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ERG protein expression as a biomarker of prostate cancer TMPRSS2–ERG is a recurrent rearrangement specific for prostate cancer, leading to the overexpression of a truncated ERG protein product that is amenable to immunohistochemical detection. Two monoclonal anti-ERG antibodies have currently been validated, with comparable sensitivity and specificity for detecting ERG rearrangement. ERG immunostaining has been applied in different settings to elucidate the role of ERG rearrangement and overexpression in prostate cancer tumorigenesis and progression, as well as to investigate potential diagnostic and prognostic applications. In this article we review the literature on the topic and suggest potential future applications. KEYWORDS: ERG immunohistochemistry n genomic rearrangements n prostate cancer n TMPRSS2–ERG
Recurrent chromosomal rearrangements leading to the fusion of the androgen receptor (AR)regulated TMPRSS2 gene and members of the ETS transcription factor family have been discovered in prostate cancer (PCa) using an unconventional analytical approach [1]. ERG is the most common fusion partner [2] detected in approximately 50% of PSA-screened PCas [3]. The genetic alteration leads to the juxtaposition of androgen-responsive regulatory elements of TMPRSS2 to the ERG sequence, with consequent overexpression of the rearranged ERG [1,4]. Oncogenic activation of other members of the ETS family (ETV1, ETV4 and ETV5) accounts for less than 10% of PCa rearrangements and demonstrate mutual exclusivity with ERG rearrangement [1,5]. Depending on factors such as cohort design (PSA screened vs population based) [6], tumor origin (transitional vs peripheral zone) [6,7] and histologic variants [8–11], ERG rearrangements occur in 15–80% of PCa.
genomic level ERG rearrangements have been extensively investigated by FISH and PCR [2,5]. A dualcolor interphase break-apart FISH assay has been applied in several studies, performed on formalin-fixed paraffin-embedded tissue to assess ERG gene rearrangement status [1,2]. Brief ly, two differentially labeled bacterial artificial chromosome (BAC) FISH probes have commonly been used: a biotin-14-dCTPlabeled BAC clone, such as RP11‑24A11 (eventually conjugated to produce a red signal), for
the neighboring 3´ centromeric region of the ERG locus and a digoxigenin-dUTP-labeled BAC clone, such as RP11‑372O17 (eventually conjugated to produce a green signal), for the neighboring 5´ telomeric region. According to this break-apart probe system, a nucleus without ERG rearrangement shows two pairs of juxtaposed red and green signals, forming yellow signals; a nucleus with ERG rearrangement through translocation shows one pair of still combined red and green signals (a yellow signal), while the other breaks into a single red and a single green signal (split signal); and a nucleus with ERG rearrangement through deletion shows one yellow signal and a single red signal, while the green signal (5´ region) is lost (Figure 1). Cases with ERG signal abnormality in >10% of the neoplastic cells have usually been scored as positive, and classified accordingly [7]. In addition, the presence of multiple copies of the ERG rea rrangement sequence, with or without retention of the green signal, has been commonly assessed [12]. Tissue hybridization, washing, and fluorescence detection have been previously described elsewhere [12]. Samples are analyzed under a 60× or 100× oil immersion objective using a fluorescence microscope equipped with appropriate filters. The corresponding hematoxylin and eosin section is used for side-by-side comparison with the FISH section. PCR detection of TMPRSS2–ERG fusion mRNAs has been used alternatively to assess ERG gene rearrangement status in PCa cell lines, tissue specimens and biologic fluids [1]. By reverse-transcriptase PCR, at least 17 distinct TMPRSS2–ERG hybrid transcripts have
10.2217/BMM.13.105 © 2013 Future Medicine Ltd
Biomarkers Med. (2013) 7(6), 851–865
ERG rearrangement detection at
Sara Moscovita Falzarano1 & Cristina Magi-Galluzzi*1 R.T. Pathology & Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, L25, Cleveland, OH 44195, USA *Author for correspondence: Tel.: +1 216 444 9251 Fax: +1 216 445 6967 [email protected] 1
part of
ISSN 1752-0363
851
Review
Falzarano & Magi-Galluzzi
Chomosome 21 q22.3
q22.2
FISH probes Spanning the 5´ end of ERG Spanning the 3´ end of ERG
No rearrangement TMPRSS2
ERG
TMPRSS2
ERG
Rearrangement through deletion
TMPRSS2 TMPRSS2
Rearrangement through translocation
TMPRSS2
ERG ERG
TMPRSS2
ERG
ERG
Chomosome ?
