Advances in Anatomic Pathology:
The Discovery of Common Recurrent Transmembrane Protease Serine 2 (TMPRSS2)-Erythroblastosis Virus E26 Transforming Sequence (ETS) Gene Fusions in Prostate Cancer: Significance and Clinical Implications
Shah, Rajal B. MD* † ‡ §; Chinnaiyan, Arul M. MD, PhD* † ‡ § ∥
Departments of *Pathology
†Michigan Center for Translational Pathology
∥Howard Hughes Medical Institute
§Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI
Reprints: Rajal B. Shah, MD, Department of Pathology, University of Michigan Medical School,1500 East Medical Center Drive, 2G337 UH, Ann Arbor, MI 48109 (e-mail: firstname.lastname@example.org).
Recurrent gene fusions and chromosomal rearrangements were previously thought to be the primary oncogenic mechanism of hematological malignancies and sarcomas. The recent discovery of recurrent gene fusions in a majority of prostate cancers represents a paradigm shift in understanding the molecular mechanisms of one of the most prevalent epithelial malignancies, with important clinical and biologic implications. The prostate cancer gene fusions that have been identified so far are characterized by 5′-genomic regulatory elements, most notably the androgen-controlled prostate specific gene, transmembrane protease serine 2, fused to members of the erythroblastosis virus E26 transforming sequence family of transcription factors, most notably ERG, leading to the overexpression of oncogenic transcription factors. The erythroblastosis virus E26 transforming sequence gene fusions most likely define a distinct class of prostate cancer with potential implications for early diagnosis, prognosis, and rational therapeutic targeting. In this review, we summarize the bioinformatics approach that led to the discovery of gene fusions, the current state of the frequency, and diversity of gene fusions that define the molecular heterogeneity of prostate cancer, their associations with prostate cancer progression and clinical outcome, the subsequent morphological characteristics, and the potential application of gene fusions as biomarkers in the diagnosis and management of prostate cancer.
BIOINFORMATICS APPROACH LEADS TO THE DISCOVERY OF COMMON PROSTATE GENE FUSIONS—A PARADIGM SHIFT
Alterations of gene expression and subsequent function, whether through the activation of oncogenes or loss of tumor suppressor genes, are a hallmark of malignancy development. These alterations often result from chromosomal translocations or deletions of genomic segments that result in modified gene expression or fusion of 2 distinct gene transcripts. Such chromosomal structural rearrangements are common in hematologic malignancies and often function as critical trigger points in oncogenesis.1–3 A classic example of this mechanism, the translocation of the BCR gene from chromosome 22 with the ABL gene of chromosome 9, yielding the “Philadelphia chromosome”, results in the production of a BCR-ABL fusion protein with a constitutively active tyrosine kinase domain that drives the development of chronic myeloid leukemia.4
Epithelial malignancies (carcinomas), which are the most common human cancers contributing to a large fraction of morbidity and mortality, comprised less than 1% of the known, disease specific chromosomal rearrangements. Mitelman et al5 in 2004, hypothesized that one of the major limitations in the discovery of recurrent rearrangements in solid tumors lies in the difficulty of growing primary tumor cells in culture, limiting their karyotype analyses. In addition, solid tumor samples harbor many nonspecific chromosomal changes that may be hindering the detection of underlying recurrent chromosomal alterations. Recent advances in genomic profiling, through the use of microarrays, spectral karyotyping, and competitive genomic hybridization, have coupled with the emerging field of bioinformatics to uncover many findings, which were not evident with standard analysis techniques.6 Recurrent chromosomal rearrangements in prostate cancer were discovered by our group through the power of an unconventional bioinformatics approach termed as the “Cancer Outlier Profile Analysis” (COPA) algorithm to analyze DNA microarray studies.7 COPA is based on 3 basic ideas: (1) chromosomal rearrangements and amplifications may result in marked overexpression of involved genes, (2) such alterations are often heterogeneous in a given cancer type, and (3) the altered gene expression in a subset of samples may be overshadowed when analyzing DNA microarray studies using standard analytical approaches (eg, a 2 class t-test method).7–9
