p53 Protein and Gene Alterations in Pathological Stage C Prostate Carcinoma

00226347/97/15824510.~ hJOURNAL OF URomY Copyright 0 1997 by AMWCANUROLOGICAL ASSOCIATION, h c .

Vol. 158,610-514,August 1997 Printed in U S A

p53 PROTEIN AND GENE ALTERATIONS IN PATHOLOGICAL STAGE C

PROSTATE CARCINOMA CAROL E. SALEM, NICKOLAS A. TOMASIC, DONALD A. ELMAJLAN, DAVID ESRIG, PETER W. NICHOLS, CLIVE R. TAYLOR, DONALD G. SKINNER, PRADIP ROY-BURMAN, GARY LIESKOVSKY AND RICHARD J. COTE* From the Departments of Pathology and Urology,University of Southern California School of Medicine and Urologic Cancer Research Laboratory, Kenneth Norris Jr. Comprehensive Cancer Center, Los Angeles, California

ABSTRACT

Purpose: We determined the extent of p53 immunoreactivity in pathological stage C prostate cancer as well as its correlation to tumor grade, substage, recurrence and proliferation rate. To define better the temporal relationship of p53 nuclear reactivity in prostate cancer p53 immunoreactivity was evaluated in all associated prostatic intraepithelial neoplasia lesions. Materials and Methods: Using immunohistochemistry p53 status and proliferation rate were determined in 96 tumors from patients with pathological stage C prostate cancer. Single strand conformational polymorphism in exons 5 to 8 was used in a subset of specimens to assess the association of p53 nuclear accumulation with mutations in the p53 gene. Results: p53 Nuclear reactivity was demonstrated in 10 tumors (10.4%),including 6 with high and 4 with low level nuclear reactivity. Of the tumors 86 (89.6%) had no evidence of p53 immunoreactivity. Each of the 6 tumors with high level p53 reactivity had associated areas of prostatic intraepithelial neoplasia that also showed p53 nuclear reactivity. Furthermore, pathological stage C substage (Cl, 2 or 3) was sigmficantly associated with p53 nuclear reactivity (p = 0.04). Proliferation rates were correlated with p53 nuclear reactivity (p = 0.091, while there was no association with tumor grade or recurrence. p53 Gene alterations were noted in 2 of the 3 p53 positive tumors versus no alterations in the p53 gene of 3 p53 negative tumors. Conclusions: p53 Nuclear accumulation is uncommon in pathological stage C prostate cancer and its presence in premalignant prostatic intraepithelial neoplasia lesions suggests that it may be an early event in a subset of prostate cancers. KEY WORDS:prostate, protein p53, prostatic neoplasms, carcinoma, genes

The p53 gene codes of a 53 kd. nuclear phosphoprotein, the rate of p53 alterations. These results were compared with which is thought to function as a regulator of cell growth. p53 gene status in a subset of cases. p53 Nuclear accumulation Expression is increased after deoxyribonucleic acid (DNA) was then correlated with proliferation rate, tumor grade, damage, resulting in an arrest in the G1 phase of the cell tumor substage and recurrence. In addition, the presence of cycle, allowing time for DNA The wild type protein p53 alterations in premalignant lesions (high grade prostatic is not detectable by standard immunohistochemical tech- intraepithelial neoplasia) was determined to assess better niques. However, mutations in the p53 gene result in nuclear the temporal relationship between p53 alterations and prosaccumulation of the altered p53 protein, allowing subsequent tate tumorigenesis. detection using immunohistochemistry.3 Mutations in the p53 gene have been described in many human tumor types, MATERIALS AND METHODS including those of the colon? breast,6 lung6 and pro~tate.~-lO Patient population. Tumors from 96 patients 46 to 79 years Previous studies have shown that p53 nuclear accumulation is rare in prostate cancer7.8.11.12 but this accumulation is old (median age 65) with pathological stage C adenocarciassociated with high histological grade and increased prolif- noma of the prostate were used in this study. Each patient eration rate.7.13 Furthermore, others have reported that p53 had undergone radical retropubic prostatectomy with limited alterations may be associated with advanced14 or metastatic bilateral pelvic lymph node dissection at our institution bedisease,7.9-l1. l3 suggesting that the gene alteration occurs as tween May 1983 and April 1989. For study purposes each a late event in prostate tumorigenesis. However, these stud- tumor was reassessed regarding Gleason grade's and subies have compared different stages of disease and to our stage by a pathologist (R. J. C.) to maintain consistency. The knowledge none has evaluated the effect of p53 and tumor extent of extraprostatic disease, that is pathological stage C substage, was categorized as described by Gibbons et all6 as recurrence in a single disease stage. We examined tumors from patients with pathological stage C1-invasion through the capsule without involvement of C prostate cancer for p53 nuclear accumulation to determine the surgical margin or seminal vesicles, C2-a positive surgical margin without seminal vesicle involvement and C3involvement of the seminal vesicle(s). Prostatic intraepithek e p t e d for publication December 27, 1996. Supported in part b Grant CA 70903 from the National Institutes lial neoplasia was evaluated and graded according to the of HealtMVational 8ancer Institute and Grant 59705 from the criteria of Bostwick and Brawer.17 Of the patients 31 (32.3%) U p e d States Public Health Service. had pathological stage C1,34 (35.4%)had pathological stage Requests for re rints: De artment of Patholo and Urology, University of SoutRern Calitrnia School of Megcine, Kenneth C2 and 31 (32.3%)had pathological stage C3 disease. Gleason N o m s Jr. Cancer Center, 1441 Eastlake Ave., Los Angeles, Cali- score was 2 to 4 in 4 cases (4.2%),5 to 7 in 64 (66.7%)and 8 fornia 90033. to 10 in 28 (29.2%).A total of 80 patients received postoper510

