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Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer.

Attard G, Clark J, Ambroisine L, Mills IG, Fisher G, Flohr P, Reid A, Edwards S, Kovacs G, Berney D, Foster C, Massie CE, Fletcher A, De Bono JS, Scardino P, Cuzick J, Cooper CS, Transatlantic Prostate Gro - Br. J. Cancer (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Research, Male Urological Cancer Research Centre, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.

ABSTRACT
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.

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ACSL3:ETV1 fusion. (A) ACSL3 (red) and ETV1 (blue) transcripts with ORFs in dark colour. Exons are numbered. A fusion transcript of ACSL3 exon 3 fused to ETV1 exon 6 was detected by 5′-RACE from exon 6 ETV1 sequences in prostate cancer sample 23. The ORF shown was predicted using software at www.dnalc.org. (B) Sequence across the ACSL3:ETV1 fusion boundary. Underlined regions indicate the position of primers used in RT–PCR to confirm the fusion. The predicted fusion gene initiation codon is indicated in red. ACSL3 sequence is in lower case and ETV1 sequence in upper case. (C) RT–PCR detection of an ACSL3:ETV1 fusion transcript in RNA extracted from formalin-fixed paraffin-embedded prostate cancer samples: lanes 1–12 are ETV1-rearranged tumour samples, lane 12: tumour sample 23, lane 13 negative control. (D) FISH assays to confirm fusion of ACSL3 with ETV1. Panel i: The ETV1 break-apart assay utilises probes corresponding to 3′-ETV1 sequences (red) and 5′-ETV1 sequences (green) (see also Figure 1). A nucleus with separated red and green probes confirming rearrangement of ETV1 is shown. Panel ii: The ACSL3 break-apart assay hybridised the same TMA slice used in the ETV1 break-apart assay to 3′-ACSL3 sequences (red) and 5′-ACSL3 sequences (green). These signals are coincident in the wild type, but are split on translocation of ACSL3. Comparison of the images in panels i and ii indicates co-localisation of 3′-ETV1 with 5′-ACSL3 and co-localisation of 5′-ETV1 and 3′-ACSL3. This is confirmed by ETV1-ACSL3 co-localisation assays (panel iii) demonstrating co-localisation of 3′-ETV1 sequences (red) and 5′-ACSL3 sequences (green) and (panel iv) demonstrating co-localisation of 3′-ACSL3 sequences and 5′-ETV1 sequences (red) in the same cell. Superimposition of the images in panels iii and iv confirms co-localisation of wild-type 3′-ETV1 (panel iii) with 5′-ETV1 (panel iv) and of wild-type 3′-ACSL3 (panel iv) with 5′-ACSL3 (panel iii). The genes and their direction of transcription are indicated by the arrowheads. (E) Map of the ACSL3 gene showing the position of the BACs used as probes in FISH assays. Probe XV: A1 (RP11-157M20) labelled with FITC. Probe XIV: A2 (RP11-136M23) and A3 (RP11-749C15) labelled with Cy3. Probes XV and probes XIV correspond, respectively, to sequences immediately 5′ (green) and 3′ (red) to the ACSL3 gene. Direction of gene transcription indicated by arrowheads.
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fig3: ACSL3:ETV1 fusion. (A) ACSL3 (red) and ETV1 (blue) transcripts with ORFs in dark colour. Exons are numbered. A fusion transcript of ACSL3 exon 3 fused to ETV1 exon 6 was detected by 5′-RACE from exon 6 ETV1 sequences in prostate cancer sample 23. The ORF shown was predicted using software at www.dnalc.org. (B) Sequence across the ACSL3:ETV1 fusion boundary. Underlined regions indicate the position of primers used in RT–PCR to confirm the fusion. The predicted fusion gene initiation codon is indicated in red. ACSL3 sequence is in lower case and ETV1 sequence in upper case. (C) RT–PCR detection of an ACSL3:ETV1 fusion transcript in RNA extracted from formalin-fixed paraffin-embedded prostate cancer samples: lanes 1–12 are ETV1-rearranged tumour samples, lane 12: tumour sample 23, lane 13 negative control. (D) FISH assays to confirm fusion of ACSL3 with ETV1. Panel i: The ETV1 break-apart assay utilises probes corresponding to 3′-ETV1 sequences (red) and 5′-ETV1 sequences (green) (see also Figure 1). A nucleus with separated red and green probes confirming rearrangement of ETV1 is shown. Panel ii: The ACSL3 break-apart assay hybridised the same TMA slice used in the ETV1 break-apart assay to 3′-ACSL3 sequences (red) and 5′-ACSL3 sequences (green). These signals are coincident in the wild type, but are split on translocation of ACSL3. Comparison of the images in panels i and ii indicates co-localisation of 3′-ETV1 with 5′-ACSL3 and co-localisation of 5′-ETV1 and 3′-ACSL3. This is confirmed by ETV1-ACSL3 co-localisation assays (panel iii) demonstrating co-localisation of 3′-ETV1 sequences (red) and 5′-ACSL3 sequences (green) and (panel iv) demonstrating co-localisation of 3′-ACSL3 sequences and 5′-ETV1 sequences (red) in the same cell. Superimposition of the images in panels iii and iv confirms co-localisation of wild-type 3′-ETV1 (panel iii) with 5′-ETV1 (panel iv) and of wild-type 3′-ACSL3 (panel iv) with 5′-ACSL3 (panel iii). The genes and their direction of transcription are indicated by the arrowheads. (E) Map of the ACSL3 gene showing the position of the BACs used as probes in FISH assays. Probe XV: A1 (RP11-157M20) labelled with FITC. Probe XIV: A2 (RP11-136M23) and A3 (RP11-749C15) labelled with Cy3. Probes XV and probes XIV correspond, respectively, to sequences immediately 5′ (green) and 3′ (red) to the ACSL3 gene. Direction of gene transcription indicated by arrowheads.