The results of COPA analysis of prostate cancer profiling studies identified erythroblastosis virus E26 transforming sequences (ETS), specifically variant gene 1 (ETV1), and v-ETS erythroblastosis virus E26 oncogene-like (ERG) as outliers in a fraction of cases with COPA scores in the top 10 of 6 independent prostate profiling studies.7,8 ETV1 (7q21.2) and ERG (21q22.3) are genes from the ETS family of transcription factors and have been previously implicated in oncogenic translocations in Ewing sarcoma and acute myeloid leukemia.10,11 They were overexpressed in the majority (50% to 70%) of prostate cancers and were mutually exclusive across several independent gene expression data sets. To determine whether the structural rearrangements responsible for the overexpression of ETV1 and ERG, the RNA from prostate cancer samples was characterized quantitatively using a polymerase-chain reaction (PCR)-based test. This technique consistently showed a loss of the 5′ region of ETV1 or ERG for cases with marked overexpression of the 3′ end. Then, using a variety of molecular techniques, it was subsequently demonstrated that the 5′ end of ETV1 or ERG was consistently replaced with the 5′-untranslated region of the androgen regulated prostate specific gene, transmembrane protease serine 2 (TMPRSS2; 21q22.2), creating the fusion of 5′ TMPRSS2 to 3′ ETS partners ETV1 or ERG in cases with overexpression of these genes.7 Most often, these fusions juxtapose a hormonally specific promoter with an oncogene. The presence of hormonally regulated promoter acts as an “on switch” for the oncogene, conferring a distinct biology to this tumor, likely in collaboration with other molecular events (Fig. 1).
The identification of recurrent gene fusions in prostate cancer has defined a new paradigm for understanding the biology of prostate cancer development, with likely implications for diagnosis and management of prostate cancer.
FREQUENCY AND DIVERSITY OF GENE FUSIONS DEFINE MOLECULAR HETEROGENEITY OF PROSTATE CANCER
Since its original discovery in 2005,7 multiple independent studies have subsequently validated the initial observation that TMPRSS2-ETS fusions are common in prostate cancer.12–26 Except for a few studies aimed at comprehensive screening of 5′ partners and 3′ ETS partners,17,27 most studies have focused on the dominant rearrangement TMPRSS2-ERG fusion, which is reported with varying frequency, ranging from a low of ∼40% to as high as ∼70% in different hospital-based surgical cohorts. In contrast to an average frequency of 50% in serum prostate-specific antigen (PSA) screened hospital-based cohorts, the reported frequency of TMPRSS2-ERG in population-based cohorts is much lower. In 2 independent Swedish and United Kingdom based population cohorts of patients who underwent expectant management, TMPRSS2-ERG rearrangement was found in only about 15% to 20% of patients.13,28 To determine whether the higher frequency observed in the surgical cohorts is owing to selection bias, the frequency of TMPRSS2-ERG fusion was prospectively determined in men with prostate cancer, detected on 12-core needle biopsies by PSA screening as part of an Early Detection Research Network study sponsored by the National Cancer Institute. In this cohort, 46% of men with prostate cancer harbored the TMPRSS2-ERG fusion, consistent with the frequency observed in the surgical cohorts. (Mosquera et al, submitted).
