STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS: RELATIONSHIP TO GRADE, IMMUNOHISTOCHEMICAL EXPRESSION OF TUMOR MARKERS, MITOTIC FREQUENCY AND DNA PLOIDY

0022-5347/01/1654-1124/0 THE JOURNAL OF UROLOGY® Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 165, 1124 –1130, April 2001 Printed in U.S.A.

STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS: RELATIONSHIP TO GRADE, IMMUNOHISTOCHEMICAL EXPRESSION OF TUMOR MARKERS, MITOTIC FREQUENCY AND DNA PLOIDY ¨ NG, PATRIK ANDIUS, HANS HEDELIN, KENNETH WESTER, CHRISTER BUSCH STEN HOLMA AND SONNY L. JOHANSSON From the Department of Urology, Sahlgrenska University Hospital, Go¨teborg, Department of Urology, Ka¨rnsjukhuset, Sko¨vde and Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden, and Department of Pathology, University Hospital, Tromso¨, Norway, and Department of Pathology and Microbiology, and Eppley Institute of Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska

ABSTRACT

Purpose: We studied 363 patients with stage Ta bladder tumors during long-term followup who were classified according to the 1998 WHO and International Society of Urological Pathology consensus classifications. We determine whether various immunohistochemical and molecular markers could predict tumor progression. Materials and Methods: A total of 680 patients in western Sweden with a first diagnosis of bladder carcinoma in 1987 and 1988 were registered and followed for at least 5 years. There were 363 (53%) tumors that were papillary stage pTa. The tumors were classified as papillary urothelial neoplasm of low malignant potential in 95 patients, low grade papillary urothelial carcinoma in 160 and high grade carcinoma in 108. Of the patients in the latter group 95 were subdivided into WHO grade 2 and 13 into WHO grade 3. Tissue from the primary tumors that progressed in stage during followup was further analyzed with immunohistochemical methods (p21, p53, Ki67 and pRb), DNA ploidy and mitotic frequency. The results were compared with those in matched controls (nonprogressors). Results: Recurrence developed in 35% of patients with papillary urothelial neoplasm of low malignant potential compared to 71% with low grade urothelial carcinoma and 73% with high grade carcinoma (p ⬍0.0001). No papillary urothelial neoplasm of low malignant potential progressed in stage. Disease progressed in 4% of patients with low grade compared to 23% with high grade carcinoma (p ⬍0.0001). Of the patients with WHO grade 3 disease progressed in 45% compared to grade 2 in 20% (p ⬍0.0011). At first diagnosis p53 score was significantly higher (p ⬍0.0022) among patients with WHO grade 2 carcinoma which later progressed compared to that in matched controls but there was no significant difference regarding the other markers. In contrast to grade 2 most grade 3 carcinoma was aneuploid, had high mitosis frequency, high p53 and Ki67 scores as well as loss of retinoblastoma gene expression. Conclusions: The 1988 WHO and International Society of Urological Pathology consensus classifications divide noninvasive papillary bladder tumors into 3 subgroups with different clinical behavior, which seems to be an advantage compared with the 1973 WHO classification. A disadvantage is that the high grade carcinoma group contains 2 subgroups with different progression rates and immunohistochemical marker profiles, corresponding to the 1999 WHO grades 2 and 3. Grade 2 tumors in patients that progressed in stage years later seem to have different immunohistochemical and molecular marker profiles compared to those in matched controls. KEY WORDS: bladder neoplasms, prognosis, disease progression

Since its introduction in 1973 the WHO system has been the most used grading system for bladder carcinoma.1 Unfortunately, criteria for the 3 different grades are rather vague, resulting in considerable interobserver variability.2 When pathologists use a 3 grade system they have a tendency to classify a majority of tumors into the middle group.3 The urologist on the other side is uncertain about how to treat patients in the middle group (WHO 2). The urologist is aware of the fact that grade 2 may imply “almost grade 1 or almost grade 3,” which means that he/she has to monitor the patient with frequent endoscopic examinations. Consequently, the urologist will have a major problem when the

pathologist classifies a high proportion of noninvasive tumors as WHO 2. Some authors have suggested a 2 tier grading system with high and low grade tumors2, 4 but this does not seem to have gained acceptance among pathologists and urologists. During 1998 the WHO and International Society of Urological Pathology (ISUP) reached a consensus on a new classification.3 Papillary lesions were categorized as papilloma, papillary urothelial neoplasm of low malignant potential, or low or high grade papillary carcinoma. The WHO classification published in 1999 uses the term WHO 1 for low grade carcinoma and allows for the separation of high grade carcinoma into WHO 2 and WHO 3.5 Histopathological grading of bladder cancer has been proved to be of considerable value for predicting clinical outcome but other more objective methods,

Accepted for publication October 6, 2000. Supported by Jubileumsklinikens forskningsfond mot cancer and Kommunalfo¨rbundet Va¨stra Go¨taland. 1124

STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS

such as morphometry and flow cytometry, may improve such predictions. Great expectations have been attributed to molecular markers and immunohistochemistry, including p53, Ki67, p21 and Rb, as prognostic indicators but they have not yet come into regular clinical use. We evaluate the clinical relevance of the new WHO/ISUP classification when applied to a large number of noninvasive bladder tumors with longterm followup. We also evaluate whether immunohistochemical expression of various molecular and other markers determined at first diagnosis could predict progression of Ta tumor. PATIENTS AND METHODS

The patient material has been described in detail in previous publications.6, 7 Briefly, all 680 patients in western Sweden (population 1.6 million) with a first diagnosis of bladder carcinoma during a 2-year period from February 1987 through January 1989, were prospectively registered. All histopathological material from the 680 primary tumors was reviewed by an author (S. L. J.). The tumors were graded according to the WHO and modified Bergkvist systems, and the WHO/ISUP consensus classification.1, 3, 8, 9 For the latter classification the term papillary urothelial neoplasm of low malignant potential is used to denote the most benign form of tumor that previously comprised approximately 30% to 50% of the old WHO grade 1, which is equivalent to modified Bergkvist grade 1. Papillary bladder carcinoma is either low grade, which is equivalent to modified Bergkvist grade 2A or 1999 WHO grade 1, or high grade, which is equivalent to modified Bergkvist grade 2B to 3 or 1999 WHO grades 2 to 3. The tumors were staged according to the 1978 TNM system.10 All original patient records were reviewed from 1994 to 1996 by an author (S. H.) resulting in at least a 5-year followup. Recurrence was defined as a tumor identified at cystoscopy that was fulgurated, which had no histopathological material, or resected, which had histopathological material that displayed urothelial malignancy. Progression in stage was defined as recurrence with invasion to or beyond the level of muscularis mucosae, which was deep lamina propria invasion and pT1b, or development of metastatic disease. All 6 patients with low grade papillary urothelial carcinoma (1999 WHO grade 1 and modified Bergkvist grade 2a), all 19 with high grade carcinoma (WHO grade 2 and Bergkvist grade 2B) in whom disease progressed and all 13 with high grade carcinoma (WHO grade 3 and Bergkvist grade 3) were selected for histochemical studies. An age and gender matched control (nonprogressor) with the same grade was selected for each patient with grades 2A and 2B disease that progressed. Immunohistochemistry. The p53, p21 and Ki67 Methods: Paraffin sections were cut 4 ␮m. thick and placed on slides. Before immunohistochemistry heat mediated antigen retrieval was performed by boiling the slides in 0.01 M. citrate buffer, pH 6.0, for 16 minutes at 750 W. in a microwave oven.11 For p53 antigen retrieval was done for 15 minutes in citrate buffer as aforementioned but in a pressure cooker designed for usage in microwave ovens. The primary antibodies were diluted, including p53 (clone D07, DAKO, Glostrup, Denmark) 1:200, p21 (clone EA10, Oncogene Science, Cambridge, Massachusetts) 1:40 and Ki67 (clone MIB1, Dianova, Hamburg, Germany) 1:100. To ensure a standardized performance, staining was done in an automated immunostainer using a diaminobenzidine kit (Ventana, Tucson, Arizona). The slides were manually counterstained in Harris hematoxylin. Finally, the slides were dehydrated through graded alcohols to xylene and mounted in organic mounting medium. Immunostaining for pRb was done manually following the

1125

same antigen retrieval protocol for p53. Both blocking for endogenous peroxidase in 0.3% hydrogen peroxide and preincubation in 10% normal rabbit serum were diluted in phosphate buffered saline and incubated for 20 minutes. The pRb antibody (clone 3C8, QED Bioscience Incorporated, San Diego, California) was diluted 1:200 and incubated for 16 hours at 4C. As link antibody a biotinylated rabbit anti-mouse was applied, followed by a peroxidase labeled streptavidin biotin complex both diluted 1:200 and incubated for 30 minutes. The slides were developed in diaminobenzidine for 6 minutes, counterstained in Harris hematoxylin and dehydrated as aforementioned. Where not otherwise stated reagents were diluted in 0.5% bovine serum albumin in phosphate buffered saline and incubations were performed at room temperature. Washings for 3 to 10 minutes between incubation steps were done in 0.05 M. Tris, pH 7.6, containing 0.3 M. sodium chloride and 0.1% Tween 20. Evaluation of immunohistochemistry. A scoring system based on both extent and intensity was used in the evaluation of the p53, p21 and Ki67 immunohistochemistry. The extent was given a score from 1 to 3. For p53 and p21 the scores were 1–less than 25%, 2–25% to 50% and 3– greater than 50% of tumor cells stained. The MIB1 immunohistochemistry was judged according to a scale in which 1–less than 10%, 2–10% to 25% and 3– greater than 25% of tumor cells stained. For all antibodies the intensity was judged as 0 –negative, 1–weak, 2–moderate and 3–strong staining. The product of the extent and intensity scores, resulting in a number between 0 and 9, was then divided into 2 classes, including I (less than 4), which is absent or low expression and II (4 or greater), which is moderate to high expression. Immunohistochemistry for pRb was judged as negative or positive. A negative staining is defined as lost pRb expression, either in the whole tumor or in extended areas of the tumor, and is paralleled by positive staining in mesenchymal cells. Evaluation of the staining was performed in collaboration by 3 authors (S. L. J., C. B. and K. W.). Only nuclear distributed staining was judged as positive. DNA ploidy. For determination of DNA ploidy in archival material 100 ␮m. thick sections were cut, deparaffinized in xylol, and rehydrated in alcohol and distilled water. The tissue was minced into small pieces, and enzymatic dispersion and digestion were achieved with 0.2% protease type VIII (Sigma, St. Louis, Missouri) for 1 hour at 37C with intermittent vortexing. The suspension was filtered through 70 ␮m. nylon mesh and centrifuged. The supernatant was removed and the pellet was resuspended in 750 ␮l. propidium iodide. The cellular DNA content was measured with a flow cytometer. Details regarding the method have been described previously.12, 13 The ratio between the position of the G2/M and G0/G1 peaks was used to define the ploidy pattern. Samples within the ratio 1.90:2.10 were regarded as diploid provided that the G2/M peak did not exceed 10% of the total number of nuclei. Samples with additional peaks outside this ratio were regarded as nondiploid. Mitotic frequency. There were 4 ␮m. sections of formalin fixed and paraffin embedded tumor tissue stained with a modified Gomori silver methenamine stain.14 Five microscopic fields in the areas with the highest mitotic activity were examined at 400x magnification with the aid of a computerized image analysis system. Low mitotic frequency was designated as that with less than 14 and as high with 14 or greater. Details regarding the method and choice of cutoff level have been described previously.12, 13 Statistics. The probability of recurrence or progression was calculated using the Kaplan-Meier estimate and compared with the log-rank test. The paired t-test was used when analyzing immunohistochemistry results from cases that progressed in stage and the respective matched control.

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STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS RESULTS

A total of 680 patients were diagnosed with bladder carcinoma during the 2-year period. Of these cases 363 (53%) were noninvasive papillary neoplasms (stage Ta). The mean age (plus or minus standard deviation) of the 95 patients with papillary urothelial neoplasm of low malignant potential was 64.6 ⫾ 13.9 years, the 160 with low grade carcinoma was 69.2 ⫾ 11.7 and the 103 with high grade carcinoma was 70.4 ⫾ 11.9. The latter group may be subdivided into 95 patients with new WHO grade 2 and 13 with new WHO grade 3. The difference in age between patients with papillary urothelial neoplasm of low malignant potential, and low and high grade carcinoma, respectively, was significant (p ⬍0.005 and 0.001) All patients underwent diagnostic transurethral resection, which totaled 383 operations in the 363 patients. Multiple tumors at diagnosis were more common in the high grade carcinoma group (table 1.). Additional treatment after initial diagnostic resection as well as after treatment for 1 or more recurrences is found in table 2. It is noteworthy that 83% of all patients were treated with transurethral resection of bladder tumor only. No patient was treated with bacillus Calmette-Guerin (BCG) after diagnostic resection but of the 35 who were treated later with intravesical instillation 13 received BCG. Patients with high grade carcinoma underwent an average number of 4.3 transurethral operations during followup compared to 3.1 in those with low grade carcinoma and 1.5 in those with papillary urothelial neoplasm of low malignant potential. During followup 35% of patients with papillary urothelial neoplasm of low malignant potential were operated on for recurrence compared to 71% with low and 73% with high grade carcinoma (p ⬍0.001, fig. 1, A.). Median time to first recurrence was 8 months for patients with high grade carcinoma and 16 months for those with low grade carcinoma (difference not statistically significant). During followup no papillary urothelial neoplasm of low malignant potential progressed in stage. Disease progressed in 6 of the 160 (4%) patients with low grade carcinoma compared to 25 of the 108 (23%) with high grade carcinoma (p ⬍0.0001, fig. 1, B). The progression rate was also significantly different among patients with WHO grades 2 and 3 (p ⬍0.0011, fig. 2). Progression in stage or metastatic disease was diagnosed in 6 patients with initial low grade carcinoma after 12 to 79 months (median 48) or after treatment for 0 to 16 recurrences (median 7). In the 19 patients with grade 2 progression in stage was diagnosed after 3 to 86 months (median 40) or after treatment for 1 to 16 recurrences (median 5). There were 6 patients with initial grade 3 in whom disease progressed after 5 to 44 months (median 25) or after treatment for 2 to 7 recurrences (median 5). Of the 363 patients with initial noninvasive bladder cancer disease progressed in 31 (8.5%), using deep lamina propria invasion or metastatic disease as criterion for progression. Of those 31 patients disease progressed to muscle invasion in 20 and deep lamina propria invasion in 5. Generalized disease later developed in 4 of the latter 5 patients. Metastatic dis-

TABLE 1. Relationship between number and grade of tumors at first diagnosis Tumor Grade 1

2–3

4 or More

No. papillary neoplasm low malignant potential (%) No. Ca (%): Low grade High grade

81 (85)

10 (11)

4 (4)

124 (78) 80 (74)

23 (14) 17 (16)

13 (8) 11 (10)

Total No. (%)

285 (78)

50 (14)

28 (8)

TABLE 2. Additional treatment after diagnostic transurethral resection initially or later during 5-year followup

No. cases No. intravesical therapy: Initially Late No. external beam radiotherapy: Initially Late No. cystectomy: Initially Late

Papillary Urethral Neoplasm Low Malignant Potential

Low Grade Ca

High Grade Ca

95

160

108

1 3

0 16

2 16

3 (1) 35 (9.6)

0 0

0 0

1 11

1 (0.3) 11 (3)

0 0

1 1

0 10

1 (0.3) 11 (3)

Total No. (%) 363

ease developed in another 6 patients who did not have previous documented invasion in the bladder (table 3). Of the histochemical markers only p53 expression was significantly different among patients with WHO grade 2 in whom disease later progressed and matched controls (p ⫽ 0.0022). Of the 9 patients with a p53 score of 4 or greater disease later progressed in 8. Grade 2 tumor in progressors were more often aneuploid, had higher mitosis frequency and a Ki67 score of 4 or greater compared with nonprogressors but these results were not statistically significant (tables 4 and 5). There was no difference between groups nor between grades for p21, and results are not shown in the tables. Tables 4 and 5 show that there is a considerable difference in marker profiles among patients with grades 2 and 3 disease, although analyses were performed in those with grades 1 and 2 (progressors and matched controls), and in all patients with grade 3. DISCUSSION

Our patient material is rather unique for 2 reasons. It is population based and encompasses all cases diagnosed in a geographic area in contrast to most reports from large treatment centers in which there is a selection of cases.7 Few patients were treated with intravesical therapy or radiotherapy during the first years after diagnosis. In a previous paper we reported on progression rates in patients with low grade papillary urothelial carcinoma and papillary urethral neoplasm of low malignant potential.6 In our present report we also comprise high grade tumors, and include evaluation of the association between various molecular markers and progression. The WHO/ISUP consensus classification was applied to our patient material of noninvasive bladder tumor resulting in 3 subgroups with presumed different clinical behavior. The advantage with the WHO/ISUP classification system as compared with the 1973 WHO system is that the pathologist has to place carcinoma in either the low or high grade group. The middle group simply does not exist anymore. The disadvantage is that the high grade group is composed of 2 subgroups with different clinical behavior corresponding to WHO grades 2 and 3. Noninvasive WHO grade 3 carcinoma is an uncommon neoplasm. In our population based study in western Sweden (population 1.6 million) only 13 of 363 cases were diagnosed during a 2-year period.7 Consequently, it accounts for only 4% of all noninvasive carcinoma and 6% of all grade 3 bladder carcinoma. Of our 13 patients 7 had carcinoma in situ, either at initial diagnosis or later during followup. The reported progression rate in grade 3 pTa varied from 11% to 45%, which may be related to the low number of patients in previous series, as well as selection factors and differences in treatment, in particular, the rate of those treated with cys-

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STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS

FIG. 1. Relationship between papillary urothelial neoplasm of low malignant potential, low and high grade carcinoma and interval to first recurrence (A), and interval to progression (B). PUNLMP, papillary urothelial neoplasm of low malignant potential.

TABLE 4. Results of DNA analysis and mitosis count among patients with and without progression during 5 years of observation WHO Grade

No. mitotic frequency greater than 14% (%): No progression Progression No. tetraploidy (%): No progression Progression No. aneuploidy (%): No progression Progression

1

2

3

5 (20) 5 (0)

19 (16) 17 (29)

5 (71) 4 (83)

5 (0) 5 (0)

16 (19) 17 (12)

7 (43) 6 (33)

5 (0) 5 (0)

16 (19) 17 (41)

7 (57) 6 (66)

TABLE 5. Immunohistochemistry results among patients with and without progression during 5 years of observation WHO Grade

FIG. 2. Relationship between WHO grades 2 and 3 and interval to progression (p ⬍0.0011). TABLE 3. Status at last followup 5 to 7 years after diagnosis Papillary Low Grade High Grade Neoplasm Low Papillary Papillary Malignant Ca Ca Potential No. survived (%): Without bladder tumor With bladder tumor No. dead (%): No bladder tumor at last control Bladder tumor at last control Bladder or upper urinary tract Ca Not examined after diagnosis Total No.

Total

75 (79)

91 (57)

52 (48)

218 (60)

3 (3)

16 (10)

10 (9)

29 (8)

14 (15)

31 (19)

20 (19)

65 (18)

1 (1)

10 (6)

8 (7)

19 (5)

0

6 (4)

17 (16)

23 (6)

2 (2)

6 (4)

1 (1)

9 (2)

95

160

108

363

tectomy immediately after diagnosis.15 The optimal treatment has yet to be determined but the clinical behavior of noninvasive grade 3 carcinoma is likely to be more similar to that of stage pT1 grade 3 than that of stage pTa grade 2.16 In patients with stage pTa grade 3 carcinoma mapping biopsy to determine the presence of concomitant carcinoma in situ seems important, and intravesical BCG treatment should probably be offered to the majority of them. Consequently, it is important to report to the urologist when stage Ta high grade carcinoma is WHO grade 3 carcinoma. There were considerable differences in the marker profiles between grades 2 and 3 carcinoma. Most grade 3 carcinoma was aneuploid, had a high mitosis frequency, high p53 and Ki67 scores as well as loss of retinoblastoma gene expression. Differences between grades 2 and 3 were more pronounced

No. p53 score 4 or greater (%): No progression Progression No. retinoblastoma gene loss (%): No progression Progression No. Ki67 score 4 or greater (%): No progression Progression

1

2

3

4 (25) 4 (50)

15 (7) 18 (44)

7 (57) 4 (75)

5 (0) 5 (20)

19 (5) 19 (5)

7 (57) 6 (50)

5 (20) 4 (50)

19 (21) 19 (42)

7 (71) 6 (66)

than those between grades 1 and 2. This result further underscores the fact that grades 2 and 3 are distinctly different from each other and is another argument against them grouped together in a “high grade carcinoma group.” The WHO/ISUP consensus classification is a step forward but an important improvement would be to report grades 2 and 3 high grade carcinoma separately. We examine whether it is possible to predict future progression already at initial diagnosis. Encouraging reports have suggested p53 as a useful marker for future progression.17 The difference in p53 score between progressors and nonprogressors with initial WHO grade 2 carcinoma reached statistical significance (p ⬍0.0022) as well as in our control study. A p53 score of 4 or greater at diagnosis of stage Ta grade 2 carcinoma is apparently a strong predictor of future progression since 8 of 9 cases progressed during followup. The results confirm observations made by Hudson et al,17 and it would seem logical to give additional treatment to such patients. Unfortunately, intravesical chemotherapy does not prevent progression and the benefit of BCG is small regarding progression.18 Furthermore, the majority of patients with grade 2 carcinoma in whom disease later progressed had values less than 4. It must also be considered that all analyses were performed in 1 laboratory during a limited time. Better standardization of immunohistochemical techniques

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STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS

are urgently needed. Consequently, neither analysis of p53, Ki67, p21, nor determination of mitotic frequency or aneuploidy can currently be recommended for clinical use. We have recently published a technique modification for standardization of immunohistochemistry suggested by Battifora using bladder cancer cell lines with defined mutations, and fibroblasts defined regarding position in the cell cycle for assessment of p53 immunohistochemistry and proliferation, respectively.19, 20 We also agree with Adshead et al who stated that the arrival of new techniques combined with the accessibility of fresh frozen urothelial tissue banks will allow determination of the molecular profile of bladder cancer in the near future. 21 It is likely that multigene analyses of tumors in combination with grade and stage will result in better predictive and prognostic information and will allow better tailoring of treatment. ¨ stra, Carlanderska, Sahlgrenska University Hospital, O Lundby, Mo¨lndal, Kunga¨lv, Uddevalla,, Lysekil, Stro¨mstad, ¨ L, Sa¨ffle, Kungsbacka, Varberg, Halmstad, Borås, Skene, NA Alingsås, Sko¨vde, Lidko¨ping and Falko¨ping contributed to the studies. Ulla-Britt Wallgren and Karin Karlsson provided valuable assistance. REFERENCES

1. Mostofi, F. K., Sorbin, L. H., Torloni, H. et al: Histological Typing of Urinary Bladder Tumours. Geneva: World Health Organization, 1973 2. Schapers, R. F., Pauwels, R. P., Wijnen, J. T. et al: A simplified grading method of transitional cell carcinoma of the urinary bladder: reproducibility, clinical significance and comparison with other prognostic parameters. Br J Urol, 73: 625, 1994 3. Epstein, J. I., Amin, M. B., Reuter, V. R. et al: The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee. Am J Surg Pathol, 22: 1435, 1998 4. Jordan, A. M., Weingarten, J. and Murphy, W. M.: Transitional cell neoplasms of the urinary bladder. Can biologic potential be predicted from histologic grading? Cancer, 60: 2766, 1987 5. Mostofi, F. K., Davis, C. J. and Sesterhenn, I.: Histological Typing of Urinary Bladder Tumours, 2nd ed. Berlin: Springer, 1999 6. Holma¨ng, S., Hedelin, H., Anderstro¨m, C. et al: Recurrence and progression in low grade papillary urothelial tumors. J Urol, 162: 702, 1999 7. Holma¨ng, S., Hedelin, H., Anderstro¨m, C. et al: Prospective registration of all patients in a geographical region with newly diagnosed bladder carcinomas during a two-year period. Scand J Urol Nephrol, 34: 95, 2000 8. Bergkvist, A., Ljungqvist, A. and Moberger, G.: Classification of bladder tumours based on the cellular pattern. Preliminary report of a clinical-pathological study of 300 cases with a minimum follow-up of eight years. Acta Chir Scand, 130: 371, 1965 9. Malmstro¨m, P. U., Busch, C. and Norlen, B. J.: Recurrence, progression and survival in bladder cancer. A retrospective analysis of 232 patients with greater than or equal to 5-year follow-up. Scand J Urol Nephrol, 21: 185, 1987 10. Harmer, M. H.: TNM Classification of Malignant Tumours, 3rd ed. Geneva: International Union Against Cancer, 1978 11. Cattoretti, C., Pileri, S., Parravicini, C. et al: Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol, 171: 83, 1993 12. Malmstrom, P. U., Vasko, J., Wester, K. et al: Flow cytometric analysis of DNA content of deparaffinized nuclei in urinary bladder carcinomas. Comparison of different isolation methods and relation to histological grade and stage. APMIS, 97: 811, 1989 13. Vasko, J., Malmstro¨m, P.-U., Wester, K. et al: Prognostic value of a systematized method for determination of mitotic frequency in bladder cancer assisted by image analysis. J Urogenital Pathol, 1: 53, 1991 14. Busch, C. and Vasko, J.: Differential staining for mitoses in tissue sections and cultured cells by a modified methenamine-

silver method. Lab Invest, 59: 876, 1988 15. Lebret, T., Bohin, D., Kassardjian, Z. et al: Recurrence, progression and success in stage Ta grade 3 bladder tumors treated with low dose Bacillus Calmette-Guerin instillations. J Urol, 163: 63, 2000 16. Herr, H. W.: Tumor progression and survival of patients with high grade, noninvasive papillary (TaG3) bladder tumors: 15year outcome. J Urol, 163: 60, 2000 17. Hudson, M., Liss, A., Swanson, P. E. et al: P53 protein accumulation in superficial bladder cancer is a predictor of subsequent muscle invasion. J Urol Pathol, 2: 307, 1994 18. Lamm, D. L., Blumenstein, B. A., Crissman, J. D. et al: Maintenance bacillus Calmette-Guerin immunotherapy for recurrent Ta, T1 and carcinoma in situ transitional cell carcinoma of the bladder: a randomized Southwest Oncology Group Study. J Urol, 163: 1124, 2000 19. Wester, K., Wahlund, E., Sundstrom, C. et al: Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen. Appl Immunohistochem Molecul Morphol, 8: 61, 2000 20. Wester, K., Andersson, A. C., Ranefall, P. et al: Cultured human fibroblasts in agarose gel as a multi-functional control for immunohistochemistry. Standardization of Ki67 (MIB1) assessment in routinely processed urinary bladder carcinoma tissue. J Pathol, 190: 503, 2000 21. Adshead, J. M., Kessling, A. M. and Ogden, C. W.: Genetic initiation, progression and prognostic markers in transitional cell carcinoma of the bladder: a summary of the structural and transcriptional changes, and the role of developmental genes. Br J Urol, 82: 503, 1998 EDITORIAL COMMENTS The WHO/ISUP reclassification of grading for urothelial (transitional cell) neoplasms in 1998 addressed problems with the 1973 WHO grading system1 but the system was accepted without validation of the prognostic significance of the various grades. Urologists have considerable experience with the former grading system but to my knowledge none with the new system. Therefore, the study by Holmang et al provides valuable information for urologists who must now make management decisions on the basis of the new grading system. It is noteworthy that all patients with stage Ta tumor in an entire population are included, and complete long-term followup for at least 5 years is provided. Important results of this study are that 53% of patients present with stage Ta disease and 78% of them have a solitary tumor. Of patients with stage Ta disease 26% had papillary urothelial neoplasm of low malignant potential, of whom only 35% had tumor recurrence, and no progression to lamina propria invasion occurred. A total of 44% of patients had low grade papillary urothelial carcinoma, and disease recurred in 71% and progressed in 4%. There were 28% of patients who had high grade urothelial carcinoma, and disease recurred in 73% and progressed in 23%. This 28% incidence of high grade carcinoma in patients with stage Ta disease is higher than that reported with the former system, as might be expected with the inclusion of some patients formerly included in the grade 2 category. In the review by Bostwick of 693 patients with Ta disease in 6 series only 41 (6%) had grade 3 transitional cell carcinoma.1 With further dividing of the patients with high grade carcinoma into WHO grades 2 and 3, the risk of progression is more refined. Of those patients with grade 3 disease progressed in 45% compared with grade 2 in 20%. Many molecular markers have been proposed to improve risk assessment but in this study only p53 correlated with risk of progression, and it added further significant information only in patients with grade 2 carcinoma. This important study must be considered in light of the treatment given to this population. Intravesical therapy was used initially in only 1% of patients, none of whom received BCG, and only 13 ever received BCG. Therefore, the statistics reported reflect the untreated natural history. While the authors state that “the benefit of BCG is small regarding progression,” I believe this conclusion is an artifact way in which the data were presented in my study. There were 3 randomized studies that compared suboptimal BCG treatment regimens with surgery alone or surgery plus intravesical chemotherapy that showed a statistically significant reduction in progression overall from 28% to 14%.2 In the study by Milla´n-Rodrı´guez et al in 1,529 patients BCG therapy was associated with a reduction in the hazard

STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS ratio for progression to 0.3 (p ⬍ 0.01).3 With 3 weeks of maintenance BCG progression, or more correctly disease worsening, was more significantly reduced. In this study of high risk patients who had 2 or more stage Ta or T1 tumors within 1 year, 3 or more tumors within the most recent 6 months and/or carcinoma in situ, maintenance BCG resulted in 5-year survival of more than 82% of patients. This result is remarkable considering the present study included only patients with stage Ta disease, 78% of whom presented with a solitary tumor and resulted in 5-year survival of only 68%, 14% less than that of those selected as high risk and given maintenance BCG in the Southwest Oncology Group study. Donald L. Lamm Department of Urology Robert C. Byrd Health Science Center West Virginia University Morgantown, West Virginia 1. Bostwick, D. G.: Natural history of early bladder cancer. J Cell Biochem, suppl., 16I: 31, 1992 2. Lamm, D. L.: BCG in perspective: advances in the treatment of superficial bladder cancer. Eur Urol, 27: 2, 1995 3. Milla´n-Rodrı´guez, F., Che´chile-Toniolo, G., Salvador-Bayarri, J. et al: Multivariate analysis of the prognostic factors of primary superficial bladder cancer. J Urol, 163: 73, 2000 This article is the second in a 2-part exposition of the personal experience of Holma¨ng et al with patients having bladder neoplasms, and both parts should be read together (reference 6 in article). The main theme is apparently a justification for the 1999 WHO classification of urothelial neoplasms. The authors report clinically unusable differences in the expression of select markers in tumors at the extreme end of the histological spectrum of high grade carcinoma but base their conclusions primarily on differences in progression among patients presenting with noninvasive papillary neoplasms after 5-year followup. After stating that 1973 WHO uses criteria to separate 3 grades of carcinoma that are “. . . rather vague . . . ,” the authors imply that they know exactly where the cutoffs lie. They know by using definitions, which they developed for the modified Bergkvist system (reference 8 in article). Grade 1 (1973 WHO) transitional cell carcinoma comprises papillary neoplasm of low malignant potential and low grade carcinoma (1998 WHO/ISUP), whereas grades 2 and 3 (1973 WHO) are combined into high grade carcinoma (1998 WHO/ISUP). Since the progression rates of papillary urethral neoplasm of low malignant potentials and low grade carcinoma are 0% and 4%, respectively, 1998 WHO/ISUP is a step in the right direction. Because high grade carcinoma (1998 WHO/ISUP) can be divided using their modified Bergkvist criteria into a large group (95 of 108) and a small group (13 of 108) with progression rates of 20% and 45%, respectively, why not simply revert to 1973 nomenclature and call all lesions with the potential to progress carcinoma and grade them 1, 2 and 3? Lesions without the potential to progress can be termed papillary urethral neoplasm of low malignant potential. The papilloma in both systems, separated because of the inability to progress, is not discussed. This procedure sounds pretty simple. However the problems are that the perception of the authors of the extrapolations between 1998 WHO/ISUP and 1999 WHO are not generally accepted. Many pathologists would include a considerable number of 1973 WHO grade 2 transitional cell carcinoma lesions in the low grade carcinoma category and most of the 1973 grade 1 carcinoma in the low malignant potential category of 1998 WHO/ISUP. In fact, the cumbersome term papillary urethral neoplasm of low malignant potential evolved from a concerted effort to remove the word “carcinoma” from histologically benign lesions previously called “grade 1.” The definition by the authors of progression is not generally accepted, and the actual rate of progression among their patients cannot be determined because many new lesions were cauterized rather than biopsied for pathological evaluation. The categorization of lesions in this study apparently represents the perception of just 1 person (S. L. J.) and cannot be considered representative of what other pathologists might do (reference 5 in article). In most cases it is not the primary neoplasm that progresses, but new tumors occurring subsequent to complete excision of the index lesion. Patients presenting with papillary urethral neoplasm of low malignant potentials (1999 WHO grade 1) who subsequently die of bladder cancer do not die of deeply invasive or metastatic papillary urethral neoplasm of low malignant potentials, but die of high grade carcinoma. Therefore, in my opinion

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the term “carcinoma” is inappropriate to describe grade 1 tumors. Splitting off a small group at the highest end of any histological spectrum creates a situation in which the denominator of any equation is so small that whatever happens to that group is magnified in comparison. For example had only 2 patients with grade 3 transitional cell carcinoma not had progression in this study, there would have been no difference from grade 2. Readers who might be confused by all of this data are not alone. If we are to be guided by the personal experience of 1 group then there is no need for consensus. The facts will speak for themselves. Of the authors 2 were signatories to the 1998 WHO/ISUP formulation. If consensus is to be disclaimed by its signatories, whom or what are others to believe? When we read that a pathologist subscribes to WHO, is it 1973, 1998 or 1999? Can we accept personal definitions of progression (pT1a is not, pT1b, c are)? What about extrapolations from 1 classification scheme to another? Would all urological pathologists agree? Is a variance of 25% in outcome established on the basis of 1 or even 2 pathologist categorizations important enough to alter an international consensus, even assuming that the variance could be verified? What effect will these results have on patient care? Under the circumstances, urologists, pathologists and patients can despair of any true consensus on the classification of urothelial neoplasms. However, maybe adding genetic profiling will clear up everything. William M. Murphy Department of Pathology and Laboratory Medicine University of Florida Gainesville, Florida REPLY BY AUTHORS Of 363 patients with initial stage Ta disease only 6% died of bladder cancer, and 23% died of other diseases during the observation period. We also wish to emphasize that the purpose of our report was to evaluate the prognostic implication of the 1998 WHO/ISUP and the 1999 WHO classifications in a large prospective study rather than to justify either of them. The statement that the 2 articles quoted in the editorial comment represent an exposition of our “personal experience with patients having bladder neoplasms” is true. Therefore, we cautiously remind readers that careful presentation and analysis of personal experience are the foundation and essence of science, and we welcome further analysis of independent clinical materials regarding the implications of the classification systems. The efforts of the WHO/ISUP group and WHO panel to reform the 1973 classification had the same 3 major incentives. 1) The criteria used in the 1973 WHO classification were not as clearly defined as they became in the 1998 WHO/ISUP and the 1999 WHO classification. As a matter of fact, the criteria are outlined on 6 lines establishing that “grade 1 tumors have the least degree of anaplasia compatible with the diagnosis of malignancy; grade 3 applies to tumors with the most severe degrees of cellular anaplasia and grade 2 lies in between.” 2) The notion that all papillary tumors are cancers except for papilloma does not correspond with international experience regarding the progression rates and mortality of low grade urothelial neoplasms. Thus, the papillary neoplasm of low malignant potential category was defined in WHO/ISUP and WHO 1999 systems. 3) The 1973 WHO classification was poorly reproducible, mainly because of poorly defined criteria, and as a consequence the large poorly defined intermediate group (grade 2). It is suggested that one should simply revert to 1973 nomenclature and lesions without the potential to progress can be called papillary neoplasm of low malignant potential, which is essentially what the 1999 WHO classification does. We agree with the statement that the 1973 grade 2 probably used to be included in what is now labeled low grade or grade I tumors at some centers (see table). Since we found no papillomas in our series we had no reason to discuss this entity which is rare if strict criteria are used (AFIP Fascicle 11 by Koss 1975). The WHO/ISUP 1998 and WHO 1999 classifications are identical and easily “translated” into one another except that WHO 1999 subclassifies the high grade tumors. The WHO/ISUP Consensus classification provides a simpler and possibly more reproducible stratification of the low and high grade categories. Both systems can be easily reported, for example “TUR-B with fragments of low grade urothelial carcinoma, WHO 1999 grade 1” and so forth or “TUR-B

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STAGE PROGRESSION IN TA PAPILLARY UROTHELIAL TUMORS Malignancy grading of bladder carcinoma: old and new systems

Modified Bergkvist 1987 Grade Grade Grade Grade Grade

0 papilloma 1 papilloma with atypia 2a urothelial Ca 2b urothelial Ca 3 urothelial Ca

WHO 1973 Papilloma Grade 1 transitional Grade 1 transitional Grade 2 transitional Grade 3 transitional

WHO 1999 cell cell cell cell

Ca Ca Ca Ca

with fragments of high grade urothelial carcinoma, WHO 1999 grade II” or “TUR-B with fragments of high grade urothelial carcinoma, WHO 1999 grade III.” We agree that our definition of progression is unusual but the difference would have been small (30 instead of 31 patients) if we had used muscle invasion or metastatic disease as a criterion for progression. We use this definition since one of us (S. L. J.) was responsible for the pathological evaluation in the study by Anderstrom et al1 and, consequently, experienced the assessment of T1 tumors. Although it was not mentioned in our report, Busch reviewed 100 cases with no knowledge of Johansson’s interpretation. There was agreement in 98 cases and the 2 discrepancies concerned papillary neoplasm of low malignant potential versus grade I carcinoma. The statement “the term carcinoma is inappropriate to describe grade 1 tumors” is valid and equally so for the WHO/ISUP 1998 (low grade) and WHO 1999 (low grade I) classifications. However, using the detailed descriptions of criteria, including order/disorder and variation/no variation distinctions, there is a consensus in both working groups of how to distinguish the low grade/grade I category. We agree completely that the high grade III category is small and, therefore, welcome further studies. However, the results in our report support previous results, which showed a significant difference in survival rates between categories 2b and 3 (equivalent to WHO 1999 grades II and III) and corresponding to WHO grades II and III (reference 9 in article). In previous morphometric studies numerous parameters were analyzed and revealed differences between these categories.2 These facts added to the marker profiles in our study indicate that it will be worthwhile to compare all categories in the

WHO/ISUP 1998 Consensus

Papilloma Papilloma Papillary urothelial neoplasm of low malignant potental Grade I urothelial Ca Low grade urothelial Ca Grade II urothelial Ca High grade urothelial Ca Grade III urothelial Ca High grade urothelial Ca

WHO 1999 classification with each other, especially if modern genetic techniques are used, for example micro-array or differential display methods. We do not believe that genetic profiling will resolve everything but would argue that genetic information will add to morphology, and functional and genetic markers, including the DNA ploidy profile which already supports the validity of the new classifications as shown in our report. Finally, there is obviously a need for pedagogical efforts (web sites, workshops) to practice the “new” principles for grading. It should be recognized that there are no differences between the WHO/ISUP and WHO 1999 systems except for subdivision of the high grade tumors, and we strongly recommend use of pattern recognition features of order/disorder, variation/no variation for distinction of grades in the diagnostic process. In the future the new technologies using inference networks including case based reasoning may add to the diagnostic arsenal.3 1. Anderstrom, C., Johansson, S. L. and Nilsson, S.: The significance of lamina propria invasion on the prognosis on patients with bladder tumors. J Urol, 124: 23, 1980 2. Choi, H. K., Jarkrans, T., Bengtsson, E. et al: Image analysis based grading of bladder carcinoma. Comparison of object, textures and graph based methods and their reproducibility. Anal Cell Path, 15: 1, 1997 3. Mazzucchelli, R., Santinelli, A., Colanzi, P. et al: Urothelial papillary lesions. Development of a Bayesian belief network for diagnosis and grading. Unpublished data