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p63 protein expression is rare in prostate adenocarcinoma: implications for cancer diagnosis and carcinogenesis
BASIC SCIENCE
p63 PROTEIN EXPRESSION IS RARE IN PROSTATE ADENOCARCINOMA: IMPLICATIONS FOR CANCER DIAGNOSIS AND CARCINOGENESIS J. KELLOGG PARSONS, WESLEY R. GAGE, WILLIAM G. NELSON,
AND
ANGELO M. DE MARZO
ABSTRACT Objectives. To examine the expression of the p63 protein in normal, preneoplastic, and neoplastic human prostatic tissue. The p63 gene, a member of the p53 gene family, is expressed in the basal epithelial cells of multiple organs. Irregularities in p63 expression have been associated with epithelial carcinogenesis. Methods. We performed immunohistochemistry with an anti-p63 antibody on specimens from radical prostatectomies, prostate needle biopsies, and metastatic prostate adenocarcinoma. We analyzed p63 expression in regions of normal prostate, benign prostatic hyperplasia, proliferative inflammatory atrophy (PIA), high-grade intraepithelial neoplasia, and adenocarcinoma. Results. Basal epithelial cells in normal, benign prostatic hyperplasia, and high-grade intraepithelial neoplasia tissue stained intensely for the p63 polypeptide, but the vast majority of adenocarcinoma specimens from 233 patients—66 (94%) of 70 radical prostatectomies, 132 (89%) of 148 prostate needle biopsies, and 14 (93%) of 15 metastases—did not. In tumors in which the adenocarcinoma cells were positive, the staining intensity was weak and occurred in less than 1% of the cells. Tumors that stained positive for p63 were more likely to be high grade than those that did not (P ⬍0.0001). Basal cells in PIA expressed p63, but these cells were sparsely distributed relative to the basal cells in the normal glands. Luminal cells in PIA were, in general, negative for p63. Conclusions. In contrast to normal and preneoplastic prostatic tissue, the vast majority of prostate adenocarcinomas do not express p63. Therefore, p63 immunohistochemistry represents a potential novel adjuvant method for facilitating the pathologic diagnosis of prostate cancer in prostate needle biopsies. The selective expression of p63 in normal basal cells, coupled with the finding that p63 null mice fail to develop prostates, provides strong evidence that the basal cells represent prostatic epithelial stem cells. In addition, these findings suggest that p63 may protect prostatic epithelial cells against neoplastic transformation and support the hypothesis that intermediately differentiated cells in the luminal epithelium of PIA are the targets of neoplastic transformation in the prostate. UROLOGY 58: 619–624, 2001. © 2001, Elsevier Science Inc.
T
he recently identified p63 gene is expressed in the regenerative epithelial compartments of several organs, and shares extensive homology with p53.1–3 In normal epidermis, hair follicles, and stratified squamous cell cultures, the p63 protein is This study was funded in part by Public Health Services grant K08 CA78588-01, NIDDK grant T32DK07522, and NIH/NCI Specialized Program in Research Excellence (SPORE) in Prostate Cancer grant P50CA58236. From the Departments of Urology, Oncology, and Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland Reprint requests: Angelo M. De Marzo, M.D., Ph.D., Division of Genitourinary Pathology, Department of Pathology, Bunting/ Blaustein Cancer Research Building, Room 153, 1650 Orleans Street, Baltimore, MD 21231-1000 Submitted: March 2, 2001, accepted (with revisions): May 29, 2001 © 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
restricted to cells with a high proliferative potential and is absent in cells undergoing terminal differentiation.4 Mice homozygous for a disrupted p63 gene demonstrate profound abnormalities in limb, craniofacial, and squamous epithelial development; they die on the first day after birth and lack normal hair, skin, and teeth.1,5,6 The p63 gene is thus probably associated with tissue renewal— possibly as a regulator of the tissue stem cell phenotype. As with p53, irregularities of p63 expression have been linked to epithelial carcinogenesis. The p63 gene is often amplified in human squamous cell carcinomas of the head and neck,4,7 and bladder carcinoma is associated with decreased (albeit heterogeneous) expression of p63.8 Yang et al.1 observed that p63 protein is present 0090-4295/01/$20.00 PII S0090-4295(01)01311-5 619
TABLE I. Distribution of the Gleason grades and immunostaining results of the adenocarcinomas in the prostatectomy and needle biopsy specimens Gleason Sum 5–6 7 8–9 Total
p63Negative Tumors (n) 126 63 9 198
p63Positive Tumors (n) 2 11 7 20
Total Tumors (n)
(1.6) (15) (44) (9)
128 74 16 218
Positive p63 staining in tumor cells correlated with high Gleason grade (P ⬍0.0001, Fisher’s exact test). Numbers in parentheses are percentages.
in the basal cells of normal human prostatic epithelium. Recently, this same group reported that p63 is absent in most adenocarcinomas in radical prostatectomy specimens. They also determined that the prostate is absent in p63 null mice, underscoring the importance of this gene to normal prostate development.9 Using an anti-p63 monoclonal antibody cocktail, we performed immunohistochemistry of p63 expression in normal, preneoplastic, and neoplastic prostatic tissue from surgical specimens, needle biopsies, and metastases. In addition, we co-localized the expression of p63 and cytokeratin 5 with double-label immunofluorescence. We discuss the implications of our results with respect to the pathologic diagnosis of adenocarcinoma, stem cell differentiation, and carcinogenesis in the prostate. MATERIAL AND METHODS SURGICAL SPECIMENS Specimens from 70 radical retropubic prostatectomies (patient age range 44 to 68 years, pathologic tumor Stage T2N0MX to T3bN0MX) and 148 prostate needle biopsies (patient age range 47 to 85 years) performed at our institution were studied. The Gleason sums are summarized in Table I. We also studied tissue from 15 patients with metastatic prostate adenocarcinoma (11 with pelvic lymph node metastases and 4 with bone metastases, none of whom had undergone hormonal ablation therapy).