As TMPRSS2 and ERG are located ∼3 Mb apart on chromosome 21, the rearrangement between these 2 partners occurs through either a translocation between chromosome 21s or through interstitial deletion (Edel).20,26 In several independent studies, the frequency of TMPRSS2-ERG rearrangement through the Edel has been reported to range from 39% to 80%.17,20,23,26,29,30 Initial observations suggest that the TMPRSS2-ERG rearrangement through Edel represents an aggressive molecular subtype of prostate cancer.20,30
Although high-throughput fluorescent in situ hybridization (FISH) assays using tissue microarray sections have been useful in screening large number of samples to determine the prevalence of recurrent gene fusions in prostate cancer, the characterization of fusion transcripts by Rapid Amplification of cDNA End and reverse transcription (RT) PCR has revealed a range of fusion transcript variants. The most common variants involve TMPRSS2 exon 1 or 2 fused to ERG exon 2, 3, 4, or 5. Less frequent combinations include TMPRSS2 exon 4 or 5 fused to ERG exon 4 or 5. Although the clinical significance of these variants is poorly understood, some TMPRSS2-ERG fusion variants have been associated with prognostic outcomes.22–24,31
In addition to the most common TMPRSS2-ERG rearrangements, several other novel 5′ and 3′ partners have been discovered, which comprise about 5% to 10% of all prostate cancers.7,17,27,31–35 The frequency of these gene fusions and their respective 5′ and 3′ partners are summarized in Table 1. We recently generated a comprehensive profile of the rearrangement status of all 27 ETS family genes and all 5 of the known 5′ fusion partners in prostate cancer using FISH split-probe hybridizations on a transcription-mediatedamplificationcomposedof 110 cases of clinically localized prostate cancer. Rearrangement of ERG, ETV1, and ETV4 was found in 43%, 5%, and 5% of prostate cancers, respectively. Overall these observations suggest that the majority of prostate cancers (∼50%) detected by PSA-screening harbor either the common TMPRSS2-ERG fusion or one of the less common ETS fusions involving TMPRSS2 or other 5′ partners (5% to 10%). In addition, these results also suggest that a large fraction of prostate cancers (∼40%) can not be ascribed to an recurrent nonETS aberrations in prostate cancer.27,36
TMPRSS2-ERG FUSIONS IN MULTIFOCAL PROSTATE CANCER SUGGESTS THAT MULTIFOCAL PROSTATE CANCER IS AN INDEPENDENT GROUP OF DISEASES
Localized prostate cancer is typically a multifocal disease, generally consisting of a dominant (index) tumor and 1 or multiple separate smaller tumors. Multifocal prostate cancers frequently display histologic and molecular heterogeneity.29,37 We evaluated TMPRSS2 rearrangement status in 93 multifocal tumor foci from 43 radical prostatectomy specimens. Overall, 70% of these cases were found to have TMPRSS2 rearrangement, 63% through deletion, 27% through translocation, and 10% through both mechanisms. Individual tumor cells within each tumor focus were homogeneous for the TMPRSS2 rearrangement. Similarly, Perner et al20 reported that 243 out of 246 prostate cancer cases demonstrated homogeneity within a discrete tumor nodule. In another study, examining prostate cancer progression, we reported that multiple, microdissected foci of cancer from individual patients examined by RT-PCR for gene fusion had either all or no foci that expressed ERG and its family members, ETV1 and ETV4.38 In contrast, further attesting to the heterogeneity of multifocal cancers, 70% of the cases, rearranged for TMPRSS2 demonstrated divergent rearrangements in the different tumor foci, suggesting that multifocal prostate cancer is a heterogeneous group of diseases arising from multiple, independent clonal expansions.29 The heterogeneity of multifocal prostate cancer was also observed in 2 other independent studies.39,40
Understanding the clinical and molecular heterogeneity of prostate cancer is critical to the future development of adequate diagnostic and prognostic biomarkers and potentially represents a hurdle in drug development.
THE ASSOCIATION OF TMPRSS2-ETS GENE FUSIONS DURING PROSTATE CANCER PROGRESSION SUGGEST THAT IT IS AN EARLY GENETIC EVENT, AND THAT METASTATIC PROSTATE CANCER ARISES THROUGH CLONAL EXPANSION OF A SINGLE FOCUS OF PRIMARY PROSTATE CANCER
Prostate cancer, like other cancers, develops in the context of multiple, complex molecular events of initiation, unregulated growth, invasion, and metastasis.38,41 Distinct sets of key genes and proteins likely dictate progression from the putative precursor lesion, termed as high-grade prostatic intraepithelial neoplasia (HGPIN), to hormone naive clinically localized cancer, and finally to androgen independent metastatic disease that is invariably fatal.38 The molecular basis of prostate cancer progression is complex and likely involves several key pathways in which the androgen receptor plays a central role.38
Emerging data strongly suggests that the TMPRSS2-ERG fusion is an early event in prostate cancer and plays an important role in carcinogenesis in vitro and in vivo.7,12,20,42–45 Several studies using FISH assays have consistently demonstrated that gene fusions are invariably observed only in the neoplastic nuclei, but not in nuclei from benign epithelial cells or stromal cells.7,20,45 Using a large cohort of a spectrum of benign prostate samples, Perner et al43 observed the TMPRSS2-ERG fusion in approximately 20% of HGPIN lesions intermingled with prostate cancer that carried the same fusion pattern. Cerveira et al12 using an RT-PCR based assay, reported a similar frequency. TMPRSS2-ERG fusion was not observed in HGPIN lesions geographically distant to prostate cancer, even if the prostate cancer from the same individual demonstrated the TMPRSS2-ERG fusion.12 Similarly, they did not observe any fusions in samples of benign prostate hyperplasia or atrophy lesions, including other putative precursor lesions termed as “proliferative inflammatory atrophy.”43 These data strongly suggests that these HGPIN lesions are a subset of true neoplastic precursors for TMPRSS2-ERG positive prostate cancer (Fig. 1).
