Context.—Fusions of androgen-regulated genes and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) occur in approximately 50% of prostate cancers, encoding a truncated ERG product. In prostatectomy specimens, ERG rearrangements are greater than 99% specific for prostate cancer or high-grade prostatic intraepithelial neoplasia adjacent to ERG-rearranged prostate cancer by fluorescence in situ hybridization and immunohistochemistry.

Objective.—To evaluate ERG staining by immunohistochemistry on needle biopsies, including diagnostically challenging cases.

Design.—Biopsies from a retrospective cohort (n  =  111) enriched in cores requiring diagnostic immunohistochemistry and a prospective cohort from all cases during 3 months (n  =  311) were stained with an anti-ERG antibody (clone EPR3864).

Results.—Among evaluable cores (n  =  418), ERG staining was confined to cancerous epithelium (71 of 160 cores; 44%), high-grade prostatic intraepithelial neoplasia (12 of 68 cores; 18%), and atypical foci (3 of 28 cores; 11%), with staining in only 2 of 162 cores (1%) diagnosed as benign. The ERG was expressed in about 5 morphologically benign glands across 418 cores and was uniformly expressed by all cancerous glands in 70 of 71 cores (99%).

Conclusions.—ERG staining is more prostate cancer–specific than α-methylacyl-coenzyme A racemase, and staining in an atypical focus supports a diagnosis of cancer if high-grade prostatic intraepithelial neoplasia can be excluded. Thus, ERG staining shows utility in diagnostically challenging biopsies and may be useful in molecularly subtyping prostate cancer and in stratifying isolated high-grade prostatic intraepithelial neoplasia by risk of subsequent cancer.

Although the diagnosis of prostate carcinoma (PCa) on needle biopsy cores can typically be made on morphology using standard hematoxylin-eosin staining, atypical foci, particularly those that are small, can pose diagnostic difficulty with discordant diagnoses among even expert pathologists.1 Hence, immunohistochemistry (IHC), most commonly with basal cell markers (p63 and high molecular weight cytokeratin) and α-methylacyl-coenzyme A racemase (AMACR), is performed to render a diagnosis or support morphologic impressions. Unfortunately, lack of basal cell markers and positive AMACR staining can occur in small foci of benign mimickers of PCa, including adenosis (atypical adenomatous hyperplasia) and partial atrophy, and AMACR is positive in only about 80% of limited PCa foci on needle biopsy.2 α-Methylacyl-coenzyme A racemase also stains almost all high-grade prostatic intraepithelial neoplasia (HGPIN),24 the presumed precursor lesion of PCa, limiting its utility for molecular stratification of subsequent cancer risk. Hence, robust immunohistochemical markers of PCa may have utility in diagnosis, molecular subtyping, and risk stratification.

In 2005, chromosomal rearrangements were identified in prostate cancer that fuse the 5′ untranslated region of the androgen-regulated gene transmembrane protease, serine 2 (TMPRSS2), with v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) or ets variant 1 (ETV1), 2 members of the ETS transcription factor family.5 Subsequent studies confirmed ETS gene fusions in approximately 50% of prostate-specific antigen–screened prostate cancers.6,7 Fusions between TMPRSS2 (or more rarely, solute carrier family 45, member 3 [SLC45A3] or N-myc downstream regulated 1 [NDRG1]8,9) and ERG represent approximately 90% of all ETS gene fusions and cause marked overexpression of the fusion transcript.7 Importantly, rearrangements of ERG at the chromosomal level (as determined by fluorescence in situ hybridization [FISH]) are essentially 100% specific for the presence of prostate cancer or HGPIN immediately adjacent to cancer in tissue studies.7,10,11 ,ERG rearrangements appear highly clonal because, when present, nearly all cells in a given cancer focus are positive, although distinct cancer foci in a single prostate may have discordant ERG rearrangement status.1215 Multiple studies have also demonstrated that ERG rearrangements are detectable in approximately 15% of HGPIN lesions, invariably adjacent to ERG-rearranged PCa.1618 In vitro and in vivo studies have also demonstrated a functional role for ERG gene fusions in prostate cancer oncogenesis,7,17,19,20 and ERG rearrangement–positive and ERG rearrangement–negative tumors have distinct molecular profiles.21,22 Taken together, ERG gene fusions are the most prostate cancer–specific biomarker yet identified and likely define a specific molecular subtype of prostate cancer.