Mentions: We performed 5′-RACE to identify novel partners that are fused to 3′-ETV1 sequences. Our studies were severely limited by the small amounts (50–200 ng) of poor quality RNA that could be prepared from the formalin-fixed tissue in this series. As obtainable RT–PCR products from these paraffin tissues were limited to ∼100–150 bp and the ETV1 exon breakpoint in each sample was unknown, 5′-RACE–PCR had to be independently initiated from each of the known ETV1 exon breakpoints in each sample, that is, exons 2, 4, 5 and 6. Using this strategy we successfully obtained a 5′-RACE fusion product from one RNA sample that contained an ex6 ETV1 sequence fused to a 51 bp sequence of ACSL3 ex3 sequence identifying ACSL3 as a novel ETV1 fusion partner. The structure of this ACSL3 ex3:ETV1 ex6 fusion is predicted to encode a truncated ETV1 protein as shown in Figure 3A. The presence of the ACSL3:ETV1 fusion was confirmed in this specimen by RT–PCR using 5′-ACSL3 and 3′-ETV1 primers (Figure 3B, C) and co-localisation by FISH of BAC probes corresponding to 5′-ACSL3 sequences (green) and 3′-ETV1 sequences (red) (Figure 3D, panel iii). An ACSL3 break-apart FISH assay screen of the entire TMA containing the 23 cancers with rearrangement of the ETV1 gene failed to identify additional cancers with this particular fusion. Like fusion to TMPRSS2, HNRPA2B1, HERV-K or SLC45A5/Prostein the fusion of 3′-ETV1 sequences to 5′-ACSL3 sequences is not a common event in this patient cohort. We have also demonstrated fusion of 5′-ETV1 sequences with 3′-ACSL3 sequences by FISH, indicating that the mechanism underlying formation of this fusion gene is a balanced translocation (Figure 3D, panel iv).


Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer.

Attard G, Clark J, Ambroisine L, Mills IG, Fisher G, Flohr P, Reid A, Edwards S, Kovacs G, Berney D, Foster C, Massie CE, Fletcher A, De Bono JS, Scardino P, Cuzick J, Cooper CS, Transatlantic Prostate Gro - Br. J. Cancer (2008)

ACSL3:ETV1 fusion. (A) ACSL3 (red) and ETV1 (blue) transcripts with ORFs in dark colour. Exons are numbered. A fusion transcript of ACSL3 exon 3 fused to ETV1 exon 6 was detected by 5′-RACE from exon 6 ETV1 sequences in prostate cancer sample 23. The ORF shown was predicted using software at www.dnalc.org. (B) Sequence across the ACSL3:ETV1 fusion boundary. Underlined regions indicate the position of primers used in RT–PCR to confirm the fusion. The predicted fusion gene initiation codon is indicated in red. ACSL3 sequence is in lower case and ETV1 sequence in upper case. (C) RT–PCR detection of an ACSL3:ETV1 fusion transcript in RNA extracted from formalin-fixed paraffin-embedded prostate cancer samples: lanes 1–12 are ETV1-rearranged tumour samples, lane 12: tumour sample 23, lane 13 negative control. (D) FISH assays to confirm fusion of ACSL3 with ETV1. Panel i: The ETV1 break-apart assay utilises probes corresponding to 3′-ETV1 sequences (red) and 5′-ETV1 sequences (green) (see also Figure 1). A nucleus with separated red and green probes confirming rearrangement of ETV1 is shown. Panel ii: The ACSL3 break-apart assay hybridised the same TMA slice used in the ETV1 break-apart assay to 3′-ACSL3 sequences (red) and 5′-ACSL3 sequences (green). These signals are coincident in the wild type, but are split on translocation of ACSL3. Comparison of the images in panels i and ii indicates co-localisation of 3′-ETV1 with 5′-ACSL3 and co-localisation of 5′-ETV1 and 3′-ACSL3. This is confirmed by ETV1-ACSL3 co-localisation assays (panel iii) demonstrating co-localisation of 3′-ETV1 sequences (red) and 5′-ACSL3 sequences (green) and (panel iv) demonstrating co-localisation of 3′-ACSL3 sequences and 5′-ETV1 sequences (red) in the same cell. Superimposition of the images in panels iii and iv confirms co-localisation of wild-type 3′-ETV1 (panel iii) with 5′-ETV1 (panel iv) and of wild-type 3′-ACSL3 (panel iv) with 5′-ACSL3 (panel iii). The genes and their direction of transcription are indicated by the arrowheads. (E) Map of the ACSL3 gene showing the position of the BACs used as probes in FISH assays. Probe XV: A1 (RP11-157M20) labelled with FITC. Probe XIV: A2 (RP11-136M23) and A3 (RP11-749C15) labelled with Cy3. Probes XV and probes XIV correspond, respectively, to sequences immediately 5′ (green) and 3′ (red) to the ACSL3 gene. Direction of gene transcription indicated by arrowheads.
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fig3: ACSL3:ETV1 fusion. (A) ACSL3 (red) and ETV1 (blue) transcripts with ORFs in dark colour. Exons are numbered. A fusion transcript of ACSL3 exon 3 fused to ETV1 exon 6 was detected by 5′-RACE from exon 6 ETV1 sequences in prostate cancer sample 23. The ORF shown was predicted using software at www.dnalc.org. (B) Sequence across the ACSL3:ETV1 fusion boundary. Underlined regions indicate the position of primers used in RT–PCR to confirm the fusion. The predicted fusion gene initiation codon is indicated in red. ACSL3 sequence is in lower case and ETV1 sequence in upper case. (C) RT–PCR detection of an ACSL3:ETV1 fusion transcript in RNA extracted from formalin-fixed paraffin-embedded prostate cancer samples: lanes 1–12 are ETV1-rearranged tumour