p53 AND GENE ALTERATIONS IN PROSTATIC CARCINOMA

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ative adjuvant radiation (median 46 Gy.) and 15 underwent he proliferation rate. Of the 96 tumors studied the median early hormonal deprivation. iroliferation rate was 3%, which was used as a cutoff to Followup. All patients were seen and evaluated 1 , 2 and 6 eparate the tumors into low (less than 3%) and high (3% or months postoperatively, at 6-month intervals until postoper- Feater) proliferation groups. ative year 5 and yearly thereafter. Each visit included a Single strand conformational polymorphism. DNA from 3 complete physical examination, and determinations of pros- t umors showing intense and 3 showing no p53 immunoreactatic acid phosphatase and, after 1987, prostate specific an- t,ivity were tested for single strand conformational polytigen (PSA). PSA values were obtained using the Hybritech* Inorphism in exons 5 to 8 of the p53 gene. Single strand assay. Bone scans were performed as clinically indicated or <:onformational polymorphism analysis was performed as when PSA was elevated to 0.4 ng./ml. or greater. Biopsies Iweviously described.10 DNA from paraftin tissue sections were performed to document local recurrence or metastatic 1was extracted. Exons 5 to 8 of the p53 gene were then indidisease as clinically indicated. Clinical recurrence was de- 1tidually amplified from isolated DNA by polymerase chain fined as biopsy proved local recurrence or metastatic disease 1reaction. Aliquots (3 pl.) of the polymerase chain reaction confirmed by bone scan or biopsy. PSA recurrence was de- 1products were mixed in a 1:2 ratio with mutation detection fined as serum PSA 0.4 ng./ml. or greater without evidence of Ixhancement denaturation buffer, which were then heated clinical recurrence. Mean followup was 4.6 years. Four pa- a t 98C for 5 minutes and subsequently cooled on ice water. tients died, although only l died of prostate cancer. Aliquots (5 pl.) were loaded onto a 0.4 mm. mutation detecAntibodies. We used 2 anti-p53 monoclonal antibodies, tion enhancement nondenaturing gel and electrophoresed at BP53-12 (IgG 2a dilution 1:40) and PAb 1801 (IgAG 1 dilu- 8 W. at room temperature for 18 hours. The gels were dried tion 1:lO). BP53-12 recognizes the region between amino at 80C and autoradiographed on x-ray film at room temperacids 1and 45 of the p53 protein, while PAb 1801 recognizes ature overnight. Mutations were identified using direct DNA the denaturation resistant epitope corresponding to amino sequencing. Band shifts detected by single strand conformaacids 32 to 79. Both antibodies react with wild type and tional polymorphism were analyzed for sequence mutations mutant p53 protein. We have previously shown that p53 as described previously,10 using total DNA and DNA eluted nuclear reactivity determined using these antibodies highly from cut out abnormal single strand conformational polymorcorrelates with mutations in the p53 gene.3 The rate of cell phism bands. proliferation was assessed using antiproliferating cell nuStatistical methods. Kaplan-Meier analysis was used to clear antigen monoclonal antibody PC-10 (class IgG 2a, di- compare recurrence rates in the clinical groups.21The Fisher luted l:lO), which identifies cells in the G1, S and G2 to M exact test was performed for correlations among p53 immuphases of the cell cycle.lS.19 noreactivity, tumor grade and proliferation rate.22 The exact Zmmunohistochernistry.Sections (5 pm.) from archival, for- logistic regression test for trend was used to correlate p53 malin fxed, paraffin embedded tissue were placed on poly- immunoreactivity to pathological stage C s u b ~ t a g e s . ~ ~ L-lysine coated slides. Deparaffnization was accomplished RESULTS with iodine-xylene