Several groups have characterized ERG staining in prostatectomy specimens by IHC using monoclonal antibodies against ERG. Using tissue microarrays containing 207 PCa cores from prostatectomy specimens, Park et al11 demonstrated strong ERG staining using a monoclonal antibody against ERG (anti-C terminus, clone EPR3864; Epitomics, Inc, Burlingame, California) in 92 cores (44%), with an overall 95.7% sensitivity and 96.5% specificity compared with FISH for ERG rearrangements; no benign glands showed ERG staining. Similarly, Furstato et al,10 using a different anti-ERG monoclonal antibody on whole-mount prostatectomy specimens, demonstrated diffuse ERG staining in 117 of 261 cancer foci (45%) but only in 22 of an estimated 200 000 benign glands (0.01%). Importantly, they also demonstrated that 82 of 85 evaluable specimens (96.5%) with ERG-expressing tumor foci contained ERG-expressing HGPIN lesions, with all ERG+ prostatic intraepithelial neoplasia foci adjacent to ERG+ tumors.

More recently, studies have begun to address the utility of ERG staining in needle biopsies. For example, van Leenders et al23 evaluated the EPR3864 antibody on a consecutive series of needle biopsies containing PCa, with 51 of 83 cores (61%) demonstrating ERG staining in cancerous foci glands. The ERG staining was present in 11 of 21 foci of HGPIN (52%), invariably adjacent to ERG+ PCa (in the 10 of 11 foci [91%] with residual PCa in the core). Similarly, He et al24 evaluated EPR3864 anti-ERG staining in 103 needle biopsy cores with a diagnosis of “atypical glands suspicious for cancer” and found that 16 (15.5%) expressed ERG. Finally, Yaskiv et al25 evaluated dual staining with p63 and EPR3864 on 77 needle biopsies containing limited PCa (<1 mm involvement of only 1 core of the entire biopsy set) and observed ERG staining in 32 foci of PCa (42%).

Together, these results support the cancer specificity of ERG rearrangements, support ERG staining by IHC as a surrogate for ERG rearrangement status, and suggest diagnostic utility. However, ERG staining in the full spectrum of lesions encountered on routine diagnostic needle biopsies has not been evaluated nor has ERG staining been evaluated in a prospective series. Here, we evaluate the performance of ERG by IHC in 422 diagnostic needle biopsies, including challenging cases.

Cohort

Unstained levels from needle core biopsies of the prostate were selected from men undergoing biopsies of the prostate at a single academic institution from April 2008 through January 2011 and comprised 2 cohorts. Cores were obtained with Institutional Review Board approval. All diagnoses were made before evaluation of ERG.

The first cohort of 111 cores was identified retrospectively, with cores selected from biopsies from April 2008 to September 2010 and January 2011 and was enriched with cores requiring IHC for diagnosis (using the basal cell markers p63, high molecular weight cytokeratin [clone 34βE12], and AMACR as a triple cocktail; n  =  66) and cores with minute cancer foci (30 of 61 cores [49%] with cancer).

A second prospective cohort of 311 cores was obtained by collecting levels from all cases during diagnosis from September to December 2010. In cases with benign diagnoses, cores were randomly selected from both sides of the prostate. In cases with PCa, a core from each involved side was selected, generally representing the highest Gleason score (in cases with Gleason score >6) or smallest percentage of core involvement (for cases in which the Gleason score  =  6). All cores with a diagnosis of HGPIN or atypia (including atypical small acinar proliferation [ASAP] and HGPIN with adjacent atypical glands [PINATYP]) were selected. Finally, all cores requiring IHC for diagnosis were selected. In some cases, unstained levels were not available for all selected cores (tissue exhausted, used for additional hematoxylin-eosin staining, etc.) or were not obtained.

Immunohistochemistry on unstained, formalin-fixed, paraffin-embedded levels was performed using a monoclonal antibody against ERG (clone EPR 3864; Epitomics), using the automated Discovery XT staining platform (Ventana Medical Systems, Tucson, Arizona) as described.11 The ERG staining was evaluated by the study pathologists. Staining of vessels was used as a positive control, and slides without staining of vessels were excluded from further analysis. The ERG staining in prostatic glands was either absent or diffusely strong (2–3+), unless otherwise indicated, and was reported as present or absent.

Of the 422 total cores stained for ERG, tissue was lost on one core, the atypical focus on one core was not present (remaining benign glands showed no ERG staining), and staining failed on 2 cores, leaving 418 cores (99%) for analysis. Demographics from both the retrospective and prospective cohorts, which differed primarily by the inclusion of more benign cores in the prospective cohort, are shown in Table 1, and results for both cohorts are summarized below. As ERG (wild-type) is expressed in endothelial cells, where it has a known biologic role,26 staining in vessels was used as an endogenous positive control, and in all evaluable cores, strong nuclear staining was present in all endothelial cells. Consistent with previous results on prostatectomy sections, ERG was also expressed in a subset of lymphocytes,11 which may be due to cross-reactivity of the antibody with Friend leukemia virus integration 1 (FLI1), a similar ETS family protein expressed in both endothelial cells and lymphocytes, as shown by Furstato et al.10 In our experience, staining in vessels and a subset of lymphocytes does not cause difficulty in interpreting ERG staining in benign or cancerous glands; Figure 1, A and B, shows a representative hematoxylin-eosin core with benign prostatic glands, lymphocytes, vessels, and prostate cancer, whereas Figure 1, C and D, shows the ERG-stained core. The ERG expression across representative cores with benign glands (Figure 2, A and B), HGPIN (Figure 2, C and D), atypical foci (Figure 2, E and F), and prostate cancer (Figure 2, G and H) are also shown.