IMMUNOHISTOCHEMISTRY—SINGLE LABEL, p63 The mouse monoclonal anti-p63 antibody cocktail (dilution of 1:100), which recognizes all known p63 isoforms, was obtained from Lab Vision (Fremont, Calif). Manual immunohistochemistry was performed as follows. After paraffin removal and hydration, the slides were immersed in 10 mM citrate buffer (pH 6.0) and steamed for 14 minutes to induce epitope retrieval. They were incubated with primary antibodies for 45 minutes at room temperature, then with secondary biotin-labeled antibodies for 30 minutes at room temperature (Universal ChemMate Detection System (Ventana Medical Systems, Tucson, Ariz). Avidin-biotin complex– horseradish peroxidase was applied for 30 minutes, followed by peroxide/ diaminobenzidine substrate/Chromagen (Ventana). The slides were counterstained with hematoxylin. 620
The positive controls for p63 expression consisted of squamous epithelium from human skin and cervix specimens.1 The negative controls consisted of human intestine.5 The expression in normal prostatic epithelium (70 radical prostatectomies, 148 needle biopsies, a total of 218 different patients), adenocarcinoma (70 radical prostatectomies, 148 needle biopsies, a total of 218 different patients), proliferative inflammatory atrophy (PIA) (25 radical prostatectomies, 13 needle biopsies, a total of 38 different patients), high-grade intraepithelial neoplasia (HGPIN) (19 radical prostatectomies, 11 needle biopsies, a total of 30 different patients), and metastatic adenocarcinoma (15 patients) was evaluated by a single pathologist using an Olympus BX-40 light microscope and compared with the expression in the positive and negative controls.
IMMUNOHISTOCHEMISTRY—SINGLE LABEL, 34E12
The mouse monoclonal anti-34E12 antibody (dilution 1:50) was obtained from Enzo (Farmingdale, NY). The paraffin removal, epitope retrieval, and manual immunohistochemistry were performed as described above.
DOUBLE-LABEL IMMUNOFLUORESCENCE—p63 AND CYTOKERATIN 5 The paraffin removal and epitope retrieval were performed as described above. The slides were simultaneously incubated with rabbit polyclonal anti-cytokeratin 5 antibody (Babco, Richmond, Calif) at a dilution of 1:15,000 and with anti-p63 antibody as above for 45 minutes at room temperature. The slides were washed with phosphate-buffered saline and then incubated with secondary antibodies consisting of donkey anti-rabbit labeled with Cy3 for cytokeratin 5 (Jackson ImmunoResearch, Wesgrove, Pa) and goat anti-mouse labeled with Alexa green 488 for p63 (Molecular Probes, Eugene, Ore) for 30 minutes at room temperature. Nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI) by treatment for 2 minutes at a dilution of 1:2000. The expression was analyzed with a Zeiss Axioskop fluorescence microscope using an ⫻40 PlanApochromat objective. The red Cy3 fluorescence was visualized with a conventional rhodamine filter set, the fluorescence of the Alexa green was observed with a FITC filter set, and the DAPI fluorescence was visualized with a DAPI filter set. Digital images of the fluorescence preparations were acquired with a Micromax 1300 Interline Cooled CCD Camera (Roper Scientific, Tuscon, Ariz) using IPLab Imaging software (Scanalytics, Fairfax, Va). Three separate eight-bit images were taken from the same field using each of the different filters. Images were pseudo-colored using IPLab.
RESULTS STAINING IN NORMAL EPITHELIUM AND BENIGN PROSTATIC HYPERPLASIA Virtually all cells in the basal layer of the epithelium in normal glands in the radical prostatectomy and needle biopsy specimens (218 different patients) stained strongly for p63. The staining was confined exclusively to the nuclei of basal epithelial cells. No staining was observed in the secretory epithelial cells or in the stroma (Fig. 1A). This pattern was seen in the normal epithelium from the peripheral, central, and transition zones. Nuclear staining was also detected in the basal cells in nodules from areas of benign prostatic hyperplasia (BPH) (20 lesions from 12 patients). The location UROLOGY 58 (4), 2001
FIGURE 1. Human prostate stained with monoclonal antibody to p63. (A) Normal gland demonstrating typical two-layer epithelial architecture. Arrows indicate positively stained basal epithelial cells; arrowhead indicates normal secretory cells with lack of staining (200⫻). (B) Focus of PIA. Arrow indicates positive staining in basal epithelial cells in PIA; n denotes adjacent normal epithelium with positive staining in basal cells. Note sparse distribution of basal cells in PIA glands (100⫻). (C) Focus of adenocarcinoma demonstrating negative staining (arrows). Note positive staining in adjacent normal glands (n) (100⫻). (D) Focus of high-grade adenocarcinoma. Arrow indicates weak positive staining in the nucleus of a cell clearly identifiable as a tumor cell (200⫻). (E) Double-labeling immunofluorescence with p63 and cytokeratin 5. Arrows indicate basal epithelial layer. Left panel, expression of p63 (red); center panel, expression of cytokeratin 5 (green); and right panel, DAPI stain (blue). Note co-localization of p63 expression (nuclear specific) with that of cytokeratin 5 (cytoplasm specific). DAPI staining highlighted the luminal cells and stromal cells that lack significant immunostaining for p63 and cytokeratin 5.
of these cells in standard hematoxylin-eosin sections, their morphologic characteristics, the staining of adjacent sections with the monoclonal antibody 34E12,10 and double-labeling immunofluorescence with cytokeratin 5, a well-known marker for prostate basal epithelium,11,12 all confirmed that the UROLOGY 58 (4), 2001
positively stained cells were in fact basal cells (Fig. 1). STAINING IN PIA AND HGPIN Focal regions of prostatic atrophy (25 different prostatectomy specimens and 13 different needle 621
specimens), lesions that we had previously termed PIA,13 also stained positive for p63. The staining was generally localized to the basal layer. However, very infrequent weak staining was also detected in the luminal epithelial cells in PIA. The basal cells in most (37 of 38) of these lesions were sparsely distributed (Fig. 1B). The basilar phenotypes were verified by positive staining with the monoclonal antibody 34E12. In a few of the PIA lesions that contained sclerotic stroma, a variable number of the atrophic acini and ducts evinced no p63 staining. The expression of p63 was detected in HGPIN as well (19 of 19 different prostatectomy specimens and 11 of 11 different needle specimens). Staining occurred primarily in the nuclei of the basal epithelial cells. These cells were often sparsely distributed, but in general were not as sparsely distributed as were those in PIA, and the overall occurrence of sparse basal cell staining was less frequent than it was in PIA (20 of 30 lesions). Rarely, HGPIN cells with nuclear atypia that were located above the basal layer in the secretory epithelial compartment showed weak to moderate nuclear staining for p63. STAINING IN ADENOCARCINOMA Most adenocarcinomas from radical prostatectomies were negative for p63 immunoreactivity; 66 (94%) of 70 lesions examined were completely negative. In the non-neoplastic glands surrounding the tumors, however, the basal epithelial cells uniformly showed strong nuclear staining (Fig. 1C). Scattered cells that were morphologically identifiable as tumor cells (and not basal cells), found only in high-grade carcinomas (Gleason pattern 4 or 5) and representing less than 1% of cells in those carcinoma specimens, demonstrated very weak nuclear staining (Fig. 1D). All four of the tumors that exhibited focal weak positive staining extended beyond the prostate (three were Stage T3aN0MX and one was Stage T3bN0MX). To determine whether positively staining cells that were morphologically identified as tumor cells were also positive for basal cell cytokeratins, adjacent 5-mm sections were stained for 34E12 and p63. In general, the same cells that stained positive for p63 also stained positive for 34E12. In some cases, however, additional scattered cells were negative for p63 but positive for 34E12. None of the four tumors positive for p63 by light microscopy were positive for p63 by immunofluorescence (although one was positive for cytokeratin 5), suggesting that standard immunohistochemistry is more sensitive in the detection of the weak expression of p63 in some tumors. Most adenocarcinomas in the prostate needle biopsies—132 (89%) of 148 —also stained completely negative. As in the radical prostatectomy 622
specimens, the tumors that stained positively did so with weak intensity and in scattered cells only. Table I shows all carcinoma specimens from the prostatectomy and needle biopsy specimens listed by Gleason grade and p63 staining status. By Fisher’s exact test, the relation between the positive p63 staining and Gleason grade was highly statistically significant (P ⬍0.0001), with a much greater proportion of high-grade tumors staining positively than did low-grade tumors (Table I). Tumor cells in metastatic prostate adenocarcinoma in 14 (93%) of 15 cases stained negative for p63. One metastatic case demonstrated weak staining in approximately 20% of the tumor cell nuclei. COMMENT The results of the present study demonstrate that basal cells in normal, BPH, PIA, and HGPIN tissue selectively express p63 in their nuclei, but that most prostate adenocarcinoma cells in both primary and metastatic lesions lack expression of this protein. Our findings support the results obtained by Signoretti et al.9 in radical prostatectomy specimens. Our study adds new information in that it includes detailed analyses of p63 expression in BPH, PIA, adenocarcinoma in prostate needle biopsies, and metastatic prostate adenocarcinoma. Moreover, we found a strong correlation between the expression of p63 in the tumors and Gleason grade. Staining for p63 in cancer may augment the staining patterns displayed by more classic basal cell markers, such as cytokeratins 5 and 14, that are currently used to aid in the diagnosis of atypical prostate lesions. Thus, p63 staining can potentially increase the specificity for discerning cancer from HGPIN, adenosis, and various atrophic lesions on prostate needle biopsy, thereby reducing diagnostic uncertainty.14 Although a small fraction of prostate cancer cells in some high-grade carcinomas stained positive, this finding should in general not affect diagnostic issues, as there was no question that these cells were morphologically identifiable as tumor cells and were not basal cells entrapped in the tumor. Although prostate adenocarcinomas may occasionally stain positive for 34E12, Yang et al.15 recently showed that occasional tumor cells in metastatic prostate cancer—a lesion that cannot, by definition, contain entrapped normal prostate basal cells—stain positive for 34E12. Similarly, 1 case of metastatic prostate cancer in our study stained positive for p63. As positive staining for p63 in tumor cells occurred primarily in high-grade lesions, and as all tumors that stained positively in radical prostatectomy specimens demonstrated extraprostatic extension, our data suggest that the degree of p63 UROLOGY 58 (4), 2001