Figure 1. Schematic representation of mechanisms of ERG rearrangement in prostate cancer. TMPRSS2 and ERG are located less than 3‑Mb apart on chromosome 21, and TMPRSS2–ERG fusions can occur either through interchromosomal translocation or deletion of the intervening region on chromosome 21. In a FISH break-apart system, two differentially labeled FISH probes, one spanning the neighboring 3´ centromeric (red signal) and another the 5´ telomeric region (green signal) of the ERG locus have commonly been used. According to this break-apart probe system, a nucleus without ERG rearrangement shows two pairs of juxtaposed red and green signals, forming yellow signals; a nucleus with ERG rearrangement through translocation shows one pair of still-combined red and green signals (a yellow signal), while the other (split signal) breaks into a single red (rearranged ERG gene on chromosome 21) and a single green signal (translocated to an unknown chromosome); and a nucleus with ERG rearrangement through deletion shows one yellow signal and a single red signal, while the green signal (5´ region) is lost. ‘?’ indicates an unknown chromosome.
been identified [13], arising from different combinations of TMPRSS2 exons 1, 2 and 3 with ERG exons 2, 3, 4, 5 and 6 in various alternative splicing patterns. TMPRSS2–ERG fusion by FISH is highly specific for PCa: FISH signal patterns of ERG rearrangement have never been detected in nonneoplastic prostate tissue samples [2,14], although detection of fusion transcripts by PCR has occasionally been reported in non-neoplastic prostate tissue [13,15]. In addition, in a recent study by Scheble et al. FISH signal patterns specific for ERG rearrangements have not been found in any of 54 different tumor types other than PCa, including several samples of common epithelial and nonepithelial neoplasms, such as colonic adenocarcinomas, urothelial carcinomas, mesotheliomas, hemangiomas and Kaposi’s sarcomas [16]. Studies have also documented that rearrange ments of ERG at the chromosomal level are seen in 15–21% of high-grade prostatic intraepithelial neoplasia (HGPIN) associated with PCa, 852
Biomarkers Med. (2013) 7(6)
demonstrating gene fusions identical to the corresponding PCa [17–19]. Mosquera et al. found TMPRSS2–ERG fusion in 23 (16%) of 143 HGPIN cases, 22 of which associated with equally rearranged PCa, and one case without concurrent adenocarcinoma [18]. The isolated fusion-positive HGPIN was observed on a prostate biopsy with no follow-up available at the time of the study. Clinically localized PCa is typically a multi focal disease. At the genomic level, a higher incidence of interfocal (between different tumor foci) versus intratumoral (within a single focus) heterogeneity for TMPRSS2–ERG fusions has been reported [20,21]. Interfocal heterogeneity for the fusion status has been reported in 41% [22] to 51% of multifocal PCa [21]. Meanwhile, intratumoral homogeneity has been detected in virtually all cases [3,21] except one (3%) [22]. Perner et al. reported homogeneity in 99% of discrete PCa nodules evaluated regardless of Gleason score [2]. Falzarano and colleagues demonstrated intratumoral homogeneity for rearrangement future science group
ERG protein expression as a biomarker of prostate cancer
status in 95% of cases of single-focus PCa evaluated by FISH [20]. It has been hypothesized that, in multifocal disease, a rearranged primary focus may have a growth advantage and become capable of dissemination, giving rise to metastatic disease. All metastatic PCa retains a rearrangement status similar to the primary focus, suggesting that ETS rearrangement occurs before progression to metastatic disease [3,23]. Recently, Perner et al. investigated 26 patients who underwent prostatectomy and lymphadenectomy with at least two distinct PCa foci and one lymph node (LN) metastasis [24]. In all cases with at least one ERG rearranged focus, they found the corresponding LN metastasis harboring an ERG rearrangement. Interestingly, in a subset of cases the rearrangement status in the LN did not correspond with the largest or highest Gleason score tumor. On the other hand, Guo and colleagues found concordance of ERG rearrangement status between the index (largest) tumor and the corresponding LN metastases, suggesting that metastasis most likely arises from the index tumor in multifocal PCa [25]. Studies exploring the potential role of TMPRSS2–ERG fusion in prostate tumori genesis and tumor progression reported controversial results, with overexpression of ERG being found to induce a neoplastic phenotype in prostate cells (mouse prostatic intraepithelial neoplasia [PIN]) in some studies [26,27], but not in others [28,29]. Other molecular events in combination with ERG overexpression, such as PTEN loss and PI3K pathway activation with AKT overexpression, may be implicated in the neoplastic transformation and progression of HGPIN to invasive adenocarcinoma capable of metastatic dissemination [28,29].
