Immunoreactive p53 Protein in High-Grade Prostatic Intraepithelial Neoplasia

Immunoreactive p53 Protein in High-Grade Prostatic Intraepithelial Neoplasia

Path. Res. Pract. 191, 881- 887 (1995) Immunoreactive pS 3 Protein in High-Grade Prostatic Intraepithelial Neoplasia P. A. Humphrey and P. E. Swanson...

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Path. Res. Pract. 191, 881- 887 (1995)

Immunoreactive pS 3 Protein in High-Grade Prostatic Intraepithelial Neoplasia P. A. Humphrey and P. E. Swanson Lauren V. Ackerman Laboratory of Surgical, Pathology, Department of Pathology, Washington University Medical Center, St. Louis, MO, U. S.A.

SUMMARY p53 protein accumulation in the nuclei of prostatic carcinoma cells, as detected by immunohistochemistry, has been associated with increased cell proliferation rate, increased histologic grade and stage, androgen independence and decreased patient survival. Little is known, however, of p53 in prostatic intraepithelial neoplasia (PIN), the putative precursor proliferation for moderately to poorly differentiated peripheral zone carcinoma of the prostate. In this investigation, we utilized a panel of antibodies reactive with p53 protein to assess p53 protein accumulation in prostatic epithelial hyperplasia, PIN and prostatic carcinoma. Forty patients who had undergone radical prostatectomy were selected for study based on the presence of high grade PIN and carcinoma in the same prostate tissue block. Tissue sections were treated with microwave irradiation for antigen retrieval, and antibodies DO-7, PAb1801 and CM-l were used for immunohistochemical analysis. An intense signal for immunoreactive p53 was identified in the nuclei of 7140 (17.5%) clinically-localized prostatic carcinomas. In all 7 cases, high grade PIN also exhibited intense p53 immunoreactivity, whereas only one case of hyperplasia contained immunoreactive p53 protein. These findings support a close relationship between high grade PIN and carcinoma in a subset of primary prostatic carcinomas with high-level p53 protein accumulation.

Introduction Mutation of the p53 tumor-suppressor gene is currently considered the most common genetic abnormality in human cancer 9,19,21,23,29,42 with 37% of all types of malignancies exhibiting mutation of p53 19 . Only relatively few (n =131) cases of prostatic carcinoma have been assessed for p53 mutations by DNA sequencingll ,19. Awide incidence range of p53 mutation, from 0%26 to 42%11, has been reported for clinically localized carcinoma. The timing of p53 gene mutation is also uncertain. While many studies have concluded that p53 gene mutation is a late event in the progression of © 1995 by Gustav Fischer Verlag, Stuttgart

prostatic carcinoma2,5,20,26,37, a recently published investigation 11 documented mutations in a substantial fraction of low-grade and low-stage carcinomas, suggesting that p53 mutations might occur early in tumor formation. Immunohistochemical detection of accumulated p53 protein in the nuclei of malignant tumor cells is predictive, for some tumor types, of missense point mutation in the p53 gene B . Most p53 mutations may be detected by immunohistochemical staining for the p53 protein, but potentially 20% of all mutants may not be identified by this method. Moreover, mechanisms do exist that result in nuclear p53 protein accumulation in 0344-0338/95/0191-0881$3.50/0

882 . P. A. Humphrey and P. E. Swanson

the absence of gene mutation 23 . For prostatic carcinoma, very good to excellent concordance between p53 gene mutation and detection of immunoreactive p53 protein has been observed 5, 12,37. Immunohistochemical detection of p53 protein in prostatic carcinoma has been associated in some, but not all, studies with higher histologic grade, ONA aneuploidy, high tumor cell proliferation rate, high stage and decreased patient survivaF,5, 16,20,27,28,34,37, 43,45-48. The timing of p53 protein accumulation, as determined by immunohistochemical analysis in hyperplasia, PIN and carcinoma in the prostate gland, has not been well-established. Most studies have not identified immunoreactive p53 protein in the nuclei of hyperplastic epithelial cells, but several recent studies 15 ,27 indicate that hyperplastic epithelial cell nuclei may accumulate detectable quantities of p53. Little is known of immunoreactive p53 in prostatic intraepithelial neoplasia (PIN), the putative precursor for many invasive carcinomas of the prostate. No immunoreactive p53 was identifed in high-grade PIN in one study37, while in a second study (thus far reported only in abstract form 28 ) p53 was detected in PIN. Two additional investigations 43 ,46 have described accumulation of p53 protein in a few cases (total n =5) of PIN of unspecified grade. The specific aim of this investigation was to characterize immunoreactive p53 in high-grade PIN and compare the expression to that in usual epithelial hyperplasia and carcinoma in the same cases. We utilized a panel of antibodies directed against different epitopes on the p53 protein to detect p53 protein accumulation in radical prostatectomy tissues from 40 patients. The results show a concordance between highlevel p53 protein immunoreactivity in high-grade PIN and carcinoma, suggesting that p53 protein accumulation may be an early event, occurring at the stage of intraepithelial neoplasia, for those primary prostatic carcinomas with p53 protein accumulation. Results Monoclonal antibody 00-7 in conjunction with microwave irradiation of tissue sections yielded the highest sensitivity in detecting p53 j,rotein. This finding is consistent with a recent report 3 that found 00-7 to be one of the anti-p53 antibodies that yielded the most consistent staining. Table 1 summarizes the results for detection of immunoreactive p53 by antibody