expression in primary adenocarcinoma may correlate with primary tumor aggressiveness. In fact, 44% of tumors with Gleason sums of 8 or greater stained positive, in contrast to 1.6% of tumors with Gleason sums 6 or less (Table I). Although only 1 (7%) of 15 metastatic lesions stained positive, it is possible that p63 expression in extraprostatic carcinoma follows a different pattern than that seen in the primary tumor. In a recent study, for example, decreased E-cadherin expression in radical prostatectomy specimens was associated with a high Gleason score and advanced pathologic stage, and yet E-cadherin staining in metastases was quite strong.16 The expression of high levels of p63 exclusively in epithelial basal cells and the complete lack of prostate development in p63 null mice9 indicates that p63 expression is involved in the control of prostate growth and differentiation. Moreover, it strongly bolsters the hypothesis that the basal cells represent prostate stem cells. Loss of p63 expression appears related to the process of differentiation as cells progress into the overlying epithelium and develop into secretory cells. That most prostate adenocarcinoma and HGPIN cells did not express p63 suggests that p63 may protect prostatic epithelial cells against neoplastic transformation, perhaps through a tumor suppressor function. Since basal cells, but not secretory cells, almost uniformly express p63, it also supports the concept that basal cells are not direct targets for neoplastic transformation.12,13,17,18 Moreover, it provides additional evidence favoring our hypothesis that basal cells are protected from undergoing neoplastic transformation, at least in part, by the carcinogen detoxifying enzyme GSTP1.13,18 Indeed, these data suggest that, rather than basal cells, proliferating cells in the secretory compartment are the targets for neoplastic transformation.12,18 Normal prostatic epithelium consists of two histologically defined compartments: basal and secretory. The secretory luminal cells likely arise from differentiation of the basal cells.17–22 Our group and others have argued that there exists a population of amplifying or transiently proliferating cells that phenotypically are intermediate in differentiation between basal cells and mature, terminally differentiated secretory cells.17,18,21 Our group has further divided this population of intermediate cells into two subpopulations— basal intermediate and secretory intermediate—and postulated that basal intermediate cells give rise to BPH and secretory intermediate cells give rise to HGPIN and carcinoma.18 Up to 50% of luminal secretory cells in PIA express cytokeratin 5 (G. Vanleenders and A.M. De Marzo, unpublished results), indicating that they retain at least some feaUROLOGY 58 (4), 2001
tures of basal cells. Yet these cells in PIA also produce high levels of cytokeratins 8 and 18 and low levels of androgen receptors and prostate-specific antigen, all of which are characteristics of secretory cells.13 That most luminal cells in PIA lack expression of p63 supports the idea that most of these cells have differentiated beyond the basal cell phenotype and is consistent with the concept that luminal PIA cells represent actively dividing, secretory intermediate cells—in our model, the same cells that are the proposed targets of neoplastic transformation (for a detailed illustration of this model, refer to the second figure of De Marzo et al.18). Lending additional support to this concept, we recently reported that a morphologic transition between HGPIN and PIA frequently occurs, and that small adenocarcinomas are often associated with nearby PIA.23 CONCLUSIONS In contrast to normal and premalignant prostatic tissue, most prostate adenocarcinomas do not express p63. p63 immunostaining is thus a potentially useful modality for facilitating the pathologic diagnosis of carcinoma in prostate needle biopsies. When p63 antibody staining is positive in adenocarcinoma, it is focal and weak and correlates strongly with a high Gleason grade. Collectively, the findings in this report support the hypothesis that basal cells represent prostate stem cells and that the immature secretory cells characteristic of PIA—not basal cells—are targets for neoplastic transformation. REFERENCES 1. Yang A, Kaghad M, Wang Y, et al: p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 2: 305–316, 1998. 2. Levrero M, De Laurenzi V, Costanzo A, et al: The p53/ p63/p73 family of transcription factors: overlapping and distinct functions. J Cell Sci 113: 1661–1670, 2000. 3. Trink B, Okami K, Wu L, et al: A new human p53 homologue. Nat Med 4: 747–748, 1998. 4. Parsa R, Yang A, McKeon F, et al: Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113: 1099 –1105, 1999. 5. Yang A, Schweitzer R, Sun D, et al: p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714 –718, 1999. 6. Mills AA, Zheng B, Wang XJ, et al: p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708 –713, 1999. 7. Hibi K, Trink B, Patturajan M, et al: AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA 97: 5462–5467, 2000. 8. Park BJ, Lee SJ, Kim JI, et al: Frequent alteration of p63 expression in human primary bladder carcinomas. Cancer Res 60: 3370 –3374, 2000. 9. Signoretti S, Waltregny D, Dilks J, et al: p63 is a prostate 623
basal cell marker and is required for prostate development. Am J Pathol 157: 1769 –1775, 2000. 10. Gown AM, and Vogel AM: Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies. J Cell Biol 95: 414 – 424, 1982. 11. Yang Y, Hao J, Liu X, et al: Differential expression of cytokeratin mRNA and protein in normal prostate, prostatic intraepithelial neoplasia, and invasive carcinoma. Am J Pathol 150: 693–704, 1997. 12. Nagle RB, Ahmann FR, McDaniel KM, et al: Cytokeratin characterization of human prostatic carcinoma and its derived cell lines. Cancer Res 47: 281–286, 1987. 13. De Marzo AM, Marchi VL, Epstein JI, et al: Proliferative inflammatory atrophy of the prostate: implications for prostatic carcinogenesis. Am J Pathol 155: 1985–1992, 1999. 14. Wojno KJ, and Epstein JI: The utility of basal cell-specific anti-cytokeratin antibody (34 beta E12) in the diagnosis of prostate cancer: a review of 228 cases. Am J Surg Pathol 19: 251–260, 1995. 15. Yang XJ, Lecksell K, Gaudin P, et al: Rare expression of high-molecular-weight cytokeratin in adenocarcinoma of the prostate gland: a study of 100 cases of metastatic and locally advanced prostate cancer. Am J Surg Pathol 23: 147–152, 1999. 16. De Marzo AM, Knudsen B, Chan-Tack K, et al: E-cadherin expression as a marker of tumor aggressiveness in rou-
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tinely processed radical prostatectomy specimens. Urology 53: 707–713, 1999. 17. Verhagen AP, Ramaekers FC, Aalders TW, et al: Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. Cancer Res 52: 6182– 6187, 1992. 18. De Marzo AM, Nelson WG, Meeker AK, et al: Stem cell features of benign and malignant prostate epithelial cells. J Urol 160: 2381–2392, 1998. 19. Bonkhoff H, Stein U, and Remberger K: The proliferative function of basal cells in the normal and hyperplastic human prostate. Prostate 24: 114 –118, 1994. 20. Bonkhoff H, Stein U, and Remberger K: Multidirectional differentiation in the normal, hyperplastic, and neoplastic human prostate: simultaneous demonstration of cell-specific epithelial markers. Hum Pathol 25: 42– 46, 1994. 21. De Marzo AM, Coffey DS, and Nelson WG: New concepts in tissue specificity for prostate cancer and benign prostatic hyperplasia. Urology 53: 29 –39, 1999. 22. van Leenders G, Dijkman H, Hulsbergen-van de Kaa C, et al: Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triplestaining confocal scanning microscopy. Lab Invest 80: 1251– 1258, 2000. 23. Putzi MJ, and De Marzo AM: Morphologic transitions between proliferative inflammatory atrophy and high-grade prostatic intraepithelial neoplasia. Urology 56: 828 – 832, 2000.