and tissue was rehydrated with 95% ethanol. Endogenous peroxidase was quenched in 3% hydrogen Association of p53 nuclear accumuZation with prostate tuperoxide in methanol. Antigen retrieval was then performed mor grade, substage and recurrence. The 2 anti-p53 monocloas described by Shi et a120 with certain modifications. Each nal antibodies BP53-12 and PAb 1801 produced similar reslide was placed in Coplin jars and heated in a microwave sults. Of the 96 tumors 10 (10.4%) demonstrated nuclear oven at high power for 10 minutes in antigen retrieval solu- reactivity for the p53 protein, including 6 (6.3%) with a high tion diluted 1:4 in distilled water. Tissues were then incu- (more than 10% positive tumor cells) and 4 (4.2%) with a low bated with 5% horse serum for blocking purposes. The pri- (10%or less positive tumor cells) level of nuclear reactivity. mary antibodies (BP53-12, PAb 1801 or PC10) were Gleason score was 2 to 4 in 8 cases (4.2%),5 to 7 in 64 (66.7%) incubated overnight at 4C. The tissue was washed in phos- and 8 to 10 in 28 (29.2%). No statistically sigrdcant relaphate buffered saline followed by incubation with biotiny- tionship was noted between p53 immunoreactivity and lated horse anti-mouse secondary antibody at a concentra- Gleason score (p = 0.23, table 1).Ofthe 6 tumors with a high tion of 1:200. Visualization of reactivity with an ABC level p53 reactivity (part A of figure) 4 were pathological immunoperoxidase system according to manufacturer recom- substage C3 and 2 were pathological substage C2 (table 2). mendations was followedby 0.03% diaminobenzidine used as A n exact logistic regression test for trend showed a signifia chromogen with hematoxylin counterstaining. Bladder can- cant correlation between high level p53 immunoreactivity cer specimens with demonstrated p53 gene mutations and and higher substage within this pathological stage C group established p53 nuclear accumulation were used as positive (p = 0.04). controls. Analysis of the immunohistochemical results was Mean followup in this patient population was 4.6 years performed by 3 of us (N. A. T., D. A. E. and R. J. C.). (range 1.4 to 8.6). Disease recurred in 35 (36.5%), including Assessment of immunoreactivity. The extent of p53 nuclear 25 (26%) with PSA recurrence and 10 (10.4%) with clinical reactivity was assessed and categorized as no nuclear reac- recurrence. Overall the mean probability plus or minus stantivity, low level immunoreactivity (1to 10% positive tumor dard deviation of being disease-free a t 5 and 7 years was cell nuclei) and high level immunoreactivity (greater than estimated at 59.3 5 5.9 and 50.5 5 7.8%, respectively. Of the 10% positive tumor cell nuclei) (part A of figure). This cutoff 10 patients with p53 nuclear reactivity 3 (30%) had recurwas based on our previous results in bladder cancer, which rence, including 2 with high and 1with low level reactivity. demonstrated that cases with less than 10% positive tumor cell nuclei did not show p53 gene mutations. In contrast, the finding of 10% or greater positive tumor cell nuclei highly TABLE1. Distribution of patients with pathological stage C correlated with p53 gene mutations.3 p53 Nuclear reactivity prostate cancer correlating p53 immunoreactivity with Gleason was considered positive when there was dark brown granular score staining confined to the nuclei. Based on our previous work3 No. pts. (%) Total only nuclear immunoreactivity was assessed in this analysis. Gleason Score No. pts. ps3 Neg. p53 Paa. In cases of proliferating cell nuclear antigen immunoreactivity the positively stained tumor nuclei were counted and 2 4 4 (100) 0 4 divided by the total number of tumor cell nuclei to determine 6 7 57 (89) 7 (11) 64

* Hybritech, Inc., San Diego, California.

8-10 26 (89) Fisher's eLaet test p = 0.23.

3 (11)

28

5 12

p53

AND GENE ALTERATIONS IN PROSTATIC CARCINOMA

Formalin fixed paraffinembedded tissue section of stage C prostate cancer. A, high level p53 nuclear immunoreactivity (largearrows) with negative staining stromal cell nuclei (small arrow).3,area of prostatic iqtraepithelial neoplasia with characteristicbasal cells (large arrow) and prominent nucleoli (small arrows)with p53 nuclear immunoreactivlty. Reduced from X400.

TABLE2. Distribution ofpatients with pathological stage C Of the 86 patients with no p53 nuclear reactivity 32 (37.2%) had recurrence. Kaplan-Meier analysis revealed no associa- prostate cancer correlating p53 immunoreactivity with pathological stage C substage tion between p53 immunoreactivity and recurrence in this population of stage C prostate cancer cases (p = 0.80). In the p53 (No. pts.) Total majority of patients recurrent tumors demonstrated no p53 Substage Greater than No. Pts. 0 to 10% immunoreactivity. 10% Association of p53 nuclear accumulation with prostatic in31 0 c1 31 traepithlial neoplasia. Each of the 6 tumors with a high level 32 2 c2 34 of p53 accumulation had areas of grade I11 prostatic intrac3 27 4 31 epithelial neoplasia. p53 Nuclear reactivity was detected in Exact logistic regression p = 0.04. all prostatic intraepithelial neoplasia lesions associated with these 6 tumors (part B of figure). In no case was p53 immuTABLE3. Distribution ofpatients with pathological stage C noreactivity noted in the prostatic intraepithelial neoplasia lesions and not in the associated tumor. p53 Immunoreactiv- prostate cancer correlating p53 immunoreactivity with proliferation rate ity was not detected in the prostatic intraepithelial neoplasia lesions of p53 negative tumors. Mean Proliferating Cell Nuclear Antigen Association of p53 nuclear accumulation with proliferation (No. pts.) rate. Each of the 96 tumors had evidence of proliferative Less Than Greater Than activity as assessed by proliferating cell nuclear antigen ac3% 3% cumulation. The median proliferation rate was 3% (range of p53 Pos. 2 8 0.5 to 20). Of the 10 tumors with detectable p53 nuclear p53 Neg. 46 40 reactivity 8 had an elevated proliferation rate as measured Fisher's exact test p = 0.09. by proliferating cell nuclear antigen immunoreactivity (3% o r greater proliferating cell nuclear antigen positive nuclei). While the association of p53 alterations and increased pro- the prostate, which indicates that p53 alterations may be an liferation rate showed a trend, it did not reach statistical early event in prostate tumorigenesis in a subset of tumors. significance (p = 0.09, table 3). Mutations in the p53 gene are a common genetic alteration Association of p53 nuclear accumulation with molecular in human cancer and they develop in a wide variety of tumor changes in the p53 gene. DNA from 6 tumors was evaluated types, including those of the colon,4 breast: lungs and prosusing single strand conformational polymorphism in exons 5 tate.7-10 In addition to its presence in invasive cancers, p53 to 8 of the p53 gene. Of the 3 tumors with p53 nuclear mutations have also been demonstrated in intraductal breast reactivity 2 also had alterations in the gene. In the 3 tumors carcinomaz4 and transitional cell carcinoma in situ.25 Furwith no demonstrable p53 nuclear reactivity no alterations thermore, p53 accumulation has been reported in severe were detected in the p53 gene. dysplasia of bronchial26 and esophageaP7 specimens, while p53 point mutations have been noted in dysplastic ulcerative DISCUSSION colitis lesions.28Thus, p53 alterations have been shown to be In this study of pathological stage C adenocarcinoma of the relatively early events in tumor progression in some systems. prostate p53 protein accumulation was noted in relatively It has been postulated that DNA damage results in infew tumors. The presence of p53 nuclear accumulation as creased expression of p53, which subsequently helps to regdetected by immunohistochemistry was associated with mu- ulate the cell cycle.1V2After the accumulation of p53 there is tations in the p53 gene in prostate carcinoma. While p53 an arrest in the G1 phase, allowing time for DNA repair. If alterations were significantly associated with increasing sub- repair fails, p53 may then induce cell suicide by apoptosis.'S2 stage in patients with stage C prostate cancer, no significant Conversely, if the p53 gene is mutated or p53 protein is association was demonstrated between p53 nuclear accumu- inactivated, the cell continues to replicate without control. lation and Gleason score or disease recurrence. A higher The results of our study are consistent with this notion. proliferation rate seemed related to p53 immunoreactivity, Prostate carcinomas that have p53 alterations also tend to although statistical significance was not reached. An impor- have higher proliferation rates than those with no detectable tant finding of this study was that, when p53 alterations alterations. Furthermore, p53 protein can become inactiwere identified in the cancer lesions, they were also seen in vated by binding to the cellular oncogene MDM2 or to viral associated prostatic intraepithelial neoplasia lesions within proteins.2.29 Mutations in the p53 gene resulting in the ac-

