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Differences in Frequency ofERG Oncoprotein Expression Between IndexTumors of Caucasian and African AmericanPatients With Prostate CancerPhilip Rosen, David Pfister, Denise Young, Gyorgy Petrovics, Yongmei Chen,Jennifer Cullen, Diana Böhm, Sven Perner, Albert Dobi, David G. McLeod,Isabell A. Sesterhenn, and Shiv Srivastava

OBJECTIVE To systematically evaluate the ETS-related gene (ERG) alterations in the multifocal tumorcontext using whole-mount prostatectomy specimens from African and Caucasian Americanpatients matched for age, pathologic grade and stage. Oncogenic activation of the ERG is themost common early genomic alteration in patients with prostate cancer (CaP) in Westerncountries. However, ERG alterations have not been systematically examined in African Amer-ican patients with a known greater risk of CaP incidence and mortality.

METHODS ERG oncoprotein expression was analyzed in 91 Caucasian and 91 African American patientswith CaP, who were matched for age, Gleason score, and pathologic stage. A unique aspect ofthe present study was the evaluation of ERG in whole-mount prostatectomy sections, minimizingsampling bias and allowing the careful assessment of the ERG in the multifocal tumor contextof CaP.

RESULTS The frequency of ERG-positive prostate tumors was significantly greater among CaucasianAmericans than among African Americans when assessed in all tumor foci (41.9% vs 23.9%,P � .0001). A markedly greater frequency of ERG oncoprotein expression was noted betweenthe index tumors of the Caucasian Americans (63.3%) and those of the African Americans(28.6%). Also, in the African American patients, the higher grade index tumors were predom-inantly ERG negative.

ONCLUSION ERG typing of CaP established a major difference between the index tumors of Caucasian andAfrican American patients. ERG-negative index tumors might indicate a less favorable outcomefor African American patients. The results of the present study underscore that typing of CaP forthe ERG could enhance our understanding of the biologic differences between the examined

ethnic groups. UROLOGY 80: 749–753, 2012. © 2012 Elsevier Inc. All rights reserved.

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Carcinoma of the prostate (CaP) displays healthdisparities reflected by the differences in cancerincidence and mortality among different popula-

tions, with a greater incidence in Western countries and

Equal contribution: Philip Rosen and David PfisterFinancial Disclosure: The views expressed in this report are those of the authors anddo not reflect the official policy of the Department of the Army, Department of Defense,or the U.S. Government.

Funding Support: This research was supported by the National Cancer Institute01CA162383 grant to S. Srivastava.From the Center for Prostate Disease Research, Department of Surgery, Uniformed

ervices University of the Health Sciences, Rockville, MD; Urology Service, Waltereed National Military Medical Center, Bethesda, MD; Institute of Pathology, Uni-ersity Hospital of Bonn, Bonn, Germany; and Department of Genitourinary Pathol-gy, Joint Pathology Center, Silver Spring, MD

Reprint requests: Shiv Srivastava, Ph.D., Center for Prostate Disease Research,epartment of Surgery, Uniformed Services University of the Health Sciences, 1530

ast Jefferson Street, Rockville, MD 20852.Submitted: June 15, 2012, accepted (with revisions): July 3, 2012

© 2012 Elsevier Inc.All Rights Reserved

a lower incidence in Asian countries.1 African Ameri-cans with CaP are diagnosed with significantly worseclinical parameters, including a 2.5-fold greater risk ofdying of CaP than Caucasian Americans.2 However, theiologic reasons for these differences remain poorly un-erstood. Increased associations of a genetic variant ofYP3A4, germ line mutations of EphB2 tyrosine kinase

nd macrophage scavenger receptor 1, and single nucle-tide polymorphisms have been reported in CaP of Af-ican Americans.3 Differences in the serum levels ofitamin D, insulin-like growth factor-1, insulin-likerowth factor binding protein-3, and low-density lipopro-ein have also been reported between African Americannd Caucasian American patients with CaP.4

Frequent genomic alterations in CaP involve fusions ofregulatory sequences of androgen receptor-regulated

genes (predominantly TMPRSS2) and protein coding

0090-4295/12/$36.00 749http://dx.doi.org/10.1016/j.urology.2012.07.001

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sequences of ETS transcription factors (predominantlyERG).5 Recent evaluation of the ETS-related gene (ERG)ncoprotein from our group in whole-mounted CaP speci-ens demonstrated a 65% frequency and very high concor-

ance (96.5%) of ERG-positive prostatic intraepithelialeoplasia and CaP.6 The presence of the ERG oncoproteinlso shows a high correlation with ERG fusion.6-9 Although,ifferences in ERG alterations between African Americannd Caucasian American patients have previously beenuggested by us and others,10-12 the present study systemat-

ically examined the ERG alterations in the multifocal tu-mor context using whole mount prostatectomy specimens ofAfrican and Caucasian American patients matched for ageand pathologic grade and stage.