UROLOGY 58 (4), 2001
p63 PROTEIN EXPRESSION IS RARE IN PROSTATE ADENOCARCINOMA: IMPLICATIONS FOR CANCER DIAGNOSIS AND CARCINOGENESIS J. KELLOGG PARSONS, WESLEY R. GAGE, WILLIAM G. NELSON,
AND
ANGELO M. DE MARZO
ABSTRACT Objectives. To examine the expression of the p63 protein in normal, preneoplastic, and neoplastic human prostatic tissue. The p63 gene, a member of the p53 gene family, is expressed in the basal epithelial cells of multiple organs. Irregularities in p63 expression have been associated with epithelial carcinogenesis. Methods. We performed immunohistochemistry with an anti-p63 antibody on specimens from radical prostatectomies, prostate needle biopsies, and metastatic prostate adenocarcinoma. We analyzed p63 expression in regions of normal prostate, benign prostatic hyperplasia, proliferative inflammatory atrophy (PIA), high-grade intraepithelial neoplasia, and adenocarcinoma. Results. Basal epithelial cells in normal, benign prostatic hyperplasia, and high-grade intraepithelial neoplasia tissue stained intensely for the p63 polypeptide, but the vast majority of adenocarcinoma specimens from 233 patients—66 (94%) of 70 radical prostatectomies, 132 (89%) of 148 prostate needle biopsies, and 14 (93%) of 15 metastases—did not. In tumors in which the adenocarcinoma cells were positive, the staining intensity was weak and occurred in less than 1% of the cells. Tumors that stained positive for p63 were more likely to be high grade than those that did not (P ⬍0.0001). Basal cells in PIA expressed p63, but these cells were sparsely distributed relative to the basal cells in the normal glands. Luminal cells in PIA were, in general, negative for p63. Conclusions. In contrast to normal and preneoplastic prostatic tissue, the vast majority of prostate adenocarcinomas do not express p63. Therefore, p63 immunohistochemistry represents a potential novel adjuvant method for facilitating the pathologic diagnosis of prostate cancer in prostate needle biopsies. The selective expression of p63 in normal basal cells, coupled with the finding that p63 null mice fail to develop prostates, provides strong evidence that the basal cells represent prostatic epithelial stem cells. In addition, these findings suggest that p63 may protect prostatic epithelial cells against neoplastic transformation and support the hypothesis that intermediately differentiated cells in the luminal epithelium of PIA are the targets of neoplastic transformation in the prostate. UROLOGY 58: 619–624, 2001. © 2001, Elsevier Science Inc.
T
he recently identified p63 gene is expressed in the regenerative epithelial compartments of several organs, and shares extensive homology with p53.1–3 In normal epidermis, hair follicles, and stratified squamous cell cultures, the p63 protein is This study was funded in part by Public Health Services grant K08 CA78588-01, NIDDK grant T32DK07522, and NIH/NCI Specialized Program in Research Excellence (SPORE) in Prostate Cancer grant P50CA58236. From the Departments of Urology, Oncology, and Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland Reprint requests: Angelo M. De Marzo, M.D., Ph.D., Division of Genitourinary Pathology, Department of Pathology, Bunting/ Blaustein Cancer Research Building, Room 153, 1650 Orleans Street, Baltimore, MD 21231-1000 Submitted: March 2, 2001, accepted (with revisions): May 29, 2001 © 2001, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
restricted to cells with a high proliferative potential and is absent in cells undergoing terminal differentiation.4 Mice homozygous for a disrupted p63 gene demonstrate profound abnormalities in limb, craniofacial, and squamous epithelial development; they die on the first day after birth and lack normal hair, skin, and teeth.1,5,6 The p63 gene is thus probably associated with tissue renewal— possibly as a regulator of the tissue stem cell phenotype. As with p53, irregularities of p63 expression have been linked to epithelial carcinogenesis. The p63 gene is often amplified in human squamous cell carcinomas of the head and neck,4,7 and bladder carcinoma is associated with decreased (albeit heterogeneous) expression of p63.8 Yang et al.1 observed that p63 protein is present 0090-4295/01/$20.00 PII S0090-4295(01)01311-5 619
TABLE I. Distribution of the Gleason grades and immunostaining results of the adenocarcinomas in the prostatectomy and needle biopsy specimens Gleason Sum 5–6 7 8–9 Total
p63Negative Tumors (n) 126 63 9 198
p63Positive Tumors (n) 2 11 7 20
Total Tumors (n)
(1.6) (15) (44) (9)
128 74 16 218
Positive p63 staining in tumor cells correlated with high Gleason grade (P ⬍0.0001, Fisher’s exact test). Numbers in parentheses are percentages.