Table 1. Immunoreactive p53 protein in the prostate Epithelial Proliferation Hyperplasia High-Grade PIN Carcinoma

15 16




+ intense

15 15

6 2

1 7




- : No immunostaining, ±: Less than 5% nuclei stained, than 5% nuclei stained, + intense: Greater than 5% nuclei stained with high intensity.

+: Greater

00-7. Use of PAb 1801 was confirmatory in identification of immunoreactive p53 in the intensely positive category but this antibody was less sensitive, under the conditions employed, in detection of lesser degrees of p53 protein accumulation. Immunostains using CM1 antibody in this study produced high stromal and cytoplasmic staining and were not considered interpretable. The results presented below are those obtained with antibody 00-7. Immunoreactive p53 protein was identified in nuclei of hyperplastic epithelial cells, nuclei of high-grade PIN and nuclei of invasive carcinoma. Seven of thirty-seven cases (18.9%) of usual hyperplasia exhibited nuclear staining for p53. Most (617) of these cases were of lower-level immunostaining intensity, with one case showing intense immunoreactivity for p53. Both hyperplastic luminal and basal cell nuclei exhibited staining for p53. In the case with an intense signal, nuclei in atrophic glands were also stained. High-grade PIN had immunoreactive p53 protein in 9/40 (22.5%) cases. In contrast to usual hyperplasia, most (719) cases of high-grade PIN with immunoreactive p53 displayed intense reactivity. Immunoreactive p53 was identified in all four architectural patterns of high-grade PIN8, including micropapillary and tufted patterns (Fig. 1), cribriform pattern (Fig. 2) and flat pattern (Fig. 3). Both isolated high-grade PIN and high-grade PIN intermingled with invasive carcinoma (Figs. 1 and 4) were positive for p53 protein. Where transitions between high-grade PIN and invasive carcinoma were identified, concordance in p53 protein accumulation in the intraepithelial and invasive neoplastic components could often be identified (Fig. 4). Of significance, there was concordance between high-grade PIN and carcinoma for cases with an intense signal for p53. In the 7 cases of carcinoma with intensely stained nuclei, a similarly intense signal was identifed in high-grade PIN on the same slide. Two

Fig. 1. Immunoreactive p53 protein in micropapillary and tufted types of high-grade PIN, as defined by Bostwick et al. 8• Ad- ~ jacent small-acinar invasive adenocarcinoma also exhibits accumulation of p53 protein (Diaminobenzidine as chromogen in ABC immunoperoxidase method, with hematoxylin counterstain, x 68). - Fig. 2 Cribriform type of high-grade PIN with immunoreactive p53. Basal cells in this gland are negative for p53, as are surrounding fibromuscular cell nuclei (Diaminobenzidine as chromogen in ABC immunoperoxidase method, with hematoxylin counterstain, x 168). - Fig. 3. Partial gland involvement by flat type of high-grade PIN, with immunoreactive p53 in nuclei of PIN cells (Diaminobenzidine as chromogen in ABC immunoperoxidase method, with hematoxylin counterstain, x 168). - Fig. 4. Transition between high-grade PIN and invasive carcinoma, with p53 protein accumulation in both the intra epithelial and invasive elements (Diaminobenzidine as chromogen in ABC immunoperoxidase method, with hematoxylin counterstain, x 168).

884 . P. A. Humphrey and P. E. Swanson Table 2. Nuclear accumulation of immunoreactive p53 protein in clinically localized adenocarcinoma of the prostate: Literature surveya Article 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Tissue fixation Antibody

Immunoreactive p53

Soini et al. 1992 Thompson et al. 1992 Visakorpi et al. 1992 Fox et al. 1993

formalin acetone formalin formalin

2/34 5129 23/137

(6%) (17%) (17%)

Navone et al. 1993 b Bookstein et al. 1993 Berner et al. 1993 Loning et al. 1993 Thomas et al. 1993 Van Veldhuizen et al. 1993 Kallakury et al. 1994 b Dinjens et al. 1994 Vesalainen et al. 1994 Ittman et al. 1994 b Aprikian et al. 1994 Henke et al. 1994 b Losi et al. 1994

formalin formalin formalin formalin mercury formalin formalin ethanol formalin formalin formalin formalin formalin