MATERIAL AND METHODS

Radical prostatectomy specimens were obtained from patientsenrolled in the Center for Prostate Disease Research programand processed as whole mount specimens from 1996 to 2010under an institutional review board-approved protocol from theWalter Reed National Military Medical Center. Archived spec-imens from the 1237 cases were examined with complete dataon age at surgery (mean 59.5 � 7.7 years, median 60.4, range39.2-80.1), serum prostate-specific antigen level at diagnosis(mean 10.8 � 143.0 ng/mL, median 5.2, range 0.4-5065),elf-declared ethnicity (African American, n � 327 [26.4%];

Caucasian American, n � 876 [70.8%]), family history of CaPno for 696 patients [65.5%] and yes for 366 [34.5%]), patho-ogic T stage (pT2 in 771 [62.3%], pT3 in 466 [37.7%]), andathologic Gleason sum (�6 in 656 [53.0%], 7 in 451 [36.5%],nd 8-10 in 130 [10.5%]). Of the 1237 radical prostatectomypecimens, 91 Caucasian American and 91 African Americanatients with CaP were matched for age, Gleason sum, andathologic stage (Table 1). The average and medium number of

Table 1. Clinicopathologic characteristics

Variable

CaucasianAmericanPatients

AfricanAmericanPatients P Value

Patients (n) 91 91Age at surgery (y) .8518

Mean � SD 59.4 � 8.3 59.6 � 8.6Median 61.2 61.1Range 40.2-74.4 40.2-73.5

Diagnostic PSA(ng/mL)

.4448

Mean � SD 7.0 � 4.5 6.7 � 5.5Median 6.0 5.6Range 1.1-23.4 0.6-41.2

Pathologic T stage 1.0000T2a-T2c 42 (46.2) 42 (46.2)T3a-T3c 49 (53.8) 49 (53.8)

Gleason sum .63876 38 (41.7) 36 (39.6)3 � 4 33 (36.3) 38 (41.8)4 � 3 5 (5.5) 2 (2.2)�8 15 (16.5) 15 (16.5)

PSA, prostate-specific antigen.Data presented as numbers of patients, with percentages in

parentheses, unless otherwise noted.

umor foci among the African American and Caucasian Amer-

750

can patients were approximately 2, with no statistically signif-cant difference between the 2 groups (Table 2). One represen-ative whole mount cross-section of each specimen was selectedrom each of the 182 patients. Pathologic evaluation was per-ormed of each tumor focus.

The 4-�m sections were dehydrated and blocked in 0.6%hydrogen peroxide in methanol for 20 minutes and processedfor antigen retrieval in ethylenediaminetetraacetic acid (pH9.0) for 30 minutes in a microwave, followed by 30 minutes ofcooling in ethylenediaminetetraacetic acid buffer. The sectionswere then blocked in 1% horse serum for 40 minutes andincubated with the ERG-MAb mouse monoclonal antibody(9FY, available from Biocare Medical, Concord, CA) at adilution of 1:1280 for 60 minutes at room temperature. Thesections were incubated with the biotinylated horse anti-mouseantibody at a dilution of 1:200 (Vector Laboratories, Burlin-game, CA) for 30 minutes followed by treatment with the ABCKit (Vector Laboratories) for 30 minutes. The color was devel-oped using VIP treatment (Vector Laboratories) for 5 minutes,and the sections were counterstained with hematoxylin. ERGexpression is reported as positive or negative in the indextumors (largest tumor with highest grade within the prostate)and in the multifocal tumor context. ERG protein expressionwas correlated with the clinicopathologic features.

The ERG rearrangement status of chromosome 21q22 and 2was analyzed using the fluorescence in situ hybridization break-apart assay.8 Biotin-14-deoxycytidine triphosphate-labeled bac-terial artificial chromosome clone RP11-24A11 (red avidin-rhodamine) and digoxigenin-deoxyuridine triphosphate-labeledbacterial artificial chromosome clone RP11-137J13 (green flu-orescein-tagged anti-digoxigenin antibody) were used in theassay. (The bacterial artificial chromosome clones were pro-vided by Dr. S. C. Chandrasekharappa, National HumanGenome Research Institute and were prepared by GeneProDiagnostics, Rockville, MD.) The tissues were assayed by hy-bridization and analyzed using fluorescence microscopy.

The categorical patient clinicopathologic features were com-pared across the groups of patients using frequencies and per-centages. Continuously measured patient features were com-pared using measures of central tendency, including the meanand median, and measures of dispersion, including the standarddeviation and range. The Student t test, Wilcoxon rank-sum

Table 2. Association of ERG immunohistochemistry sta-tus with ethnicity

Variable

CaucasianAmericanPatients

AfricanAmericanPatients P Value

Patients (n) 91 91Patients with

ERG-positive indextumor* (n)

57 (63.3) 24 (28.6) � .0001

Patients with �1ERG-positivetumor

60 (65.9) 39 (42.9) .0018

Tumor foci (n) 191 205ERG-positive tumors

(n)80 (41.9) 49 (23.9) .0001

ERG, ETS-related gene.Data in parentheses are percentages.

* Index tumor absent in 1 Caucasian and 7 African Americanpatients.

test, or chi-square test was used to evaluate the difference in the

UROLOGY 80 (4), 2012

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distribution of the clinicopathologic characteristics betweenthe African American and Caucasian American patients. Thedifference in ERG immunohistochemistry status (positive vsnegative) between the African Americans and CaucasianAmericans and the association of ERG status with the Gleasonsum stratified by race/ethnicity were examined using the chi-square test. Biochemical recurrence, defined as 2 consecutivepostoperative prostate-specific antigen values �0.2 ng/mL mea-sured at �8 weeks postoperatively, was correlated with the ERGtatus. If the biochemical recurrence status could not bessessed, only patients with �24 months of follow-up were

considered for analysis. For the assessment of the differences inbiochemical recurrence-free survival across ERG status, an un-adjusted Kaplan-Meier survival analysis was conducted. Thelog-rank test was used to test for differences in the Kaplan-Meier curves by ERG status; P � .05 was considered statisticallyignificant. For all data analysis, SAS, version 9.3 (SAS Insti-ute, Cary, NC), was used.

RESULTSOverall, a greater frequency of ERG oncoprotein expres-sion was detected in the CaP specimens of the CaucasianAmerican patients than in those of the African Ameri-can patients (Table 2). Of the 91 Caucasian Americanpatients and 91 African American patients, 59 (65.9%)and 41 (42.9%) had �1 ERG-positive tumor, respec-tively (P � .0018). Moreover, 80 (41.9%) of the 191individual tumor foci were ERG positive in the Cauca-sian Americans compared with 49 of 205 foci in theAfrican Americans (23.9%; P � .0001). A high concor-dance of ERG-positive or ERG-negative prostatic intra-epithelial neoplasia with ERG-positive or ERG-negativetumors in both Caucasian Americans (95.3%) and Afri-can Americans (95.5%) was observed.

A remarkable difference was noted in the frequency ofERG-positive index tumors between the CaucasianAmerican (57 of 91; 63.3%) and African American (24of 91; 28.6%) patients (P � .0001). Consistent with thegreater frequency of ERG-negative index tumor types inthe African American patients, the correlation of ERG

Table 3. Association of index tumor ERG immunohistoche

Gleason Sum Overall

3 � 3Index tumor ERG negative 33 (48.5)Index tumor ERG positive 35 (51.5)

3 � 4Index tumor ERG negative 35 (50.7)Index tumor ERG positive 34 (49.3)

4 � 3 and 8-10Index tumor ERG negative 25 (67.6)Index tumor ERG positive 12 (32.4)

Abbreviation as in Table 2.Data presented as number of patients, with percentages in paGleason sum 4 � 3 category included 2 African American pat

combined.* Fisher’s exact test.

status with pathologic grade revealed a significant asso- (

UROLOGY 80 (4), 2012

ciation with higher grade tumors (Table 3). Althoughnot statistically significant, the data indicated a trend ofan association between prostate-specific antigen recur-rence and the frequency of ERG-negative tumors amongAfrican American patients (P � .2201) that was notfound among the Caucasian patients (P � .9057). Theevaluation of ERG rearrangement using fluorescence in situhybridization in 20 African American and 20 CaucasianAmerican cases revealed 99% concordance with ERG pro-tein frequencies noted for ERG oncoprotein expression.

COMMENTThe results of the present study have underscored astriking difference in ERG oncoprotein expression be-tween CaP of Caucasian and African American pa-tients by a comprehensive evaluation of oncoproteinexpression in multiple tumor foci of whole mountprostatectomy specimens from patients matched forage and tumor pathologic features. The differences inERG expression between African Americans and Cau-casian Americans with CaP are consistent with thedata from our initial quantitative evaluations of ERGmRNA in CaP10 and recent studies of ERG mRNA inpost-digital rectal examination urine.11 Similar differ-nces were also noted in studies of TMPRSS2-ERGusion status or ERG protein expression in CaP,6,12

including the present study. An increased associationof the TMPRSS2-ERG fusion with interstitial deletion(IDEL type) in CaP specimens from African Ameri-cans noted in a previous report12 was also observed inhe present study. Our study showed that the indexumor was most likely to express ERG if any of theumor foci were ERG positive in Caucasian Americanatients (index/any ratio 0.96). In contrast, this ratioas much lower in African American patients withaP (index/any ratio 0.67). This finding suggests aore frequent clonal expansion of ERG-positive foci

n Caucasian Americans than in African Americans

ry status with race/ethnicity stratified by Gleason sum

CaucasianAmericanPatients

AfricanAmericanPatients P Value

.003711 (31.4) 22 (66.7)24 (68.6) 11 (33.3)

.038515 (39.5) 20 (64.5)23 (60.5) 11 (35.5)

.0036*7 (41.2) 18 (90.0)

10 (58.8) 2 (10.0)

eses.and 5 Caucasian patients; thus, 4 � 3 and 8-10 groups were

mist

renthients

Fig. 1). We postulated that most index tumors in

751

erica

renth

African American patients are using distinct geneticmechanisms for progression. The greater frequency of

Figure 1. Representative images of whole-mount sections ahybridization in Caucasian and African American patients. (ACaucasian American patient outlined, with representative vieERG-positive tumor in African American patient. (C) ERG rearraERG rearrangement by deletion in section from an African Am

Table 4. Association of index tumor ERG immunohistoche

Index TumorGleason Su

3 � 4 (�

Caucasian American patients (n � 90)ERG negative 20 (33ERG positive 40 (66

African American patients (n � 84)Negative 33 (63Positive 19 (36

Abbreviation as in Table 2.Data presented as number of patients, with percentages in pa

* Worst tumor grade percentage.

ERG-negative tumors in African American patients

752

was reflected by the preponderance of high-grade tu-mors; however, this difference was not apparent in the

zed by ERG immunohistochemistry and fluorescence in situ-positive index tumor and ERG-negative secondary tumor oflds enlarged. (B) ERG-negative index tumor and secondaryent by translocation in a Caucasian American patient and (D)n patient shown by fluorescence in situ hybridization assay.

ry status with Gleason sum stratified by race

� 3,*)

Gleason Sum 3 � 4 (�25%*),4 � 3, 8-10 P Value

.353413 (43.3)17 (56.7)

.039427 (84.4)5 (15.6)

eses.

naly) ERGw fiengem

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m 325%

.3)

.7)

.5)

.5)

CaP of Caucasian American patients (Table 4).

UROLOGY 80 (4), 2012

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CONCLUSIONSThe findings we have presented have the potential tostratify CaP by the status of cancer gene alterations, suchas ERG, toward the understanding of widely observeddifferences of disease aggressiveness noted between Afri-can and Caucasian American patients.13,14 The emergingethnic differences in ERG alterations are intriguing. In alimited number of studies, much lower frequencies ofERG positivity have been reported in the CaP of Asianpatients (�20%).12,15 Issues related to sampling bias,umor biology, or treatment effects have not been ruledut for the apparent differences noted in the ERG fre-uencies in CaP specimens from Western and Asianountries. The present report considered these issueshen establishing the differences in ERG frequenciesetween Caucasian American and African Americanatients. Because ERG oncogenic alterations are beingxtensively evaluated as a biomarker and therapeuticarget in CaP, ERG typing of CaP has the potential tonravel the biologic differences in CaP in different eth-ic groups.

cknowledgment. To Dr. Ann W. Hsing, Cancer Preven-ion Institute of California, for valuable advice and Mr. Ste-hen Doyle, Center for Prostate Disease Research, for thertwork, with our sincere thanks.

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