erg protein expression has been recently suggested to be reflective of erg gene rearrangements in prostate cancer ( pca ) documenting remarkable concordance between the two [ 16 ] .
the rearrangements between the androgen receptor - regulated gene tmprss2 ( 21q22.3 ) and members of the ets family member of transcription factor gene , most commonly erg ( 21q22.2 ) , are among the most common genetic alterations detected in prostate cancer [ 711 ] .
erg gene rearrangements have been detected in roughly half ( 4060% ) of pca of surgical cohorts compared to a rate of 12%15% in incidental or watchful waiting cohorts [ 7 , 1218 ] .
previous studies investigating the prognostic significance of erg gene rearrangements have revealed mixed results [ 1922 ] .
however , it is becoming more evident that erg gene rearrangements signify a molecular subtype of pca .
some studies investigating the significance of erg protein expression in localized pca failed to show association with adverse clinical outcome [ 23 , 24 ] . however , a recent report by our group demonstrated an association of erg expression with lethal disease in patients with unsuspected and advanced / castrate resistant disease who were treated by transurethral resection of prostate . moreover , we documented significant association between erg expression and both gleason score and tumor volume .
studies from our group and others have also linked erg status to responsiveness to hormonal therapy , and longer progression time to castration resistant disease , compared to men with no erg expression [ 24 , 25 ] . in the current study , we investigated the association of erg protein expression to clinical - pathological parameters in a cohort of men with localized prostate cancer .
the study cohort consisted of 198 patients who were treated by retropubic radical prostatectomy for localized prostate cancer with a mean followup of 4.8 years ( range 015.8 ) .
clinical progression was defined as a postoperative serum psa elevation of > 0.2  ng / ml .
prostate samples were embedded onto three tissue microarray ( tma ) blocks using a manual tissue arrayer ( beecher instruments , silver spring , md , usa ) .
one to nine cores ( average 3.3 ) , 0.6  mm in diameter , were sampled including benign , high grade intraepithelial neoplasia ( hgpin ) , and prostate cancer ( pca ) . after construction ,
4  m sections were cut and stained with haematoxylin and eosin on the initial slides to verify the histological diagnosis .
briefly , 4  m thick sections from formalin - fixed paraffin - embedded tissue blocks were stained with ventana autostainer .
prior to the staining , heat - induced antigen retrieval was carried out by vegetable steamer in sodium citrate antigen retrieval buffer ( 10  mm ph 6.0 ) for 40 minutes and then cooled down to room temperature for about 20 minutes .
the slides were incubated for 60 minutes at 37c with erg rabbit monoclonal antibody ( epitomics , clone epr 3864 ) at 1  :  50 dilution . a ventana iview dab detection kit ( ventana tucson , az , usa )
the diagnoses of all tma cores were confirmed by the three study pathologists ( lht , cw , and tab ) .
for each patient , the two predominant patterns were sampled and included on the tmas for analysis .
erg protein expression was assessed semiquantitatively using 3-tiered system ( 0 , negative ; 1 , low ; 2 , high ) .
cases with either 1 or 2 intensity were considered positive based on previous correlation with erg gene rearrangement as detected by fluorescent in situ hybridization ( data not shown ) .
the erg antibody was consistently strongly expressed in endothelial cells , which acted as internal control for expression and intensity level .
patient characteristics were presented as frequencies and percentages for categorical variables and as means and ranges for continuous variables .
chi - square tests were used to test for associations between erg protein expression and gleason score , surgical margin , and pathological stage . the kaplan - meier approach along with the log - rank test was used for the survival analyses to test the association between erg expression and serum psa relapse . in all statistical tests ,
mean patients ' age of this cohort was 64 years ( range 42.780.5 years ) with average follow - up time of 4.8 years ( range 0.015.8 months ) .
table 1 demonstrates patients ' demographics of the study cohort with respect to erg expression .
overall , there were no significant differences between the two subgroups ( erg pos & erg neg ) of patients except for pathological stage , with 37% of erg positive tumors detected in pt3 versus 24% in pt2 . to investigate erg expression in different diagnostic categories , we characterized erg expression based on individual cores sampled .
when we accounted for foamy type pca morphology , the rate of erg expression was 15/84 ( 17.9% ) , compared to 302/704 ( 42.9% ) in acinar pca ( cases with no foamy type morphology ) ( p < 0.001 ) .
there was no difference in high erg intensity between foamy type and acinar pca ( data not shown ) . however , mean intensity level of erg in acinar pca was significantly higher than foamy type pca , 1.01  1.27 versus 0.37  0.83 ( p < 0.001 ) .
erg intensity levels in hgpin were comparable to those in foamy type pca but slightly lower ( 0.13  0.56 ) but significantly lower than those in acinar pca ( p < 0.001 ) ( figure 1 ) .
erg expression was noted in 106/280 ( 37.8% ) , 175/463 ( 37.8% ) , and 37/108 ( 34.2% ) of gleason scores 6 , 7 , and 810 , respectively .  
figure 2 demonstrates examples of erg expression in tissue samples and the distribution of erg in relation to gleason score .
when investigating relations between erg expression and pathological parameters , there was significant association between erg expression and higher disease stage . in this cohort , erg expression was present in 50/131 ( 38.1% ) patients with pt2 versus 30/55 ( 54.5% ) patients with pt3 ( p = 0.04 ) .
a similar association was also noted between erg expression and extra - capsular extension . in this cohort , 52/134 ( 38.8% )
erg positive patients demonstrated organ confined disease versus 29/53 ( 54.7% ) erg positive patients showing extra - capsular extension ( p = 0.04 ) .