p53 AND GENE ALTERATIONS IN PROSTATIC CARCINOMA

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CONCLUSIONS cumulation of a mutated p53 protein with an extended halflife have been detected a t the protein level by immunohistop53 Accumulation is a relatively uncommon event in proschemical methods. However, the half-life of the wild type p53 tate cancer. Furthermore, the presence of p53 immunoreacprotein is too short to allow its detection by immunohisto- tivity in premalignant prostatic intraepithelial neoplasia lechemical techniques5.30 The association between p53 nu- sions of the prostate provides us evidence that p53 clear accumulation and mutations in the p53 gene has been accumulation is an early event in a subset of prostate candescribed in breast cancer.5 Furthermore, we have demon- cers. However, such interpretations are complicated by the strated in a large series of bladder carcinomas a highly sig- evidence of heterogeneous topographical distribution of p53 It is now necessary nificant association between mutations in the p53 gene and mutations within a prostate cancer.*O~~~ p53 nuclear accumulation. The concordance of p53 mutations to demonstrate whether these observed p53 mutations in by polymerase chain reaction-single strand conformational tumors with prostatic intraepithelial neoplasia lesions are polymorphism and p53 nuclear accumulation was 76.7% (p = distributed uniformly or heterogeneously within a tumor. 0.0001). Most tumors that have p53 gene mutations also Dr. Susan Groshen, Su-Chiu Chen and Ming Diekinson demonstrate p53 nuclear immunoreactivity when our meth- provided biostatistical analysis of the data. ods are used.3 Our study as well as othersg.lo. l3.3l support these findings in prostate cancer. The presence of p53 nuREFERENCES clear immunoreactivity correlates with mutations in the p53 gene. 1. Levine, A. J., Momand, J. and Finlay, C. A.: The p53 tumour suppressor gene. Nature, 361: 453, 1991. In our series the overall prevalence of p53 nuclear accu2. Lane, D. P.: Cancer. p53 Guardian of the genome. Nature, 358: mulation was 10.5%, which is in accord with the results of 15, 1992. previous studies that have also demonstrated a low level 3. Esrig, D., Spruck, C. H., Ill, Nichols, P. W., Chaiwun, B., Steven, of p53 immunoreactivity in prostate cancer at rates of 12 K., Groshen, S., Chen, S. C., Skinner, D. G., Jones, P. A. and to 17%7-10These studies generally included tumors of all Cote, R. J.: p53 Nuclear protein accumulation correlates with stages,7.8 while another included only stage A1 prostate canmutations in the p53 gene, tumor grade, and stage in bladder cancer. Amer. J. Path., 143: 1389, 1993. cers.32 Notably Van Veldhuizen et al reported p53 immuno4. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Kern, S. E., reactivity in 79% of prostate tumors tested.33 However, they Preisinger, A. C., Leppert, M., Nakamura, Y., White, R., Smits, considered cytoplasmic as well as nuclear staining as p53 A. M. and Bos, J. L.: Genetic alterations during colorectalpositive, while all others considered only nuclear reactivity tumor development. New Engl. J. Med., 319: 525, 1988. as indicative of p53 immunoreactivity. Visakorpi et al dem5. Davidoff, A. M., Humphrey, P. A., Iglehart, J. D. and Marks, onstrated that tumors with high level p53 accumulation J. R.: Genetic basis for p53 overexpression in human breast cancer. Proc. Natl. Acad. Sci., 88: 5006, 1991. were associated with high histological grade, high prolifer6. Yokota, J., Wada, M., Shimosato, Y., Terada, M. and Sugimara, ation rate and poor survival.7 Others have suggested a T.: Loss of heterozygosity on chromosome 3, 13, and 17 in correlation between p53 immunoreactivity and advanced dissmall-cell carcinoma and on chromosome 3 in adenocarcinoma ease.9.10,11.13,14Although our study did not show a correlaof the lung. Proc. Natl. h a d . Sci., &i. 9252, 1987. tion between p53 nuclear accumulation and tumor recur7. Visakorpi, T., Kallioniemi, 0. P., Heikkinen, A, Koivula, T. and rence, this result is not necessarily contradictory. Our study Isola, J.: Small subgroup of aggressive, highly proliferative prostatic carcinomas defined by p53 accumulation. J. Natl. included a relatively low number of patients with p53 immuCancer Inst.. &L: 883, 1992. noreactivity, making any statistically significant association 8. Thompson, S . J., Mellon, K, Charlton, R. G., Marsh, C., with recurrence unlikely. Followup was relatively brief and Robinson, M. and Neal, D. E.: P53 and Ki-67 immunoreactivity included only stage C cancer. However, our results seem to in human prostate cancer and benign hyperplasia. Brit. demonstrate that, if p53 alterations have a role in prostate J. Urol., 69: 609, 1992. cancer progression, it appears that most tumors recur in the 9. Bookstein, R., MacGrogen, D., Hilsenbeck, S. G., Sharkey. F. and Allred, D. C.: p53 Is mutated in a subset of advanced-stage absence of such alterations in the primary tumor. While p53 prostate cancers. Cancer Res., 53.3369, 1993. alterations may occur as a late event after the morphological recognition of cancer and, therefore, are primarily noted in 10. Mirchandani, D., Zheng, J., Miller, G. J., Ghosh, A. K, Shibata, D. K, Cote, R. J. and Roy-Burman, P.: Heterogeneity in intrametastases?-11-13 our data indicate that p53 alterations may tumor distribution of p53 mutations in human prostate cancer. also be an early event in prostate tumorigenesis in a subset Amer. J. Path., 147: 92, 1995. of tumors. 11. Aprikian, A. G., Sarkis, A. S., Fair, W. R., Zhang, Z.-F., Fuks, Z. and Cordon-Cardo,C.: Immunohistochemical determination of It is accepted that there are phenotypic similarities bep53 protein nuclear accumulation in prostatic adenocarcitween prostatic intraepithelial neoplasia and invasive prosnoma. J. Urol., 161: 1276, 1994. tate carcinoma,34 and dysplasia is probably a direct biological H. J., Sugars, L. Y., Pretlow, T. and Gelmann, E.P.: p53 precursor of prostate carcinoma.35.36 Our study showed that 12. Voeller, Oncogene mutations in human prostate cancer specimens. all tumors with a high level of p53 immunoreactivity had J. Urol., 161: 492, 1994. areas of high grade prostatic intraepithelial neoplasia dem- 13. Navone, N. M., Troncoso, P., Pisters, L. L., Goodrow, T. L., onstrating immunoreactivity. No immunoreactivity was obPalmer, J. L., Nichols, W. W., von Eschenbach, A. C. and Conti, C. J.: $3 Accumulation and gene mutation in the served in the prostatic intraepithelial neoplasia lesions assoprogression of human prostate carcinoma. J. Natl. Cancer ciated with any of the p53 negative tumors. That p53 Inst., 85: 1657, 1993. alterations occur in prostatic intraepithelial neoplasia is not 14. Konishi, N., Hiasa, Y., Hayashi, I., Matsuda, H., Tsuzuki, T., surprising, since p53 accumulation has been shown in bronMing, T., Kitahori, Y.,Shiraishi, T., Yatani, R. and Shimazaki, chial dysplasia associated with squamous cell carcinomaz6 J.: p53 Mutations occur in clinical, but not latent, human and in Barrett’s mucosa associated with esophageal carcinoprostate carcinoma. Jap. J. Cancer Res., 86: 57, 1995. mas.27 Also, p53 point mutations have been demonstrated in 15. Gleason, D. F.: Histologic grading and clinical staging of prostatic carcinoma. In: Urologic Pathology. The Prostate. Edited dysplasia adjacent to colon carcinomas associated with ulcerby M.Tannenbaum. Philadelphia: Lea and Febiger, pp. 171ative colitis.28 The finding of p53 alterations in areas of 198, 1977. prostatic intraepithelial neoplasia M h e r supports the con- 16. Gibbons, R. P.,Cole, B. S.,Richardson, R. G., Coma, R. J., Jr., cept that prostatic intraepithelial neoplasia is a premaligBrannen, G. E., Mason,J. T., Taylor, W.J., and Hafermann, nantlpreinvasive lesion, because it has alterations similar to M.D.: Adjuvant radiotherapy following radical pmstatectorn~ results and complications. J. Uml.. 13& 66.1986. those of cancer.

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p53 AND GENE ALTERATIONS IN PROSTATE CARCINOMA

17. Bostwick, D. G. and Brawer, M. K: Prostatic intra-epithelial neoplasia and early invasion in prostate cancer. Cancer, 59: 788, 1987. 18. Garcia, R. L., Coltrera, M. D. and Gown, A M.: Analysis of proliferative grade using anti-PCNNcyclin monoclonal antibodies in fixed embedded tissues. Comparison with flow cytometric analysis. h e r . J. Path., 154: 733, 1989. 19. Bravo, R., Frank, R., Blundell, P. A, and MacDonald-Bravo,M.: CyclinlPCNA is the auxiliary protein of DNA polymerasedelta. Nature, 328:515,1987. 20. Shi, S. R., Key, M. E. and Kalra, K L.: Antigen retrieval in formalin-hed paraffinembedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem., 39: 741, 1991. 21. Kaplan, E. and Meier, P.: Non-parametric estimation h m incomplete observations. J. Amer. Stat. Ass., 63: 457, 1958. 22. Mehta, C. R. and Patel, N. R.: A network algorithm for performing Fisher’s exact test in R X C contingency tables. J. Amer. Stat. Ass., 18: 427, 1983. 23. Hiji, K F., Mehta, C. R. and Patel, N. R.: Computing distributions for exact logistic regression. J. Amer. Stat. Ass., 8 2 1110, 1987. 24. Davidoff, A M., Kerns, B. M., Iglehart, J. D. and Marks, J. R.: Maintenance of p53 alterations throughout breast cancer progression. Cancer Res., 51: 2605, 1991. 25. Spruck, C. H., 111, Ohneseit, P. F., Gonzalez-Zulueta,M., Esrig, D., Miyao, N., Tsai, Y. C., Lerner, S. P., Schmutte, C., Yang, A. S. and Cote, R.: Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res., 54: 784, 1994. 26. Nuorva, K, Soini, Y., Kamel, D., Autio-Harmainen, H., Risteli, L., Risteli, J.,Vahakangas, K and Paakko, P.: Concurrent p53 expression in bronchial dysplasias and squamua cell lung carcinomas. h e r . J. Path., 142: 725,1993. 27. Flejou, J. F., Potet, F., Muzeau, F.,Le Pelletier, F., Fekete, F. and Henin, D.: Overexpression of p53 protein in Barrett’s syndrome with malignant transformation. J. Clin. Path., 46: 330, 1993. 28. Yin, J., Harpaz, N., Tong, Y., Huang, Y., Laurin, J., Greenwald, B. D., Hontanosas, M., Newkirk, C. and Meltzer, S. J.: p53 Point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology, 104: 1633, 1993. 29. Oliner, J. D., Kinzler, K W., Meltzer, P. S., George, D. L. and Vogelstein, B.: Amplification of a gene encoding a p53associated protein in human sarcomas. Nature, 3M):80,1992. 30. Rodrigues, N. R., Rowan, A., Smith, M. E., Kerr, I. B., Bodmer, W. F., Gannon, J. V. and Lane, D. P.: p53 Mutations in colorectal cancer. Proc. Natl. Acad. Sci., 87: 7555, 1990. 31. w e n s , W. N., van der Weiden, M. M., Schroeder, F. H., Bosman, F. T. and Trapman, J.: Frequency and characterization of p53 mutations in primary and metastatic human prostate cancer. Int. J. Cancer, 66:630, 1994. 32. Fox, S. B., Persad, R. A, Royds, J., Kore, R. N., Silcocks, P. B. and Collins, C. C.: p53 And c-myc expression in stage A1 prostatic adenocarcinoma: useful prognostic determinants? J. Urol., 1M):490, 1993. 33. Van Veldhuizen, P. J., Sadasivan, R., Garcia, F., Austenfeld, M.S. and Stephens, R. L.:Mutant p53 expression in prostate carcinoma. Prostate, 22: 23, 1993. 34. Nagle, R. B.,Brawer, M. K, Kittelson, J. and Clark, V.: Phenotypic relationships of prostatic intraepithelial neoplasia to invasive prostatic carcinoma. h e r . J. Path., 138: 119, 1991. 35. Brawer, M. K: Prostatic intraepithelial neoplasia: a premalignant lesion. Hum. Path., 23: 242, 1992. 36. McNeal, J. E. and Bostwick, D. G.: Intradudal dysplasia: a

