Prognostic significance of p53 nuclear accumulation in localized prostate cancer treated with radical prostatectomy

[CANCER RESEARCH 60, 1585–1594, March 15, 2000]

Prognostic Significance of p53 Nuclear Accumulation in Localized Prostate CancerTreated with Radical Prostatectomy1

David I. Quinn, Susan M. Henshall, Darren R. Head, David Golovsky, J. David Wilson,2 Phillip C. Brenner,Jennifer J. Turner, Warick Delprado, John F. Finlayson, Phillip D. Stricker, John J. Grygiel, andRobert L. Sutherland3

Cancer Research Program, Garvan Institute of Medical Research [D. I. Q., S. M. H., D. R. H., R. L. S.], and Departments of Urology [D. G., J. D. W., P. C. B., P. D. S.],Anatomical Pathology [J. J. T.], and Medical Oncology [J. J. G.], St. Vincent’s Hospital, Darlinghurst, New South Wales 2010; Douglass Hanly Moir Pathology, North Ryde,New South Wales 2113 [W. D.]; and Sydney Diagnostic Services, North Ryde, New South Wales 2113 [J. F. F.], Australia

ABSTRACT

The role of p53 in the pathogenesis of, and as a predictive biomarkerfor, localized prostate cancer (PCa) is contested. Recent work hassuggested that patterns of p53 nuclear accumulation determined byimmunohistochemistry are prognostic, whereas studies using othermethods question the role ofp53 mutations in predicting outcome. Westudied 263 men with localized PCa treated with radical prostatectomyto determine whether p53 nuclear accumulation predicts relapse anddisease-specific mortality. We combined two p53 immunohistochemis-try scoring systems: (a) percentage of p53-positive tumor nuclei in allmajor foci of cancer within the prostate; and (b) clustering, where thepresence of 12 or more p53-positive cells within a3200 power field wasdeemed “cluster positive.” Analysis was undertaken usingx2, Kruskal-Wallis, and Mann-Whitney tests for clinicopathological variables andthe Kaplan-Meier method, log-rank test, and univariate and multiva-riate Cox regression modeling for evaluation of contribution to relapseand disease-specific survival. At mean follow-up of 55.1 months (range,4.9 –123.0 months), 39% (102 of 263) of patients had relapsed and 2.3%(6 of 253) had died of PCa. Pretreatment serum prostate-specificantigen concentration, pathological tumor stage, lymph node involve-ment, Gleason score, and p53 nuclear accumulation, as determined byeither percentage score or cluster status, were independent predictorsof relapse in multivariate analysis. Clustering of p53-positive cellsdistinguished between favorable and poor prognosis patients within thelowest p53-positive stratum (>0 to <2%) and was the most discrimi-natory threshold for predicting relapse in the entire cohort. p53 statuspredicted outcome in patients with a Gleason score of 5 and above butnot those with a score of 4 and below. In patients treated with neoad-juvant hormonal therapy, p53 cluster positivity carried a 90% (19 of21) risk of relapse by 36 months. All six patients who died from PCa inthe period of the study exhibited p53 nuclear accumulation in 20% ormore tumor nuclei. This study demonstrates strong relationships be-tween p53 nuclear accumulation and relapse and disease-specific mor-tality in a large series of localized PCas. Furthermore, the presence ofclusters of p53-positive nuclei delineates a group of patients with poorprognosis not identified by traditional scoring methods and supportsthe hypothesis that p53 dysfunction within PCa may exist in foci oftumor cells that are clonally expanded in metastases.

INTRODUCTION

PCa4 is the most common male cancer in industrialized societiesand represents a serious public health problem. Identification ofpatients with aggressive rather than indolent PCa is a major challengefor optimal management and is only partially met by current prog-nostic parameters. Delineation of patterns of gene expression in earlyPCa that correlate with an aggressive phenotype is a priority and mayallow radical treatment to be offered on a more selective basis to thosepatients with clinically localized yet aggressive disease.

Inactivation of the tumor suppressor genep53 is implicated intumorigenesis for.50% of all human cancers (1). p53 functions as atranscriptional regulator involved in G1 phase growth arrest of cells inresponse to DNA damage as well as having roles in the regulation ofthe spindle checkpoint, centrosome homeostasis, and G2-M phasetransition (2). p53 also induces apoptosis by transcription-dependentand -independent mechanisms in many cell types (1–3) and regulatestumor angiogenesis and expression of theKai1 metastasis suppressorgene (1, 4–6). Nuclear accumulation of p53 detected by IHC typicallyindicates the presence ofp53 gene mutations (7, 8), although thecorrelation between nuclear accumulation of p53 and the presence ofp53 gene mutations can vary (9). Nuclear accumulation of p53 is aprognostic indicator in several human cancers, including breast (4, 10,11), lung (12), and colorectal carcinoma (13).

The value of p53 nuclear accumulation as a prognostic factor inlocalized PCa is controversial. A number of studies have shown thatp53 nuclear accumulation detected by IHC is prognostic at a varietyof dichotomizing cutoff points based on the number of p53-positivenuclei. These studies describe either a group of poor prognosis pa-tients with 20% p53-positive nuclei (14, 15) or a group of patientswith lower percentages of positive cells in a heterogeneous, focalstaining pattern where either the presence of any nuclear accumulationor the presence of clusters of cells showing nuclear accumulation isadversely prognostic (16–18). However, other studies comparing p53nuclear accumulation with assessment ofp53 gene mutations havefailed to provide conclusive evidence for the importance of p53 inlocalized PCa or a strong correlation between nuclear accumulationandp53gene mutation (19–22). One study suggested that a chromo-some 17p locus close to thep53 gene was an important prognosticfeature when both alleles at the site were lost but found that p53 IHCis noncontributory in predicting prognosis and concluded that anothergene or genes on chromosome 17p may be involved in PCa progres-sion (19). In studying other cancers, several authors have suggestedthat assessment ofp53gene mutations and p53 expression in combi-nation may more accurately define prognostically important p53 dys-function (9, 23, 24).

Comparison of PCa metastases with primary PCas in the same

Received 9/27/99; accepted 1/19/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by grants from the National Health and MedicalResearch Council of Australia, RT Hall Trust, Laurence Freedman Trust, New SouthWales Cancer Council, Leo and Jenny Leukemia, and Cancer Foundation of Australia, St.Vincent’s Clinic Foundation, and Merck Sharp and Dohme Research Foundation. D. Q. isa National Health and Medical Research Council of Australia Medical PostgraduateResearch Scholar and recipient of the Vincent Fairfax Family Foundation Fellowship fromthe Royal Australasian College of Physicians.