samples, lane 12: tumour sample 23, lane 13 negative control. (D) FISH assays to confirm fusion of ACSL3 with ETV1. Panel i: The ETV1 break-apart assay utilises probes corresponding to 3′-ETV1 sequences (red) and 5′-ETV1 sequences (green) (see also Figure 1). A nucleus with separated red and green probes confirming rearrangement of ETV1 is shown. Panel ii: The ACSL3 break-apart assay hybridised the same TMA slice used in the ETV1 break-apart assay to 3′-ACSL3 sequences (red) and 5′-ACSL3 sequences (green). These signals are coincident in the wild type, but are split on translocation of ACSL3. Comparison of the images in panels i and ii indicates co-localisation of 3′-ETV1 with 5′-ACSL3 and co-localisation of 5′-ETV1 and 3′-ACSL3. This is confirmed by ETV1-ACSL3 co-localisation assays (panel iii) demonstrating co-localisation of 3′-ETV1 sequences (red) and 5′-ACSL3 sequences (green) and (panel iv) demonstrating co-localisation of 3′-ACSL3 sequences and 5′-ETV1 sequences (red) in the same cell. Superimposition of the images in panels iii and iv confirms co-localisation of wild-type 3′-ETV1 (panel iii) with 5′-ETV1 (panel iv) and of wild-type 3′-ACSL3 (panel iv) with 5′-ACSL3 (panel iii). The genes and their direction of transcription are indicated by the arrowheads. (E) Map of the ACSL3 gene showing the position of the BACs used as probes in FISH assays. Probe XV: A1 (RP11-157M20) labelled with FITC. Probe XIV: A2 (RP11-136M23) and A3 (RP11-749C15) labelled with Cy3. Probes XV and probes XIV correspond, respectively, to sequences immediately 5′ (green) and 3′ (red) to the ACSL3 gene. Direction of gene transcription indicated by arrowheads.
Mentions: We performed 5′-RACE to identify novel partners that are fused to 3′-ETV1 sequences. Our studies were severely limited by the small amounts (50–200 ng) of poor quality RNA that could be prepared from the formalin-fixed tissue in this series. As obtainable RT–PCR products from these paraffin tissues were limited to ∼100–150 bp and the ETV1 exon breakpoint in each sample was unknown, 5′-RACE–PCR had to be independently initiated from each of the known ETV1 exon breakpoints in each sample, that is, exons 2, 4, 5 and 6. Using this strategy we successfully obtained a 5′-RACE fusion product from one RNA sample that contained an ex6 ETV1 sequence fused to a 51 bp sequence of ACSL3 ex3 sequence identifying ACSL3 as a novel ETV1 fusion partner. The structure of this ACSL3 ex3:ETV1 ex6 fusion is predicted to encode a truncated ETV1 protein as shown in Figure 3A. The presence of the ACSL3:ETV1 fusion was confirmed in this specimen by RT–PCR using 5′-ACSL3 and 3′-ETV1 primers (Figure 3B, C) and co-localisation by FISH of BAC probes corresponding to 5′-ACSL3 sequences (green) and 3′-ETV1 sequences (red) (Figure 3D, panel iii). An ACSL3 break-apart FISH assay screen of the entire TMA containing the 23 cancers with rearrangement of the ETV1 gene failed to identify additional cancers with this particular fusion. Like fusion to TMPRSS2, HNRPA2B1, HERV-K or SLC45A5/Prostein the fusion of 3′-ETV1 sequences to 5′-ACSL3 sequences is not a common event in this patient cohort. We have also demonstrated fusion of 5′-ETV1 sequences with 3′-ACSL3 sequences by FISH, indicating that the mechanism underlying formation of this fusion gene is a balanced translocation (Figure 3D, panel iv).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Research, Male Urological Cancer Research Centre, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus