Correlation of Ki-67 and p53 With the New World Health Organization/International Society of Urological Pathology Classification System for Urothelial Neoplasia.

The World Health Organization/International Society of Urological Pathology (WHO/ISUP) consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder was developed in 1998 to create "a universally acceptable classification system for bladder neoplasia that could be used effectively by pathologists, urologists, and oncologists."[1] This system consists of 12 diagnostic entities encompassing both neoplastic and reactive flat and papillary lesions. Histologic criteria for each of these entities are clearly described in the manuscript introducing this system. However, it is unknown whether these hematoxylin-eosin--based diagnoses are associated with indicators of biologic behavior, including overexpression of p53 gene product or an increased proliferative index as measured by Ki-67 expression. Further, it is unclear whether immunohistochemical staining for Ki-67, p53, or both antigens can be of diagnostic utility in classifying problematic urothelial biopsies by the new WHO/ISUP schema.

MATERIALS AND METHODS

Samples of the entities described in the WHO/ISUP classification system were obtained from review of routine hematoxylineosin--stained glass slides from the archives of Wilford Hall Medical Center. The blocks of the corresponding formalin-fixed, paraffin-embedded urothelial biopsies were retrieved from the archives and additional 4-[micro]m-thick sections were placed on sialinated slides for batched immunohistochemical staining with antibodies to Ki-67 (MIB-1) (1:200, Dako Corporation, Carpinteria, Calif) and p53 (1:200, BioGenex, San Ramon, Calif) using standard methodology. Briefly, slides were microwaved with a citrate buffer for heat-induced epitope retrieval. All stains were performed using the HRP (horseradish peroxidase) Detection System (Cell Marque, Austin, Tex). One hundred fifty-one bladder biopsies from 81 patients were studied; these biopsies included normal urothelium (n = 34); low-grade intraurothelial neoplasia (LGIUN, "dysplasia") (n = 19); high-grade intraurothelial neoplasia (HGIUN, "carcinoma in situ") (n = 20); papillary hyperplasia (n = 4); papilloma (n = 3); papillary neoplasm of low malignant potential (LMP) (n = 12); papillary urothelial carcinoma, low grade (n = 28); and papillary urothelial carcinoma, high grade (n = 31). Two hundred cells from the most immunoreactive regions of each lesion were visually counted and the percentage of cells positive for p53 and Ki-67 (labeling index [LID was tabulated without knowledge of the WHO/ISUP diagnosis. Immunohistochemical positivity was defined as strong, homogenous nuclear staining; nonspecific staining of umbrella cells was excluded.

RESULTS

The lesions included in this study represented 8 of the entities described in the new WHO/ISUP classification system. The immunostaining characteristics are summarized in Tables 1 through 4, and the statistical relationships between the LIs for the 2 antigens among the various entities are shown in Table 5.

Table 1. Percentage of p53-Positive Cells Within Various
Papillary Entities in the WHO/ISUP Classification(*)

                          Mean p53   Median p53   Range of p53
Entity                    Index, %    Index, %     Indices, %

Papillary hyperplasia        0         0            0-0
Papilloma                    0         0            0-0
Papillary urothelial
  neoplasm of LMP            0.4       0            0-2
Papillary urothelial
  carcinoma, low grade       2.9       1            0-20.5
Papillary urothelial
  carcinoma, high grade     25.7       8.5          0-100

(*) WHO/ISUP indicates World Health Organization/International
Society of Urological Pathology; LMP, low malignant potential.
Table 2. Percentage of p53-Positive Cells Within
Various Flat (Nonpapillary) Entities in the WHO/ISUP
Classification(*)

         Mean p53   Median p53   Range of p53
Entity   Index, %    Index, %     Indices, %

Benign     1.1         0             0-29
LGIUN      3.2         0             0-22.5
HGIUN      5.9         0.25          0-70

(*) WHO/ISUP indicates World Health Organization/International
Society of Urological Pathology; LGIUN, low-grade intraurothelial
neoplasia (dysplasia); and HGIUN, high-grade intraurothelial
neoplasia (carcinoma in situ).
Table 3. Percentage of Ki-67--Positive Cells Within Various
Papillary Entities in the WHO/ISUP Classification(*)

                          Mean Ki-67   Median Ki-67   Range of Ki-67
Entity                     Index, %      Index, %       Indices, %

Papillary hyperplasia         1.1          1.2             0-2
Papilloma                     4.3          4               1-8
Papillary urothelial
  neoplasm of LMP             2.5          1             0.5-15
Papillary urothelial
  carcinoma, low grade        7.3          3.7           0.5-38.5
Papillary urothelial
  carcinoma, high grade      15.7         11               1-65

