after 1 h and the sediment was reccntrifuged at 1,500 rpm for 10 min. using a Shandon Southern Cytospin. Cell concentration, routine HE and TPA immunocytochcmical evaluations were performed for each sample. The WHO classification for UBC was applied [23] on cytologic material. Those patients who had positive cytology were submitted for cystoscopy and urinary bladder mapping. Materials Sera used for immunocytochcmical evaluations were as follows: normal swine serum (Dako, lot. No. 043); rabbit anti TPA (AB Sangtec Medical, lot. No. 5011 Cl 105; swine serum Ig to rabbit Ig (Dako. lot. No. 101 B); PAP complexes (Dako. lot. No. 083). Immunocytochemistry Prior to immunocylochemical staining with a PAP method [26], endogenous peroxidase activity was blocked with methanol for 15 min. TPA antiserum was used undiluted, swine Ig to rabbit Igat 1:50 and soluble PAP complexes at 1:100. A solution of 3-amino-9cthylcarbazolc (AEC), 0.02%, and N-N-dimethyl-formamide. 5%, in acetate buffer (0.1 M , pH 5.2) to which 0.02% hydrogen peroxide was then added. Counterstaining with Mayer's hemalum stain was performed, normal samples were considered as positive controls. Negative controls were obtained by omitting the primary antiserum and substituting with nonimmune serum. TPA staining intensity (SI) was evaluated. Negative, weak, strong and heavy immunorcactions coded from 0 to 3, were con sidered as different staining degress. SI grade I, i.e. weak, was considered as that expressed from urothelial cells with the classic cytological criteria of normality [22] observed in urine cytology of unaffected subjects.
Results Neoplastic urothelial cells during routine observa tions were found in 13 (46%) of the considered cases with differing degrees of atypia. In particular, well differentiated tumor cells were detected in 8 cases, poorly differentiated cells in 5 cases (table 1). Urothelial cells in all control cases were normal. TPA Staining Normal urothelial cells showed a weak TPA im munoreaction (SI grade 1). Within individual cells, TPA staining was granular, with heavier perinuclear or peripheral location (fig. 1). On the basis of both morphology and immunoreactivity, normal cells were easily distinguished from neoplastic cells. In par ticular, TPA staining patterns, indicating different neoplastic differentiation stages, appear to be an avail able and distinctive diagnostic tool for the detection of the degree of tumor cell atypia. Well differentiated neoplastic cells were heavily stained (SI grade 2-3) (fig. 2). Both peripheral honey comb-like and cytoplasmic intervacuolar immuno staining were observed. A globular cap-like staining has been observed around the nucleus. Although staining distribution in these cells was similar to that of normal urothelial cells, their SI was clearly different (table 2) (fig. 3). Undifferentiated neoplastic cells were less stained for TPA with a wide range of SI, from marked to weak or negative (table 2; fig. 3). TPApositive reactivity decreased consistently in anaplastic cells (fig. 4, 5). Both uneven staining patterns and SI were observed in these cells. In our study, tumor cell detection by ordinary cy tology and TPA immunocytochemical evaluation gave different results (fig. 6). In the latter, neoplastic Table 1. Cytologic degree of anaplasia Tumor cell grading
Go G, Gj Gj
Cases n
%
15 2 6 5
53.5 7.1 21.4 17.9
Go = No evidence of anaplasia; G i = well-differentiated tumor cells; G 2 = medium-differentiated tumor cells; G 3 = poorly differentiated tumor cells.
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In this study, urine cytological samples obtained by follow-up examinations of asymptomatic patients were assayed for TPA by immunocytochemistry. These patients previously underwent transurethral re section (TUR) with UBC diagnosed histologically. Both TPA immunostaining and morphological pat terns in urothelial cells were correlated to clarify their diagnostic value in cases in which individual cells were not sufficiently abnormal to give an unequivocal diag nosis of malignancy, as in residual or recurrent tumor diagnosis.
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TPA in Urinary Bladder Cancer Cytology
Fig. I. a TPA in normal urothelial cells showing a cap-like staining around the nucleus, b TPA staining with peripheral location.
Fig. 2. TPA staining patterns in well-differentiated neoplastic cells.
Fig. 3. SI related to cytologic degree of anaplasia. TPA staining patterns. Go = No evidence of anaplasia; Gi = well-differentiated tumor cells: G 2 - medium-differentiated tumor cells; G j = poorly differentiated tumor cells.
Fig. 4. TPA-positive immunoreaction in anaplastic cell.
Fig. 5. TPA quite negative staining in anaplastic cell.