6145 2/45 171140 18/91 14/42 0110 15/26 15/107 2120 21/139 0/29 9148 35173

(13%) (4.4%) (12.1%) (19.8%) (33.3%) (0%) (57.8%) (14%) (10%) (15%) (0%) (19%) (48%)

0145 2/85 2/39 12/40

(0%) (2.4%) (5.1%) (30%)

18. Voeller et al. 1994 19. Ohori et al. 1994 20. Current studyb

methanol formalin formalin

CM-l PAb240 CM-l PAb1801, D07 PAb240, CM-1 CM-1 PAb1801, CM-1 Not specified Not specified CM-1 Pab240, Pb1801 DO-1 PAb1801 CM-1 CM-1 PAb1801, CM-l DO-7 PAb1801, DO-7 CM-1 PAb1801 CM-1 DO-7 Total

aData from full-length published articles only,



200/1269 (15.8%)

Microwave irradiation used in antigen retrieval.

cases of high-grade PIN exhibited lower intensity of staining, and, in these two cases, the invasive carcinoma component also exhibited low intensity staining. For the five cases of carcinoma with weaker positive staining, the associated high-grade PIN exhibited weaker positive staining in 2 cases, while only rare cells stained in 3 cases. Twelve of 40 (30%) invasive carcinomas exhibited immunoreactive p53, seven of which exhibited intense staining. This incidence of 30% is higher than the literature incidence in aggregate (15.8%; see Table 2), but a high incidence of p53 protein accumulation in prostatic carcinoma has been found in a recent study20 which also used microwave irradiation of tissue sections for antigen retrieval and antibody DO-7 for p53 detection. Discussion Our results indicate that nuclear p53 protein accumulation, as defined by immunohistochemical reactivity with antibody DO-7, occurs in a substantial group of cases of high-grade prostatic intraepithelial neoplasia. Furthermore, the concordance of an intense p53 signal between high-grade PIN and invasive carcinoma provides further support for a close relationship between these two lesions. We found that 22.5% of 40 cases of high-grade PIN associated with carcinoma contain immunoreactive

p53. Only a few manuscripts 37,43,46 have reported p53 protein accumulation in PIN. In one study37, no staining was identified in 17 cases of high-grade PIN. This finding was obtained with the use of antibody CM-l, while our immunohistochemical protocol employed antibody DO-7 with antigen retrieval by microwave irradiation, which has been shown to enhance detection of p53 30 . In two other studies43 ,46, p5 3 protein accumulation was described (but not illustrated) in 5 cases, but the grade of PIN was not provided. Finally, detection of immunoreactive p53 in PIN of unspecified grade has been reported in abstract form 28 ; of note, the incidence of p53 protein accumulation in PIN reported in this abstract (25%) is similar to the incidence reported here (22.5%) and both studies utilized antibody DO-7. Concordance of an intense immunohistochemical signal for p53 was observed between high-grade PIN and invasive carcinoma in seven cases. In only one of these cases was an intense signal for p53 observed in usual hyperplasia. Thus, high-grade PIN is more closely linked to invasive carcinoma than usual hyperplasia when evaluated for the accumulation of p53 protein. This intense signal for p53 joins an ever-growing list of morphologic, histochemical, immunophenotypic and genetic similarities between high-grade PIN and invasive carcinoma 1, 7. The timing of p53 gene mutation and protein accumulation in the prostatic epithelial proliferation spectrum of hyperplasia, intraepithelial neoplasia and carcinoma is uncertain. p53 gene mutations, which of-