in the basal cells of normal human prostatic epithelium. Recently, this same group reported that p63 is absent in most adenocarcinomas in radical prostatectomy specimens. They also determined that the prostate is absent in p63 null mice, underscoring the importance of this gene to normal prostate development.9 Using an anti-p63 monoclonal antibody cocktail, we performed immunohistochemistry of p63 expression in normal, preneoplastic, and neoplastic prostatic tissue from surgical specimens, needle biopsies, and metastases. In addition, we co-localized the expression of p63 and cytokeratin 5 with double-label immunofluorescence. We discuss the implications of our results with respect to the pathologic diagnosis of adenocarcinoma, stem cell differentiation, and carcinogenesis in the prostate. MATERIAL AND METHODS SURGICAL SPECIMENS Specimens from 70 radical retropubic prostatectomies (patient age range 44 to 68 years, pathologic tumor Stage T2N0MX to T3bN0MX) and 148 prostate needle biopsies (patient age range 47 to 85 years) performed at our institution were studied. The Gleason sums are summarized in Table I. We also studied tissue from 15 patients with metastatic prostate adenocarcinoma (11 with pelvic lymph node metastases and 4 with bone metastases, none of whom had undergone hormonal ablation therapy).
IMMUNOHISTOCHEMISTRY—SINGLE LABEL, p63 The mouse monoclonal anti-p63 antibody cocktail (dilution of 1:100), which recognizes all known p63 isoforms, was obtained from Lab Vision (Fremont, Calif). Manual immunohistochemistry was performed as follows. After paraffin removal and hydration, the slides were immersed in 10 mM citrate buffer (pH 6.0) and steamed for 14 minutes to induce epitope retrieval. They were incubated with primary antibodies for 45 minutes at room temperature, then with secondary biotin-labeled antibodies for 30 minutes at room temperature (Universal ChemMate Detection System (Ventana Medical Systems, Tucson, Ariz). Avidin-biotin complex– horseradish peroxidase was applied for 30 minutes, followed by peroxide/ diaminobenzidine substrate/Chromagen (Ventana). The slides were counterstained with hematoxylin. 620
The positive controls for p63 expression consisted of squamous epithelium from human skin and cervix specimens.1 The negative controls consisted of human intestine.5 The expression in normal prostatic epithelium (70 radical prostatectomies, 148 needle biopsies, a total of 218 different patients), adenocarcinoma (70 radical prostatectomies, 148 needle biopsies, a total of 218 different patients), proliferative inflammatory atrophy (PIA) (25 radical prostatectomies, 13 needle biopsies, a total of 38 different patients), high-grade intraepithelial neoplasia (HGPIN) (19 radical prostatectomies, 11 needle biopsies, a total of 30 different patients), and metastatic adenocarcinoma (15 patients) was evaluated by a single pathologist using an Olympus BX-40 light microscope and compared with the expression in the positive and negative controls.
IMMUNOHISTOCHEMISTRY—SINGLE LABEL, 34E12
The mouse monoclonal anti-34E12 antibody (dilution 1:50) was obtained from Enzo (Farmingdale, NY). The paraffin removal, epitope retrieval, and manual immunohistochemistry were performed as described above.
DOUBLE-LABEL IMMUNOFLUORESCENCE—p63 AND CYTOKERATIN 5 The paraffin removal and epitope retrieval were performed as described above. The slides were simultaneously incubated with rabbit polyclonal anti-cytokeratin 5 antibody (Babco, Richmond, Calif) at a dilution of 1:15,000 and with anti-p63 antibody as above for 45 minutes at room temperature. The slides were washed with phosphate-buffered saline and then incubated with secondary antibodies consisting of donkey anti-rabbit labeled with Cy3 for cytokeratin 5 (Jackson ImmunoResearch, Wesgrove, Pa) and goat anti-mouse labeled with Alexa green 488 for p63 (Molecular Probes, Eugene, Ore) for 30 minutes at room temperature. Nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI) by treatment for 2 minutes at a dilution of 1:2000. The expression was analyzed with a Zeiss Axioskop fluorescence microscope using an ⫻40 PlanApochromat objective. The red Cy3 fluorescence was visualized with a conventional rhodamine filter set, the fluorescence of the Alexa green was observed with a FITC filter set, and the DAPI fluorescence was visualized with a DAPI filter set. Digital images of the fluorescence preparations were acquired with a Micromax 1300 Interline Cooled CCD Camera (Roper Scientific, Tuscon, Ariz) using IPLab Imaging software (Scanalytics, Fairfax, Va). Three separate eight-bit images were taken from the same field using each of the different filters. Images were pseudo-colored using IPLab.