2 Deceased.3 To whom requests for reprints should be addressed, at Cancer Research Program,

Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010,Australia. Phone: 612-9295-8322; Fax: 612-9295-8321; E-mail: [email protected].

4 The abbreviations used are: PCa, prostate cancer; IHC, immunohistochemistry; PSA,prostate-specific antigen; RP, radical prostatectomy; NHT, neoadjuvant hormonal ther-apy; DRE, digital rectal examination; TNM, Tumor-Node-Metastasis; SVI, seminal ves-icle involvement.

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patients suggest that foci withp53mutations are clonally expanded inmetastases (25, 26), perhaps explaining the high frequency of IHCpositivity and the presence of gene mutations in hormone-refractoryand metastatic PCa (20–22, 27, 28). Two studies have demonstratedsignificant heterogeneity in the distribution ofp53mutations betweenand within foci of carcinoma in the same prostate (29, 30). Otherstudies have documented heterogeneity for other genes and suggestedthat clones responsible for metastases do not always originate fromwithin the dominant tumor focus (31). The possibility exists that inlocalized PCa, p53 overexpression and mutations as well as othergenetic aberrations may be limited to subgroups of prognosticallyimportant malignant cells.

In the present study, we evaluated p53 nuclear accumulation byIHC in a series of 263 primary PCas. Nuclear accumulation wasscored for overall number of positive nuclei and for the presence ofclusters of 12 or more cells within a3200 magnification field show-ing accumulation of p53. Assessment included sections from allidentified major foci of cancer within an individual prostate. Thesefindings were correlated with clinicopathological features, includingPCa relapse and death from PCa.

MATERIALS AND METHODS

Study Population. After Research Ethics Committee approval, we studied278 prostates from patients treated with radical retropubic prostatectomy forclinically localized adenocarcinoma of the prostate between May 1989 andDecember 1995 at St. Vincent’s Hospital in Sydney, Australia. Staging wasundertaken by clinical examination, transrectal ultrasound, pelvic computer-ized tomography, and whole-body bone scanning. The 278 cases for studywere selected from 409 RPs undertaken during this period based on theavailability of archival tissue and were not stratified for known preoperative orpathological prognostic factors. Fifteen patients were excluded either becauseof a lack of a sufficient cancer to allow adequate study with contiguoussections (4 patients), inadequate follow-up information (5 patients), and recur-rent technical problems related to IHC that made interpretation inconsistent,e.g.,lifting of the section on antigen retrieval (6 patients). Analysis showed nostatistical difference for pretreatment serum PSA, Gleason score, pathologicalstage, or treatment given between the 263 patients studied and the remainderof patients treated in the same time period.

Of these 263 patients, 164 were treated with RP alone with no neoadjuvantor adjuvant therapy of any kind. Thirty-nine patients received preoperativeNHT (34 with goserelin and flutamide, 4 with flutamide alone, 1 with cypro-terone alone) for between 1 and 8 months. Sixty-nine patients had postoper-ative adjuvant therapy with hormonal therapy, radiation therapy, or both (seeTable 1).

Patients were followed postoperatively by their surgeons on a monthly basisuntil satisfactory urinary continence was obtained and then at 3-month inter-vals until the end of the first year, at 6-month intervals to 5 years, and yearlythereafter. Each visit included history and physical examination including DREand measurement of serum PSA concentration using a Hybritech assay. Theduration of follow-up ranged from 4.9 to 123.0 months (mean, 55.5 months),with 90% of the patients having follow-up for at least 36 months. Loss tofollow-up was defined as no clinical contact and serum PSA test for.12months at the censure date in February 1999, but no patients fulfilled thisdefinition. Relapse was defined by the criteria: biochemical disease progres-sion with a serum PSA concentration$0.4 ng/ml rising over a 3-month period;local recurrence on DRE confirmed by biopsy or by subsequent rise in PSA;or institution of long-term hormonal therapy or orchidectomy. All patientsfulfilling the last criterion commenced long-term hormonal therapy or weretreated with orchidectomy based on pathological features in the prostatectomyspecimen.

Pathological Examination. RP specimens were step sectioned at 2-mmintervals and completely embedded in paraffin after fixation in neutral-buff-ered 10% formalin. Up to three (mean, 1.74; median, 2) blocks from each casewere obtained to provide representative material from the major foci of cancerwithin each prostate. The mean number of cancer foci sampled in this way foreach case was 1.96 (median, 2; range, 1–4). The pathological staging as per the

TNM classification, involvement of surgical margins, Gleason score, andWHO classification for nuclear grade and glandular differentiation was re-ported contemporaneously by one of three histopathologists (J. F. F., W. D., orJ. J. T.) and confirmed at review with consensus where reported parametersdiffered (32–34).

IHC. IHC was performed on formalin-fixed paraffin-embedded blockssectioned at 5mm, mounted on SuperFrost Plus slides (Menzel-Glaser, Braun-schweig, Germany) and processed within 10 days of sectioning. The mousemonoclonal antibody DO-7 (DAKO Corporation, Carpinteria, CA) and avidin-biotin-peroxidase and diaminobenzidine kits (Vector Laboratories, Burling-ham, CA) were used according to the manufacturers’ instructions. Briefly,sections were deparaffinized in xylene, rehydrated through graded ethanol, andthen heated in a pressure cooker in 0.01M citrate buffer (pH 6.0) for 10 minto enhance antigen retrieval. The sections were then treated with 2% H2O2 for10 min at room temperature to inactivate endogenous peroxidase activity. Aftera blocking step with 10% normal horse serum, the sections were incubatedwith DO-7 antibody diluted to 1:200 in 2% BSA/PBS overnight at 4°C.Subsequently, sections were sequentially incubated with a biotinylated horseantimouse IgG, avidin-biotinylated complex, and diaminobenzidine. Counter-staining was undertaken with Whitlock’s hematoxylin and light green beforedehydration through graded ethanol and xylene and coverslipping. A contig-uous section was stained with H&E. Positive controls for p53 nuclear accu-mulation used with each run of staining included a paraffin-embedded pellet ofthe PCa cell line DU145 (35), which has a documentedp53mutation; a coloncancer specimen withp53 missense mutation; and a tongue cancer specimenwith p53 nuclear accumulation. Negative controls included a paraffin-embed-ded pellet of the PCa cell line PC3 (35), which does not express p53 protein;and the above described positive controls processed with the substitution of anon-immune mouse monoclonal antibody for the DO-7 antibody.