Figure 1. 

Staining of ERG in prostate needle biopsies with hematoxylin-eosin (A and B) or evaluated for ERG staining by immunohistochemistry (C and D). A representative core containing benign glands (green arrows), cancerous glands (red), vessels (blue), and lymphocytes (black) is shown. Inset regions of A and C (indicated by boxes) are shown in B and D (original magnifications ×2.5 [A and C] and ×20 [B and D]).

Figure 1. 

Staining of ERG in prostate needle biopsies with hematoxylin-eosin (A and B) or evaluated for ERG staining by immunohistochemistry (C and D). A representative core containing benign glands (green arrows), cancerous glands (red), vessels (blue), and lymphocytes (black) is shown. Inset regions of A and C (indicated by boxes) are shown in B and D (original magnifications ×2.5 [A and C] and ×20 [B and D]).

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Figure 2. 

Representative ERG staining across diagnostic lesions. Prostate needle biopsies were stained by hematoxylin-eosin (A, C, E, and G) or evaluated for ERG staining by immunohistochemistry (IHC) (B, D, F, and H). A and B, Negative ERG staining in a benign focus of adenosis requiring IHC with basal cell markers (p63 and high molecular weight cytokeratin [34βE12], brown) and α-methylacyl coenzyme A racemase (red) for diagnosis (inset of A). C and D, Positive ERG staining in a focus of high-grade prostatic intraepithelial neoplasia. Inset in C demonstrates nuclear atypia, including prominent nucleoli. E and F, Positive ERG staining in a focus of atypical small acinar proliferation. Inset in E demonstrates focal nuclear atypia. G and H, Positive ERG staining in a focus of Gleason 3 + 3 prostate cancer. Inset regions are indicated by boxes (original magnifications ×20 [A through H and inset A] and ×40 [insets C and E]).

Figure 2. 

Representative ERG staining across diagnostic lesions. Prostate needle biopsies were stained by hematoxylin-eosin (A, C, E, and G) or evaluated for ERG staining by immunohistochemistry (IHC) (B, D, F, and H). A and B, Negative ERG staining in a benign focus of adenosis requiring IHC with basal cell markers (p63 and high molecular weight cytokeratin [34βE12], brown) and α-methylacyl coenzyme A racemase (red) for diagnosis (inset of A). C and D, Positive ERG staining in a focus of high-grade prostatic intraepithelial neoplasia. Inset in C demonstrates nuclear atypia, including prominent nucleoli. E and F, Positive ERG staining in a focus of atypical small acinar proliferation. Inset in E demonstrates focal nuclear atypia. G and H, Positive ERG staining in a focus of Gleason 3 + 3 prostate cancer. Inset regions are indicated by boxes (original magnifications ×20 [A through H and inset A] and ×40 [insets C and E]).

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Table 1. 

Clinicopathologic Data for Patients With Prostate Biopsy Cores Evaluable for ERG Staining (N  =  418)

Clinicopathologic Data for Patients With Prostate Biopsy Cores Evaluable for ERG Staining (N  =  418)
Clinicopathologic Data for Patients With Prostate Biopsy Cores Evaluable for ERG Staining (N  =  418)

ERG Staining in PCa

Among all evaluable cores (n  =  418) diagnosed before evaluation of ERG staining, ERG was expressed in cancerous glands in 71 of 160 cores (44%) as shown in Table 2 and Figure 2, G and H. In cores with PCa where diagnostic IHC was performed, ERG was expressed in cancerous glands in 11 of 39 cores (28%), as shown in Table 3 and Figure 3, A through F. When positive, ERG showed strong (2–3+) nuclear staining in cancerous glands; no benign glands showed ERG staining (except in a single core as described below). The ERG was expressed diffusely in all cancerous glands in 70 of 71 cores (99%). A single core (1%), shown in Figure 4, A and B, showed ERG staining in a few cancerous glands (Figure 4, C), with no appreciable morphologic difference between ERG+ and ERG glands. Although we could not definitively identify the ERG staining glands with FISH for evaluation of ERG rearrangement status, given the strongly clonal nature of the ERG rearrangements and the staining in individual tumor foci, this core may represent a collision of ERG+ and ERG tumors, which we have observed in prostatectomy studies (S. A. Tomlins and L. P. Kunju, unpublished data, 2011).

Figure 3. 

