Anne Fletcher

Duplication of the fusion of TMPRSS2 to ERG sequences identifies fatal human prostate cancer

New predictive markers for managing prostate cancer are urgently required because of the highly variable natural history of this disease. At the time of diagnosis, Gleason score provides the gold standard for assessing the aggressiveness of prostate cancer. However, the recent discovery of TMPRSS2 fusions to the ERG gene in prostate cancer raises the possibility of using alterations at the ERG locus as additional mechanism-based prognostic indicators. Fluorescence in situ hybridization (FISH) assays were used to assess ERG gene status in a cohort of 445 prostate cancers from patients who had been conservatively managed. The FISH assays detected separation of 5′ (labelled green) and 3′ (labelled red) ERG sequences, which is a consequence of the TMPRSS2–ERG fusion, and additionally identify interstitial deletion of genomic sequences between the tandemly located TMPRSS2 and ERG gene sequences on chromosome 21. Cancers lacking ERG alterations exhibited favourable cause-specific survival (90% survival at 8 years). We identify a novel category of prostate cancers, characterized by duplication of the fusion of TMPRSS2 to ERG sequences together with interstitial deletion of sequences 5′ to ERG (called ‘2+Edel’), which by comparison exhibited extremely poor cause-specific survival (hazard ratio=6.10, 95% confidence ratio=3.33–11.15, P<0.001, 25% survival at 8 years). In multivariate analysis, ‘2+Edel’ provided significant prognostic information (P=0.003) in addition to that provided by Gleason score and prostate-specific antigen level at diagnosis. Other individual categories of ERG alteration were associated with intermediate or good prognosis. We conclude that determination of ERG gene status, including duplication of the fusion of TMPRSS2 to ERG sequences in 2+Edel, allows stratification of prostate cancer into distinct survival categories.

Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer

Background:The discovery of ERG/ETV1 gene rearrangements and PTEN gene loss warrants investigation in a mechanism-based prognostic classification of prostate cancer (PCa). The study objective was to evaluate the potential clinical significance and natural history of different disease categories by combining ERG/ETV1 gene rearrangements and PTEN gene loss status.Methods:We utilised fluorescence in situ hybridisation (FISH) assays to detect PTEN gene loss and ERG/ETV1 gene rearrangements in 308 conservatively managed PCa patients with survival outcome data.Results:ERG/ETV1 gene rearrangements alone and PTEN gene loss alone both failed to show a link to survival in multivariate analyses. However, there was a strong interaction between ERG/ETV1 gene rearrangements and PTEN gene loss (P<0.001). The largest subgroup of patients (54%), lacking both PTEN gene loss and ERG/ETV1 gene rearrangements comprised a ‘good prognosis’ population exhibiting favourable cancer-specific survival (85.5% alive at 11 years). The presence of PTEN gene loss in the absence of ERG/ETV1 gene rearrangements identified a patient population (6%) with poorer cancer-specific survival that was highly significant (HR=4.87, P<0.001 in multivariate analysis, 13.7% survival at 11 years) when compared with the ‘good prognosis’ group. ERG/ETV1 gene rearrangements and PTEN gene loss status should now prospectively be incorporated into a predictive model to establish whether predictive performance is improved.Conclusions:Our data suggest that FISH studies of PTEN gene loss and ERG/ETV1 gene rearrangements could be pursued for patient stratification, selection and hypothesis-generating subgroup analyses in future PCa clinical trials and potentially in patient management.

Transcription factor E2F3 overexpressed in prostate cancer independently predicts clinical outcome.

E2F transcription factors, including E2F3, directly modulate expression of EZH2. Recently, overexpression of the EZH2 gene has been implicated in the development of human prostate cancer. In tissue microrarray studies we now show that expression of high levels of nuclear E2F3 occurs in a high proportion (98/147, 67%) of human prostate cancers, but is a rare event in non-neoplastic prostatic epithelium suggesting a role for E2F3 overexpression in prostate carcinogenesis. Patients with prostate cancer exhibiting immunohistochemically detectable nuclear E2F3 expression have poorer overall survival (P=0.0022) and cause-specific survival (P=0.0047) than patients without detectable E2F3 expression. When patients are stratified according to the maximum percentage of E2F3-positive nuclei identified within their prostate cancers (up to 20, 21–40%, etc.), there is an increasingly significant association between E2F3 staining and risk of death both for overall survival (P=0.0014) and for cause-specific survival (P=0.0004). Multivariate analyses select E2F3 expression as an independent factor predicting overall survival (unstratified P=0.0103, stratified P=0.0086) and cause-specific survival (unstratified P=0.0288, stratified P=0.0072). When these results are considered together with published data on EZH2 and on the E2F3 control protein pRB, we conclude that the pRB–E2F3–EZH2 control axis may have a critical role in modulating aggressiveness of individual human prostate cancer.

Amplification and overexpression of E2F3 in human bladder cancer

We demonstrate that, in human bladder cancer, amplification of the E2F3 gene, located at 6p22, is associated with overexpression of its encoded mRNA transcripts and high levels of expression of E2F3 protein. Immunohistochemical analyses of E2F3 protein levels have established that around one-third (33/101) of primary transitional cell carcinomas of the bladder overexpress nuclear E2F3 protein, with the proportion of tumours containing overexpressed nuclear E2F3 increasing with tumour stage and grade. When considered together with the established role of E2F3 in cell cycle progression, these results suggest that the E2F3 gene represents a candidate bladder cancer oncogene that is activated by DNA amplification and overexpression.

Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer

A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5′-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5′-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.

Overexpression of RAD51 occurs in aggressive prostatic cancer.

Aims:  To test the hypothesis that, in a matched series of prostatic cancers, either with or without BRCA1 or BRCA2 mutations, RAD51 protein expression is enhanced in association with BRCA mutation genotypes.

Immunohistochemistry for p16, but not Rb or p21, is an independent predictor of prognosis in conservatively treated, clinically localised prostate cancer

Aims: Treatment decisions are difficult in clinically localised prostate cancer and further biomarkers of aggressive behaviour are required. We investigated the hypothesis that the tissue expression of three cell cycle markers, Rb, p21 and p16, would provide helpful prognostic information in a well characterised series of prostate cancers which were clinically localised and treated conservatively. Methods: The immunohistochemical staining expression of these markers was assessed in tissue microarrays and correlated with 10 year prostate cancer survival and overall survival and then compared with pathological data including contemporary Gleason score, age, measures of tumour extent and initial serum prostate specific antigen (PSA) level. Results: Rb overexpression did not show any significant association with Gleason score or prostate cancer survival. p21 protein expression showed a significant association with prostate cancer survival (p = 0.02) and overall survival (p = 0.01) in a univariate model but not in a multivariate model with pathological and serum PSA data. There was a significant association between p16 cytoplasmic expression and prostate cancer survival (HR = 2.52, 95%CI = 1.79–3.55, p < 0.001) and overall survival (HR = 1.54, 95% CI = 1.20–1.98, p = 0.001) in a univariate model. p16 expression remained an independent prognostic factor for prostate cancer survival (HR = 1.50, 95%CI = 1.05–2.14, p = 0.03). Conclusion: We conclude that p16 cytoplasmic expression can be used as a predictor of outcome in conservatively treated prostate cancer. Rb and p21 show no independent association with outcome and therefore further research is not warranted.

The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer

Our previous work identified a chromosomal translocation t(4;6) in prostate cancer cell lines and primary tumors. Using probes located on 4q22 and 6q15, the breakpoints identified in LNCaP cells, we performed fluorescence in situ hybridization analysis to detect this translocation in a large series of clinical localized prostate cancer samples treated conservatively. We found that t(4;6)(q22;q15) occurred in 78 of 667 cases (11.7%). The t(4;6)(q22;q15) was not independently associated with patient outcome. However, it occurs more frequently in high clinical T stage, high tumor volume specimens and in those with high baseline PSA (P=0.001, 0.001 and 0.01, respectively). The t(4;6)(q22;q15) occurred more frequently in samples with two or more TMPRSS2:ERG fusion genes caused by internal deletion than in samples without these genomic alterations, but this correlation is not statistically significant (P=0.0628). The potential role of this translocation in the development of human prostate cancer is discussed.

Overexpression of TP53 is Associated with Aggressive Prostate Cancer butdoes not Distinguish Disease in BRCA1 or BRCA2 Mutation Carriers froma Control Group

We have shown that prostate cancer occurring in men with germline BRCA1 and BRCA2 mutations is more ag- gressive. In an attempt to identify an associated immunohistochemical phenotype, we have studied TP53 immunostaining in prostate cancers in mutation carriers versus prostate cancers occurring in a control group of men. There was a significantly higher expression of TP53 protein in prostate cancer with a higher Gleason score (p< 0.001). Twenty four per cent of prostate cancer occurring in BRCA1/2 mutation carriers and 19% of those from controls stained positively for the TP53 protein; this difference was not significant. Cases and controls were combined and matched for benign and malignant disease within the same individual. There were 152 men who had a sample of each within the tissue samples. Thirty one (20%) stained positively within the malignant tissue alone; none had positive staining in benign tissue, p<0.001. Over expression of TP53 cannot distinguish prostate cancer on a background of BRCA1/2 mutation, but it is associated with prostate cancer malignant tissue per se, in particular aggressive disease.

Abstract 662: Molecular characterisation of ERG, ETV-1 and PTEN- gene loci identifies patients at low and high risk of death from prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: The discovery of ERG/ETV-1-gene rearrangements and PTEN-gene loss suggests their use in a mechanism-based prognostic classification of prostate cancer (PCa). Purpose: To evaluate the potential clinical significance and natural history of different disease categories by combining ERG/ETV-1-gene rearrangements and PTEN-gene loss status. Methods: We utilized fluorescence in situ hybridization (FISH) assays to detect PTEN-gene loss and ERG/ETV-1-gene rearrangements in 308 conservatively managed PCa patients with survival outcome data. Results: ERG/ETV-1-gene rearrangements alone and PTEN-gene loss alone each failed to show a link to survival in multivariate analyses. However, there was a strong interaction between ERG/ETV-1-gene rearrangements and PTEN-gene loss (p<0.001). The largest subgroup of patients (54%), lacking both PTEN-gene loss and ERG/ETV-1-gene rearrangements comprised a ‘good prognosis’ population exhibiting favourable cancer-specific survival (85.5% alive at 11 years). The presence of PTEN-gene loss in the absence of ERG/ETV-1-gene rearrangements identified a patient population (6%) with poorer cancer-specific survival that was highly significant (HR = 4.87, p<0.001 in multivariate analysis, 13.7% survival at 11 years) when compared to the ‘good prognosis’ group. ERG/ETV-1-gene rearrangement and PTEN-gene loss status should now prospectively be incorporated into a predictive model to establish whether predictive performance is improved. Conclusions: Our data suggest that FISH studies of PTEN-gene loss and ERG/ETV-1-gene rearrangements could be pursued for patient stratification, selection and hypothesis-generating sub-group analyses in future PCa clinical trials and potentially in patient management. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 662.