Scoring for p53 nuclear accumulation required assessment of all cancer inselected sections from an individual patient. Counting of a minimum of 200cancer cells in each cancer (mean, 812; range, 210-2000 cells) was undertakento determine the percentage of nuclei showing accumulation across all areas ofcancer present. The target cell count was 500 per case where possible, butcancers with fewer cells were not excluded because of the potential forselection bias. Where the cancers had multiple foci, or were extensive orheterogeneous, more cells were counted and selected from areas of varying p53nuclear accumulation to provide a sample representative of staining across theentirety of the cancer. Additionally, assessors scored the cancers clusterpositive if 12 or more cancer cell nuclei within any3200 power microscopicfield showed p53 accumulation. In arriving at the cluster definition, we initiallyrelied on observations of other workers using clusters of 15 cells (18, 25) andtested a variety of thresholds for the definition of a cluster within a range of6–20 cells in the p53 score stratum with.0 to ,2% p53 nuclear positivity(see below). Below 10 cells per3200 field, there was no correlation withprognosis, and positive cells tended to be dispersed throughout the cancerrather than clustered within the area of a single field, whereas there were fewcases with clusters of.15 cells within the.0 to ,2% p53 score stratum. Thisdefinition differentiated for outcome when between 10 and 15 cells (log-rank,P 5 0.04 andP 5 0.05, respectively, within the.0 to ,2% stratum, whereasuse of 9 cells producedP 5 0.12, and 16 cells producedP 5 0.24) wereincluded as thresholds; therefore 12 cells (log-rank,P , 0.001) within the fieldwas selected as a midpoint in this range. Sections were scored independentlyfor p53 by two assessors (D. I. Q. and S. M. H.) and one pathologist (J. F. F.,W. D., or J. J. T.), all of whom were blinded to patient outcome. Theinterobserver Spearman rank coefficients for p53 score were between 0.92 and0.96, signifying close agreement between scorers. Thex2 test for the p53cluster status initially assigned by different scorers producedP values of 0.87and 0.76. Specifically, the two assessors identified 12 p53 cluster-positivecases not identified on initial assessment by the pathologist, whereas thepathologist identified 2 cases not initially identified by the assessors. All ofthese cases were deemed cluster positive at consensus review.

Statistical Analysis. Data were evaluated for relapse and disease-specificmortality prediction using the Kaplan-Meier product limit method and log-ranktest, and by univariate and multivariate analysis in a Cox proportional hazardsmodel for p53 cluster status and score, and other recognized clinical andpathological predictors of outcome (36, 37). To produce multivariate modelsrelevant to clinical practice, variables including factors previously described aspredictive of outcome and found to be statistically significant on univariate

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analysis, but excluding p53 status, were modeled as dichotomized or contin-uous variables to determine their independent prognostic value. Further mod-eling with independent variables and p53 score was then undertaken. Allstatistical analyses were performed using StatView 4.5 software (AbacusSystems, Berkeley, CA). Statistical significance in this study was set atP , 0.05. All reportedP values are two-sided.

RESULTS

Clinical and Pathological Characteristics. The mean age of thepatients at surgery was 63.2 years (range, 43.8–76.7 years). The meanpreoperative serum PSA concentration was 19.7 ng/ml (n 5 260;range, 1.0–280 ng/ml; median, 11.7 ng/ml). The original diagnostic

indications for prostatectomy were as follows: unknown,n 5 2(0.8%); restaging of PCa diagnosed on transurethral resection,n 5 18(6.8%); abnormal DRE alone,n 5 27 (10.3%); PSA elevation alone,n 5 93 (35.5%); and abnormal DRE with PSA elevation,n 5 122(46.6%). The distribution of PCa clinical stage according to the TNMclassification is presented in Table 1.

Of 263 patients, 115 had organ-confined disease; 139 had extrapros-tatic extension, 36 had SVI; 142 had no surgical margin involvement,whereas 62 had a single margin positive and 58 had multiple positivemargins; and 5 had pelvic lymph node metastases. The pathological stageand correlation with a series of pathological variables are shown in Table2. Forty-five (17%) patients had well-differentiated, 151 (57%) hadmoderately differentiated, and 67 (26%) had poorly differentiated tumorsaccording to the WHO classification (34).

Clinical, treatment, and outcome data for the cohort are summa-rized in Table 1. One hundred two (38.8%) patients relapsed duringthe period of follow-up (mean, 55.1 months; range, 4.9–123.0months). Mean time to relapse was 19.6 months (range, 0.1–59.5months). Ninety-two patients who relapsed developed elevated serumPSA measurements in the period of study either as the first sign ofrelapse or subsequent to evidence of local recurrence. Twelve patientsdeveloped evidence of local recurrence either with or without PSAelevation as the first sign of recurrence, but all developed a simulta-neous or subsequent rise in PSA. Ten patients were deemed to haverelapsed on the basis of commencement of continuous postoperativehormonal therapy or orchidectomy based on adverse features withinthe histopathology report. Omission of these 10 patients from theanalyses described below yielded results essentially the same as forthe overall cohort in each instance. Fourteen patients in the cohortdied during the study period, 6 of them from PCa.

Significant predictors of relapse on univariate analysis in this seriesof clinically localized PCas treated with RP were preoperative serumPSA, clinical stage, Gleason score, worst single Gleason grade, sur-gical margin involvement, overall pathological stage, pathologicaltumor stage, extraprostatic extension, SVI, and lymph node involve-ment (see Tables 3 and 4). Patients undergoing NHT or any form ofadjuvant therapy had a significantly worse prognosis compared withpatients treated with RP alone (see Tables 3 and 4).

Multivariate analysis was initially undertaken using representative

Table 1 Clinical, treatment, and outcome data for 263 patients with clinically localizedPCa treated with RP

Characteristic Number Percentage

Age (n 5 263).65 years 119 43.5

Clinical stagea (n 5 263)T1A 7 2.7T1B 14 5.3T1C 74 28.1T2A 73 27.8T2B 59 22.4T2C 23 8.7T3 14 5.3

Treatment (n5 263)b

RP alone 164 62.4NHT 39 14.8Total adjuvant therapy 69 26.2

Adjuvant radiation therapy 15 5.7Adjuvant hormonal therapy 46 17.5Adjuvant radiation therapy and hormonal therapy 7 2.7Continuous postoperative hormonal therapyc 10 3.8

Relapsed 102 38.8Death from PCa 6 2.3Death from any cause 14 5.3a Tumor stage according to TNM (32).b Nine NHT patients received adjuvant therapy (4 radiation therapy, 5 hormonal

therapy#3 months), and therefore, total adds to more than 100%.c Orchidectomy or continuous postoperative hormonal therapy (defined as therapy

intended to be long-term) and undertaken or commenced on the basis of pathologiccharacteristics of the PCa at RP.

d Relapse defined as serum PSA concentration at or above 0.4 ng/ml, rising over a3-month period; local recurrence on DRE confirmed by biopsy or by subsequent rise inPSA; or institution of long-term hormonal therapy or orchidectomy.

Table 2 Association of p53 immunoreactivity with tumor Gleason score, stage, and pretreatment PSA levels

No. ofsubjects

p53 nuclear accumulation strataa (% of patients within each group) p53 clusteraMean p53

scoreb

0

.0 to ,2%,cluster

negative

.0 to ,2%,clusterpositive

$2 to,5%

$5 to,20% $20% P

%positive P P

All 263 20.5 27.8 12.2 13.3 11.0 12.9 50.2 7.4Gleason scorec (n 5 258) 2–4 23 39.1 26.1 4.3 13.0 13.0 4.3 34.8 2.8

5–7 194 17.0 29.4 16.5 11.9 10.8 11.3 0.02 47.9 0.009 6.7 0.00078–10 41 4.9 24.4 9.8 22.0 12.2 26.8 70.7 13.8

Pathological staged (n 5 263) PT2N0 114 28.1 29.8 12.2 11.4 8.8 9.7 43.0 4.7PT3N0 108 19.4 29.6 15.7 14.8 13.9 6.5 50.0 4.9PT3CN0 29 13.8 24.1 3.4 27.6 6.9 24.1 ,0.0001 62.1 0.01 13.7,0.0001PT4N0 7 14.3 0 0 0 0 85.7 85.7 43.6PTXN1 5 0 0 0 0 40 60 100.0 36.0

Pretreatment PSA concentration(n 5 260)e

,4 14 35.7 21.4 0.0 21.4 0.0 21.4 42.8 12.94–10 96 28.1 39.6 8.3 6.3 10.4 7.3 32.3 4.2

10.1–20 86 17.4 24.4 17.4 12.8 14.0 14.0 0.003 58.1 ,0.0001 8.0 0.007.20 64 15.6 17.2 14.1 25.0 10.9 17.2 67.2 9.9

a Immunohistochemical detection of p53 nuclear accumulation was performed using 5-mm thick sections of tumor tissue and DO-7 monoclonal p53 antibody (see “Materials andMethods”). Stratification of tumors was based on percentage of tumor cell nuclei demonstrating immunoreactivity. Cases where tumor cells demonstrated.0 but ,2% nucleiimmunoreactivity were further subdivided for the presence or absence of clusters of 12 or more immunoreactive nuclei with a3200 magnification field (cluster negative or clusterpositive): 0%;.0 to ,2% and cluster negative;.0 to ,2% and cluster positive,$2 to ,5%;$5 to ,20%; and$20% of immunoreactive nuclei.

b Mean p53 score as a percentage of tumor cells with p53 nuclear accumulation. For the purpose of calculation of a mean p53 score for each group, p53 scores,1% were assumedto be 0.5%.

c Tumor grade according to Gleason criteria (33).d Tumor stage according to TNM (32).e PSA concentrations as described in “Materials and Methods.”

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prognostic variables of univariate significance and excluding thosepatients who commenced long-term hormonal therapy or who hadorchidectomy performed based on the results of histopathology be-cause these patients were deemed to have relapsed at institution ofsuch therapy. This analysis showed that NHT (P 5 0.33) and adjuvanttherapy (P5 0.1) become nonsignificant when other predictive vari-

ables were introduced, as did surgical margin involvement (P 5 0.46)and SVI (P5 0.09; Table 4).

p53 Nuclear Accumulation and Relapse.The pattern of p53nuclear accumulation seen was consistent with that described inprevious studies where there was considerable heterogeneity betweendifferent areas of cancer within the same prostate and within single

Table 3 Univariate analysis for clinicopathologic and biological variables with disease-free survival following RP in 263 patients with clinically localized PCa

Variable Hazards ratio 95% confidence interval P

Age .65 vs.,65 years 0.79 0.53–1.18 0.25NHT plus RPvs.RP alone 2.07 1.19–3.60 0.01Adjuvant therapyvs.no adjuvant therapy (n5 253)a 1.63 1.05–2.53 0.03Clinical stageb

T1 1.00T2 1.51 0.96–2.35 0.71T3 4.25 2.06–8.76 0.003

Pretreatment PSAc,d (n 5 260) 1.012 1.008–1.017 ,0.0001Pretreatment PSA.10 vs.,10 ng/mlc 3.05 1.93–4.82 ,0.0001pT stageb

pT2 1.00pT3 2.08 1.29–3.34 0.003pT3C 3.92 2.19–6.99 0.02pT4 11.16 5.20–23.91 0.03

SVI 3.08 1.97–4.81 ,0.0001Lymph node involvement 58.84 19.97–173.41 ,0.0001Margins

Nil 1.00Single margin 1.64 1.01–2.66 0.04Multiple margins 2.65 1.67–4.20 0.09

Pathological stagec

pT2N0 1.00pT3N0 2.02 1.26–3.23 0.004pT3CN0 3.50 1.93–6.33 0.47pT4N0 7.61 3.13–18.49 0.17pTXN1 108.76 35.13–336.69 0.023

Gleason graded,e (n 5 258) 1.59 1.38–1.83 ,0.0001p53 scored,f 1.033 1.024–1.041 ,0.0001p53 score strataf

0 1.00.0 to ,2%, N 1.99 0.71–5.57 0.19.0 to ,2%, P 7.08 2.59–19.34 0.004$2 to ,5% 7.88 2.97–20.92 0.9$5 to ,20% 11.03 4.13–29.44 0.17$20% 16.63 6.39–43.28 0.2

p53 cluster status negativevs.positivef 6.42 3.85–10.70 ,0.0001a Excludes 10 patients treated with orchidectomy or continuous postoperative hormonal therapy defined as therapy intended to be long-term and undertaken or commenced based

on pathologic characteristics of the PCa at RP.b Tumor stage according to TNM (32).c PSA concentrations determined as described in “Materials and Methods.”d Calculations use continuous variables so that for each unit (ng/ml) increase in pretreatment PSA, there is a 1.2% increase in risk of relapse; for each unit increase in Gleason score,

there is a 59% increase in risk of relapse; and for each percentage point increase in p53 score, there is 3.3% increase in risk of relapse. For the purpose of calculation of a mean p53score for each group, p53 scores below 1% were assumed to be 0.5%. Note thatP values presented in more than two tiered variables compare the category on the same line with theone above so that, for example, when T3N0 tumors are compared to pT2N0 tumors,P 5 0.004, when pT3CN0 tumors are compared with pT3N0 tumors,P 5 0.47 and so on.