Representative ERG staining in lesions requiring diagnostic immunohistochemistry (IHC) with basal cell markers and α-methylacyl coenzyme A racemase (AMACR). Prostate needle biopsies were stained with hematoxylin-eosin (A and B), evaluated for staining of basal cell markers p63 and high molecular weight cytokeratin (34βE12, brown), and AMACR (red) by IHC as part of the diagnostic workup (C and D), or evaluated for ERG staining by IHC (E and F). A, C, and E, Negative ERG staining in a benign focus of adenosis requiring diagnostic IHC; B, D, and F, Positive ERG staining in a minute focus of Gleason 3 + 3 PCa requiring diagnostic IHC (original magnifications ×20).

Figure 3. 

Representative ERG staining in lesions requiring diagnostic immunohistochemistry (IHC) with basal cell markers and α-methylacyl coenzyme A racemase (AMACR). Prostate needle biopsies were stained with hematoxylin-eosin (A and B), evaluated for staining of basal cell markers p63 and high molecular weight cytokeratin (34βE12, brown), and AMACR (red) by IHC as part of the diagnostic workup (C and D), or evaluated for ERG staining by IHC (E and F). A, C, and E, Negative ERG staining in a benign focus of adenosis requiring diagnostic IHC; B, D, and F, Positive ERG staining in a minute focus of Gleason 3 + 3 PCa requiring diagnostic IHC (original magnifications ×20).

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Figure 4. 

A single core with focal ERG staining in prostate carcinoma (PCa). Prostate needle biopsies were stained with hematoxylin-eosin or evaluated for ERG staining by immunohistochemistry. In 62 of 63 cores (98%) with PCa expressing ERG, staining was present in all cancerous glands. A, A single core with PCa (top) showed focal staining of ERG in a subset of cancerous glands (bottom). Inset regions of hematoxylin-eosin and ERG staining (indicated by boxes) are shown in B and C, respectively (original magnifications ×2.5 [A] and ×10 [B and C]).

Figure 4. 

A single core with focal ERG staining in prostate carcinoma (PCa). Prostate needle biopsies were stained with hematoxylin-eosin or evaluated for ERG staining by immunohistochemistry. In 62 of 63 cores (98%) with PCa expressing ERG, staining was present in all cancerous glands. A, A single core with PCa (top) showed focal staining of ERG in a subset of cancerous glands (bottom). Inset regions of hematoxylin-eosin and ERG staining (indicated by boxes) are shown in B and C, respectively (original magnifications ×2.5 [A] and ×10 [B and C]).

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Table 2. 

ERG Expression in Prostate Needle Biopsy Cores (N  =  418)

ERG Expression in Prostate Needle Biopsy Cores (N  =  418)
ERG Expression in Prostate Needle Biopsy Cores (N  =  418)
Table 3. 

ERG Expression in Prostate Needle Biopsy Cores Requiring Diagnostic Immunohistochemistry (N  =  101)

ERG Expression in Prostate Needle Biopsy Cores Requiring Diagnostic Immunohistochemistry (N  =  101)
ERG Expression in Prostate Needle Biopsy Cores Requiring Diagnostic Immunohistochemistry (N  =  101)

When positive, ERG showed strong nuclear staining regardless of its Gleason pattern; as shown in Figure 5, among cancerous cores, ERG was positive in 56 of 91 cores (62%) with Gleason scores of 6 cores (Figure 5, A though D), in 30 of 52 cores (58%) with Gleason scores of 7 (Figure 5, E through H), and in 6 of 17 cores (35%) with Gleason scores of 8 to 10 (Figure 5, I through L). Given the diagnostic difficulty posed by small atypical foci, we preferentially selected cores with minute cancerous foci (defined as ≤5% of the core involved) for evaluation by ERG staining. Overall, 60 of 160 cancerous cores (38%) had minute cancerous foci, with 22 of 60 minute cancerous cores (37%) expressing ERG, compared with 49 of 100 nonminute cancerous cores (51%).

Figure 5. 

ERG staining in prostate cancer across Gleason scores. Consecutive levels from diagnostic prostate needle biopsies were stained with hematoxylin-eosin or evaluated for ERG staining by immunohistochemistry. Representative hematoxylin-eosin staining (A, C, E, G, I, and K) and ERG staining (B, D, F, H, J, and L) in cores with Gleason scores of 6 (A through D), 7 (E through H), and 9 (I through L) prostate cancer. Inset regions of A, B, E, F, I, and J, (indicated by boxes) are shown in C, D, G, H, K, and L, respectively, (original magnifications ×2.5 [A, B, E, F, I, and J], ×10 [K and L], and ×20 [C, D, G, and H]).

Figure 5. 

ERG staining in prostate cancer across Gleason scores. Consecutive levels from diagnostic prostate needle biopsies were stained with hematoxylin-eosin or evaluated for ERG staining by immunohistochemistry. Representative hematoxylin-eosin staining (A, C, E, G, I, and K) and ERG staining (B, D, F, H, J, and L) in cores with Gleason scores of 6 (A through D), 7 (E through H), and 9 (I through L) prostate cancer. Inset regions of A, B, E, F, I, and J, (indicated by boxes) are shown in C, D, G, H, K, and L, respectively, (original magnifications ×2.5 [A, B, E, F, I, and J], ×10 [K and L], and ×20 [C, D, G, and H]).

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In cases in which multiple cancerous cores were evaluated from the same side (right or left) of the prostate, 10 of 10 cases (100%) showed concordant ERG staining (5 ERG+, 5 ERG). In cases in which multiple cancerous cores were evaluated from both sides of the prostate, 24 of 30 cases (80%) showed concordant ERG staining (9 ERG+, 15 ERG), with 6 of 30 cases (20%) showing at least one ERG+ and one ERG core.

ERG Staining in HGPIN

The ERG was expressed in 12 of 68 cores (18%) diagnosed as HGPIN; in cores in which diagnostic IHC was performed, ERG was expressed in 2 of 9 cores (22%) diagnosed as HGPIN (Tables 2 and 3; Figure 2, C through D). In all positive cores, ERG staining was limited to HGPIN foci, with no staining in adjacent benign glands. In cases in which multiple cores with HGPIN were evaluated from the same side (right or left) of the prostate, 9 of 11 cases (82%) showed concordant ERG staining (1 ERG+, 8 ERG). In cases in which multiple cores with HGPIN were evaluated from both sides of the prostate, 4 of 6 cases (67%) showed concordant ERG staining (1 ERG+, 3 ERG), with 2 cases showing at least one ERG+ and one ERG core. Of 15 cases with at least one cancerous core and one core with HGPIN evaluated for ERG staining, 6 (40%) showed entirely concordant ERG staining (all 6 with ERG HGPIN and PCa) and 9 (60%) showed discordant ERG staining (Figure 6).

Figure 6. 

ERG staining in cases with cores containing high-grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PCA). Prostate needle biopsies were stained with hematoxylin-eosin or evaluated for ERG staining by immunohistochemistry. Heat-map visualization of 15 cases with at least one core with HGPIN and one core with PCA. Side of the prostate (right or left) is indicated for each core. ERG HGPIN or PCA is indicated in white or light gray, respectively, and ERG+ HGPIN or PCA is indicated in dark gray or black, respectively, as indicated in the key.

Figure 6. 

ERG staining in cases with cores containing high-grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PCA). Prostate needle biopsies were stained with hematoxylin-eosin or evaluated for ERG staining by immunohistochemistry. Heat-map visualization of 15 cases with at least one core with HGPIN and one core with PCA. Side of the prostate (right or left) is indicated for each core. ERG HGPIN or PCA is indicated in white or light gray, respectively, and ERG+ HGPIN or PCA is indicated in dark gray or black, respectively, as indicated in the key.

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ERG Staining in Atypical Foci

In 28 cores with foci diagnosed as atypical (including ASAP and PINATYP), the atypical focus in 3 (11%) expressed ERG (all diagnosed as ASAP), with ERG staining in 1 of 17 cores (6%) diagnosed as atypical after diagnostic IHC (Figure 2, E and F; Tables 2 and 3). In all positive cores, ERG staining was limited to the atypical foci, with no staining in adjacent benign glands. Of 5 cases with at least one cancerous core and one core with an atypical focus evaluated for ERG staining, ERG staining was concordant in 4 (80%) (1 ERG+, 3 ERG).

ERG Staining in Benign Prostatic Tissue

In cores diagnosed as benign, ERG was expressed in 2 of 162 cores (1%), as shown in Table 2 and Figure 7, A through F . Among benign cores requiring diagnostic IHC (most commonly for foci of partial atrophy or adenosis), ERG was expressed in 1 of 35 cores (3%) as shown in Table 3 and Figure 7, A through F. The ERG+ benign core that required diagnostic IHC was stained for a focus of small, suspicious glands adjacent to a large gland with nuclear enlargement, hyperchromasia, and occasional nucleoli, and was, upon review, borderline between low-grade prostatic intraepithelial neoplasia and HGPIN (Figure 7, A and B). Diagnostic IHC showed weak AMACR staining and a complete basal layer around the large gland suspicious for HGPIN and lack of AMACR and an incomplete patchy basal layer around the small glands (Figure 7, C). The ERG was expressed in the prostatic intraepithelial neoplasia gland, the adjacent small glands, and a larger gland at the edge of the biopsy (Figure 7, D). Thus, on review, this core may better be classified as PINATYP with positive ERG staining. The patient with this core did not have HGPIN, atypia, or PCa on any other core in the biopsy. The second ERG+ core diagnosed as benign showed ERG staining in a large gland with slight nuclear enlargement and hyperchromasia without prominent nucleoli (consistent with what was previously termed low-grade prostatic intraepithelial neoplasia), as shown in Figure 7, E and F. The patient with this core had 2 additional cores with cancer in the biopsy. Finally, a single core showed ERG staining in 4 morphologically benign glands adjacent to a minute focus of PCa (Gleason 3 + 3), which also expressed ERG (Figure 7, G and H). Thus, in total, ERG was expressed in only about 5 morphologically benign glands (1%) from 2 foci across 418 cores.

Figure 7. 

ERG staining in cores diagnosed as benign and in morphologically benign glands. A and B, A core diagnosed as benign contained a gland with nuclear enlargement and hyperchromasia but with only occasional nucleoli, along with adjacent suspicious small glands. Inset region of A (indicated by box) is shown in B. Inset of B indicated by box. C, Diagnostic immunohistochemistry showed weak α-methylacyl coenzyme A racemase (AMACR) staining and a complete basal layer around the larger gland, lack of AMACR, and an incomplete, patchy basal layer around the small glands. D, ERG was expressed in the large gland, the adjacent small glands, and a large gland at the edge of the specimen. Thus, on review, this core may better be classified as high-grade prostatic intraepithelial neoplasia with adjacent small atypical glands with positive ERG staining. E through H, On rereview of all cores (n  =  418), 2 cores (0.5%) contained morphologically benign glands that expressed ERG. E and F, One core contained a large gland with nuclear enlargement and hyperchromasia but insufficient nucleolar enlargement for a diagnosis of high-grade prostatic intraepithelial neoplasia, which expressed ERG. G, A second core (bottom panel) contained a focus of benign glands (green arrow) adjacent to a minute focus of cancer (red arrow), both of which expressed ERG (top panel). H, Inset of similar morphologically benign glands (indicated by box in G) that were positive (top glands) or negative (bottom gland) for ERG staining (original magnifications ×10 [A], ×20 [B through F], ×4 [G], and ×40 (H and inset B).

Figure 7. 

ERG staining in cores diagnosed as benign and in morphologically benign glands. A and B, A core diagnosed as benign contained a gland with nuclear enlargement and hyperchromasia but with only occasional nucleoli, along with adjacent suspicious small glands. Inset region of A (indicated by box) is shown in B. Inset of B indicated by box. C, Diagnostic immunohistochemistry showed weak α-methylacyl coenzyme A racemase (AMACR) staining and a complete basal layer around the larger gland, lack of AMACR, and an incomplete, patchy basal layer around the small glands. D, ERG was expressed in the large gland, the adjacent small glands, and a large gland at the edge of the specimen. Thus, on review, this core may better be classified as high-grade prostatic intraepithelial neoplasia with adjacent small atypical glands with positive ERG staining. E through H, On rereview of all cores (n  =  418), 2 cores (0.5%) contained morphologically benign glands that expressed ERG. E and F, One core contained a large gland with nuclear enlargement and hyperchromasia but insufficient nucleolar enlargement for a diagnosis of high-grade prostatic intraepithelial neoplasia, which expressed ERG. G, A second core (bottom panel) contained a focus of benign glands (green arrow) adjacent to a minute focus of cancer (red arrow), both of which expressed ERG (top panel). H, Inset of similar morphologically benign glands (indicated by box in G) that were positive (top glands) or negative (bottom gland) for ERG staining (original magnifications ×10 [A], ×20 [B through F], ×4 [G], and ×40 (H and inset B).

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In this study, we have demonstrated the clinical utility of a monoclonal antibody against the C-terminus of ERG across a wide spectrum of lesions encountered in diagnostic prostate core biopsies, including challenging lesions. Gene fusions between androgen-regulated genes (most commonly TMPRSS2) and ERG occur in about 50% of prostate cancers. By FISH, the ERG gene rearrangements have been shown to be nearly 100% specific for the presence of PCa (or HGPIN immediately adjacent to ERG-rearranged PCa) across more than 2000 samples, including prostatectomy and needle biopsy specimens.7 As FISH can be difficult to perform in routine diagnostic settings, alternative techniques to detect ERG gene rearrangements are desirable and may be more clinically applicable. Although common ERG gene fusions do not encode chimeric proteins, a monoclonal antibody against the C-terminus of ERG (retained in all known ERG gene fusions) has previously been shown to be highly sensitive and specific for detecting ERG-rearranged PCa (as assessed by FISH) on prostatectomy specimens.11 Although the antibody used in our study has also been shown to react with FLI1,10 our results demonstrate that FLI1 staining is very rare in prostate cancer (given the previously demonstrated concordance between the antibody and ERG rearrangement status by FISH11) and benign prostate glands (given only 5 benign glands across 418 cores [1%] with any staining in the current study).

We evaluated ERG staining in 2 cohorts of diagnostic needle biopsy cores. The first retrospective cohort was enriched with cores most likely to pose diagnostic difficulty, including cores subjected to diagnostic IHC (with basal cell markers and AMACR) and cores with minute cancer foci. The second cohort consisted of cores from all cases signed out during 3 months at an academic center with subspecialty-based sign-out. In this cohort, cores selected for ERG evaluation from each case were again enriched with those requiring diagnostic IHC and minute cancer foci, but the cohort also included HGPIN cores, benign cores from men without a diagnosis of PCa, and PCa cores with a full spectrum of Gleason scores. Hence, our study assessed ERG staining across a large spectrum of lesions encountered in diagnostic needle biopsy specimens.

In this study, ERG was expressed in 71 of 160 cores (44%) with cancer, including 11 of 39 cores (28%) requiring IHC for diagnosis and 22 of 60 cores (37%) with minute cancer foci. In positive cores, ERG showed strong nuclear staining, and in 70 of 71 cores (98%) was diffusely expressed in all cancerous glands, regardless of Gleason pattern, consistent with the highly clonal nature of ERG rearrangements. Because not all cores with minute cancer foci or the highest Gleason scores were selected from each case, we did not formally compare rates of ERG staining in minute versus nonminute cancers or across Gleason scores, which has been addressed in prior studies using FISH for ERG rearrangement.7,27 

Overall, our results were highly concordant with those of Park et al,11 who performed IHC on prostatectomy specimens using the same antibody, where 44% of PCa foci expressed ERG (all of which showed diffuse staining), as well as those of van Leenders et al,23 who, using the same antibody, reported ERG staining in 51 of 83 cores (61%) with cancerous foci glands. Because the antibody used in our study has been shown to have greater than 95% sensitivity and specificity between FISH-determined ERG rearrangement and ERG staining by IHC,11 our results here demonstrate that IHC can be used to determine ERG rearrangement status on biopsy specimens, which may facilitate molecular subtyping of prostate cancer in the clinical setting. However, concordant ERG status in cancerous cores evaluated from both sides of the prostate was observed in only 79% of evaluated cases, consistent with the multifocal nature of prostate cancer and supporting previous FISH- and IHC-based studies demonstrating heterogeneous ERG status in different cancer foci. Thus, future efforts correlating ERG status in cancer on biopsy, index, and secondary lesions in prostatectomy samples and in metastatic cancer can likely be used to track lesion progression. The TMPRSS2 and ERG genes are located approximately 3 Mb apart on chromosome 21, and TMPRSS2:ERG gene fusions can develop either through insertion or deletion of the intervening region.7 Hence, FISH, which can differentiate these subtypes of TMPRSS2:ERG gene fusions, may have increased utility compared with IHC concerning issues of clonality or lesion tracking.

In the current study, ERG was expressed in 18% of HGPIN foci, consistent with prior FISH studies from independent groups on prostatectomy specimens, which have shown that approximately 15% of HGPIN lesions harbor ERG rearrangements, invariably adjacent to ERG rearranged PCa.1618 Similarly, Zhang et al28 showed that HGPIN lesions from 10 of 60 patients (17%) harbored TMPRSS2 rearrangements, invariably associated with TMPRSS2-rearranged PCa. Furstato et al,10 using a different ERG monoclonal antibody, showed similar concordance of ERG-expressing HGPIN with adjacent ERG-expressing PCa. Finally, in their study of limited cancer (77 needle biopsies), Yaskiv et al25 identified ERG staining in 5 of 17 cores (29%) with HGPIN, with all ERG+ HGPIN adjacent to ERG+ PCa.

van Lenders et al23 reported ERG staining in 11 of 21 foci of HGPIN (52%), attributing that higher frequency to difficulties in identifying HGPIN on slides used for previous FISH-based studies. Based on that higher frequency of ERG+ HGPIN, they concluded that ERG was not involved in the transition of HGPIN to invasive carcinoma and, instead, drives the development of HGPIN from benign glands.23 However, all HGPIN foci evaluated by van Lenders et al23 were selected from cores that contained cancer. In the FISH-based study by Han et al,16 11 of 15 foci of HGPIN (73%) adjacent to carcinoma had ERG rearrangements (all 11 [100%] adjacent to ERG-rearranged carcinoma), whereas 0 of 10 foci of HGPIN [0%] distant to carcinoma had ERG rearrangements (even though 8 of 10 carcinomas [80%] were ERG rearranged). If ERG is instrumental in the transition of benign glands to HGPIN, ERG rearrangements and protein expression should be equally prevalent in HGPIN lesions adjacent and distal to the carcinoma, which has not been observed. Hence, we feel the 18% prevalence of ERG staining in HGPIN in our study, which included cores with HGPIN from both benign and PCA cases, more accurately represents the true prevalence.

Presently, the risk of cancer on rebiopsy after a diagnosis of isolated HGPIN is approximately 25%, and clinicopathologic parameters are unable to reliably identify men with increased risk of cancer on rebiopsy.2 Based on the association of ERG- (or TMPRSS2-) rearranged or expressing HGPIN and similarly rearranged or expressing PCa, we hypothesize that ERG+ HGPIN indicates unsampled PCa or HGPIN that will inevitably progress to invasive disease. Thus, we predict that ERG staining may be useful for risk stratification of isolated HGPIN, with ERG+-isolated HGPIN having an increased risk of cancer on rebiopsy.

Also supporting previous IHC results,10,11,2325 we confirm here that ERG staining in benign glands, including foci requiring diagnostic IHC, is exceedingly rare (2 foci of approximately 5 glands [1%] in 397 cores). In our opinion, because morphologically benign glands (or foci consistent with what was previously termed low-grade prostatic intraepithelial neoplasia) rarely express ERG, those that do express it are molecularly neoplastic. Unlike AMACR, which can be negative in about 20% of unequivocal PCa; is positive in a subset of benign mimics of PCa, including adenosis and partial atrophy; and may show focal staining in up to 20% of morphologically benign glands,2931 our results here confirm that ERG staining is highly specific for PCa and is exceedingly rare in benign glands (including mimickers of PCa). Hence, we advocate that in a focus with atypical small acini (ASAP), where HGPIN or PINATYP can be excluded and basal cell markers are negative, ERG staining strongly supports a diagnosis of PCa, regardless of AMACR staining.

In our study, ERG staining was noted in 3 of 28 cores (11%) with atypical foci (all 3 [100%] diagnosed as ASAP). Similarly, He et al24 identified ERG staining in 16 of 103 atypical biopsies (15.5%). However, all cores in both studies were diagnosed before evaluation of ERG staining. Hence, these studies do not directly address the ability of ERG staining to add to current diagnostic IHC in the workup of challenging cases. Additionally, an individual's thresholds for calling lesions atypical or PCa (both with and without diagnostic IHC) complicate assessment of the usefulness of a novel biomarker. However, the consistency of our results compared with previous IHC- and FISH-based studies, which demonstrate exceptionally high specificity for ERG rearrangements and staining for PCa (or adjacent HGPIN), in combination with the ease of staining interpretation, suggests immediate diagnostic utility.

He et al24 did not find significantly different rates of cancer in follow-up biopsies from patients with atypical biopsies and either positive or negative ERG staining. However, our results, and previous FISH- and IHC-based studies, suggest that ERG+ atypical or HGPIN foci only indicate risk for developing or harboring unsampled cancer immediately adjacent to that ERG+ focus, and hence, prospective studies with targeted rebiopsy may be required to assess the utility of ERG staining for risk stratification. Similar studies will likely be required to determine the significance of isolated morphologically benign glands that are ERG+.

In summary, our study evaluating ERG staining by IHC in a large cohort of prostate biopsies demonstrated positivity in 44% of PCa, 18% of HGPIN, and 11% of atypical foci. In positive cancer foci, ERG is expressed uniformly in almost all cases, and ERG staining is exceedingly rare in benign glands. Overall, ERG appears to be more specific than AMACR for PCa; hence, ERG staining in an atypical focus (where HGPIN or PINATYP can be excluded) supports a diagnosis of PCa, irrespective of AMACR staining. We recommend adding ERG to standard diagnostic IHC (basal markers and AMACR) in the workup of atypical lesions in prostate core biopsies as well as for molecular profiling of PCa. Large multi-institutional studies will be required to characterize the risk-stratification benefit of ERG+ isolated HGPIN and to better define rebiopsy strategy and clinical management.

We thank Gary Pestano (Ventana Medical Systems) for providing the ERG antibody and IHC reagents. Supported in part by the National Cancer Institutes Early Detection Research Network (U01 CA111275 and U01 CA113913) and the National Institutes of Health Specialized Programs of Research Excellence (P50 CA69568).

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Author notes

From the Michigan Center for Translational Pathology, Department of Pathology, University of Michigan Health System, Ann Arbor. Dr Chinnaiyan is also with the Comprehensive Cancer Center and the Howard Hughes Medical Institute at the University of Michigan.

The University of Michigan has been issued a patent on the detection of ETS gene fusions in prostate cancer, on which S.A.T. and A.M.C. are listed as coinventors. The University of Michigan licensed the diagnostic field of use to Gen-Probe, Inc, which sublicensed rights to Ventana Medical Systems, Inc. Neither company played a role in data collection, interpretation, or analysis and neither participated in the study design, drafting, or revision of the manuscript or the decision to submit the manuscript for publication. S.A.T. has received honoraria from Ventana Medical Systems and has served as a consultant to Cougar Biotechnology, AstraZeneca, and Compendia Biosciences. N.P. has served as a consultant for Ventana Medical Systems. A.M.C. has served as consultant to Gen-Probe, Inc. and Ventana Medical Systems. J.S. and L.P.K. have no relevant financial interest in the products or companies described in this article.

Presented in part at the 100th Annual Meeting of the United States and Canadian Academy of Pathology, San Antonio, Texas, February 28, 2011.