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Proliferating Cell Nuclear Antigen: A New Prognostic Indicator in Renal Cell Carcinoma
0022-5347 /94/1523-0834$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1994 by AMERICAN UROLOGICAL AssoCJATION, INC.
Vol. 152, 834-836, September 1994 Printed in U.S.A.
Original Articles PROLIFERATING CELL NUCLEAR ANTIGEN: A NEW PROGNOSTIC INDICATOR IN RENAL CELL CARCINOMA K. J. CRONIN, N.N. WILL IAMS, M. J. KERIN, T. A CREAGH, P.A. DE RVAN, J. M. SMITH AND J. M. FITZPATRICK From the Departments of Urology and Surgery, Mater Misericordiae Hospital and University College Dublin, Dublin, Ireland
ABSTRACT Renal cell carcinoma is a tumor, the prognosis and behavior of which remain poorly understood. Proliferating cell nuclear antigen levels have been shown to act as an independent prognostic variable in a variety of malignancies. Proliferating cell nuclear antigen was evaluated in 59 cases of renal cell carcinoma, and the results were correlated with existing clinicopathological variables and survival. Proliferating cell nuclear antigen index (percentage of tumor cells positive for proliferating cell nuclear antigen) did not correlate with stage, grade or ploidy. To assess survival, tumors with proliferating cell nuclear antigen indexes of greater than and less than 60% were compared. The 24 patients with a high index (greater than 60%) had a significantly worse survival than did 35 with a low index (less than 60%, p <0.001). Therefore, the prognostic potential of proliferating cell nuclear antigen in renal cell carcinoma is promising and may be of clinical value in the management of patients with renal cell carcinoma. KEY WORDS: carcinoma, renal cell; kidney neoplasms; prognosis; antigens, neoplasm
Renal cell carcinoma is an uncommon neoplasm, the tumor biology of which remains poorly understood and the progno sis of which is unpredictable. Standard histopathological cri teria, such as tumor stage, nodal status and tumor grade, have proved to be of limited value in the evaluation of renal cell carcinoma in the clinical setting, and to date only tumor stage has been shown to correlate strongly with survival and has achieved widespread clinical recognition.1 In neoplastic lesions the rate of cell proliferation is believed to be a major influence of tumor behavior, and it has been shown that proliferative activity correlates with metastatic potential, tumor recurrence and, in some instances, overall prognosis.2 Several methods have been used to evaluate pro liferative rates in various malignancies, including flow cy tometry, KI67 staining (an immunohistochemical marker) and argyrophilic nucleolar organizer scores.2-5 However, technical problems have limited their use and they have not as yet found a role in routine clinical practice.6 Proliferating cell nuclear antigen is an auxillary protein of deoxyribonucleic acid polymerase delta that was first discov ered in patients with systemic lupus erythematosis who pro duce an autoantibody to it.7 It is synthesized during the Gl and S phases of the cell cycle, and proliferating cell nuclear antigen levels have been shown to correlate with the cell proliferative status.8· 9 It is recognized by PClO, a monoclo nal antibody, in routinely processed paraffin wax embedded tissues and it has been suggested that proliferating cell nu clear antigen levels may be of value as an independent prog nostic variable in a variety of tumors.6' 10-12 We evaluated the role of proliferating cell nuclear antigen index as a prog nostic indicator in renal cell carcinoma in a retrospective study of 59 patients and report our results.
Accepted for publication February 25, 1994.
PATIENTS, MATERIALS AND METHODS
Between 1981 and 1991, 59 patients underwent radical nephrectomy for renal cell carcinoma at our institution. There were 33 men and 26 women (ratio 1.27:1, mean age 60 years, range 23 to 84 years). Tumor stage was established from operative notes and pathological reports. Tumor grade was reviewed by a single pathologist (P.A. D.) in each case and when heterogeneity of grade was present in a tumor the worst grade present was assigned to that specimen. Followup ranged from 12 to 132 months (mean 64.1) and 5-year fol lowup was available on 31 of 59 patients. Specimens were routinely processed and embedded in par affin wax blocks. Sections of 4 µ. were cut onto Poly-L-Lysine coated glass slides and air dried overnight (since oven drying diminishes the immunoreactivity of proliferating cell nuclear antigen).10 Proliferating cell nuclear antigen staining was performed with the standard ABC immunoperoxidase tech nique. Positive controls of tonsillar tissue were used with each batch. Any variation in the proliferating cell nuclear antigen staining in the positive control from the norm auto matically meant that the batch was repeated, thus minimiz ing interbatch variation (fig. 1). Negative controls were also used by performing the identical assay except for the omis sion of the primary antibody, thus allowing us to control for artifactual staining. Each section was counted manually at high power (XlOO) using an eyepiece graticule. Approximately 200 cells per slide were counted from areas designated by the pathologist as being representative of assigned grade, and away from areas of hemorrhage, necrosis and artifactual staining, thus con trolling these factors. The proliferating cell nuclear antigen index was expressed as the percentage of cells positive for proliferating cell nuclear antigen in each specimen. All identifiable staining was regarded as positive. Ten cases were counted by 2 independent blinded observers and 10 were counted twice by 1 observer to assess interobserver and in tra-observer error.
834
835
PROLIFERATING CELL NUCLEA_R ANTIGEN IN RENAL CELL CANCER
Fm. 1. Proliferating cell nuclear antigen in renal cell carcinoma. A, low power shows proliferating cell nuclear antigen as diffuse staining within nucleus. Reduced from X40. B, high power demonstrates proliferating cell nuclear antigen as intranuclear pigment. Reduced from XlOO. TABLE
Variable Age Proliferative cell nuclear antigen index
1. Simple statistics No. Pts. 59 59
RESULTS ( 11)
Mean :+: SD (range)
0
60 ± 12.5 (25-83) 45.17 ± 24.67 (5-95)
1 <60%
90
2 2: 60%
80
%
Survival
Multivariate statistical analysis was performed using the Cox proportional hazards model. 13 The effect of various pa rameters on clinical outcome was assessed by plotting actu arial survival curves and comparing the groups using the log-rank test. 14
Actuarial Survival Curve
100
70
60 50 40 30
20 10
RESULTS
Time ( years )
The mean proliferating cell nuclear antigen index was 45.2% ± 24. 7 (standard deviation, range 5 to 95%, median 50%). The mean pooled standard deviation for 10 cases for interobserver error was 6. 7 and for intra-observer error it was 3.8. Proliferating cell nuclear antigen index did not cor relate with tumor stage, or nodal or metastatic status (tables l and 2) with some correlation between proliferating cell nuclear antigen index and tumor grade. However, this was not statistically significant (Pearson's coefficient 0.51). To compare proliferating cell nuclear antigen index and survival, patients were divided into 2 groups: group 1-24 patients with a proliferating cell nuclear antigen index of 60% or greater and group 2-35 with an index of less than 60%. There was a statistically significant difference in sur vival between the 2 groups (p <0.001), with patients who had a higher proliferating cell nuclear antigen index having a worse outcome (fig. 2). The stratum of 60% was selected on reviewing the data. A difference in prognosis was noted at the median level of 50% but this was not statistically signif icant. However, significance was noted at a 60% cutoff level retaining relatively large populations in each group. DISCUSSION
Clinical outcome in patients with renal cell carcinoma re mains unpredictable. To date, there is no reliable prognostic TABLE
Variable Age Stage Nodal status Metastasis status Grade Proliferating cell nuclear antigen index Survival
FIG. 2. Actuarial survival curve shows difference in survival be tween group 1 (35 patients, proliferating cell nuclear antigen index less than 60%) and group 2 (24 patients, proliferating cell nuclear antigen index greater than 60%). Followup was available at 1, 2, 3 and 5 years in 35, 27, 24 and 18 patients, respectively, in group 1, and 24, 20, 16 and 13, respectively, in group 2. marker that allows prediction of which patients will have relapse and, thus, there is delay in instituting adjuvant ther apies in such patients. This is particularly true in patients with early stage disease who might benefit most from such therapies. Cytogenetic abnormalities have been shown to be more frequent in patients with locally invasive tumors but the prognostic significance of this remains unclear. 15 Similarly, deoxyribonucleic acid flow cytometry in renal cell carcinoma may be of value in predicting prognosis but only in patients with stage I disease.16 To date, no prognostic marker is available that accurately predicts prognosis in all cases of renal cell carcinoma. Several reports have suggested that proliferating cell nu clear antigen may have clinical applications in the histo logical grading of tumor and as an independent prognostic variable in certain malignancies.2' 6' 11' 12 These reports prompted us to examine proliferating cell nuclear antigen in
2. Correlation statistics (Pearson's coefficient)
Pt. Age
Stage
Nodal Status
Metastasis Status
Grade
Proliferating Cell Nuclear Antigen Index
Survival
1.00 0.03 0.28 0.02 -0.10 -0.21
0.03 1.00 0.52 0.34 0.23 0.07
0.28 0.52 1.00 0.38 0.83 -0.32
0.02 0.34 0.38 1.00 -0.14 0.09
-0.10 0.960 0.23 0.08 1.00 0.51
0.21 0.07 -0.03 0.08 0.51 1.00
0.38 0.54 0.38 0.20 0.25 0.17
0.04
0.54
0.38
0.20
0.25
0.17
1.00
836
PROLIFERATING CELL NUCLEAR ANTIGEN IN RENAL CELL CANCER
renal cell carcinoma to determine its value in this tumor. In our study proliferating cell nuclear antigen index did not correlate with any of the existing histological variables, such as stage or grade. This has been the case in other proliferat ing cell nuclear antigen studies and studies involving an other proliferative marker antibody, KI67. As Jain et al noted, this finding probably suggests that factors other than proliferative activity are important in the control of tumor behavior. 6 What is of greater interest is the fact that proliferating cell nuclear antigen index correlated significantly with clinical outcome. This finding was independent of stage and grade. Patients with a proliferating cell nuclear antigen index of 60% or greater had a significantly worse survival, which may be of clinical significance in that it may allow urologists to identify patients who have a worse prognosis and enable them to institute adjuvant therapies at the initial operation rather than at relapse. This elimination of delay may im prove the efficacy of current adjuvant therapies, since dis ease bulk will be at minimum and, thus, survival may be improved. Dr. M. Codd provided advice and assisted with the statis tical analysis of the data. REFERENCES
1. Siminovitch, J.M. P., Montie, J. E. and Straffon, R. A.: Prognos tic indicators in renal adenocarcinoma. J. Urol., 130: 20, 1983. 2. Hall, P. A. and Levison, D. A.: Review: assessment of cell prolif eration in histological material. J. Clin. Path., 43: 184, 1990. 3. Hall, P. A. and Woods, A. L.: Immunohistochemical markers of cellular proliferation: achievements, problems and prospects. Cell Tiss. Kin., 23: 505, 1990. 4. Dervan, P. A., Gilmartin, L. G., Loftus, B.M. and Carney, D. N.: Breast carcinoma kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67 scores. Amer. J. Clin. Path., 92: 401, 1989. 5. Pich, A., Valente, G., Azzoni, L., Stramignoni, A., Margaria, E. and Tasso, M.: Argyr ophilic nucleolar organizer region counts and Ki-67 scores in human renal cell carcinoma. Path. Res.
Pract., 187: 482, 1991. 6. Jain, S., Filipe, M. I., Hall, P. A., Waseem, N., Lane, D. P. and Levison, D. A.: Prognostic value of proliferating cell nuclear antigen in gastric carcinoma. J. Clin. Path., 44: 655, 1991. 7. Asero, R., Origgi, L., Crespi, S., Bertetti, E., D'Agostino, P. and Riboldi, P.: Autoantibody to proliferating cell nuclear antigen (PCNA) in SLE: a clinical and serological study. Clin. Exp. Rheumatol., 5: 241, 1987. 8. Prelich, G., Tan, C. K., Kotsura, M., Mathews, M. B., So, A. G., Downey, K. M. and Shilman, B.: Functional identity of prolif erating cell nuclear antigen and a DNA polymerase-delta aux illary protein. Nature, 326: 517, 1987. 9. Waseem, N. H. and Lane, D. P.: Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural con servation and the detection of a nucleolar form. J. Cell. Sci., part 1, 96: 121, 1990. 10. Hall, P. A., Levison, D. A., Woods, A. L., Yu, C. C., Kellock, D. B., Watkins, J. A., Barnes, D. M., Gillett, C. E., Camplejohn, R. and Dover, R.: Proliferating cell nuclear antigen (PCNA) im munolocalization in paraffin sections: an index of cell prolifer ation with evidence of deregulated expression in some neo plasms. Pathology, 162: 285, 1990. 11. Yu, C., Hall, P. A. and Fletcher, C. D.: Immunohistochemical staining with a monoclonal antibody to PCNA may be a good indicator of prognosis in haemangiopericytomas. J. Path., 161: 342a, 1990. 12. Woods, A. L., Hanby, A.M. and Hall, P. A.: The prognostic value of CNA immunostaining in gastrointestinal lymphomas. J. Path., 161: 342a, 1990. 13. Cox, D. R.: Regression models and life tables. J. Roy. Stat. Soc. B., 34: 187, 1972. 14. Peto, R., Pike, M. C., Armitage, P., Breslow, N. E., Cox, D. R., Howard, S. V., Mantel, N., McPherson, K., Peto, J. and Smith, P. G.: Design and analysis of randomised clinical trials requir ing prolonged observation of each patient. II. Analysis and examples. Brit. J. Cancer, 35: 1, 1977. 15. Maloney, K.M., Norman, R. W., Lee, C. L. Y., Millard, 0. H. and Welch, J. P.: Cytogenic abnormalities associated with renal cell carcinoma. J. Urol., 146: 692, 1991. 16. Ljunberg, B., Larsson, P., Stenling, R. and Roos, G.: Flow cyto metric deoxyribonucleic acid analysis in stage I renal cell carcinoma. J. Urol., 146: 697, 1991.
Vol. 152, 834-836, September 1994 Printed in U.S.A.
Original Articles PROLIFERATING CELL NUCLEAR ANTIGEN: A NEW PROGNOSTIC INDICATOR IN RENAL CELL CARCINOMA K. J. CRONIN, N.N. WILL IAMS, M. J. KERIN, T. A CREAGH, P.A. DE RVAN, J. M. SMITH AND J. M. FITZPATRICK From the Departments of Urology and Surgery, Mater Misericordiae Hospital and University College Dublin, Dublin, Ireland
ABSTRACT Renal cell carcinoma is a tumor, the prognosis and behavior of which remain poorly understood. Proliferating cell nuclear antigen levels have been shown to act as an independent prognostic variable in a variety of malignancies. Proliferating cell nuclear antigen was evaluated in 59 cases of renal cell carcinoma, and the results were correlated with existing clinicopathological variables and survival. Proliferating cell nuclear antigen index (percentage of tumor cells positive for proliferating cell nuclear antigen) did not correlate with stage, grade or ploidy. To assess survival, tumors with proliferating cell nuclear antigen indexes of greater than and less than 60% were compared. The 24 patients with a high index (greater than 60%) had a significantly worse survival than did 35 with a low index (less than 60%, p <0.001). Therefore, the prognostic potential of proliferating cell nuclear antigen in renal cell carcinoma is promising and may be of clinical value in the management of patients with renal cell carcinoma. KEY WORDS: carcinoma, renal cell; kidney neoplasms; prognosis; antigens, neoplasm
Renal cell carcinoma is an uncommon neoplasm, the tumor biology of which remains poorly understood and the progno sis of which is unpredictable. Standard histopathological cri teria, such as tumor stage, nodal status and tumor grade, have proved to be of limited value in the evaluation of renal cell carcinoma in the clinical setting, and to date only tumor stage has been shown to correlate strongly with survival and has achieved widespread clinical recognition.1 In neoplastic lesions the rate of cell proliferation is believed to be a major influence of tumor behavior, and it has been shown that proliferative activity correlates with metastatic potential, tumor recurrence and, in some instances, overall prognosis.2 Several methods have been used to evaluate pro liferative rates in various malignancies, including flow cy tometry, KI67 staining (an immunohistochemical marker) and argyrophilic nucleolar organizer scores.2-5 However, technical problems have limited their use and they have not as yet found a role in routine clinical practice.6 Proliferating cell nuclear antigen is an auxillary protein of deoxyribonucleic acid polymerase delta that was first discov ered in patients with systemic lupus erythematosis who pro duce an autoantibody to it.7 It is synthesized during the Gl and S phases of the cell cycle, and proliferating cell nuclear antigen levels have been shown to correlate with the cell proliferative status.8· 9 It is recognized by PClO, a monoclo nal antibody, in routinely processed paraffin wax embedded tissues and it has been suggested that proliferating cell nu clear antigen levels may be of value as an independent prog nostic variable in a variety of tumors.6' 10-12 We evaluated the role of proliferating cell nuclear antigen index as a prog nostic indicator in renal cell carcinoma in a retrospective study of 59 patients and report our results.
Accepted for publication February 25, 1994.
PATIENTS, MATERIALS AND METHODS
Between 1981 and 1991, 59 patients underwent radical nephrectomy for renal cell carcinoma at our institution. There were 33 men and 26 women (ratio 1.27:1, mean age 60 years, range 23 to 84 years). Tumor stage was established from operative notes and pathological reports. Tumor grade was reviewed by a single pathologist (P.A. D.) in each case and when heterogeneity of grade was present in a tumor the worst grade present was assigned to that specimen. Followup ranged from 12 to 132 months (mean 64.1) and 5-year fol lowup was available on 31 of 59 patients. Specimens were routinely processed and embedded in par affin wax blocks. Sections of 4 µ. were cut onto Poly-L-Lysine coated glass slides and air dried overnight (since oven drying diminishes the immunoreactivity of proliferating cell nuclear antigen).10 Proliferating cell nuclear antigen staining was performed with the standard ABC immunoperoxidase tech nique. Positive controls of tonsillar tissue were used with each batch. Any variation in the proliferating cell nuclear antigen staining in the positive control from the norm auto matically meant that the batch was repeated, thus minimiz ing interbatch variation (fig. 1). Negative controls were also used by performing the identical assay except for the omis sion of the primary antibody, thus allowing us to control for artifactual staining. Each section was counted manually at high power (XlOO) using an eyepiece graticule. Approximately 200 cells per slide were counted from areas designated by the pathologist as being representative of assigned grade, and away from areas of hemorrhage, necrosis and artifactual staining, thus con trolling these factors. The proliferating cell nuclear antigen index was expressed as the percentage of cells positive for proliferating cell nuclear antigen in each specimen. All identifiable staining was regarded as positive. Ten cases were counted by 2 independent blinded observers and 10 were counted twice by 1 observer to assess interobserver and in tra-observer error.
834
835
PROLIFERATING CELL NUCLEA_R ANTIGEN IN RENAL CELL CANCER
Fm. 1. Proliferating cell nuclear antigen in renal cell carcinoma. A, low power shows proliferating cell nuclear antigen as diffuse staining within nucleus. Reduced from X40. B, high power demonstrates proliferating cell nuclear antigen as intranuclear pigment. Reduced from XlOO. TABLE
Variable Age Proliferative cell nuclear antigen index
1. Simple statistics No. Pts. 59 59
RESULTS ( 11)
Mean :+: SD (range)
0
60 ± 12.5 (25-83) 45.17 ± 24.67 (5-95)
1 <60%
90
2 2: 60%
80
%
Survival
Multivariate statistical analysis was performed using the Cox proportional hazards model. 13 The effect of various pa rameters on clinical outcome was assessed by plotting actu arial survival curves and comparing the groups using the log-rank test. 14
Actuarial Survival Curve
100
70
60 50 40 30
20 10
RESULTS
Time ( years )
The mean proliferating cell nuclear antigen index was 45.2% ± 24. 7 (standard deviation, range 5 to 95%, median 50%). The mean pooled standard deviation for 10 cases for interobserver error was 6. 7 and for intra-observer error it was 3.8. Proliferating cell nuclear antigen index did not cor relate with tumor stage, or nodal or metastatic status (tables l and 2) with some correlation between proliferating cell nuclear antigen index and tumor grade. However, this was not statistically significant (Pearson's coefficient 0.51). To compare proliferating cell nuclear antigen index and survival, patients were divided into 2 groups: group 1-24 patients with a proliferating cell nuclear antigen index of 60% or greater and group 2-35 with an index of less than 60%. There was a statistically significant difference in sur vival between the 2 groups (p <0.001), with patients who had a higher proliferating cell nuclear antigen index having a worse outcome (fig. 2). The stratum of 60% was selected on reviewing the data. A difference in prognosis was noted at the median level of 50% but this was not statistically signif icant. However, significance was noted at a 60% cutoff level retaining relatively large populations in each group. DISCUSSION
Clinical outcome in patients with renal cell carcinoma re mains unpredictable. To date, there is no reliable prognostic TABLE
Variable Age Stage Nodal status Metastasis status Grade Proliferating cell nuclear antigen index Survival
FIG. 2. Actuarial survival curve shows difference in survival be tween group 1 (35 patients, proliferating cell nuclear antigen index less than 60%) and group 2 (24 patients, proliferating cell nuclear antigen index greater than 60%). Followup was available at 1, 2, 3 and 5 years in 35, 27, 24 and 18 patients, respectively, in group 1, and 24, 20, 16 and 13, respectively, in group 2. marker that allows prediction of which patients will have relapse and, thus, there is delay in instituting adjuvant ther apies in such patients. This is particularly true in patients with early stage disease who might benefit most from such therapies. Cytogenetic abnormalities have been shown to be more frequent in patients with locally invasive tumors but the prognostic significance of this remains unclear. 15 Similarly, deoxyribonucleic acid flow cytometry in renal cell carcinoma may be of value in predicting prognosis but only in patients with stage I disease.16 To date, no prognostic marker is available that accurately predicts prognosis in all cases of renal cell carcinoma. Several reports have suggested that proliferating cell nu clear antigen may have clinical applications in the histo logical grading of tumor and as an independent prognostic variable in certain malignancies.2' 6' 11' 12 These reports prompted us to examine proliferating cell nuclear antigen in
2. Correlation statistics (Pearson's coefficient)
Pt. Age
Stage
Nodal Status
Metastasis Status
Grade
Proliferating Cell Nuclear Antigen Index
Survival
1.00 0.03 0.28 0.02 -0.10 -0.21
0.03 1.00 0.52 0.34 0.23 0.07
0.28 0.52 1.00 0.38 0.83 -0.32
0.02 0.34 0.38 1.00 -0.14 0.09
-0.10 0.960 0.23 0.08 1.00 0.51
0.21 0.07 -0.03 0.08 0.51 1.00
0.38 0.54 0.38 0.20 0.25 0.17
0.04
0.54
0.38
0.20
0.25
0.17
1.00
836
PROLIFERATING CELL NUCLEAR ANTIGEN IN RENAL CELL CANCER
renal cell carcinoma to determine its value in this tumor. In our study proliferating cell nuclear antigen index did not correlate with any of the existing histological variables, such as stage or grade. This has been the case in other proliferat ing cell nuclear antigen studies and studies involving an other proliferative marker antibody, KI67. As Jain et al noted, this finding probably suggests that factors other than proliferative activity are important in the control of tumor behavior. 6 What is of greater interest is the fact that proliferating cell nuclear antigen index correlated significantly with clinical outcome. This finding was independent of stage and grade. Patients with a proliferating cell nuclear antigen index of 60% or greater had a significantly worse survival, which may be of clinical significance in that it may allow urologists to identify patients who have a worse prognosis and enable them to institute adjuvant therapies at the initial operation rather than at relapse. This elimination of delay may im prove the efficacy of current adjuvant therapies, since dis ease bulk will be at minimum and, thus, survival may be improved. Dr. M. Codd provided advice and assisted with the statis tical analysis of the data. REFERENCES
1. Siminovitch, J.M. P., Montie, J. E. and Straffon, R. A.: Prognos tic indicators in renal adenocarcinoma. J. Urol., 130: 20, 1983. 2. Hall, P. A. and Levison, D. A.: Review: assessment of cell prolif eration in histological material. J. Clin. Path., 43: 184, 1990. 3. Hall, P. A. and Woods, A. L.: Immunohistochemical markers of cellular proliferation: achievements, problems and prospects. Cell Tiss. Kin., 23: 505, 1990. 4. Dervan, P. A., Gilmartin, L. G., Loftus, B.M. and Carney, D. N.: Breast carcinoma kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67 scores. Amer. J. Clin. Path., 92: 401, 1989. 5. Pich, A., Valente, G., Azzoni, L., Stramignoni, A., Margaria, E. and Tasso, M.: Argyr ophilic nucleolar organizer region counts and Ki-67 scores in human renal cell carcinoma. Path. Res.
Pract., 187: 482, 1991. 6. Jain, S., Filipe, M. I., Hall, P. A., Waseem, N., Lane, D. P. and Levison, D. A.: Prognostic value of proliferating cell nuclear antigen in gastric carcinoma. J. Clin. Path., 44: 655, 1991. 7. Asero, R., Origgi, L., Crespi, S., Bertetti, E., D'Agostino, P. and Riboldi, P.: Autoantibody to proliferating cell nuclear antigen (PCNA) in SLE: a clinical and serological study. Clin. Exp. Rheumatol., 5: 241, 1987. 8. Prelich, G., Tan, C. K., Kotsura, M., Mathews, M. B., So, A. G., Downey, K. M. and Shilman, B.: Functional identity of prolif erating cell nuclear antigen and a DNA polymerase-delta aux illary protein. Nature, 326: 517, 1987. 9. Waseem, N. H. and Lane, D. P.: Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural con servation and the detection of a nucleolar form. J. Cell. Sci., part 1, 96: 121, 1990. 10. Hall, P. A., Levison, D. A., Woods, A. L., Yu, C. C., Kellock, D. B., Watkins, J. A., Barnes, D. M., Gillett, C. E., Camplejohn, R. and Dover, R.: Proliferating cell nuclear antigen (PCNA) im munolocalization in paraffin sections: an index of cell prolifer ation with evidence of deregulated expression in some neo plasms. Pathology, 162: 285, 1990. 11. Yu, C., Hall, P. A. and Fletcher, C. D.: Immunohistochemical staining with a monoclonal antibody to PCNA may be a good indicator of prognosis in haemangiopericytomas. J. Path., 161: 342a, 1990. 12. Woods, A. L., Hanby, A.M. and Hall, P. A.: The prognostic value of CNA immunostaining in gastrointestinal lymphomas. J. Path., 161: 342a, 1990. 13. Cox, D. R.: Regression models and life tables. J. Roy. Stat. Soc. B., 34: 187, 1972. 14. Peto, R., Pike, M. C., Armitage, P., Breslow, N. E., Cox, D. R., Howard, S. V., Mantel, N., McPherson, K., Peto, J. and Smith, P. G.: Design and analysis of randomised clinical trials requir ing prolonged observation of each patient. II. Analysis and examples. Brit. J. Cancer, 35: 1, 1977. 15. Maloney, K.M., Norman, R. W., Lee, C. L. Y., Millard, 0. H. and Welch, J. P.: Cytogenic abnormalities associated with renal cell carcinoma. J. Urol., 146: 692, 1991. 16. Ljunberg, B., Larsson, P., Stenling, R. and Roos, G.: Flow cyto metric deoxyribonucleic acid analysis in stage I renal cell carcinoma. J. Urol., 146: 697, 1991.