e Tumor grade according to Gleason criteria (33).f Immunohistochemical detection of p53 nuclear accumulation was performed using 5-mm thick sections of tumor tissue and DO-7 monoclonal p53 antibody (see “Materials and

Methods”). p53 score refers to the percentage of tumor cells demonstrating p53 nuclear accumulation. Stratification of tumors was based on percentage of tumor cell nucleidemonstrating immunoreactivity and, in cases where tumor cells demonstrated.0 but,2% nuclei immunoreactivity, were further subdivided for the presence or absence of clustersof 12 or more immunoreactive nuclei within a3200 magnification field [cluster negative (N) or cluster positive (P)]: 0;.0 to ,2% and cluster negative;.0 to ,2% and clusterpositive;$2 to ,5%,$5 to ,20%; and$20% of immunoreactive nuclei.

Table 4 Univariate and multivariate analysis of described prognostic factors and treatment as dichotomized or continuous variables in determining outcome (n 5 245)a

Variable

Univariate analysis Multivariate analysis

Hazards ratio (95% confidence interval) P Hazards ratio (95% confidence interval) P

NHT plus RPvs.RP alone 2.07 (1.19–3.60) 0.01 1.36 (0.73–2.52) 0.33Adjuvant therapyvs.no adjuvant therapy 1.63 (1.05–2.53) 0.03 0.63 (0.36–1.09) 0.10Clinical stageb T3 vs.T1 and T2 3.25 (1.70–6.26) 0.0004 1.22 (0.51–2.91) 0.66Pretreatment PSAc .10 vs.,10 ng/ml 3.05 (1.93–4.82) ,0.0001 2.52 (1.55–4.09) 0.0002Tumor stageb pT2 vs.pT3 or greater 2.67 (1.72–4.14) ,0.0001 1.75 (1.02–3.01) 0.04SVI positivevs.negative 3.08 (1.97–4.81) ,0.0001 1.62 (0.92–2.84) 0.09Lymph node positivevs.negative 54 (19.97–173) ,0.0001 10.88 (1.26–93.94) 0.03Margins multiplevs.nil & single 1.87 (1.18–2.96) 0.008 1.22 (0.71–2.08) 0.46Gleason scored 1.59 (1.38–1.83) ,0.0001 1.40 (1.17–1.68) 0.0003

a Excludes patients treated with continuous postoperative hormonal therapy or adjuvant orchidectomy.b Tumor stage according to TNM (32).c PSA concentrations determined as described in “Materials and Methods.”d Tumor grade according to Gleason criteria (33). Calculations use Gleason score as a continuous variable so that for each unit increase in Gleason score there is a 40% increase

in risk of relapse.

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foci of carcinoma. Homogeneous p53 nuclear accumulation was un-usual, with heterogeneous staining containing foci of varying sizeencompassing a variable number of p53-positive cells being thecommonest pattern (Fig. 1). The p53 percentage score and p53 clusterstatus were significantly correlated with Gleason score, pathologicalstage, and pretreatment serum PSA concentration (Table 2). Univari-

ate analysis demonstrated that p53 score as a continuous variable wasprognostic (Table 3).

Kaplan-Meier product limit analysis for relapse demonstrated dif-ferences in relapse for strata based on the percentage of cells showingp53 nuclear accumulation at the levels of 0%,.0–2%, .2–5%,

Fig. 1. Representative photomicrographs demonstrating p53 nuclear accumulation inPCa. Immunohistochemical detection of p53 nuclear accumulation was performed using5-mm thick sections of tumor tissue and DO-7 monoclonal p53 antibody with hematoxylinand light green counterstain (see “Materials and Methods”).A, cluster-positive case withGleason 4 PCa acinar formation with.12 cells demonstrating p53 nuclear accumulation(brown;3400 magnification).B, PCa Gleason single grade 4 with no nuclei demonstrat-ing p53 nuclear accumulation (3200 magnification).C, PCa Gleason single grade 4 with.70% of nuclei demonstrating p53 nuclear accumulation (3200 magnification).

Fig. 2.A, relapse-free survival for patients with clinical localized PCa treated with RP withor without neoadjuvant or adjuvant therapy categorized by p53 IHC score into strata: 0 (3);.0 to ,2% (l); $2 to ,5% (ƒ); $5 to ,20% (L); $20% (Œ) of immunoreactive nuclei.Survival curves were generated according to the Kaplan-Meier method, and statistical com-parisons were made by use of the log-rank method (n 5 263; overall log-rank,P , 0.0001;intergroup log-rank: 0versus.0 to ,2%, P 5 0.01; .0 to ,2% versus$2 to ,5%,P 5 0.002;$2 to ,5%versus$5% to,20%,P 5 0.24; and$5% to,20%versus.20%,P 5 0.19).B, relapse-free survival for patients with clinically localized PCa treated with RPwith or without neoadjuvant or adjuvant therapy categorized by p53 IHC strata incorporatingp53 cluster status: 0 (3); .0 to ,2% and cluster negative (f); .0 to ,2% and clusterpositive (�); $2 to ,5% (ƒ), $5 to ,20% (L); $20% (‰) of immunoreactive nuclei(n 5 263, overall log-rank,P , 0.0001; intergroup log-rank: 0versus.0 to ,2%, clusternegative,P 5 0.19; .0 to ,2%, cluster negativeversus.0 to ,2%, cluster positive,P 5 0.0004;.0 to ,2%, cluster positiveversus$2 to ,5%,P 5 0.73;$2 to ,5% versus$5% to,20%,P 5 0.24;$5% to,20%versus$20%,P 5 0.19; and$2 to ,5% versus$20%,P 5 0.009).C, relapse-free survival for patients with localized PCa treated with RPalone by p53 IHC strata as forB, demonstrating a similar relationship between p53 score andrelapse in this subset of patients (n 5 164; overall log-rank,P , 0.0001; all intergrouplog-rankP values were not significant except that for.0 to ,2%, cluster negativeversus.0to ,2%, cluster positive:P 5 0.048).

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.5–20%, and.20% positive cells, respectively (Fig. 2A; overalllog-rank, P , 0.0001). Differences between individual strata weresignificant by log-rank calculation between 0 and.0 to ,2%(P 5 0.01),.0 to ,2% and$2 to ,5% (P5 0.0002), and$2–5%and$20% (P5 0.009).

p53 Clustering and Relapse.The use of p53 cluster status pro-duced the most discriminatory demarcation point for relapse in thecohort when analyzed by Kaplan-Meier and univariate analysis (seeFig. 3, A andB, and Tables 3 and 4).

p53 cluster status was discriminatory for cases with a Gleason score$5 (log-rank, P , 0.0001) but not for those with a score#4

(log-rank, P 5 0.61), and for those cases designated moderately(log-rank, P , 0.0001) and poorly differentiated (log-rank,P , 0.0001) on WHO criteria but not for those designated welldifferentiated (log-rank,P 5 0.28). p53 cluster status was statisticallydiscriminatory for relapse in all pathological staging groups includingcases where the cancer was organ confined (pT2N0; log-rank,P , 0.0001).

In comparing the separate scoring systems, all cancers with p53nuclear accumulation.2% were also p53 cluster positive. Thosecancers with,2% p53 nuclear accumulation exhibited the presenceof clusters in a proportion of cases with no strict relationship to theoverall percentage of tumor nuclei positive,i.e.,a single cluster couldbe present as the only p53-positivity in the entire cancer. Given thisobservation, the stratum for a p53 nuclear accumulation score between0 and 2% was further subdivided according to the presence or absenceof clusters. Subsequently, Kaplan-Meier analysis demonstrated thatcluster-positive cases within this stratum relapsed at a similar rate to

Fig. 3. Relapse-free survival for patients with clinically localized PCa treated with RPcategorized by p53 IHC cluster status: “positive” refers to those cancers with a cluster ofimmunoreactive nuclei within the strata.0 to ,2%, and all patients with.2% of nucleiscored positive; “negative” refers to patients in the strata.0 to ,2% without clusters aswell as those patients with 0 nuclei showing immunoreactivity.f, negative;�, positive.Survival curves were generated according to the Kaplan-Meier method, and statisticalcomparisons were made by use of the log-rank method.A, all patients treated with RP withor without other therapy. (n5 263; P , 0.0001).B, patients treated with RP alone(n 5 164; P 5 0.0001). C, patients treated with NHT followed by RP (n5 39;P 5 0.0001).

Fig. 4. Disease-specific survival for patients with clinically localized PCa treated withRP categorized by p53 IHC score,20% (1) or $20% (3) of tumor nuclei showing p53accumulation: Survival curves were generated according to the Kaplan-Meier method, andstatistical comparisons were made by use of the log-rank method (n5 263;P , 0.0001).

Table 5 Multivariate analyses incorporating prognostic variables of significance withinthe model tested in Table 4 modeled with p53 score, p53 strata, and p53 cluster status

(n 5 255)

Variable

Hazards ratio (95% confidence interval)

p53 score model p53 strata model p53 cluster model

Pretreatment PSAa

,10, .10 ng/ml2.54 (1.60–4.05) 2.11 (1.32–3.38) 2.01 (1.25–3.21)

Tumor stageb pT2 vs.pT3 or greater

1.76 (1.10–2.81) 1.78 (1.10–2.87) 1.67 (1.03–2.69)

Lymph node negativevs.positive

13.51 (3.81–47.86) 15.88 (5.12–49.21) 19.97 (6.66–59.88)

Gleason scorec 1.35 (1.16–1.57) 1.33 (1.13–1.55) 1.38 (1.18–1.62)p53 scored 1.022 (1.012–1.032)p53 stratad

0% 1.00.0 to ,2%;

cluster negative1.67 (0.60–4.70)

.0 to ,2%;cluster positive

5.12 (1.87–14.06)

$2 to ,5% 4.94 (1.84–13.28)$5 to ,20% 7.55 (2.78–20.41)$20% 10.44 (3.93–27.74)

p53 clusterd positivevs.negative

4.67 (2.76–7.88)

a PSA concentrations determined as described in “Materials and Methods.”b Tumor stage according to TNM (32).c Tumor grade according to Gleason criteria (33). Calculations use Gleason score as a

continuous variable so that for each unit increase in Gleason score, there is a 33–38%increase in risk of relapse.

d Immunohistochemical detection of p53 nuclear accumulation was performed using5-mm thick sections of tumor tissue and DO-7 monoclonal p53 antibody (see “Materialsand Methods”). For details of p53 score, p53 score strata, and p53 cluster status, see Table3 and “Materials and Methods.” Calculations use p53 score as a continuous variable sothat for each percentage point increase in p53 staining, the risk of relapse increases by2.2%.

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those with$2 to ,5% p53 nuclear accumulation (Fig. 1B; log-rank,P 5 0.73; and Fig. 1C; log rank,P 5 0.46), and this was confirmedby univariate analysis (Tables 3 and 4) and maintained in multivariatemodels (Table 5). There was no significant difference in outcomebetween those cases deemed cluster negative within the.0 to ,2%stratum and those in the 0 stratum (log-rank,P 5 0.19), althoughthose patients within the 0 stratum had significantly better relapse-freesurvival compared with all other patients,i.e., those with any p53nuclear accumulation (log-rank,P , 0.0001).

When representative independently prognostic variables were thenmodeled with p53 cluster status, p53 score as a continuous variable,and p53 strata, p53 nuclear accumulation proved to be an independentprognostic indicator of relapse (Table 5). When stepwise multivariateanalyses were constructed with backward elimination in the Coxproportional hazard regression model, the factors most predictive ofrelapse were, in descending order: lymph node involvement, Gleasonscore, p53 status measured by either score or cluster status, pretreat-ment PSA less than or greater than 10 ng/ml, and pathological stagepT3 or greater against pT2.

p53 Nuclear Accumulation and Relapse in Patients Treatedwith NHT. Thirty-nine patients received NHT prior to RP, 23 ofwhom relapsed, and 1 of whom died of PCa during the study period.p53 score (Mann-Whitney,P 5 0.0008), p53 strata (x2, P 5 0.005),and p53 cluster status (Fisher’s exact test,P 5 0.0002) predictedrelapse in the NHT group. Ninety percent (19 of 21) of NHT patientswho were p53 cluster positive relapsed within 36 months of prosta-tectomy (Fig. 4; log-rank,P , 0.0001) compared with 22% (4 of 18)of those who were p53 cluster negative. Univariate analysis in thisgroup revealed that p53 cluster status and pretreatment PSA concen-tration were significant predictors of outcome (Table 6). Multivariateanalysis extended to include Gleason score stratified at the 4–7 and8–10 levels and pathological T stage stratified between pT2 and pT3or greater showed that all were significant predictors of outcome inthe model (Table 6). However, with stepwise regression analysis, thefactors most predictive of relapse were, in descending order: p53cluster positivity, pretreatment PSA level, Gleason score of 8–10, andpathological T stage. In bivariate analysis using each of these vari-ables with p53 cluster status, pretreatment PSA level maintainedstatistical significance, whereas Gleason score and pathological Tstage did not. Hence, p53 cluster status was the strongest predictor ofoutcome in patients treated with NHT prior to RP.

p53 Nuclear Accumulation and Survival. Six patients died fromPCa at a mean of 52.3 months (range, 13.5–104.4 months) followingsurgery. These patients had a mean time to PSA relapse of 10.9months (range, 0.5–37.0 months) compared with 20.2 months (range,0.1–59.5 months) in the group of patients that had relapsed but not

died of PCa (P5 0.14). The mean time from surgery to developmentof clinical or bone scan-detected metastatic disease in the six patientsto die from PCa was 28.7 months (range, 8–88 months). All patientsthat died from PCa had a p53 score of$20%. Kaplan-Meier analysisof the $20% stratum against the,20% strata was highly significantin predicting death from PCa (log-rank,P , 0.0001; Fig. 4). Evalu-ation of p53 status in predicting overall survival failed to reachsignificance (data not shown).

DISCUSSION

Our study illustrates a strong relationship between p53 nuclearaccumulation detected by IHC in clinically localized PCa and relapseand PCa-related death in a large well-characterized cohort. Univariateanalysis for clinicopathological factors described as predicting out-come following RP demonstrates that the cohort has characteristicsconsistent with previous large studies of outcome (Refs. 38–41, andTable 3). The novel findings of this study are as follows: that increas-ing p53 score carries with it an increased risk of relapse and death;that the presence of a cluster of p53-positive tumor cells within a3200 magnification field provides a discriminatory point predictiveof relapse; and that p53 nuclear accumulation is the most powerfulpredictor of outcome in patients given NHT prior to RP. These datasupport the findings of previous studies that demonstrated relation-ships between outcome and p53 IHC at thresholds of no nuclearaccumulationversus any accumulation (16, 17), the presence ofclusters of p53-positive cellsversustheir absence (18, 42), and be-tween$20% p53-positive nuclei and,20% (14, 15). In predictingearly death from PCa following RP, p53 positivity in$20% of nucleidefines a group of patients with highly aggressive disease thatprogresses much more rapidly than is the case for most patientsexperiencing PSA relapse (41). Our data also support previouslydescribed relationships between p53 nuclear accumulation and path-ological tumor stage and Gleason score, and define a positive corre-lation between p53 score and pretreatment serum PSA concentration.These data run counter to a number of studies that found p53 nuclearaccumulation to be uncommon and/or nonprognostic in localized PCa(19–22).

p53 status was not predictive of outcome in patients with betterdifferentiated tumors,i.e., those that were well differentiated or witha Gleason score#4. The outcome following surgery for patients withbetter differentiated tumors was good, and there was a low event ratein these groups. It may be that an effect of p53 clustering has not beenapparent within our follow-up period. Alternatively, it may be thatthese cancers are intrinsically indolent and that p53 status is not ofimportance within this subset because of a lack of other genetic and

Table 6 Univariate and multivariate analysis of prognostic factors in determining relapse-free survival of 39 patients treated with NHT prior to RP

Parameter

Univariate analysis Multivariate analysis

Hazards ratio 95% confidence interval P Hazards ratio 95% confidence interval P

pT stagea

pT2 1.00 1.00pT3 or greater 2.57 0.87–7.57 0.087 4.17 0.95–18.35 0.06

Gleason scoreb

4–7 1.00 1.008–10 3.43 0.76–15.56 0.11 22.06 2.70–180.13 0.007

Pretreatment PSAc 1.025 1.005–1.014 0.0012 1.020 1.004–1.036 0.016p53 clusterd

Negative 1.00 1.00Positive 6.98 2.30–21.17 0.0006 6.35 2.05–19.69 0.0014

a Tumor stage according to TNM (32).b Tumor grade according to Gleason criteria (33).c PSA concentrations determined as described in “Materials and Methods.”d Immunohistochemical detection of p53 nuclear accumulation was performed using 5-mm thick sections of tumor tissue and DO-7 monoclonal p53 antibody and assessed for the

presence of p53 clusters (see “Materials and Methods”).

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epigenetic factors contributing to cancer progression. Conversely, p53status predicted outcome in all subgroups based on pathological stageand pretreatment serum PSA strata. One potential limitation of ourstudy is that although biochemical relapse correlates with subsequentdevelopment of clinical metastases, the rate of development of thesemetastases varies greatly; further evaluation of this and other cohortsis needed to determine whether p53 status predicts clinical relapse.

p53 cluster status was the most predictive factor in determiningoutcome in patients who received NHT prior to RP (Table 6). Grignonet al. (15), in reporting on 129 patients with clinically localized PCatreated with radiation therapy in the RTOG 8610 trial, found that p53nuclear accumulation in pretreatment diagnostic material predictedreduced time to distant metastases in those patient given NHT but notin those treated with radiation therapy alone. The predictive value ofp53 IHC in the NHT group raises interesting questions about theeffect of hormonal therapy on PCa cells with and without p53 nuclearaccumulation. Following androgen ablation, a small proportion ofPCa cells undergo apoptosis, but the majority of cells respondingundergo cell cycle arrest mediated, at least in part, by p53, entering G0

phase and losing cell volume (43, 44). Cells with dysfunctional p53may be resistant to hormonal therapy and fail to undergo cell cyclearrest or apoptosis (15, 45), conferring a relative growth advantageand greater prominence when evaluated for p53 nuclear accumulation.Such a postulated mechanism may explain the observation that p53dysfunction is associated with hormonal resistance in some studies ofbreast cancer and PCa (15, 45).

