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It is well established that prostate cancer (PCA) is one of the most heterogeneous cancers, which has made it difficult to identify and validate significant molecular changes associated with disease progression. However, common molecular changes with established prognostic value include loss of 8p, 6q, 10q, 13q, 16q and 18q, and gain of 8q [1,2].
The recent discovery of the common ERG gene rearrangements in PCA  suggests that a molecular classification based on gene fusion status might be possible. Recent expression-profiling studies characterizing genomic events in ERG-rearranged tumours support this hypothesis [4–6].
PTEN, a phosphoinositide 3-phosphatase which negatively regulates the PI3K signalling pathway, is located on chromosome 10 and acts as tumour suppressor gene . Reduction in PTEN function has been associated with disease progression and the androgen-independent state [8–15]. Using in vitro research, it has been shown by several groups that the ERG gene rearrangements by themselves may not be capable of transforming normal cells into cancers and that this requires interaction with other genetic changes [16–18]. Using transgenic mouse models, it is becoming more obvious that PCA initiation requires several genetic hits and might not be fully explained by the presence of TMPRSS2–ERG gene fusion alone [17,18]. Two recent studies showed that PTEN genomic deletions and TMRPSS2–ERG gene fusion (the most common gene fusion resulting from ERG gene rearrangement) interact in driving PCA disease development and progression [16,19,20]. In the study by Carver et al. , transgenic mice showing aberrant ERG in conjunction with PTEN heterozygosity showed invasive tumours compared to high-grade prostatic intra-epithelial neoplasia (HGPIN) -like lesions observed in PTEN+/- mice without ERG aberrations. Similar results were obtained by King et al. . Recent reports from our group have identified associations between poor outcome in localized and androgen-independent tumours and PTEN genomic deletion [21,22]. In the study by Yoshimoto et al. , localized PCA tumours harbouring TMPRSS2–ERG gene fusion and PTEN genomic deletions in combination were associated with a worse prognosis than tumours without any of those genetic alterations, which had the best prognosis in terms of PSA relapse .
However, there is still controversy as to the significance and sequence of events in the human prostate. Whereas all studies agree that ERG gene rearrangements occur early at the HGPIN stage [24–26], only two previous studies reported PTEN losses in HGPIN [27,28], while recent in vivo studies have suggested that the development and progression into invasive cancer would start initially by PTEN deregulation causing higher levels of instability and activation of the AKT pathway. Cells with PTEN haplo-insufficiency have been found to be more unstable  and prone to undergo additional genomic alterations, such as ERG gene rearrangements and concomitant aberrant expression of ERG, to fully establish disease development and progression. However, others consider that PTEN genomic deletions probably occur later, at the metastatic stage [16,19,28].
Fluorescence in situ hybridization (FISH) has substantial advantages over other methods for determining clonal presence of genomic aberrations at the cellular level, enabling direct correlation with histologies such as Gleason score (GS) and HGPIN. In the present study, we used FISH to interrogate a large cohort of patients with localized PCA to determine the prevalence of ERG rearrangements and to annotate several PTEN genomic aberrations distinguishing among PTEN gain and monzygous, hemi- and homozygous PTEN genomic deletions at early, intermediate and later steps in the carcinogenic process.
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To date, the ERG gene rearrangement has been investigated in several studies, all of which confirmed its specification to HGPIN and invasive cancer, with the TMPRSS2–ERG gene fusion representing the most common gene fusion resulting from ERG rearrangement in localized PCA . PTEN genomic deletions have also been widely studied as a significant contributor to PCA progression and several studies have confirmed its association with a worse clinical outcome [21,33]. In vitro studies showed that ERG gene rearrangements by themselves are not capable of transforming normal epithelial cell lines (RWPE) but increases its invasion capabilities , whereas PTEN genomic deletions caused increased transformation and proliferation . Using in vivo mouse models, it is evident that PCA development requires the interaction between PTEN genomic deletions and ERG gene rearrangements, with PTEN genomic deletions causing AKT activation and HGPIN-like lesions followed by ERG gene rearrangements to allow for the establishment of PCA [16,19]. However, the association and sequence incidence between those two genetic aberrations in human PCA development and progression are not yet fully characterized.
In the present study, we systematically analysed the status of ERG gene rearrangements and PTEN deletions across various stages of disease progression, starting from benign prostatic tissue and moving on to HGPIN and invasive PCA. We confirmed the specificity and significant association of ERG gene rearrangements with PCA, as well as its significant association with PTEN genomic deletions.
We have also confirmed that the two distinct genomic aberrations are detected in a subset of HGPIN, as was recently shown by Han et al. . However, in contrast to these results, we have detected homozygous deletions in ≈5% of HGPIN lesions, compared with 0% in the study by Han et al. . Furthermore, benign prostatic tissue showed evidence of PTEN genomic aberrations as well, albeit at much lower rates than HGPIN and PCA. Although we cannot fully exclude the possibility that some deletions in benign prostatic tissue could represent background nuclear truncations from slide sectioning or early neoplastic changes at molecular levels that are not obvious morphologically at the microscopic level, the observation of PTEN homozygous deletions within a subset of HGPIN (5.3%) coupled with the increased incidence of this aberration in localized cancers with higher GS would argue that PTEN deletions are occurring at the HGPIN stage in co-operation with ERG gene rearrangements and are not always a later event in PCA progression as hypothesized by Han et al. . Although it is possible that some of the HGPIN lesions sampled here could represent intra-ductal spread of cancer, and from these observations we cannot firmly ascertain which of those two genetic aberrations is the initial event in PCA development, the present results are in keeping with the recent in vivo studies of Carver et al.  and King et al. , which showed that double transgenic mice for PTEN and ERG aberrations develop PCA possibly through AKT activation [16,20].
We hypothesize that a subset of PCA may be driven initially by PTEN genomic hemizygous loss, causing HGPIN lesions. Thereafter, PTEN haplo-insufficiency leads to genomic instability [29,35], which may facilitate the chromosomal rearrangement leading to gene fusion formation and progression to cancer. Subsequent PTEN deregulation by homozygous deletion (or other inactivating mechanisms) could then occur to induce PCA progression (Fig. 3). This hypothetical sequence of events is supported here by the observation that hemizygous deletions were more prevalent in HGPIN and localized PCA than homozygous deletions, with the latter showing significant and increased association with disease progression as evidenced by higher incidence of homozygous deletions in poorly differentiated PCA (GS 8–10) than in well and moderately differentiated tumours (GS 6–7). In the present study, there was significant and higher association between ERG gene rearrangements and homozygous PTEN deletions than with hemizygous deletions in PCA (71.4 vs 44%), suggesting that the interaction between the two genetic aberrations is stronger when there is complete lack of PTEN activity, and this association would signify the highest risk for PCA development and progression. Whether HGPIN lesions with homozygous PTEN deletions are associated with accelerated rate of disease progression is beyond the scope of the present study, and needs to be confirmed in larger cohorts to identify HGPIN lesions at highest risk for disease progression.
Figure 3. Schematic model of hypothetical sequence of genomic events in PCA progression. The acquisition of a PTEN haplo-insufficiency in benign prostatic precursors may represent an early event in a subset of PCAs. Reduced PTEN protein levels may facilitate genomic instability, leading to acquisition of ETS (erythroblastosis virus E26 transformation-specific) rearrangements. Synergistic co-operation between PTEN and ETS is associated with early steps of PCA formation. Continuing instability generates genotypic heterogeneity and diversity such that subclones bearing ‘null PTEN’ (PTEN homozygous deletions) have increased selective advantage for tumour progression through activation of the AKT pathway.
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It is becoming evident that ERG gene rearrangements signify a distinct molecular subtype of PCA that may have potential clinical implications. However, the prognostic value of these markers are still unclear. While several studies have shown an association with an unfavourable prognosis and aggressive clinical behaviour (such as PSA relapse or cancer-specific mortality) [36–38], others have failed to show such association, or have even pointed towards a better prognosis [25,39,40]. Similarly, only a few reports (including the present study) were able to confirm a significant association with GS [32,41]. These discrepancies could be a reflection of the different methods used in assessing the ERG gene rearrangement status (FISH vs reverse transcription PCR), including detecting variable versions of TMPRSS2–ERG gene fusion. Other potential causes of discrepancy include variable cohort sizes, background and population studied (radical prostatectomy vs watchful waiting) as well as variation in clinical outcome endpoints measured (after radical PSA relapse vs cancer-specific mortality) [25,36,38,40,42–44]. In the present study, there was a significant association between ERG rearrangements and GS, with tumours of GS 6 and 7 more likely to be ERG-rearranged than those of GS > 7. This latter finding would allow us to subtype and differentiate among two tumours with similar GS, should ERG rearrangements be confirmed as a prognostic or therapeutic marker, specifically those with GS ≤ 7, which includes most patients seeking medical advice for PCA diagnosis. A recent report by Attard et al.  suggested a significant association between patients with castration-resistant PCA with ERG rearrangements and abiraterone acetate. The prognostic value of PTEN genomic deletions and haplo-insufficieny of the protein in PCA is well documented for patients with homozygous PTEN deletions and this is true for other cancers as well. We and others have confirmed a significant association of homozygous PTEN genomic deletions and ERG gene rearrangements with lymph node metastasis, hormone-refractory PCA and cancer-specific death [21,33,37,46,47].
Recently, our group has shown that tumours with TMPRSS2–ERG gene fusion and PTEN deletions are associated with even more aggressive clinical behaviour than those with any of the genetic aberrations alone . Although the significance of this later finding still needs validation in larger cohorts, the data presented in the present study, showing a strong and significant association between ERG gene rearrangement and PTEN genomic deletions, confirm that those tumours represent distinct molecular subsets of PCA that require more detailed definition and characterization.
These data also provide further evidence signifying the importance of targeting the two genetic aberrations and their pathways as potential markers for disease progression and targets for PCA therapy in the hope of altering or slowing the development of hormone refractory PCA and preventing cancer-specific mortality.
In conclusion, the present study highlights the co-operation and importance of ERG gene rearrangements and PTEN deletions as significant drivers of PCA development and progression. It also confirms that PTEN genomic deletions occur in a subset of HGPIN lesions and show subsequent accumulating genetic aberrations with PCA progression, as evidenced by a greater incidence of homozygous deletions in higher-grade tumours.