Immunohistochemical detection of ERG rearrangement protein product In the most commonly detected fusion transcripts of TMPRSS2–ERG -positive cases, TMPRSS2 contributes only untranslated sequences, thus resulting in the overproduction of a truncated ERG protein product, rather than a chimeric protein [1,26]. Several independent studies have addressed the immunohistochemical (IHC) detection of the fusion product as surrogate for TMPRSS2–ERG gene fusions by testing two different monoclonal antibodies (mAbs; Table 1) showing positive nuclear immunostaining [12,30–36]. Furusato and colleagues developed a mouse anti-ERG mAb targeting an epitope at the future science group
Review
N‑terminus of ERG protein (CPDR, clone 9FY) [32]. The antibody was generated against an immunizing polypeptide GQTSKMSPRV PQ QDWL SQPPARVTI (amino acids 49–73). There was no significant homology of the ERG mAb peptide antigen with 29 other protein sequences belonging to the human ETS family. Notably, the FLI1 protein sequence, which showed 48% identity with the ERG-immunizing peptide, was not recognized by the ERG mAb [32]. The authors tested the antibody on wholemount sections of 132 prostates and found it to be highly specific in distinguishing PCa foci from benign glands. The authors also noticed a strong concordance rate of 82.8% between mRNA levels of fusion transcripts detected by branched-chain DNA signal amplification (35 cases) and ERG immunohistochemistry, as well as between ERG rearrangement detected by FISH (ten cases) and ERG protein expression (100% specificity and sensitivity). Park et al. characterized a novel rabbit antiERG mAb (clone EPR3864, also know as EP111) directed against the C‑terminus of the most common gene fusion product [33]. In this study, the antibody epitope was identified in the C-terminal amino acids 393–479 of the ERG protein [33]. They found that ERG protein expression determined by IHC with this antibody highly correlated (95.7% sensitivity and 96.5% specificity) with the ERG rearrangement status determined by FISH on PCa tissue microarrays from two PCa patient cohorts. These results were subsequently confirmed by other studies, as summarized in Table 1 [12,30,31,34–36]. Braun et al. compared the ERG protein expression detected by the two antibodies against each other and against the ERG rearrangement status assessed by FISH [30]. They found a highly significant correlation between the ERG protein expression and the gene rearrangement status for both the rabbit and the mouse anti-ERG antibodies (Pearson’s correlation coefficient: 0.971 and 0.956, respectively; p
ERG protein expression as a biomarker of prostate cancer TMPRSS2–ERG is a recurrent rearrangement specific for prostate cancer, leading to the overexpression of a truncated ERG protein product that is amenable to immunohistochemical detection. Two monoclonal anti-ERG antibodies have currently been validated, with comparable sensitivity and specificity for detecting ERG rearrangement. ERG immunostaining has been applied in different settings to elucidate the role of ERG rearrangement and overexpression in prostate cancer tumorigenesis and progression, as well as to investigate potential diagnostic and prognostic applications. In this article we review the literature on the topic and suggest potential future applications. KEYWORDS: ERG immunohistochemistry n genomic rearrangements n prostate cancer n TMPRSS2–ERG
Recurrent chromosomal rearrangements leading to the fusion of the androgen receptor (AR)regulated TMPRSS2 gene and members of the ETS transcription factor family have been discovered in prostate cancer (PCa) using an unconventional analytical approach [1]. ERG is the most common fusion partner [2] detected in approximately 50% of PSA-screened PCas [3]. The genetic alteration leads to the juxtaposition of androgen-responsive regulatory elements of TMPRSS2 to the ERG sequence, with consequent overexpression of the rearranged ERG [1,4]. Oncogenic activation of other members of the ETS family (ETV1, ETV4 and ETV5) accounts for less than 10% of PCa rearrangements and demonstrate mutual exclusivity with ERG rearrangement [1,5]. Depending on factors such as cohort design (PSA screened vs population based) [6], tumor origin (transitional vs peripheral zone) [6,7] and histologic variants [8–11], ERG rearrangements occur in 15–80% of PCa.
genomic level ERG rearrangements have been extensively investigated by FISH and PCR [2,5]. A dualcolor interphase break-apart FISH assay has been applied in several studies, performed on formalin-fixed paraffin-embedded tissue to assess ERG gene rearrangement status [1,2]. Brief ly, two differentially labeled bacterial artificial chromosome (BAC) FISH probes have commonly been used: a biotin-14-dCTPlabeled BAC clone, such as RP11‑24A11 (eventually conjugated to produce a red signal), for
the neighboring 3´ centromeric region of the ERG locus and a digoxigenin-dUTP-labeled BAC clone, such as RP11‑372O17 (eventually conjugated to produce a green signal), for the neighboring 5´ telomeric region. According to this break-apart probe system, a nucleus without ERG rearrangement shows two pairs of juxtaposed red and green signals, forming yellow signals; a nucleus with ERG rearrangement through translocation shows one pair of still combined red and green signals (a yellow signal), while the other breaks into a single red and a single green signal (split signal); and a nucleus with ERG rearrangement through deletion shows one yellow signal and a single red signal, while the green signal (5´ region) is lost (Figure 1). Cases with ERG signal abnormality in >10% of the neoplastic cells have usually been scored as positive, and classified accordingly [7]. In addition, the presence of multiple copies of the ERG rea rrangement sequence, with or without retention of the green signal, has been commonly assessed [12]. Tissue hybridization, washing, and fluorescence detection have been previously described elsewhere [12]. Samples are analyzed under a 60× or 100× oil immersion objective using a fluorescence microscope equipped with appropriate filters. The corresponding hematoxylin and eosin section is used for side-by-side comparison with the FISH section. PCR detection of TMPRSS2–ERG fusion mRNAs has been used alternatively to assess ERG gene rearrangement status in PCa cell lines, tissue specimens and biologic fluids [1]. By reverse-transcriptase PCR, at least 17 distinct TMPRSS2–ERG hybrid transcripts have
10.2217/BMM.13.105 © 2013 Future Medicine Ltd
Biomarkers Med. (2013) 7(6), 851–865
ERG rearrangement detection at
Sara Moscovita Falzarano1 & Cristina Magi-Galluzzi*1 R.T. Pathology & Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, L25, Cleveland, OH 44195, USA *Author for correspondence: Tel.: +1 216 444 9251 Fax: +1 216 445 6967 [email protected] 1
part of
ISSN 1752-0363
851
Review
Falzarano & Magi-Galluzzi
Chomosome 21 q22.3
q22.2
FISH probes Spanning the 5´ end of ERG Spanning the 3´ end of ERG
No rearrangement TMPRSS2
ERG
TMPRSS2
ERG
Rearrangement through deletion
TMPRSS2 TMPRSS2
Rearrangement through translocation
TMPRSS2
ERG ERG
TMPRSS2
ERG
ERG
Chomosome ?
Figure 1. Schematic representation of mechanisms of ERG rearrangement in prostate cancer. TMPRSS2 and ERG are located less than 3‑Mb apart on chromosome 21, and TMPRSS2–ERG fusions can occur either through interchromosomal translocation or deletion of the intervening region on chromosome 21. In a FISH break-apart system, two differentially labeled FISH probes, one spanning the neighboring 3´ centromeric (red signal) and another the 5´ telomeric region (green signal) of the ERG locus have commonly been used. According to this break-apart probe system, a nucleus without ERG rearrangement shows two pairs of juxtaposed red and green signals, forming yellow signals; a nucleus with ERG rearrangement through translocation shows one pair of still-combined red and green signals (a yellow signal), while the other (split signal) breaks into a single red (rearranged ERG gene on chromosome 21) and a single green signal (translocated to an unknown chromosome); and a nucleus with ERG rearrangement through deletion shows one yellow signal and a single red signal, while the green signal (5´ region) is lost. ‘?’ indicates an unknown chromosome.
been identified [13], arising from different combinations of TMPRSS2 exons 1, 2 and 3 with ERG exons 2, 3, 4, 5 and 6 in various alternative splicing patterns. TMPRSS2–ERG fusion by FISH is highly specific for PCa: FISH signal patterns of ERG rearrangement have never been detected in nonneoplastic prostate tissue samples [2,14], although detection of fusion transcripts by PCR has occasionally been reported in non-neoplastic prostate tissue [13,15]. In addition, in a recent study by Scheble et al. FISH signal patterns specific for ERG rearrangements have not been found in any of 54 different tumor types other than PCa, including several samples of common epithelial and nonepithelial neoplasms, such as colonic adenocarcinomas, urothelial carcinomas, mesotheliomas, hemangiomas and Kaposi’s sarcomas [16]. Studies have also documented that rearrange ments of ERG at the chromosomal level are seen in 15–21% of high-grade prostatic intraepithelial neoplasia (HGPIN) associated with PCa, 852
Biomarkers Med. (2013) 7(6)