Our study reports a higher rate of p53 positivity than some previousstudies, with 79% of cases showing at least occasional p53 positivityand 52% being p53 cluster positive (Table 2). The most obviousexplanation for this relates to a modified definition of p53 positivitythat integrates p53 cluster status into the p53 accumulation score.Another potential reason is the inclusion of a greater proportion ofpatients with adverse features not as prevalent in other series, such aspathological stage (57% pT3N0 or greater), tumor differentiation (16%with Gleason score$8 and 26% poorly differentiated by WHOcriteria), and pretreatment serum PSA concentration (25% greaterthan 20 ng/ml), that contribute to a relapse rate of 38.8% at the meanfollow-up of 55.1 months. In a study of 175 RP cases evaluated forp53 nuclear accumulation, 65% of cases were reported to demonstrateoccasional or greater (i.e.,.0) p53 positivity (17) with a correspond-ing relapse of 37.7% at a mean follow-up of 55.2 months. Within thatseries (17), 57.1% of cases were pathological stage pT3N0 or greater,10.8% had a Gleason score$8, and 5.7% were poorly differentiated;pretreatment serum PSA concentrations were not reported. This sug-gests that although the stage of tumors studied was similar in bothstudies, the tumors in our study had a higher Gleason score, weremore poorly differentiated on WHO criteria, and had a slightly higherchance of relapse. The inclusion of patients receiving neoadjuvant andadjuvant therapy is likely to have resulted in a cohort with relativelymore aggressive or advanced cancer as indicated by pathologicalstage, Gleason score, and preoperative serum PSA concentrations.Further comparison between our cohort and a recently reported largesingle-surgeon case series, where 54.4% of cases were pathologicalstage pT3N0 or greater, 7.8% had a Gleason score$8, and 5.5% ofpatients had a pretreatment serum PSA concentration$20 ng/ml, alsosuggests that our group contains patients with more adverse featuresthan others (41). Given these differences and the trend toward screen-ing-detected cancers treated with RP being of lower stage, furtherevaluation of p53 nuclear accumulation in cohorts with less aggres-sive features is desirable. Finally, it may be that the antigen retrievaland IHC techniques used in our laboratory are more sensitive thanthose reported in some other studies.

Methodological factors may also have contributed to our higher

p53-positivity rate because of the assessment of all major foci withineach individual prostate and the use of a p53 antibody directed at theDO7 epitope, which has a high correlation withp53 gene mutationcompared with other antibodies (46). Other researchers have foundthat a cocktail of DO1 and DO7 epitope-directed antibodies is slightlymore sensitive than DO7 alone (47). We achieved equivalent resultswith DO7 antibody, which detects both wild-type and mutant p53protein, having noted increased background staining in initial exper-iments with the described cocktail (data not shown). In determiningp53status, it appears that IHC is sensitive but may suffer from lack ofspecificity for detecting mutation, whereas methods of direct sequenc-ing are highly specific in detectingp53mutation but may lack sensi-tivity when such mutations are present in only a small percentage ofcells within a population containing wild-type p53. This may in partexplain differences in p53 status based on the technique used.

Accumulation of p53 protein may occur in response to a number ofstimuli independent ofp53 gene mutation, including DNA damage,hypoxia, and redox stress (48). Elledgeet al. (49, 50) have suggestedthat even low levels of p53 protein accumulation are prognostic inbreast cancer specimens regardless of whetherp53 gene mutationcould be detected concurrently, whereas Silvestriniet al. (51) haveshown that an increasing percentage of p53-positive nuclei between 0and 12% has a corresponding adverse “dose” effect on prognosis in alarge series of breast cancer patients. Our study describes a similarrelationship between percentage of p53-positive nuclei and relapse inPCa, but it also illustrates that clusters of p53-positive cells areprognostic when,2% of all tumor cells are positive. The prognosticsignificance of p53 clusters suggests that cells in such clusters haveimportant characteristics such as p53 mutation or are affected by otherfactors capable of producing local p53 nuclear accumulation and areassociated with a poorer outcome. In this regard, p53 clusters mayrepresent foci of cells withp53gene mutations that expand within theprostate to increase the p53 score with time so that more advancedand/or higher Gleason grade cancer is associated with a higher p53score. It also is possible that micrometastases occur at a similar timein PCa progression to the development of clusters with a criticalnumber of cells showing p53 nuclear accumulation. This hypothesissuggests a focal dose-response threshold for p53 dysfunction andgenesis of metastases (see below).

PCa is usually multifocal. The clone responsible for human PCametastases may reside in smaller tumor foci within the prostate glandrather than the largest (31, 52, 53). Heterogeneity between and withindifferent foci of PCa within the same gland is inferred by studies ofDNA flow cytometry (54) and allelic loss (52, 55). Heterogeneity ofp53 mutations and p53 protein nuclear accumulation within andbetween individual foci of early-stage localized cancer in the sameprostate has been demonstrated previously (29, 30). p53 is crucial indetermining metastatic potential in models of skin carcinogenesiswhere p53 protein dose does not contribute to initiation and/or pro-motion but is associated with metastasis (56). Similarly, one studyfound that in ras1myc-initiated prostate carcinoma metastasis isconcurrent with loss of expression of the wild-typep53allele (57). Inthat study, comparative DNA analysis between primary tumors andmetastases demonstrated that metastases did not necessarily derivefrom the most abundant clone but seeded from small subpopulationsfrom within the primary tumor (57). The clonal expansion of p53dysfunctional cells (57) has been confirmed in small series of primaryhuman PCas and metastases within the same patients (25, 26, 58).These studies add to others that demonstrate increased p53 nuclearaccumulation in metastatic, recurrent, and/or androgen-insensitivePCa compared with clinically localized disease (20–22, 27, 28). In astudy of 50 metastatic PCa foci in 19 men with lethal PCa,p53genemutations demonstrated homogeneity for mutation in virtually all

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metastases assessed from individual patients, in contrast toPTEN/MMAC1mutations, which demonstrated intermetastasis heterogeneity(59). Taken together, these studies suggest that prostate tumor cellsharboringp53 mutations and perhaps other genetic aberrations areclonally expanded in metastases. Our study extends this concept bysuggesting that a “p53 dose” effect across the entire cancer and at agiven focal threshold within clusters is important in the metastaticprocess.

The work presented in this study and that of others (16–18, 60)suggests that p53 has an important prognostic role in approximatelyhalf of all patients with clinical localized PCa. Given the finding thatp53 is the most important predictor of outcome in patients given NHTbefore RP and similar observations in patients given NHT prior toradiation therapy (15), p53 status may have significant implicationsfor patients treated with hormonal therapy as part of these regimens.The genetic and epigenetic basis for clustered p53 nuclear accumu-lation and its effect on clinical as well as biochemical relapse requirefurther investigation.

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2000;60:1585-1594. Cancer Res David I. Quinn, Susan M. Henshall, Darren R. Head, et al. Localized Prostate Cancer Treated with Radical ProstatectomyPrognostic Significance of p53 Nuclear Accumulation in

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Prognostic significance of p53 nuclear accumulation in localized prostate cancer treated with radical prostatectomy