(*) WHO/ISUP indicates World Health Organization/International
Society of Urological Pathology; LMP, low malignant potential.
Table 4. Percentage of Ki-67--Positive Cells Within Various
Flat (Nonpapillary) Entities in the WHO/ISUP Classification(*)

         Mean Ki-67   Median Ki-67   Range of Ki-67
Entity    Index, %      Index, %       Indices, %

Benign       0.62         0              0-5.5
LGIUN        3.3          1              0-30
HGIUN       11.6          9.2            0-36

(*) WHO/ISUP indicates World Health Organization/International
Society of Urological Pathology; LGIUN, low-grade intraurothelial
neoplasia (dysplasia); and HGIUN, high-grade intraurothelial
neoplasia (carcinoma in situ).
Table 5. Discriminatory Power of Ki-67 and p53 for Various
Entities in the WHO/ISUP Classification by Rank Sum Test(*)

Lesions                               p53     Ki-67

Benign vs LGIUN                        NS     .035
Benign vs HGIUN                      .008    <.00001
LGIUN vs HGIUN                         NS     .0008
Papillary hyperplasia vs papillary
  carcinoma, low grade               .025        NS
Papillary hyperplasia vs papillary
  carcinoma, high grade              .005     .003
Papillary neoplasm of LMP vs
  papillary carcinoma, low grade     .027     .02
Papillary neoplasm of LMP vs
  papillary carcinoma, high grade    .0001   <.00001
Papillary carcinoma, low grade vs
  papillary carcinoma, high grade    .0012    .0013
Papilloma vs papillary carcinoma,
  high grade                         .013        NS
Papilloma vs papillary carcinoma,
  low grade                          .049        NS

(*) Other comparisons yielded results that were not statistically
significant. WHO/ISUP indicates World Health Organization/
International Society of Urological Pathology; LGIUN, low-grade
intraurothelial neoplasia (dysplasia); HGIUN, high-grade
intraurothelial neoplasia (carcinoma in situ); LMP, low malignant
potential; and NS, not significant.

Benign Urothelium

Immunoreactivity to p53 did not allow for discrimination of benign urothelium from dysplasia (LGIUN) (Table 2). In one case, p53 antibody labeled 29% of benign urothelial cells (Figure 1). In this case, HGIUN was present elsewhere in the bladder. Expression of Ki-67 was limited in benign urothelium (Table 4); the mean LI was 0.62% of cells, and none of the Lis exceeded 5.5%.

[ILLUSTRATION OMITTED]

Low-Grade Intraurothelial Neoplasia

Expression of p53 was limited in most cases of LGIUN (mean LI, 3.2%; range, 0%-22.5%) (Table 2). Interestingly, in 2 of 3 cases with elevated p53 staining ([is greater than] 10% positive cells), HGIUN was found elsewhere in the bladder. In contrast, only 1 of 16 cases of LGIUN with a p53 LI of less than 10% was associated with concurrent HGIUN. Surprisingly, the p53 Lis in the associated HGIUN lesions in these 3 cases were 0%. LGIUN had an increased proliferative index (mean Ki-67 LI, 3.3%; range, 0%-30%) compared with benign urothelium (mean LI, 0.62%; range, 0%-5.5%) (Table 4 and Figure 2, a).

[ILLUSTRATION OMITTED]

High-Grade Intraurothelial Neoplasia

Elevated p53 expression (mean LI, 5.9%; range, 0%-70%) discriminated HGIUN from benign urothelium (mean LI, 1.1%; range; 0%-29%) but not from LGIUN (Table 2). In HGIUN, the increased nuclear staining with antibodies to Ki-67 (mean LI, 11.6%; median, 9.2%; range, 0%-36%) was significantly different from the patterns noted in both LGIUN and benign urothelium (P = .0008 and P = .00001, respectively) (Table 4 and Figure 2, b).

Papillary Hyperplasia

Overexpression of p53 was not noted in 4 of 4 cases of papillary hyperplasia (Table 1), and Ki-67 staining was negligible (mean LI, 1.1%; median, 1.2%; range, 0%-2%) in these neoplasms (Table 3).

Papilloma

Papillomas are rare lesions in the new WHO/ISUP classification system. None of the 3 cases of papilloma in this study were immunoreactive with antibodies to p53 (Table 1). The proliferative index was slightly elevated in papillomas (mean Ki-67 LI, 4.3%; median, 4%; range, 1%-8%) (Table 3).

LMP Papillary Urothelial Neoplasm

In LMP papillary urothelial neoplasms, p53 expression was rarely noted (mean LI, 0.4%; median, 0%; range, 0%-2%) (Table 1); However, these lesions had increased Ki-67 positivity (mean LI, 2.5%; median, 1%; range, 0.5%-15%) compared with papillary hyperplasia and decreased expression compared with papilloma (Table 3), although these differences were not statistically significant.

Papillary Urothelial Carcinoma, Low Grade

In the low-grade papillary urothelial carcinomas, p53 expression (mean LI, 2.9%; median, 1%; range, 0%-20.5%) (Table 1) effectively discriminated low-grade carcinoma (Figure 3, a) from papillary hyperplasia, LMP neoplasms, and high-grade carcinoma (Figure 3, b). Expression of Ki-67 (mean LI, 7.3%; median, 3.7%; range, 0.5%-38.5%) also distinguished low-grade carcinomas from high-grade carcinomas and LMP tumors (Table 3 and Figure 4, a through c).

[ILLUSTRATIONS OMITTED]

Papillary Urothelial Carcinoma, High Grade

In the high-grade papillary urothelial carcinomas, p53 overexpression was common (mean LI, 25.7%; median, 8.5%; range, 0%-100%) (Table 1) though not always seen. These neoplasms showed an increased proliferative index as measured by Ki-67 expression (mean LI, 15.7%; median, 11%; range, 1-65%) (Table 3).

Summary

Expression of the p53 gene product by more than 30% of cells was noted only in high-grade urothelial carcinoma (11/31 biopsies) and a single case of HGIUN (1/20 biopsies) (Figure 5). Among the 53 lesions of benign neoplasms and LGIUN, a Ki-67 LI greater than 10% was seen in only one case of LGIUN (Figure 6). In contrast, 9 of 20 cases of HGIUN had Ki-67 Lis greater than 10%. Among papillary lesions, Ki-67 Lis greater than 20% were noted in only low-grade (3/28 biopsies) and high-grade (10/31 biopsies) papillary urothelial carcinomas.

[ILLUSTRATIONS OMITTED]

COMMENT

The WHO/ISUP classification of urothelial neoplasms represents a consensus that resulted from the recognized need among pathologists, urologists, and oncologists for a universally acceptable and clinically useful classification of bladder neoplasia.[1] The current study is an attempt to correlate this histologic subdivision with previously studied markers of neoplastic progression in urothelial carcinoma.

Cytogenetic and molecular studies have shown the existence of a strong relationship between urothelial carcinoma and alterations involving specific chromosomes.[2-5] It has been determined that chromosomal loss and inactivation of tumor suppressor genes play a significant role in the development and progression of these tumors.

The p53 gene is a tumor suppressor gene that maps to the human chromosome 17p13.1.[4,6] The product of the gene is a cellular phosphoprotein that has been shown to have tumor suppressive properties.[7] Compared with the wild type protein, mutant p53 protein is more stable with a prolonged half-life and more likely to be detected by immunohistochemical analysis.[8] Esrig et al[9] demonstrated a strong association between the immunohistochemical detection of p53 nuclear accumulation in formalin-fixed, paraffin-embedded tissue and the presence of a mutation in the p53 gene as demonstrated by the method of single-strand conformational polymorphism analysis. Heterogeneity in nuclear immunoreactivity of monoclonal antibodies to p53 has been previously discussed.[9,10] Regardless of the mechanism for nuclear reactivity, accumulation of the protein is indicative of a change in the cell state, and detection of this change by immunohistochemistry has been shown to aid the diagnosis of malignant disease.[11]

Mutations involving the p53 gene have been found in a wide variety of malignancies including urothelial carcinomas.[8,12-14] Mutations of the p53 gene and immunohistochemical positivity for the p53 protein have been found in 40% to 60% of urothelial carcinomas.[13-15] Many investigators have demonstrated a positive correlation between tumor expression of p53 and pathologic indicators of progression in urothelial carcinoma, including high grade and stage[16-18] Fujimoto et al[12] reported that the incidence of p53 mutations is much higher in invasive and high-grade urinary bladder cancers than in superficial and low-grade tumors. Likewise, Harano et al[19] found p53 mutations to be associated with higher grades and later stages of bladder tumors. Although these studies have suggested that mutations of the p53 gene are involved in late events of tumorigenesis, other studies have demonstrated changes in p53 expression in premalignant noninvasive lesions, including those of the oral cavity,[20] bronchial mucosa,[20] and esophagus[21] Soini et al[17] demonstrated a strong association of p53 staining between dysplasias and urothelial carcinomas, indicating that similar p53 mutations may be seen in the preinvasive stage of bladder neoplasia.

In the present study, p53 expression was a feature of papillary carcinomas. The staining pattern allowed a statistically significant separation of the 2 categories of papillary carcinoma (low and high grade) as classified in the WHO/ISUP consensus classification (discriminatory power, .0012). These findings are in agreement with earlier studies, which have demonstrated the association of p53 expression with tumor grade. With respect to flat (non-papillary) lesions, p53 expression was also seen with increasing grade; however, the discriminatory power was only significant for the comparison of benign versus high-grade intraurothelial neoplasia. Expression of p53 was identified (LI, 29%) in one case of benign urothelium that happened to be adjacent to a high-grade intraurothelial lesion. The significance of finding positivity for p53 in benign urothelium is uncertain; however, using p53 immunolabeling, Xu et al[22] demonstrated monoclonality of multifocal urothelial carcinoma and long-term persistence of tumor clones in the bladder in the absence of obvious tumor growth. The presence of p53 within benign urothelium may be indicative of the presence of abnormal molecular events with the potential for neoplastic progression and/or may serve as a marker for the concurrent presence of dysplasia or carcinoma elsewhere in the bladder.

Ki-67 is a nuclear protein that is present during G1, S, G2, and M phases of cycling cells. Studies have consistently shown that cell proliferative activity correlates with the growth of many human neoplasms, including urothelial carcinoma.[23,24] The Ki-67 antigen is sensitive to most fixatives, thus requiring frozen tissue sections for analysis. The MIB-1 monoclonal antibody detects a formalin-resistant epitope of the Ki-67 antigen and is suitable for use on formalin-fixed paraffin-embedded tissue[25] Previous studies have shown an association between cell proliferation and tumor grade, stage, recurrence, and prognosis in bladder carcinoma.[26,27] Additional studies demonstrated a significant correlation between the MIB-1 index and the tumor grade and stage of urinary bladder cancer.[28,29] In a study of radical cystectomy specimens in patients treated for urothelial carcinoma of the bladder, Tsuji et al[30] showed that a high Ki-67 index ([is greater than] 32%) correlated with p53 expression and a significantly worse prognosis. In the present study, the proliferative index as demonstrated by nuclear immunoreactivity with the MIB-1 monoclonal antibody was increased in the papillary carcinoma group with good discriminatory power between low- and high-grade lesions (discriminatory power, .0013). These findings are similar to those observed with p53 staining and correlate with previous studies that have demonstrated an increased proliferative index with increasing grade of tumor. In this study, papillomas showed a slightly increased proliferative index (mean Ki-67 LI, 4.3%). Previous studies have demonstrated similar proliferative activity in papillomas.[31] Interestingly, papillary neoplasms of LMP seemed to show a lower proliferative index than papillomas. Despite this finding, we feel that the increased atypia noted in LMP tumors compared with papillomas merits separation of these 2 entities. The histologic features noted in LMP tumors are associated with an increased risk of bladder carcinoma; this does not appear to be true of the cytologically bland papillomas.[1] This study also represents the first immunohistochemical study of papillary hyperplasia, a lesion characterized by an absence of p53 gene product accumulation and a very low proliferative index; however, a larger series is required to better understand the biologic potential of this recently described entity. Lastly, increasing Ki-67 positivity also separated LGIUN lesions from benign urothelium (discriminatory power, .035) and from HGIUN lesions (discriminatory power, .0008).

This study clearly shows that many benign papillary lesions and tumors of LMP may be separated from papillary carcinomas by their immunohistochemical staining properties. Comparison with the staining patterns noted in previous studies for papillary carcinomas is difficult because grading was rather subjective, and the grading in previous studies may have been based on either a 3- or 4-tiered system. Regarding flat urothelial lesions, a literature search revealed several studies that examined p53 and Ki-67 expression in flat urothelial lesions of the bladder; however, the histologic criteria used to discriminate "dysplasia" from "severe atypia" and "carcinoma in situ" are unclear. Using WHO/ISUP histologic criteria, there is a clear increase in the proliferative index between LGIUN and HGIUN, as indicated by Ki-67 immunoreactivity, and, further, these worrisome lesions may be readily distinguished from benign urothelium in most cases.

In summary, Ki-67 and p53 stains may be used as adjuncts to routine histologic sections in the diagnosis of urothelial biopsies. We believe that these stains may be used to support histologic impressions or may be employed to suggest the correct diagnosis in problematic cases. Useful scenarios for application of this immunostain battery include establishing the diagnosis of "denuding cystitis" (HGIUN) and discriminating between benign urothelium and LGIUN based on the proliferative index. The histologic entities described in the new WHO/ISUP system demonstrate an increase in proliferative activity and p53 gene product accumulation with progression from benign urothelium through intermediate entities to high-grade carcinoma.

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Accepted for publication December 21, 2000.

From the Department of Pathology, Wilford Hall Medical Center, LAFB, San Antonio, Tex (Drs Cina and Lancaster-Weiss) and the Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Md (Ms Lecksell and Dr Epstein).

Reprints: Jonathan I. Epstein, MD, Meyer 7-181, Department of Pathology, The Johns Hopkins Medical Institutions, 600 N. Wolfe Street, Baltimore, MD 21287 (e-mail: [e-mail]).