similar trends were noted between erg expression and seminal vesicle invasion , but this was not statistically significant ( p = 0.10 ) ( data not shown ) .
no significant association was noted between overall erg positivity and positive surgical margins ( table 1 ) .
similarly , no association was observed with postsurgical psa levels when assessed by univariate or multivariate analysis ( figure 3 ) . although not informative due to limited patients ' numbers , the association between erg expression and higher stage disease was more pronounced in patients with higher gs . in this cohort ,
none of the patients with gs > 7 and erg positive ( 0/7 ) were of stage pt2 compared to 47% ( 7/15 ) of erg negative patients who were of pt2 stage ( p = 0.02 ) ( table 2 ) .
this study reports on the potential significance of erg protein expression in localized prostate cancer .
erg gene rearrangements and erg expression have been documented in roughly 50% of localized prostate and locally advanced castrate resistant prostate cancer compared to 12%15% in watchful waiting or incidental cohorts [ 16 , 17 , 22 , 25 , 29 ] . published reports on the significance of erg expression to patients ' outcome are conflicting with some showing association with adverse outcome , while others document no association .
some suggest that it indicates a better prognosis [ 16 , 22 , 25 , 2933 ] .
however , it is proposed that erg signifies a molecular class of prostate cancer and may play a role in disease progression within those tumors .
this pathway is closely linked to increased rate of pten genomic deletions as well as increased erg expression or erg gene rearrangements [ 34 , 35 ] .
erg gene rearrangements and erg expression are associated with adverse outcome and lethal disease in watchful waiting or expectant cohorts [ 16 , 25 ] .
moreover , patients with erg overexpression demonstrated shorter progression times to castrate resistance , needing surgical intervention ( channel turp ) .
in localized prostate cancer , the majority of the reported data suggest no prognostic implication for erg rearrangements or erg overexpression in relation to psa relapse or the recurrence of local disease [ 22 , 36 , 37 ] .
however , two earlier reports suggested adverse association of erg rearrangements with psa relapse after radical prostatectomy [ 20 , 31 ] . in nam 's study ,
yashimoto 's group noted that the adverse prognostic association was linked to pten genomic deletions . of note , a study by reid et al .
documented that erg gene rearrangements in addition to pten genomic deletions had actually favorable outcomes compared to pten deletion alone .
these observations suggest that the method utilized in determining erg status , the inclusion or exclusion of other genomic aberration , and the cohort chosen may be the reason for the different results obtained in various studies investigating the role of erg in prostate cancer .
our results support previous observations showing no prognostic relationship between erg overexpression and clinical outcome in localized prostate cancer .
however , in contrast to earlier studies , we document significant association between erg expression and some pathological parameters . in this cohort , patients whose tumors were erg positive were at higher risk of exhibiting extra - prostatic extension and increased disease stage compared to patients whose tumors did not express erg .
specifically , all patients with gs > 7 and erg expression showed pt3 stage disease compared to none of pt2 . however , this data is limited by the number of patients within each gs subgroup and need further confirmation .
more importantly , in addition to not documenting any clinical prognostic significance for erg expression , we did not observe any association between erg expression and other pathological parameters such as gleason score or surgical margins , which further diminishes any potential prognostic significance for erg expression , at least in localized pca .
another issue worth mentioning regarding erg is that , erg gene rearrangements have been previously associated with specific histopathological features and detected more frequently in some morphologic variants of prostate cancer than others . in this study , we confirm lower rates of erg expression in tumors with foamy / xanthomatous morphology which demonstrated lower mean expression intensities compared to acinar pca .
the significance of this is not yet known , but suggests that those two types of tumors may be different at the molecular level . in conclusion , this study demonstrates significant association between erg expression and extra - prostatic extension and higher pathological stage in localized prostate cancer .
moreover , the lack of association with any other pathological parameters also significantly diminishes any potential clinical application of erg expression , at least in men with localized prostate cancer .
 
 background . 
 the prognostic significance of erg expression in prostate cancer ( pca ) has generated mixed results . 
 we sought to investigate the prognostic significance of erg expression in a localized cohort of men with pca . 
 material and methods . 
 we investigated erg protein expression in a cohort of 198 men with localized pca . 
 erg expression was correlated with patients ' clinical outcome and several pathological parameters , including gleason score ( gs ) , pathological stage , surgical margin , and extra - capsular extension . results . 
 erg expression was detected in 86/198 ( 43.4% ) patients exclusively in neoplastic epithelium . 
 overall , erg mean expression intensity was 1.01  1.27 versus 0.37  0.83 in acinar pca compared to foamy type pca ( p < 0.001 ) . in hgpin , erg intensity levels were comparable to those in foamy type pca ( 0.13  0.56 ) but significantly lower than those in acinar pca ( p < 0.001 ) . 
 erg expression was significantly associated with extra - prostatic extension and higher pathological stage and showed a trend toward seminal vesicle invasion . 
 herein , erg expression was documented in 50/131 ( 38.1% ) patients with pt2 versus 30/55 ( 54.5% ) patients with pt3 ( p = 0.04 ) . 
 erg association with higher pathological stage was more pronounced in patients with gs > 7 . 
 grouping patients into those with gs  7 versus > 7 , there was no significant association between erg expression and gs . 
 similarly , no association was present in relation to either surgical margins or postsurgical serum psa levels . conclusion . 
 we report significant association between erg protein levels and extra - prostatic extension and higher pathological stage . 
 erg expression is not associated with adverse clinical outcome and is of limited prognostic value in localized pca . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion