D. Berney

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.

Prognostic value of a cell cycle progression signature for prostate cancer death in a conservatively managed needle biopsy cohort

Background:The natural history of prostate cancer is highly variable and it is difficult to predict. We showed previously that a cell cycle progression (CCP) score was a robust predictor of outcome in a conservatively managed cohort diagnosed by transurethral resection of the prostate. A greater need is to predict outcome in patients diagnosed by needle biopsy.Methods:Total RNA was extracted from paraffin specimens. A CCP score was calculated from expression levels of 31 genes. Clinical variables consisted of centrally re-reviewed Gleason score, baseline prostate-specific antigen level, age, clinical stage, and extent of disease. The primary endpoint was death from prostate cancer.Results:In univariate analysis (n=349), the hazard ratio (HR) for death from prostate cancer was 2.02 (95% CI (1.62, 2.53), P<10−9) for a one-unit increase in CCP score. The CCP score was only weakly correlated with standard prognostic factors and in a multivariate analysis, CCP score dominated (HR for one-unit increase=1.65, 95% CI (1.31, 2.09), P=3 × 10−5), with Gleason score (P=5 × 10−4) and prostate-specific antigen (PSA) (P=0.017) providing significant additional contributions.Conclusion:For conservatively managed patients, the CCP score is the strongest independent predictor of cancer death outcome yet described and may prove valuable in managing clinically localised prostate cancer.

Complex patterns of ETS gene alteration arise during cancer development in the human prostate

An ERG gene ‘break-apart’ fluorescence in situ hybridization (FISH) assay has been used to screen whole-mount prostatectomy specimens for rearrangements at the ERG locus. In cancers containing ERG alterations the observed pattern of changes was often complex. Different categories of ERG gene alteration were found either together in a single cancerous region or within separate foci of cancer in the same prostate slice. In some cases the juxtaposition of particular patterns of ERG alterations suggested possible mechanisms of tumour progression. Prostates harbouring ERG alterations commonly also contained cancer that lacked rearrangements of the ERG gene. A single trans-urethral resection of the prostate specimen examined harboured both ERG and ETV1 gene rearrangements demonstrating that the observed complexity may, at least in part, be explained by multiple ETS gene alterations arising independently in a single prostate. In a search for possible precursor lesions clonal ERG rearrangements were found both in high grade prostatic intraepithelial neoplasia (PIN) and in atypical in situ epithelial lesions consistent with the diagnosis of low grade PIN. Our observations support the view that ERG gene alterations represent an initiating event that promotes clonal expansion initially to form regions of epithelial atypia. The complex patterns of ERG alteration found in prostatectomy specimens have important implications for the design of experiments investigating the clinical significance and mechanism of development of individual prostate cancers.

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.

Prognostic value of PTEN loss in men with conservatively managed localised prostate cancer

Background:The natural history of prostate cancer is highly variable and difficult to predict. We report on the prognostic value of phosphatase and tensin homologue (PTEN) loss in a cohort of 675 men with conservatively managed prostate cancer diagnosed by transurethral resection of the prostate.Methods:The PTEN status was assayed by immunohistochemistry (PTEN IHC) and fluorescent in situ hybridisation (PTEN FISH). The primary end point was death from prostate cancer.Results:The PTEN IHC loss was observed in 18% cases. This was significantly associated with prostate cancer death in univariate analysis (hazard ratio (HR)=3.51; 95% CI 2.60–4.73; P=3.1 × 10−14). It was highly predictive of prostate cancer death in the 50% of patients with a low risk score based on Gleason score, PSA, Ki-67 and extent of disease (HR=7.4; 95% CI 2.2–24.6; P=0.012) ), but had no prognostic value in the higher risk patients. The PTEN FISH loss was only weakly associated with PTEN IHC loss (κ=0.5). Both PTEN FISH loss and amplification were univariately predictive of death from prostate cancer, but this was not maintained in the multivariate analyses.Conclusion:In low-risk patients, PTEN IHC loss adds prognostic value to Gleason score, PSA, Ki-67 and extent of disease.

Prognostic value of Ki-67 for prostate cancer death in a conservatively managed cohort

Background:Standard clinical parameters cannot accurately differentiate indolent from aggressive prostate cancer. Our previous work showed that immunohistochemical (IHC) Ki-67 improved prediction of prostate cancer death in a cohort of conservatively treated clinically localised prostate cancers diagnosed by transurethral resection of the prostate (TURP). Here, we present results in a more clinically relevant needle biopsy cohort.Methods:Biopsy specimens were microarrayed. The percentage of Ki-67 positively stained malignant cells per core was measured and the maximum score per individual used in analysis of time to death from prostate cancer using a Cox proportional hazards model.Results:In univariate analysis (n=293), the hazard ratio (HR) (95% confidence intervals) for dichotomous Ki-67 (⩽10%, >10%) was 3.42 (1.76, 6.62) χ2 (1 df)=9.8, P=0.002. In multivariate analysis, Ki-67 added significant predictive information to that provided by Gleason score and prostate-specific antigen (HR=2.78 (1.42, 5.46), χ2 (1 df)=7.0, P=0.008).Conclusion:The IHC Ki-67 scoring on prostate needle biopsies is practicable and yielded significant prognostic information. It was less informative than in the previous TURP cohort where tumour samples were larger and more comprehensive, but in more contemporary cohorts with larger numbers of biopsies per patient, Ki-67 may prove a more powerful biomarker.

Validation of an RNA cell cycle progression score for predicting death from prostate cancer in a conservatively managed needle biopsy cohort

Background:The natural history of prostate cancer is highly variable and difficult to predict accurately. Better markers are needed to guide management and avoid unnecessary treatment. In this study, we validate the prognostic value of a cell cycle progression score (CCP score) independently and in a prespecified linear combination with standard clinical variables, that is, a clinical-cell-cycle-risk (CCR) score.Methods:Paraffin sections from 761 men with clinically localized prostate cancer diagnosed by needle biopsy and managed conservatively in the United Kingdom, mostly between 2000 and 2003. The primary end point was prostate cancer death. Clinical variables consisted of centrally reviewed Gleason score, baseline PSA level, age, clinical stage, and extent of disease; these were combined into a single predefined risk assessment (CAPRA) score. Full data were available for 585 men who formed a fully independent validation cohort.Results:In univariate analysis, the CCP score hazard ratio was 2.08 (95% CI (1.76, 2.46), P<10−13) for one unit change of the score. In multivariate analysis including CAPRA, the CCP score hazard ratio was 1.76 (95% CI (1.44, 2.14), P<10−6). The predefined CCR score was highly predictive, hazard ratio 2.17 (95% CI (1.83, 2.57), χ2=89.0, P<10−20) and captured virtually all available prognostic information.Conclusions:The CCP score provides significant pretreatment prognostic information that cannot be provided by clinical variables and is useful for determining which patients can be safely managed conservatively, avoiding radical treatment.

Hsp-27 expression at diagnosis predicts poor clinical outcome in prostate cancer independent of ETS-gene rearrangement

Background:This study was performed to test the hypothesis that expression of small heat shock protein Hsp-27 is, at diagnosis, a reliable predictive biomarker of clinically aggressive prostate cancer.Methods:A panel of tissue microarrays constructed from a well-characterised cohort of 553 men with conservatively managed prostate cancer was stained immunohistochemically to detect Hsp-27 protein. Hsp-27 expression was compared with a series of pathological and clinical parameters, including outcome.Results:Hsp-27 staining was indicative of higher Gleason score (P<0.001). In tissue cores having a Gleason score >7, the presence of Hsp-27 retained its power to independently predict poor clinical outcome (P<0.002). Higher levels of Hsp-27 staining were almost entirely restricted to cancers lacking ERG rearrangements (χ2 trend=31.4, P<0.001), although this distribution did not have prognostic significance.Interpretation:This study has confirmed that, in prostate cancers managed conservatively over a period of more than 15 years, expression of Hsp-27 is an accurate and independent predictive biomarker of aggressive disease with poor clinical outcome (P<0.001). These findings suggest that apoptotic and cell-migration pathways modulated by Hsp-27 may contain targets susceptible to the development of biologically appropriate chemotherapeutic agents that are likely to prove effective in treating aggressive prostate cancers.

Interactive digital slides with heat maps: a novel method to improve the reproducibility of Gleason grading

Our aims were to analyze reporting of Gleason pattern (GP) 3 and 4 prostate cancer with the ISUP 2005 Gleason grading and to collect consensus cases for standardization. We scanned 25 prostate biopsy cores diagnosed as Gleason score (GS) 6–7. Fifteen genitourinary pathologists graded the digital slides and circled GP 4 and 5 in a slide viewer. Grading difficulty was scored as 1–3. GP 4 components were classified as type 1 (cribriform), 2 (fused), or 3 (poorly formed glands). A GS of 5–6, 7 (3 + 4), 7 (4 + 3), and 8–9 was given in 29%, 41%, 19%, and 10% (mean GS 6.84, range 6.44–7.36). In 15 cases, at least 67% of observers agreed on GS groups (consensus cases). Mean interobserver weighted kappa for GS groups was 0.43. Mean difficulty scores in consensus and non-consensus cases were 1.44 and 1.66 (p = 0.003). Pattern 4 types 1, 2, and 3 were seen in 28%, 86%, and 67% of GP 4. All three coexisted in 16% (11% and 23% in consensus and non-consensus cases, p = 0.03). Average estimated and calculated %GP 4/5 were 29% and 16%. After individual review, the experts met to analyze diagnostic difficulties. Areas of GP 4 and 5 were displayed as heat maps, which were helpful for identifying contentious areas. A key problem was to agree on minimal criteria for small foci of GP 4. In summary, the detection threshold for GP 4 in NBX needs to be better defined. This set of consensus cases may be useful for standardization.

Association between DNA methylation of HSPB1 and death in low Gleason score prostate cancer

Background:Heat shock protein 27 (Hsp-27) encoded by gene HSPB1 is a critical regulator of the behavioral phenotype of human prostate cancer (PCa) cells, enhanced expression being associated with highly aggressive disease and poor clinical outcome. In contrast, the protein is not expressed in PCas of low malignant potential. To gain insight into the mechanism regulating its expression, we tested the hypothesis that differential methylation of CpG islands within HSPB1 controls transcription and subsequent translation of the gene.Methods:We studied prostate epithelial cell lines and tissue biopsies, including 59 BPH and 415 PCas, of which 367 were a cohort of men with up to 20 years of follow-up. Methylation across the gene (DNA methylation (DNAme)) was assayed by pyrosequencing. Hsp-27 expression was assessed by western blot and immunohistochemistry.Results:In cancer tissues, methylation increased in a 3′ direction (P<0.0001) whereas in benign hyperplasia methylation was constantly below 5%, a cutoff giving a specificity of 100% and sensitivity of 50%. Although methylation of the promoter region was significantly discriminating between benign and malignant prostatic epithelia, it compared poorly with methylation of the first intron. The prognostic value of HSPB1 DNAme was confirmed by both univariate (hazard ratio 1.77 per 50% increment, P=0.02) and multivariate models. Interaction between HSPB1 methylation and Gleason score revealed high DNAme to be a reliable prognostic marker of poor outcome in men with low Gleason score (P=0.014).Conclusions:Our data indicate CpG methylation of the first HSPB1 intron to be an important biomarker that identifies aggressive PCas otherwise regarded as low risk by current clinical criteria but that, biologically, require immediate active management.