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4
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Senatore Zizzi Blasi Alfieri Saccani Jotti/Gabrielli/Luccarelli
Table 2. TPA findings related to cytologic degree of anaplasia Tumor cell grading
Stained cases, %
Stainig intensity
Well-stained cells < 25%
100 100 100 100
Go G, G,
Gj
+ 1 + 2- + 3 + 2 -+ 3 + 1 —1-2
< 50%
Cases > 50%
+ + + +
11
%
7 7 9 5
25 25 32.1 17.9
Fig. 6. Cytologic accuracy in both routine (—) and TPA immunocytochcmical evaluations ( —). Tumor grading as in figure 3.
urothelial cells were found in 21 (75%) of the considered cases. Morpho-functional abnormalities were assessed in 16 cases exhibiting differentiated tumor cells and in 5 with poorly differentiated cells (table 2). In those cases showing different cytologic degrees of anaplasia and immunoreactivity in the urinary samples, the one with the high grade was considered. The accuracy of our morpho-functional evaluations, compared with histopathological diag nosis on renewed biopsies at subsequent follow-up examinations, was of 95.2%, i.e. 20 positive cases.
Discussion Conventional urine cytology is often inadequate for distinguishing between cells derived from benign bladder papillomas [18, 22, 24, 27, 28] and those from well-differentiated UBC [16]. In particular, it was ob served that urine cytology was less often positive in first grade PTCT than in second or third grades [29],
In these conditions, several routine cytological evalua tions of urinary samples from each patient need to be requested to provide a suitable diagnosis in the man agement and follow-up of UBC patients [24, 25], In our follow-up study, both cytological and immunocytochemical evaluations were performed to increase the diagnostic accuracy of cytology alone. Previous studies demonstrated that TPA im munoreactivity in PTCT was different from that of normal urothelium and was related to the tumor grad ing [12]. TPA findings indicated that tumor grading and stage of invasiveness were unrelated [12]. TPA, a tissue-related [2, 3], differentiation/proliferation marker [4, 5], was employed as a highly available diagnostic tool in UBC [12]. Our TPA findings in urine cytology seem to indicate different patterns of tumor cell biology, i.e. differentiation and/or proliferative patterns. In addition, TPA is abundant in cells undergoing mitosis, while interphase cells contain less TPA [14], On this basis, tumor cell TPA expres sion seems to be a valuable functional index of the cell status [12]. Both TPA staining patterns and intensity may represent a diagnostic parameter indicating the morpho-functional tumor growth rate [14, 30]. Such an immunoreaction clearly indicates different tumor cell populations. In our experience, there is no definite relationship between cytological and TPA immunocytochemical findings (table 1,2; fig. 6). Welldifferentiated tumor cells were easily distinguished from normal cells by a heavier TPA immunoreaction. Such a finding could be due to a modified cell cycle, with a reduction of the interphase period and an increase of DNA replication and mitosis rate. These TPA immunostaining patterns are in accordance with serum TPA values in bladder cancer evolution [31]. Occasionally, these staining patterns exhibited by neo plastic cells could be observed in apparently normal
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Tumor grading as in table 1.
cells. In these cases, we suggest that only neoplastic cells having both morphofunctional variable pa rameters be considered. In this study, cytological and immunocytochemical diagnostic conclusions were similar in poorly-differen tiated tumor cells. The particular TPA distribution observed in clearly anaplastic urothelial cells was probably related to their complex neoplastic cell changes. Moreover, both the complex mechanism of TPA release from tumor cells [32] and its absence after cell death [33] must be considered. In these cells, im paired methabolic pathways, e.g. protein synthesis, can be involved [12]. These staining patterns, which could reflect an increase in the interphase period in anaplastic cells, indicate different stages of neoplastic cell disruption. Both an increased production or an impaired se cretion could explain the TPA ‘engulfed' picture in well-differentiated tumor cells wich is highly diagnos tic for low-grade neoplasms. Neoplastic cell auton omy, antigen masking and other functional disruption in the cell, e.g. those regarding tumor cell cycle, could explain the TPA-‘depleted' picture in anaplastic cells, diagnostic for high-grade tumors. TPA detection in urine cytology aids in the follow-up of UBC patients, focusing on the actual functional status of the neo plastic urothelial cells [12, 30, 33] derived from un classified tumors. On the basis of our results, the limitations of conventional urinary cytology [16, 20] could be successfully reduced by assaying for TPA in well-differentiated tumors. Samples collected at follow-up, as well as those obtained from high-risk patients for UBC, should be assayed for TPA to ac quire more information concerning the biology and behavior of the tumor for a better management of cancer patients. Acknowledgments This work was supported in part by a grant from the Reggio Emilia Section of the Italian League Against Cancer and in part by the Gruppo di Studi in Oncologia; the authors wish to thank Mr. G. Capano and Mr. P. Libetta for assistance in the preparation of the manuscript.
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TPA in Urinary Bladder Cancer Cytology
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Senatore/Zizzi/Blasi, Alfieri/Saccani Jotti/Gabrielli/Luccarelli
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Dr. Silvano Scnatorc Morbid Anatomy ‘Di Summa' Hospital USE BR/4 1-72100 Brindisi (Italy)
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