although prostate cancer is the second leading cause of cancer - related deaths in men in the us , early detection with serum prostate - specific antigen ( psa ) has led to the over - detection and over - treatment of indolent prostate cancer(13 ) .
recently , active surveillance , where men newly diagnosed with prostate cancer undergo serial biopsy , psa and/or imaging to delay intervention until prompted by clinicopathological evidence of disease progression ( or patient decision ) , has emerged as management strategy for low risk prostate cancer that does not significantly decrease prostate cancer specific mortality compared to immediate treatment(46 ) . despite advances in imaging and prognostic expression / protein assays ,
serum psa and clinicopathological parameters are the only factors routinely used to assess prognosis at diagnosis ( 4 , 611 ) .
although pathological inclusion criteria for active surveillance protocols vary by institution or group , almost all include gleason score , number ( or percentage ) of positive cores and the tumor measurement / length or maximum percentage of tumor involvement in any core ( 4 , 5 , 1115 ) .
in particular , more than 50% of tumor involvement in any given core is commonly considered as an exclusion criterion for most active surveillance protocols ( 4 , 5 , 1115 ) . however , when two or more foci of prostate cancer separated by intervening benign tissue are present in a single core biopsy , there is currently no consensus dictating the optimal method to report such a tumor involvement percentage
. the pathologist can either 1 ) measure discontinuous foci as if they were one continuous tumor by including the benign intervening tissue , assuming they represent two sections of a unique tumor , or 2 ) only measure the areas actually involved by a tumor focus .
the first option , which would report a higher percentage of tumor involvement of a core , has been proposed to be the optimal method by suggesting it is more representative of tumor volume at prostatectomy ( 16 ) .
prostate cancer is known to be a multifocal disease ( 17 ) , with most radical prostatectomy specimens actually harboring clonally distinct tumor foci , as supported by single marker molecular subtyping and next generation sequencing ( 1824 ) . of note ,
approximately 50% of psa - screened prostate cancer foci in predominantly caucasian populations harbor chromosomal rearrangements that result in the fusion of the 5 untranslated region of tmprss2 to erg , an ets transcription factor , which can be detected by fluorescence in situ hybridization ( fish ) or immunohistochemistry ( 2527 ) .
additionally , about 10% of prostate cancers show marked over - expression of spink1 , which can be evaluated concurrently with erg by dual immunohistochemistry ( 28 , 29 ) .
importantly , in these studies , erg fusion status has been shown to be clonal in a given tumor focus , and spink1 and erg show essentially mutually exclusive expression .
hence , dual erg / spink1 immunohistochemistry represents a simple , rapid and inexpensive method to assess tumor clonality in routine specimens . in this study
, we hypothesized that spatially distinct tumor foci in a given biopsy core may arise from separate clones , and sought to determine the frequency of such multiclonality .
thus , we used erg / spink1 dual immunohistochemistry to determine whether discontinuous cancer foci truly represent the same tumor clone ( uniform erg / spink1 status in separate foci ) , or multiclonal disease ( discordant erg / spink1 status between foci ) .
results of this pilot study demonstrate multiclonality in 25% of discontinuously involved cores , supporting additional studies on whether clonality impacts the prognostic ability of tumor volume at biopsy and prostatectomy .
surgical pathology databases were searched for prostatic needle core biopsies containing  discontinuous prostatic adenocarcinoma  diagnosed between 2010 and 2013 at two institutions , weill medical college of cornell university and university of michigan .
two genitourinary pathologists reviewed the h&e - stained slides for each case ; biopsies with confirmed discontinuous foci of prostatic adenocarcinoma were selected . only biopsies with intact cores ( measuring at least 1 cm )
, in this study we considered biopsies with tumor foci separated by at least 2.5 mm or 25% of total core volume as discontinuously involved ( figure 1a  c ) . for each core , the gleason score was verified , and we determined the maximum percentage of tumor involvement by 1 ) routine histomorphology ( inclusive of discontinuous foci ) , as well as 2 ) tumor involvement measured after evaluating erg / spink1 status ( if discontinuously staining foci were present , we added the percentage of both foci [ figure 1a  c ] .
dual erg / spink1 immunohistochemistry staining ( 28 ) was performed on 5m - thick unstained slides .
these corresponded to intervening unstained levels generated as part of the standard diagnostic workup , or de - stained h&e slides when unstained slides were not available or tissue was exhausted from the block .
immunohistochemistry was performed either on the benchmark ultra or the discovery xt automated staining systems ( ventana medical system , inc . ,
tucson , az ) . a monoclonal rabbit anti - erg primary antibody from ventana medical systems ( epr3864 ) and a mouse primary anti - spink1 4d4 antibody ( abnova , taiwan ) were used , as previously described ( 26 , 2830 ) .
tumor foci with intense ( 2 + or 3 + ) and diffuse ( at least 90% of cancer cells ) nuclear erg staining were considered as erg positive ( erg ) .
tumor foci with intense ( 2 + or 3 + ) cytoplasmic spink1 staining in more than 10% of cancer cells were considered spink1 positive ( spink1 ) .
biopsies were considered multiclonal if they harbored spatially distinct foci with discrepant erg and/or spink1 status ( i.e. one erg / spink1 focus and one erg / spink1 focus ; one erg / spink1 focus and one erg / spink1 focus ; or one erg / spink1 focus and one erg / spink1 focus ) .
biopsies with uniform erg or spink1 staining of tumor foci were considered clonal ( figure 1a  d ) .
surgical pathology databases were searched for prostatic needle core biopsies containing  discontinuous prostatic adenocarcinoma  diagnosed between 2010 and 2013 at two institutions , weill medical college of cornell university and university of michigan .
two genitourinary pathologists reviewed the h&e - stained slides for each case ; biopsies with confirmed discontinuous foci of prostatic adenocarcinoma were selected . only biopsies with intact cores ( measuring at least 1 cm )
, in this study we considered biopsies with tumor foci separated by at least 2.5 mm or 25% of total core volume as discontinuously involved ( figure 1a  c ) . for each core , the gleason score was verified , and we determined the maximum percentage of tumor involvement by 1 ) routine histomorphology ( inclusive of discontinuous foci ) , as well as 2 ) tumor involvement measured after evaluating erg / spink1 status ( if discontinuously staining foci were present , we added the percentage of both foci [ figure 1a  c ] .
dual erg / spink1 immunohistochemistry staining ( 28 ) was performed on 5m - thick unstained slides .
these corresponded to intervening unstained levels generated as part of the standard diagnostic workup , or de - stained h&e slides when unstained slides were not available or tissue was exhausted from the block .
immunohistochemistry was performed either on the benchmark ultra or the discovery xt automated staining systems ( ventana medical system , inc . ,
tucson , az ) . a monoclonal rabbit anti - erg primary antibody from ventana medical systems ( epr3864 ) and a mouse primary anti - spink1 4d4 antibody ( abnova , taiwan ) were used , as previously described ( 26 , 2830 ) .
tumor foci with intense ( 2 + or 3 + ) and diffuse ( at least 90% of cancer cells ) nuclear erg staining were considered as erg positive ( erg ) .
tumor foci with intense ( 2 + or 3 + ) cytoplasmic spink1 staining in more than 10% of cancer cells were considered spink1 positive ( spink1 ) .
biopsies were considered multiclonal if they harbored spatially distinct foci with discrepant erg and/or spink1 status ( i.e. one erg / spink1 focus and one erg / spink1 focus ; one erg / spink1 focus and one erg / spink1 focus ; or one erg / spink1 focus and one erg / spink1 focus ) .
biopsies with uniform erg or spink1 staining of tumor foci were considered clonal ( figure 1a  d ) .
ninety - seven prostate needle biopsies from 80 patients fulfilled our criteria for discontinuous involvement by prostatic adenocarcinoma ( table 1 ) .
. maximum tumor involvement , including benign intervening tissue , ranged from 20 to 100% of core length .
overall , erg and spink1 frequency ( assessed per core ) was 38% ( 37/97 ) and 12% ( 12/97 ) , respectively .
erg / spink1 immunohistochemistry status , gleason score , and maximum tumor involvement for all evaluated cores are shown in a heatmap in figure 1e .
benign prostate tissue was negative for erg or spink1 expression , consistent previous reports of exceptionally rare staining for either antigen in benign prostate tissue(23 , 24 , 26 , 28 , 30 ) .
of the 97 biopsies with discontinuous tumor , 24 ( 25% ) showed discrepant erg / spink1 status between spatially distinct foci ( table 2 ) . within these 24 multiclonal biopsies ,
14 ( 58% ) harbored one erg / spink focus and one erg / spink1 focus ; 7 ( 29% ) harbored one erg / spink1 focus and one erg / spink1 focus ; and 3 ( 13% ) showed one erg / spink1 focus and one erg / spink1 focus ( examples of discordant foci are shown in figure 2 ) .
when determining clonality by only considering erg status , 17 ( 17.5% ) had discordant erg status between foci , consistent with multiclonal disease .
in the 24 multiclonal biopsies , gleason scores were 3 + 3=6 in 54% ( 13/24 ) , 3 + 4=7 in 21% ( 5/24 ) , 4 + 3=7 in 17% ( 4/24 ) and 4 + 4=8 in 12% ( 2/24 ) of cores .
maximum tumor involvement percentage in multiclonal biopsies , including intervening benign tissue , ranged from 20 to 100% . after incorporating multiclonal assessment
( collapsing involvement to the summed tumor lengths of foci with discordant erg / spink1 status ) , maximum tumor involvement percentage fell under under 50% in 22/24 biopsies ( 92% ) .
recent consensus statements recommend reporting the amount of cancer in prostate biopsies by estimating the percentage of tumor involvement in the core with the greatest tumor extent ( 11 , 31 ) .
importantly , this pathology parameter has been shown to be correlated with tumor volume at prostatectomy ( 32 ) .
although measuring the percentage of involvement in a core may seem straightforward , it can be challenging for the pathologist when assessing biopsy cores with discontinuous tumor separated by benign intervening tissue , which is a well - recognized finding on prostate biopsies .
there is currently no consensus on the best method  including or excluding intervening benign tissue  when reporting tumor percent involvement in a discontinuously involved core .
the choice of method remains controversial between pathologists , and both techniques are being utilized clinically ( 3335 ) .
brimo et al . reported no significant difference using either tumor content estimation technique on predicting pathologic outcome at prostatectomy , although they included cases in which the amount of intervening benign tissue was less than 5 mm ( 34 ) .
however , recent studies propose reporting the maximum tumor involvement , including the benign intervening tissue , assuming that the separate foci represent the same tumor coming in and out of the core section .
showed that this measurement method better correlated with stage and margin positivity in cases with gleason 6 on biopsy with no upgrade at radical prostatectomy ( 16 ) .
shultz et al . further supported this finding in a private practice setting ( 35 ) .
multiclonality / multifocality is well recognized at prostatectomy , as spatially distinct tumor foci are routinely appreciated .
importantly , molecular evidence supporting true multiclonality of distinct tumor foci has been demonstrated by our groups and others using erg status ( by fish or immunohistochemistry as a surrogate ) to identify distinct foci ( 1820 , 23 , 24 ) .
a recent study assessed clonality between biopsies in patients with multiple positive biopsy cores , using erg immunohistochemistry as a clonal marker . in that study ,
mertz et al . showed that approximately 12% of prostate needle biopsy sets in their cohort of patients with indolent prostate cancer showed heterogeneous erg staining ( 36 ) . in this study , we show that 25% of discontinuously involved prostate biopsies harbor clonally distinct tumors , revealed by discrepant erg and/or spink1 immunohistochemistry status between foci .
to our knowledge , this represents the first study to interrogate prostate cancer clonality in core needle biopsies with discontinuous tumor involvement .
although associations with disease burden at prostatectomy must be established in cases where the discontinuously involved core drives management , we hypothesize that reporting tumor volume by excluding intervening benign tissue ( instead of including intervening benign tissue ) will be more appropriate in cores with multiclonal discontinuous involvement .
hence , erg / spink1 evaluation in such cases would be expected to increase the number of men eligible for active surveillance by identifying those with multiclonal involvement . in our study , the frequency of erg and spink1 overexpression was comparable to previous studies at 38% ( 37/97 ) and 12% ( 12/97 ) , respectively .
as expected , erg status was therefore the major driver in determining clonality , since 17/24 ( 71% ) of multiclonal cases showed at least one erg focus .
however , incorporating spink1 in a dual staining with erg adds further data to support distinct clonal origin , since erg and spink1 represent distinct molecular prostate cancer subclasses and their expression is essentially mutually exclusive ( 23 , 29 , 3739 ) .
although other molecular subtypes have been identified ( e.g. 
 spop mutated ) , routine assays to identify them in situ ( or surrogate tissue - based markers ) are not yet available ( 40 , 41 ) .
although heterogeneous spink1 status has been observed within the same tumor focus ( 42 ) , in our experience with prostatectomy specimens this usually represents discrepant staining between the leading edge and center of a given focus ( 23 , 28 ) .
thus , although we interpret discordant spink1 staining between two discontinuous foci on biopsy as evidence of multiclonality ( as a leading edge of both can be appreciated ) , this will require further validation using alternative techniques .
given that this study represents , to our knowledge , the first assessment of multiclonality in discontinuously involved cores , we included cores from cases where tumor content of the discontinuous core would not have impacted potential active surveillance inclusion ( e.g. the discontinuously involved core or other core had high grade / volume disease or numerous other cores were positive ) .
however , our cohort did include cases where evaluation of the discontinuously involved core may have played a key role in clinical management .
for example , one core was from a patient diagnosed with gleason 3 + 3=6 prostate cancer in 1 of 12 cores , with cancer discontinuously involved 80% of the core ( figure 3 ) .
this case was reviewed by an expert genitourinary pathologist at an outside institution , who agreed with high volume discontinuous involvement , and the patient underwent surgery . at radical prostatectomy ,
only a single , small ( 0.7 cm in greatest dimension ) focus of gleason score 6 , organ confined prostate cancer was present , consistent with clinically insignificant disease by standard epstein criteria ( 43 ) .
erg / spink1 dual immunohistochemistry on the involved biopsy core demonstrated discordant erg / spink1 status between the foci ( figure 3 ) , and hence clonality assessment would support this patient as having two small foci ( occupying a total of ~20% [ ~15% and < 5% individually ] of the core ) , warranting consideration for active surveillance . of interest , arias - stella et al .
( when including intervening benign tissue ) represented the maximally involved core and patients underwent prostatectomy ( 44 ) . in their cohort , although 78% of patients had a single tumor focus in the corresponding region at prostatectomy , 22% had multiple small cancer foci ( 44 ) . these results ,
when combined with our identification of multiclonality in ~25% of discontinuous foci on biopsy , further supports the need to determine whether clonality assessment at biopsy can be used to stratify disease involvement prior to prostatectomy .
first , we are likely underestimating the true rate of multiclonality , given that concordant discontinuous foci may still represent distinct tumor clones that can not be resolved using currently available in situ assays . for example , in a biopsy with two discontinuous tumor foci that are ergand spink1 , they may still represent two different clones ( as would be supported if spop mutant and spop wild - type ) .
second , we assessed a relatively small cohort , which may reflect that not all pathologists at our institutions have adopted the approach of reporting  discontinuous  cancer involvement on needle core biopsies , particularly in cases where it is unlikely to drive management .
likewise , we included cases where the discontinuous core did not drive management ( e.g. the discontinuous core or other positive cores had gleason score > 6 , there were multiple other positive cores or other high volume involved cores ) .
our study is a proof of principle study assessing the frequency of multiclonality across prostate biopsies with discontinuous tumor foci .
therefore , further validation on larger study sets including prospectively collected cohorts ( e.g. active surveillance ) is essential .
confirmation of our hypothesis would provide compelling evidence that multifocal disease is common in biopsies with discontinuous tumor , supporting the opportunity to refine key histopathological parameters through incorporating multiclonality assessment to provide more accurate prognostic information .
likewise , assessment of the prognostic impact on assigned gleason scores in discontinuously involved cores may also be of interest ( e.g. a discontinuous gleason score 3 + 4=7 foci may actually represent separate gleason score 4 + 4=8 and 3 + 3=6 foci ) .
we expect that assessment of erg / spink1 for clonality may also have utility in selected scenarios at prostatectomy .
for example , we are assessing whether incorporation of erg / spink1 in gleason score 7 tumors may improve the prognostic ability of routine pathologic parameters ( e.g. gleason score and tumor size ) through identifying tumors that are collisions of foci with distinct erg / spink1 status .
examples of prostatectomy cases where a single tumor focus by morphology are composed of foci with discordant erg / spink1 status are shown in figure s1 . in summary , in our study , nearly 25% of prostate core biopsies with discontinuous tumor involvement harbored spatially molecularly distinct cancer foci , as indicated by discrepant erg and/or spink1 status . here
we demonstrate that assessment of tumor clonality by erg / spink1 dual immunohistochemistry in biopsies with discontinuous involvement of prostate cancer is an effective tool to refine histopathological parameters , specifically estimation of tumor involvement when discontinuous foci are present in prostate biopsies .
incorporation of this inexpensive ancillary test may have significant impact on routine practice as it could potentially increase the number of eligible patients for active surveillance .
clinical application of erg / spink1 dual immunohistochemistry is an important area for continued study .
 the presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well - recognized finding on prostate biopsy . 
 cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area . 
 importantly , this parameter is a common enrollment criterion for active surveillance protocols . 
 we hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones , impacting cancer involvement assessment . 
 hence , we used dual erg / spink1 immunohistochemistry to determine the frequency of separate clones  when separate tumor foci showed discordant erg and/or spink1 status  in discontinuously involved prostate biopsy cores from two academic institutions . in our cohort of 97 prostate biopsy cores with spatially discrete tumor foci ( from 80 patients ) , discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and gleason scores ranged from 6 to 9 . twenty four ( 25% ) of 97 
 discontinuously involved cores harbored clonally distinct cancer foci by discordant erg and/or spink1 expression status : 58% ( 14/24 ) had one erg+ focus , and one erg/spink1 focus ; 29% ( 7/24 ) had one spink1 + focus and one erg/spink1 focus ; and 13% ( 3/24 ) had one erg+ focus and one spink1 + focus . 
 erg and spink1 overexpression were mutually exclusive in all tumor foci . 
 in summary , our results demonstrate that ~25% of discontinuously involved prostate biopsy cores showed tumor foci with discordant erg / spink1 status , consistent with multiclonal disease . 
 the relatively frequent presence of multiclonality in discontinuously involved prostate biopsy cores warrants studies on the potential clinical impact of clonality assessment , particularly in cases where tumor volume in a discontinuous core may impact active surveillance eligibility . 
Introduction
Material and Methods
Patient population and specimen collection
Determining tumor involvement
Evaluation of tumor clonality by ERG/SPINK1 dual immunohistochemistry
Results
Discussion
Supplementary Material