Recently, to better understand the role of ERG and another commonly altered genomic event, phosphatase and tension homolog (PTEN), in prostate cancer progression, we analyzed the frequency of ERG and PTEN aberrations in a cohort of 73 benign prostate tissues, 89 HGPIN foci, 282 localized prostate cancers, and 47 androgen-independent metastatic prostate cancer patients. Overall ERG rearrangement was present in 15% of HGPIN, 47% of localized prostate cancer, and 35% of metastases, whereas PTEN deletion was identified in 9% of HGPIN, 17% of localized prostate cancer, and 54% of metastasis. Similar to previous observations, 100% of HGPIN lesions rearranged for ERG were located adjacent to a cancer focus demonstrating a similar gene fusion type. Isolated HGPIN lesions were uniformly negative for gene fusion. In addition, despite well-known morphologic, immunophenotypic, and genotypic heterogeneity of metastatic prostate cancers,46 all foci from individual cases of metastatic cancer were uniformly ERG-rearrangement negative or positive, similar to primary prostate cancer, indicating that ERG rearrangement occurs before progression to metastatic disease (Han Bo et al, submitted).
In summary, emerging data suggests that ETS rearrangements, in collaboration with other critical molecular events, play an important role in prostate cancer initiation and progression. Clonal expansion of a single focus of primary prostate cancer capable of dissemination eventually gives rise to metastatic prostate cancer.30 The potential role of TMPRSS2-ETS gene fusions in prostate cancer progression pathways in collaboration with other potential molecular events is summarized in Figure 1.
ASSOCIATION WITH PROSTATE CANCER OUTCOMES
With the widespread use of PSA screening, over 90% of prostate cancers diagnosed in American men were clinically localized.47 These clinically localized cancers are biologically diverse, ranging from clinically indolent tumors to a subset of aggressive tumors with a potential for recurrence and metastasis.48,49 Biomarkers that can accurately identify and stratify these prognostic subsets of clinically localized cancers currently remain as a major field of investigation. Similar to hematologic malignancies, molecular subtypes of gene fusion prostate cancers are likely to provide significant prognostic information, and recently the influence of fusion status on cancer outcome has become a subject of investigation.
TMPRSS2-ERG has been frequently, but not unequivocally, associated with poor prognosis or aggressive prostate cancers. TMPRSS2-ERG rearrangement was variously associated with high pathologic stage17 and higher rate of PSA recurrence19 in independent cohorts of surgically treated localized prostate cancer cases, and in some studies the presence of gene fusion was scored as the single most important prognostic factor.19
In a FISH based analysis of 445 cancer cases, the absence of the ERG fusion was found to be a good prognostic factor (90% survival at 8 y), as compared with cancers with a duplication of TMPRSS2-ERG in combination with deletion of 5′-ERG (2+ Edel), which exhibited poor cause specific survival (25% survival at 8 y).50 This novel category of 2+ Edel status was found to be independent of other prognostic factors, including Gleason scores and serum PSA levels. In a population-based, watchful waiting cohort of men with localized prostate cancer, tumors from just 15% of the patients were found to harbor the TMPRSS2-ERG fusion. Remarkably, this fusion-positive subset was significantly associated with prostate cancer specific death.13 In another study involving primary prostate cancers and hormone naive lymph node metastasis, a significant association was observed between TMPRSS2-ERG rearranged tumors through Edels and higher tumor stage, as well as the presence of metastatic disease involving pelvic lymph nodes.20 Similarly Rajput et al21 observed more frequent TMPRSS2-ERG fusions in moderately to poorly-differentiated tumors compared with well-differentiated tumors. In a cohort of patients treated for clinically localized prostate cancer, Nami et al19 observed that the TMPRSS2-ERG fusion-positive subgroup of patients had a significantly higher risk of recurrence (58.4% at 5 y) than fusion-negative patients (8.1%). Wang et al24 suggested that the clinical significance of gene fusions might be related to the splice variants of expressed TMPRSS2/ERG transcripts, rather than presence of rearrangements alone. They observed a total of 8 different isoforms, of which certain isoforms were associated with clinical and pathological variables of aggressive disease. Recently, we comprehensively characterized TMPRSS2-ETS gene aberrations in androgen-independent metastatic lethal prostate cancers. The most common prostate cancer gene fusion, TMPRSS2-ERG, has been reported to be generated by the mechanism of Edel approximately 39% to 60% of the time in clinically localized prostate cancers. Interestingly, we observed that all of the androgen-independent metastatic prostate cancer sites harboring TMPRSS2-ERG were associated with Edel.30 These findings suggest that TMPRSS2-ERG with Edel is an aggressive molecular subtype of prostate cancer.
Conversely, many studies have also reported an absence of such a clinical correlation or even negative correlation between the fusions and prognosis. In a report preceding the discovery of gene fusions, Petrovics et al51 had associated ERG-overexpressing prostate cancers with several positive prognosticators, such as long recurrence-free survival, well and moderately differentiated tumors, lower pathologic stage, and negative surgical margins. Winnes et al25 recently made similar observations with respect to gene fusion-positive prostate cancers, and they reported “a clear tendency” for fusion-positive tumors to be associated with lower Gleason grade and better survival than fusion-negative tumors. Yoshimoto et al26 found no correlation between clinical outcome and presence of gene fusions, and Lapointe et al16 found no significant association between the presence of a TMPRSS2-ERG fusion and tumor stage, Gleason grade or recurrence free survival.
It must be noted that these negative studies have relatively small sample sizes. Intertumor heterogeneity observed in multifocal tumors, as discussed earlier, could be another possible explanation for the discrepancy observed between positive and negative studies. Clearly, more studies with larger patient cohorts and a standardized approach to this biomarker analysis would help to resolve specific prognostic association of the gene fusions. A summary of the published studies demonstrating prognostic associations of TMPRSS2-ERG fusion is shown in Table 2.
MORPHOLOGIC FEATURES OF GENE FUSION-POSITIVE PROSTATE CANCERS
It is well documented that carcinomas with specific germline or somatic alterations show a combination of recognizable histopathologic features. For example, microsatellite unstable colon cancer, as seen in hereditary nonpolyposis colorectal cancer, is characterized by poorly differentiated tumor cells, an expanding growth pattern with pushing borders, a pronounced lymphocytic reaction with tumor infiltrating lymphocytes, and the lack of dirty necrosis.52,53 Breast cancers associated with BRCA1 or BRCA2 germline mutations frequently show morphologic features that can predict the presence of underlying genetic aberrations.54,55 Similarly, renal-cell carcinomas associated with Xp11 translocation demonstrate a combination of morphologic features that may predict the presence of the underlying molecular genetic abnormality.56,57
Mosquera et al18 identified 5 morphologic features: blue-tinged mucin, cribriform growth pattern, macro nucleoli, intraductal tumor spread, and signet-ring cell morphology to be significantly associated with prostate cancer samples with TMPRSS2-ERG fusion. Samples that harbored 3 or more of these features were almost always (93%) fusion-positive and only 24% of fusion-positive samples did not display any of these morphological features. These features were recently validated as part of a prospective Early Detection Research Network study to explore the clinical role of TMPRSS2-ETS gene fusions in prostate cancer. (Mosquera et al, submitted). Tu et al23 also noted a significantly higher frequency of TMPRSS2-ETS gene fusions in mucin-positive carcinomas than mucin-negative tumors. Additional larger follow-up studies may help to further establish the phenotypic associations of the gene fusions.