demonstrating gene fusions identical to the corresponding PCa [17–19]. Mosquera et al. found TMPRSS2–ERG fusion in 23 (16%) of 143 HGPIN cases, 22 of which associated with equally rearranged PCa, and one case without concurrent adenocarcinoma [18]. The isolated fusion-positive HGPIN was observed on a prostate biopsy with no follow-up available at the time of the study. Clinically localized PCa is typically a multi focal disease. At the genomic level, a higher incidence of interfocal (between different tumor foci) versus intratumoral (within a single focus) heterogeneity for TMPRSS2–ERG fusions has been reported [20,21]. Interfocal heterogeneity for the fusion status has been reported in 41% [22] to 51% of multifocal PCa [21]. Meanwhile, intratumoral homogeneity has been detected in virtually all cases [3,21] except one (3%) [22]. Perner et al. reported homogeneity in 99% of discrete PCa nodules evaluated regardless of Gleason score [2]. Falzarano and colleagues demonstrated intratumoral homogeneity for rearrangement future science group
ERG protein expression as a biomarker of prostate cancer
status in 95% of cases of single-focus PCa evaluated by FISH [20]. It has been hypothesized that, in multifocal disease, a rearranged primary focus may have a growth advantage and become capable of dissemination, giving rise to metastatic disease. All metastatic PCa retains a rearrangement status similar to the primary focus, suggesting that ETS rearrangement occurs before progression to metastatic disease [3,23]. Recently, Perner et al. investigated 26 patients who underwent prostatectomy and lymphadenectomy with at least two distinct PCa foci and one lymph node (LN) metastasis [24]. In all cases with at least one ERG rearranged focus, they found the corresponding LN metastasis harboring an ERG rearrangement. Interestingly, in a subset of cases the rearrangement status in the LN did not correspond with the largest or highest Gleason score tumor. On the other hand, Guo and colleagues found concordance of ERG rearrangement status between the index (largest) tumor and the corresponding LN metastases, suggesting that metastasis most likely arises from the index tumor in multifocal PCa [25]. Studies exploring the potential role of TMPRSS2–ERG fusion in prostate tumori genesis and tumor progression reported controversial results, with overexpression of ERG being found to induce a neoplastic phenotype in prostate cells (mouse prostatic intraepithelial neoplasia [PIN]) in some studies [26,27], but not in others [28,29]. Other molecular events in combination with ERG overexpression, such as PTEN loss and PI3K pathway activation with AKT overexpression, may be implicated in the neoplastic transformation and progression of HGPIN to invasive adenocarcinoma capable of metastatic dissemination [28,29].
Immunohistochemical detection of ERG rearrangement protein product In the most commonly detected fusion transcripts of TMPRSS2–ERG -positive cases, TMPRSS2 contributes only untranslated sequences, thus resulting in the overproduction of a truncated ERG protein product, rather than a chimeric protein [1,26]. Several independent studies have addressed the immunohistochemical (IHC) detection of the fusion product as surrogate for TMPRSS2–ERG gene fusions by testing two different monoclonal antibodies (mAbs; Table 1) showing positive nuclear immunostaining [12,30–36]. Furusato and colleagues developed a mouse anti-ERG mAb targeting an epitope at the future science group
Review
N‑terminus of ERG protein (CPDR, clone 9FY) [32]. The antibody was generated against an immunizing polypeptide GQTSKMSPRV PQ QDWL SQPPARVTI (amino acids 49–73). There was no significant homology of the ERG mAb peptide antigen with 29 other protein sequences belonging to the human ETS family. Notably, the FLI1 protein sequence, which showed 48% identity with the ERG-immunizing peptide, was not recognized by the ERG mAb [32]. The authors tested the antibody on wholemount sections of 132 prostates and found it to be highly specific in distinguishing PCa foci from benign glands. The authors also noticed a strong concordance rate of 82.8% between mRNA levels of fusion transcripts detected by branched-chain DNA signal amplification (35 cases) and ERG immunohistochemistry, as well as between ERG rearrangement detected by FISH (ten cases) and ERG protein expression (100% specificity and sensitivity). Park et al. characterized a novel rabbit antiERG mAb (clone EPR3864, also know as EP111) directed against the C‑terminus of the most common gene fusion product [33]. In this study, the antibody epitope was identified in the C-terminal amino acids 393–479 of the ERG protein [33]. They found that ERG protein expression determined by IHC with this antibody highly correlated (95.7% sensitivity and 96.5% specificity) with the ERG rearrangement status determined by FISH on PCa tissue microarrays from two PCa patient cohorts. These results were subsequently confirmed by other studies, as summarized in Table 1 [12,30,31,34–36]. Braun et al. compared the ERG protein expression detected by the two antibodies against each other and against the ERG rearrangement status assessed by FISH [30]. They found a highly significant correlation between the ERG protein expression and the gene rearrangement status for both the rabbit and the mouse anti-ERG antibodies (Pearson’s correlation coefficient: 0.971 and 0.956, respectively; p