RESULTS STAINING IN NORMAL EPITHELIUM AND BENIGN PROSTATIC HYPERPLASIA Virtually all cells in the basal layer of the epithelium in normal glands in the radical prostatectomy and needle biopsy specimens (218 different patients) stained strongly for p63. The staining was confined exclusively to the nuclei of basal epithelial cells. No staining was observed in the secretory epithelial cells or in the stroma (Fig. 1A). This pattern was seen in the normal epithelium from the peripheral, central, and transition zones. Nuclear staining was also detected in the basal cells in nodules from areas of benign prostatic hyperplasia (BPH) (20 lesions from 12 patients). The location UROLOGY 58 (4), 2001
FIGURE 1. Human prostate stained with monoclonal antibody to p63. (A) Normal gland demonstrating typical two-layer epithelial architecture. Arrows indicate positively stained basal epithelial cells; arrowhead indicates normal secretory cells with lack of staining (200⫻). (B) Focus of PIA. Arrow indicates positive staining in basal epithelial cells in PIA; n denotes adjacent normal epithelium with positive staining in basal cells. Note sparse distribution of basal cells in PIA glands (100⫻). (C) Focus of adenocarcinoma demonstrating negative staining (arrows). Note positive staining in adjacent normal glands (n) (100⫻). (D) Focus of high-grade adenocarcinoma. Arrow indicates weak positive staining in the nucleus of a cell clearly identifiable as a tumor cell (200⫻). (E) Double-labeling immunofluorescence with p63 and cytokeratin 5. Arrows indicate basal epithelial layer. Left panel, expression of p63 (red); center panel, expression of cytokeratin 5 (green); and right panel, DAPI stain (blue). Note co-localization of p63 expression (nuclear specific) with that of cytokeratin 5 (cytoplasm specific). DAPI staining highlighted the luminal cells and stromal cells that lack significant immunostaining for p63 and cytokeratin 5.
of these cells in standard hematoxylin-eosin sections, their morphologic characteristics, the staining of adjacent sections with the monoclonal antibody 34E12,10 and double-labeling immunofluorescence with cytokeratin 5, a well-known marker for prostate basal epithelium,11,12 all confirmed that the UROLOGY 58 (4), 2001
positively stained cells were in fact basal cells (Fig. 1). STAINING IN PIA AND HGPIN Focal regions of prostatic atrophy (25 different prostatectomy specimens and 13 different needle 621
specimens), lesions that we had previously termed PIA,13 also stained positive for p63. The staining was generally localized to the basal layer. However, very infrequent weak staining was also detected in the luminal epithelial cells in PIA. The basal cells in most (37 of 38) of these lesions were sparsely distributed (Fig. 1B). The basilar phenotypes were verified by positive staining with the monoclonal antibody 34E12. In a few of the PIA lesions that contained sclerotic stroma, a variable number of the atrophic acini and ducts evinced no p63 staining. The expression of p63 was detected in HGPIN as well (19 of 19 different prostatectomy specimens and 11 of 11 different needle specimens). Staining occurred primarily in the nuclei of the basal epithelial cells. These cells were often sparsely distributed, but in general were not as sparsely distributed as were those in PIA, and the overall occurrence of sparse basal cell staining was less frequent than it was in PIA (20 of 30 lesions). Rarely, HGPIN cells with nuclear atypia that were located above the basal layer in the secretory epithelial compartment showed weak to moderate nuclear staining for p63. STAINING IN ADENOCARCINOMA Most adenocarcinomas from radical prostatectomies were negative for p63 immunoreactivity; 66 (94%) of 70 lesions examined were completely negative. In the non-neoplastic glands surrounding the tumors, however, the basal epithelial cells uniformly showed strong nuclear staining (Fig. 1C). Scattered cells that were morphologically identifiable as tumor cells (and not basal cells), found only in high-grade carcinomas (Gleason pattern 4 or 5) and representing less than 1% of cells in those carcinoma specimens, demonstrated very weak nuclear staining (Fig. 1D). All four of the tumors that exhibited focal weak positive staining extended beyond the prostate (three were Stage T3aN0MX and one was Stage T3bN0MX). To determine whether positively staining cells that were morphologically identified as tumor cells were also positive for basal cell cytokeratins, adjacent 5-mm sections were stained for 34E12 and p63. In general, the same cells that stained positive for p63 also stained positive for 34E12. In some cases, however, additional scattered cells were negative for p63 but positive for 34E12. None of the four tumors positive for p63 by light microscopy were positive for p63 by immunofluorescence (although one was positive for cytokeratin 5), suggesting that standard immunohistochemistry is more sensitive in the detection of the weak expression of p63 in some tumors. Most adenocarcinomas in the prostate needle biopsies—132 (89%) of 148 —also stained completely negative. As in the radical prostatectomy 622
specimens, the tumors that stained positively did so with weak intensity and in scattered cells only. Table I shows all carcinoma specimens from the prostatectomy and needle biopsy specimens listed by Gleason grade and p63 staining status. By Fisher’s exact test, the relation between the positive p63 staining and Gleason grade was highly statistically significant (P ⬍0.0001), with a much greater proportion of high-grade tumors staining positively than did low-grade tumors (Table I). Tumor cells in metastatic prostate adenocarcinoma in 14 (93%) of 15 cases stained negative for p63. One metastatic case demonstrated weak staining in approximately 20% of the tumor cell nuclei. COMMENT The results of the present study demonstrate that basal cells in normal, BPH, PIA, and HGPIN tissue selectively express p63 in their nuclei, but that most prostate adenocarcinoma cells in both primary and metastatic lesions lack expression of this protein. Our findings support the results obtained by Signoretti et al.9 in radical prostatectomy specimens. Our study adds new information in that it includes detailed analyses of p63 expression in BPH, PIA, adenocarcinoma in prostate needle biopsies, and metastatic prostate adenocarcinoma. Moreover, we found a strong correlation between the expression of p63 in the tumors and Gleason grade. Staining for p63 in cancer may augment the staining patterns displayed by more classic basal cell markers, such as cytokeratins 5 and 14, that are currently used to aid in the diagnosis of atypical prostate lesions. Thus, p63 staining can potentially increase the specificity for discerning cancer from HGPIN, adenosis, and various atrophic lesions on prostate needle biopsy, thereby reducing diagnostic uncertainty.14 Although a small fraction of prostate cancer cells in some high-grade carcinomas stained positive, this finding should in general not affect diagnostic issues, as there was no question that these cells were morphologically identifiable as tumor cells and were not basal cells entrapped in the tumor. Although prostate adenocarcinomas may occasionally stain positive for 34E12, Yang et al.15 recently showed that occasional tumor cells in metastatic prostate cancer—a lesion that cannot, by definition, contain entrapped normal prostate basal cells—stain positive for 34E12. Similarly, 1 case of metastatic prostate cancer in our study stained positive for p63. As positive staining for p63 in tumor cells occurred primarily in high-grade lesions, and as all tumors that stained positively in radical prostatectomy specimens demonstrated extraprostatic extension, our data suggest that the degree of p63 UROLOGY 58 (4), 2001
expression in primary adenocarcinoma may correlate with primary tumor aggressiveness. In fact, 44% of tumors with Gleason sums of 8 or greater stained positive, in contrast to 1.6% of tumors with Gleason sums 6 or less (Table I). Although only 1 (7%) of 15 metastatic lesions stained positive, it is possible that p63 expression in extraprostatic carcinoma follows a different pattern than that seen in the primary tumor. In a recent study, for example, decreased E-cadherin expression in radical prostatectomy specimens was associated with a high Gleason score and advanced pathologic stage, and yet E-cadherin staining in metastases was quite strong.16 The expression of high levels of p63 exclusively in epithelial basal cells and the complete lack of prostate development in p63 null mice9 indicates that p63 expression is involved in the control of prostate growth and differentiation. Moreover, it strongly bolsters the hypothesis that the basal cells represent prostate stem cells. Loss of p63 expression appears related to the process of differentiation as cells progress into the overlying epithelium and develop into secretory cells. That most prostate adenocarcinoma and HGPIN cells did not express p63 suggests that p63 may protect prostatic epithelial cells against neoplastic transformation, perhaps through a tumor suppressor function. Since basal cells, but not secretory cells, almost uniformly express p63, it also supports the concept that basal cells are not direct targets for neoplastic transformation.12,13,17,18 Moreover, it provides additional evidence favoring our hypothesis that basal cells are protected from undergoing neoplastic transformation, at least in part, by the carcinogen detoxifying enzyme GSTP1.13,18 Indeed, these data suggest that, rather than basal cells, proliferating cells in the secretory compartment are the targets for neoplastic transformation.12,18 Normal prostatic epithelium consists of two histologically defined compartments: basal and secretory. The secretory luminal cells likely arise from differentiation of the basal cells.17–22 Our group and others have argued that there exists a population of amplifying or transiently proliferating cells that phenotypically are intermediate in differentiation between basal cells and mature, terminally differentiated secretory cells.17,18,21 Our group has further divided this population of intermediate cells into two subpopulations— basal intermediate and secretory intermediate—and postulated that basal intermediate cells give rise to BPH and secretory intermediate cells give rise to HGPIN and carcinoma.18 Up to 50% of luminal secretory cells in PIA express cytokeratin 5 (G. Vanleenders and A.M. De Marzo, unpublished results), indicating that they retain at least some feaUROLOGY 58 (4), 2001
tures of basal cells. Yet these cells in PIA also produce high levels of cytokeratins 8 and 18 and low levels of androgen receptors and prostate-specific antigen, all of which are characteristics of secretory cells.13 That most luminal cells in PIA lack expression of p63 supports the idea that most of these cells have differentiated beyond the basal cell phenotype and is consistent with the concept that luminal PIA cells represent actively dividing, secretory intermediate cells—in our model, the same cells that are the proposed targets of neoplastic transformation (for a detailed illustration of this model, refer to the second figure of De Marzo et al.18). Lending additional support to this concept, we recently reported that a morphologic transition between HGPIN and PIA frequently occurs, and that small adenocarcinomas are often associated with nearby PIA.23 CONCLUSIONS In contrast to normal and premalignant prostatic tissue, most prostate adenocarcinomas do not express p63. p63 immunostaining is thus a potentially useful modality for facilitating the pathologic diagnosis of carcinoma in prostate needle biopsies. When p63 antibody staining is positive in adenocarcinoma, it is focal and weak and correlates strongly with a high Gleason grade. Collectively, the findings in this report support the hypothesis that basal cells represent prostate stem cells and that the immature secretory cells characteristic of PIA—not basal cells—are targets for neoplastic transformation. REFERENCES 1. Yang A, Kaghad M, Wang Y, et al: p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 2: 305–316, 1998. 2. Levrero M, De Laurenzi V, Costanzo A, et al: The p53/ p63/p73 family of transcription factors: overlapping and distinct functions. J Cell Sci 113: 1661–1670, 2000. 3. Trink B, Okami K, Wu L, et al: A new human p53 homologue. Nat Med 4: 747–748, 1998. 4. Parsa R, Yang A, McKeon F, et al: Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113: 1099 –1105, 1999. 5. Yang A, Schweitzer R, Sun D, et al: p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714 –718, 1999. 6. Mills AA, Zheng B, Wang XJ, et al: p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708 –713, 1999. 7. Hibi K, Trink B, Patturajan M, et al: AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA 97: 5462–5467, 2000. 8. Park BJ, Lee SJ, Kim JI, et al: Frequent alteration of p63 expression in human primary bladder carcinomas. Cancer Res 60: 3370 –3374, 2000. 9. Signoretti S, Waltregny D, Dilks J, et al: p63 is a prostate 623
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