TMPRSS2-ETS GENE FUSIONS IN UNUSUAL VARIANT MORPHOLOGIES OF PROSTATE CANCER
Histologic variants of prostate carcinoma such as mucinous, large duct, foamy, and small-cell carcinoma account for about 5% to 10% of all prostate cancers and are typically seen in association with ordinary acinar prostate cancer. These variants often differ from the latter in clinical, immunophenotypic, and genetic features as well as biologic potential. Recently, to understand the frequency, molecular subtype, and clonality of common gene fusions in histologic variants in comparison to conventional acinar prostate cancer, we comprehensively analyzed gene rearrangement for the major ETS family members and their known 5′-fusion partner TMPRSS2 in these variant cancer morphologies. (Han Bo et al, submitted). Overall 55% of variant cancers demonstrated TMPRSS2-ETS aberrations. ERG, the most common genetic rearrangement observed in acinar prostate cancer, was identified in 82%, 71%, 56%, 33%, and 29% of mucinous, small cell, large duct, glomeruloid, and foamy variant morphologies, respectively. This data again emphasize possible association of phenotype and underlying genetic abnormality. ERG rearrangement through intronic deletion of the 5′ end was observed exclusively (100%) in small cell prostate cancer, providing additional evidence that TMPRSS2-ERG rearrangement through Edel may represent an aggressive molecular subtype of prostate cancer. The concordance of gene rearrangement between variant morphology and paired prostate cancer was observed in 91% of cases, suggesting that variant morphologies represent a clonal expansion of primary acinar prostate cancer.
APPLICATION IN THE EARLY DIAGNOSIS AND MANAGEMENT OF PROSTATE CANCER
PSA has been used extensively to screen for prostate cancer in the United States, based on early studies showing that PSA levels >4 ng/mL have predictive value for detecting prostate cancer.58,59 Although PSA testing has led to a dramatic increase in prostate cancer detection,60 PSA has substantial drawbacks. As PSA is also produced by benign prostate glands, it is often elevated in benign conditions, such as benign prostate hyperplasia and prostatitis, likely accounting for the poor specificity of the PSA test, which has been reported to be only 20% at a sensitivity of 80%. Further, even in patients with PSA levels <4 ng/mL, >15% had biopsy-detectable prostate cancer.61 The diagnosis of indolent prostate cancer has also added to a significant burden of avoidable cancer. Together, these data support the need for biomarkers that can supplement PSA as a diagnostic test, to reduce the number of unnecessary biopsies, and to distinguish indolent from clinically significant prostate cancer.
As prostate cancer cells can be isolated from blood or urine through PCR-based methods, the detection of gene fusions has been envisioned as a biomarker that can supplement the PSA test for the early detection of prostate cancer. Mao et al62 reported the detection of gene fusion in circulating tumor cells.6 As prostate tumor cells are also shed in urine, PCR-based detection of fusion transcripts from urine sediments could provide a noninvasive adjunct to diagnose prostate cancer. We initially reported both sensitive and specific detection of the TMPRSS2-ERG fusion transcript from urine sediments of prostate cancer patients,63 and subsequently analyzed this fusion transcript in combination with other putative prostate cancer biomarkers in urine sediments using quantitative PCR.64 In this follow-up study, multiplexed detection of GOLPH2, SPINK1, PCA3, and TMPRSS2-ERG fusion transcripts outperformed PSA or PCA3 alone in the detection of prostate cancer.64 In another study, combined detection of PCA3 and gene fusion transcripts in urine improved the sensitivity of detection of prostate cancer.65 Such refinements might lead to a clinical supplement to serum PSA to aid in the selection of those patients who should undergo biopsies and in triaging patients who might need a repeat biopsy after first negative biopsy. The diagnostic and clinical management implications of gene fusions through various sources are summarized in Figure 2.
Identification of recurrent gene fusions in prostate cancer represents a paradigm shift in our understanding of common epithelial cancers, and arguably represents the most common mutation class in human cancers, despite the fact that they were previously relegated to rare hematologic and soft tissue malignancies. Fusion proteins and subsequent alterations in gene expression in collaboration with other pathways such as PTEN deletion, act as trigger points in malignant transformation and progression. Similar to hematologic malignancies, the application of gene fusions in prostate cancer will likely change the classification, detection, and treatment of prostate cancer and may eventually rival the discovery of PSA as a prostate cancer biomarker.
The authors thank Robin Kunkel for assistance with images, Christine Betts and Jill Granger for assistance in editing the manuscript, and Bo Han and Sooryanarayana Varambally for reviewing the components of this article.
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