premalignant lesion of the prostate. Hum. Path., 17:64, 1986. 37. Konishi, N., Hiasa, Y., Matsuda, H., Tao, M., Tsuzulii, T., Hayashi, I., Kitahori, Y., Shiraishi, T., Yatani, R., Shimazab, J. and Lin, J.: Intratumor cellular heterogeneity and alterations in ras and p53 tumor suppressor gene in human prostate carcinoma. Amer. J. Path., 147: 1112, 1996. EDITORIAL COMMENT In regard to prostate cancer p53 status remains unclear and problematic, and results tend to be confusing. For example, reports of ~ 5 3 mutation in primary untreated prostate cancer range from 0 to 80% with metastatic cancer even higher. It is important to try to unravel this problem because, as the authors point out, p53 is an extremely important molecule with respect to malignant transformation. They report a logical step in the search, which is to evaluate a single stage, such as stage C or T3. Unfortunately, no real answers are forthcoming. There is no apparent association with the biological behavior of the disease with respect to Gleason score, recurrence, or proliferation and only a weak correlation with T substage. The real problem may be a general lack of understanding of all of the components of p53 reactivity, making measurement difficultand perhaps unreliable. For example, there are several methods of immunostaining that may produce sampling artifacts. Beyond that, approximately 20 to 30% of mutations do not stain and approximately 25% stain without a corresponding mutation by a partial sequence! As the authors point out, intranuclear staining is the usual index for mutation. However, p53 may also accumulate abnormally in the cytoplasm, as in neuroblastoma and some breast cancers.’ This is often called background but it probably represents more. For example, it is known that p53 enters the nucleus as a function of its nuclear localization signal, which is largely affected by other proteins expressed in the ce11.2 Our knowledge of this function with respect to nuclear staining is still rudimentary. Other interesting and yet poorly defined clinical parameters may have a significant role. For example, suggestive evidence exists that the level of apoptosis within prostate cancers may be variable and perhaps predictive of recurrence.s The cause is unknown but it may be related to alterations in androgen stimulation. Nevertheless, a p optosis may be mediated by p53 with associated increased levels within prostate cancer cells. If that were the case, then increased immunostaining would be purely artifactual in regard to p53 mutation. In all likelihood we are measuring heterogeneous p53 events. Not until our understanding of p53 and its mechanics is more refined w i l l it become a reliable clinical tool. William C. DeWolf 330 Brookline Awe. Boston, Massachusetts 02215 1. Moll, U. M., LaQuaglia, M., Benard, J. and Riou, G.: Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc. Natl. Acad. Sci., 9 2 4407, 1995. 2. Shaulsky, G., Goldfinger, N., Ben-Ze’ev, A., Rotter, V.: Nuclear accumulation of p53 protein is mediated by several localization signals and plays a role in tumorigenesis. Mol. Cell Biol., 10: 6565,1990. 3. Stapleton, A. M. F., Zbell, P., Kattan, M. W., Yang, G., Wheeler, T. M., Scardino, T. P. and Thompson, T. C.: Assessment of the biological markers p53, Ki67, and apoptic index as predictive indicators of cancer recurrence following surgery. J. Urol., part 2,157: 347, abstract 1355, 1997.