p53 in PIN . 885

ten but not always result in nuclear p53 protein accumulation 23 , have been detected in usual hyperplasia 11,35 and carcinoma in the prostateS, 12-14, 19,25,31,37,45,49. p53 protein accumulation, as detected by immunohistochemistry, has been identified in usual hyperplasia 15,27, PIN28,43,46 and carcinoma (Table 2). Several studies have reported an association of p53 gene mutation and protein accumulation with advanced stage prostatic carcinomas 2,5,20,26,37,43 such that the highest incidence of p53 protein accumulation occurs in metastatic sites, particularly in metastatic growths in the bone marrow. However, other inquiries ll ,12,34,43,47 have failed to demonstrate such an association with advanced stage. Indeed, in a recent reportll based on the finding of p53 mutations in low-stage and low-grade tumors, it was proposed that p53 mutations might occur early in tumor formation. Our data suggest that high-level p53 protein accumulation occurs early, at the level of high-grade PIN, in that subset (17.5%) of primary prostatic carcinomas that exhibit intense staining for p53. That intraepithelial neoplastic proliferations may harbor altered p53 has been well-documented in other anatomic sites such as the breast24 ,39. It is not known if p53 protein accumulation is predictive of missense mutation in the p53 gene in highgrade PIN. To address this, future studies should entail microdissection of hyperplasia, PIN and carcinoma, with PCR-SSCP screening for p53 gene mutation followed by direct sequencing of identified mutants. It will be of particular interest to define the relationship between the presence and degree of p53 protein accumulation in these various epithelial proliferations with the presence or absence of mutations in DNA. Such a parallel analysis has not yet been performed for prostatic usual hyperplasia and high-grade PIN. However, it should be noted that p53 protein accumulation, as detected by immunohistochemistry, may be by itself an important indicator of altered p53 function. In summary, this immunohistochemical analysis of immunoreactive p53 has provided evidence of concordance of high-level p53 protein accumulation in highgrade PIN and associated invasive carcinoma, suggesting a role of p53 in early development of that subset of primary prostatic carcinomas with p53 protein alteration. Materials and Methods This study included 40 patients who had undergone radical prostatectomy for clinically organ-confined (clinical stage B or T2) adenocarcinoma of the prostate. Patients were selected based on the presence of high-grade PIN and carcinoma on the same slide. The rationale for this selection was that immunohistochemical staining for p53 protein in high-grade PIN and carcinoma could be directly compared on the same slide. In 37 of 40 cases, foci of usual epithelial hyperplasia were also available for assessment of p53 immunoreactivity. Highgrade PIN (also known as PIN grade 2 and 3, or moderate and severe dysplasia) was morphologically defined as previously described 6,33. Gleason's histologic grade was deter-

mined 17,18 and pathologic stage was assigned 40 • The pathological features of the carcinomas in the radical prostatectomy specimens were as follows: mean Gleason score =6.1 (range, 3-9); mean percentage gland involvement carcinoma =11 % (range, 1-24%). Most cases were pathologic stage T2 (48%) or T3 (44%), and 2 cases were pathologic stage T4. Immunohistochemical detection of p53 protein was performed on 5-micron sections from formalin-fixed, paraffinembedded tissue blocks. The sections were mounted on acid-cleaned glass slides and heated for 1 h at 55°C. Slides were dewaxed and dehydrated and then immersed for 30 min in absolute methanol containing 0.6% (v/v) hydrogen peroxide to quench endogenous peroxidase activity. After rehydration through graded ethanols and distilled water, sections were immersed in 10 mM citrate buffer (10 mM citrate monohydrate in distilled water, pH 6.0) in plastic Coplin jars and subjected to microwave irradiation (600 W; 2,450 MHz) for 4 min. After cooling to room temperature, slides were removed to phosphate-buffered saline (PBS, pH 7.4) for 10 min. Serial sections were then overlaid with mouse monoclonal antibody DO-7 (Dako Corporation, Carpinteria CA; diluted 1: 150 from commercial stock), mouse monoclonal antibody PAb1801 (BioGenex Laboratories, Dublin CA; diluted 1 : 2 from commercial stock), or rabbit polyclonal antibody CM-l (Oncogene Science, Manhassett, NY; diluted 1: 1600 from commercial stock) and incubated for 18 h in a moisture chamber at 4°C. Monoclonal antibody DO-7 and monoclonal antibody PAb1801 bind to epitopes at the amino-terminal end of the p53 protein 3,50, while polyclonal antibody CM-llikely binds to multiple epitopes on the p53 protein 36 • The avidin-biotin-peroxidase complex (ABC) method was performed in all cases (VectaStain ABC Elite Mouse Universal Kit; Vector Laboratories, Burlingame, CA, U.S.A.)22. Chromogenic development resulted from immersion of slides in 3,3'-diaminobenizidine tetrahydrochloride [Sigma Chemicals, St. Louis, MO, U.S.A.; 0.5 mg/ml, containing 0.003% (v/v) hydrogen peroxide] for ::; 10 min. The reaction product was enhanced by brief exposure to 0.125% (w/v) osmium tetroxide. Slides were counterstained in Harris's hematoxylin and covers lipped in a synthetic mounting medium. The positive control for p53 immunoreactivity consisted of formalin-fixed sections from an adenocarcinoma of the small intestine with known accumulation of the p53 protein. Negative internal controls were stromal cell and lymphocytic nuclei. A stain was interpreted as positive only if nuclear reactivity was obtained in > 5% of the nuclei of neoplastic cells. Cases were designated as ± if only rare (less than 5%) nuclei were stained. Negative controls were performed by substituting primary antibody with nonimune murine ascites fluid or nonimmune rabbit immunoglobulin.

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Received December 20, 1994 . Accepted in revised form June 10, 1995

Key words: p53 - Prostate - Cancer - Intraepithelial neoplasia Peter A. Humphrey, M.D ., Ph.D., Division of Surgical Pathology, Department of Pathology, Box 8118, One Barnes Hospital Plaza, Washington University Medical Center, St. Louis, MO 63110, U.S.A.