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ABSTRACT: Formalin-fixed prostate biopsies are frequently the only tissue collected at the time of prostate cancer diagnosis. There is therefore a requirement for techniques that allow the use of these prostate biopsy specimens in a high-throughput analysis of immunohistochemical and fluorescence-in-situ-hybridisation-detected biomarkers.
The authors have previously described methods that allow tissue microarray (TMA) construction from prostate biopsies. Here, we describe significant technical innovations that provide an easier and more robust system of biopsy-TMA construction.
The TMAs produced are of a high density (up to 104 cores each, 8 × 13) and allow a multiplex analysis of biomarkers in the context of clinical trials.
Journal of clinical pathology 11/2010; 64(1):88-90. · 2.43 Impact Factor
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A H M Reid,
G Attard,
L Ambroisine,
G Fisher,
G Kovacs,
D Brewer,
J Clark, P Flohr,
S Edwards,
D M Berney,
C S Foster,
A Fletcher,
W L Gerald,
H Møller,
V E Reuter,
P T Scardino,
J Cuzick,
J S de Bono,
C S Cooper
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ABSTRACT: 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.
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.
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.
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.
British Journal of Cancer 02/2010; 102(4):678-84. · 5.04 Impact Factor
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L Shan,
L Ambroisine,
J Clark,
R J Yáñez-Muñoz,
G Fisher,
S C Kudahetti,
J Yang,
S Kia,
X Mao,
A Fletcher, [......],
B D Young,
C S Foster,
V Reuter,
H Moller,
T D Oliver,
D M Berney,
P Scardino,
J Cuzick,
C S Cooper,
Y-J Lu
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ABSTRACT: 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.
Prostate cancer and prostatic diseases 02/2010; 13(2):117-25. · 2.10 Impact Factor
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A H M Reid,
G Attard,
L Ambroisine,
G Fisher,
G Kovacs,
D Brewer,
J Clark, P Flohr,
S Edwards,
D M Berney,
C S Foster,
A Fletcher,
W L Gerald,
H M|[oslash]|ller,
V E Reuter,
P T Scardino,
J Cuzick,
J S de Bono,
C S Cooper
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ABSTRACT: Background: Methods: Results: Conclusions: Patients and methods Results Discussion Conclusions References Acknowledgements Figures and TablesBackground: 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.
British Journal of Cancer 01/2010; 102(4):678-684. · 5.04 Impact Factor
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F McCarthy,
A Fletcher,
N Dennis,
C Cummings,
H O'Donnell,
J Clark, P Flohr,
R Vergis,
S Jhavar,
C Parker,
C S Cooper
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ABSTRACT: Prostate cancer diagnosis is routinely made by the histopathological examination of formalin fixed needle biopsy specimens. Frequently this is the only cancer tissue available from the patient for the analysis of diagnostic and prognostic biomarkers. There is, therefore, an urgent need for methods that allow the high-throughput analysis of these biopsy samples using immunohistochemical (IHC) markers and fluorescence in situ hybridisation (FISH) analysis based markers.
A method that allows the construction of tissue microarrays (TMAs) from diagnostic prostate needle biopsy cores has previously been reported. However, the technique only allows the production of low-density biopsy TMAs with a maximum of 20 cores per TMA. Here two methods are presented that allow the rapid and uniform production of biopsy TMAs containing between 54 and 72 biopsy cores. IHC and FISH techniques were used to detect biomarker status.
Biopsy TMAs were constructed from prostate needle biopsy specimens taken from 102 patients entered into an active surveillance trial and 201 patients in a radiotherapy trial. The detection rate for cancer in slices of these biopsy TMAs was 66% and 79% respectively. Slices of a biopsy TMA prepared from biopsies from active surveillance patients were used to detect multiple IHC markers and to score TMPRSS2-ERG fusion status in a FISH-based assay.
The construction of biopsy TMAs provides an effective method for the multiplex analysis of IHC and FISH markers and for their assessment as prognostic biomarkers in the context of clinical trials.
Journal of clinical pathology 09/2009; 62(8):694-8. · 2.43 Impact Factor
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G Attard,
J Clark,
L Ambroisine,
I G Mills,
G Fisher, P Flohr,
A Reid,
S Edwards,
G Kovacs,
D Berney,
C Foster,
C E Massie,
A Fletcher,
J S De Bono,
P Scardino,
J Cuzick,
C S Cooper
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ABSTRACT: Results Discussion Materials and methods References Acknowledgements Figures and TablesA 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.Keywords: prostate cancer, ETV1, ACSL3, ACSL3:ETV1 fusion
British Journal of Cancer 06/2008; 99(2):314-320. · 5.04 Impact Factor
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J Clark,
G Attard,
S Jhavar, P Flohr,
A Reid,
J De-Bono,
R Eeles,
P Scardino,
J Cuzick,
G Fisher,
M D Parker,
C S Foster,
D Berney,
G Kovacs,
C S Cooper
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ABSTRACT: 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.
Oncogene 04/2008; 27(14):1993-2003. · 6.37 Impact Factor
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G Attard,
J Clark,
L Ambroisine,
G Fisher,
G Kovacs, P Flohr,
D Berney,
C S Foster,
A Fletcher,
W L Gerald,
H Moller,
V Reuter,
J S De Bono,
P Scardino,
J Cuzick,
C S Cooper
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ABSTRACT: 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.
Oncogene 02/2008; 27(3):253-63. · 6.37 Impact Factor
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J Clark,
S Merson,
S Jhavar, P Flohr,
S Edwards,
C S Foster,
R Eeles,
F L Martin,
D H Phillips,
M Crundwell,
T Christmas,
A Thompson,
C Fisher,
G Kovacs,
C S Cooper
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ABSTRACT: TMPRSS2-ERG gene fusions have recently been reported to be present in a high proportion of human prostate cancers. In the current study, we show that great diversity exists in the precise structure of TMPRSS2-ERG hybrid transcripts found in human prostates. Fourteen distinct hybrid transcripts are characterized, each containing different combinations of sequences from the TMPRSS2 and ERG genes. The transcripts include two that are predicted to encode a normal full-length ERG protein, six that encode N-terminal truncated ERG proteins and one that encodes a TMPRSS2-ERG fusion protein. Interestingly, distinct patterns of hybrid transcripts were found in samples taken from separate regions of individual cancer-containing prostates, suggesting that TMPRSS2-ERG gene fusions may be arising independently in different regions of a single prostate.
Oncogene 05/2007; 26(18):2667-73. · 6.37 Impact Factor
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S Edwards,
C Campbell, P Flohr,
J Shipley,
I Giddings,
R Te-Poele,
A Dodson,
C Foster,
J Clark,
S Jhavar,
G Kovacs,
C S Cooper
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ABSTRACT: In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT-PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types.
British Journal of Cancer 02/2005; 92(2):376-81. · 5.04 Impact Factor
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Y-F Lee,
M John,
S Edwards,
J Clark, P Flohr,
K Maillard,
M Edema,
L Baker,
D C Mangham,
R Grimer,
R Wooster,
J M Thomas,
C Fisher,
I Judson,
C S Cooper
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ABSTRACT: In this study, we have used genome-wide expression profiling to categorise synovial sarcomas, leiomyosarcomas and malignant fibrous histiocytomas (MFHs). Following hierarchical clustering analysis of the expression data, the best match between tumour clusters and conventional diagnosis was observed for synovial sarcomas. Eight of nine synovial sarcomas examined formed a cluster that was characterised by higher expression of a set of 48 genes. In contrast, sarcomas conventionally classified as leiomyosarcomas and MFHs did not match the clusters defined by hierarchical clustering analysis. One major cluster contained a mixture of both leiomyosarcomas and MFHs and was defined by the lower expression of a set of 202 genes. A cluster containing a subgroup of MFHs was also detected. These results may have implications for the classification of soft tissue sarcomas, and are consistent with the view that sarcomas conventionally defined as MFHs do not represent a separate diagnostic category.Keywords: synovial sarcoma, microarray, hierarchical clustering, leiomyosarcoma, malignant fibrous histiocytoma, soft tissue sarcoma
British Journal of Cancer 02/2003; 88(4):510-515. · 5.04 Impact Factor
