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

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.

Figure 1

FISH detection of ERG gene breakpoints. Top right: Principle of detection of ERG gene status. Interphase nuclei are hybridized to probes that detect sequences 5′ to the ERG gene (green) and 3′ to the ERG gene (red). The red and green signals are separated when an ERG gene rearrangement occurs. (a) Signals from normal unrearranged ERG loci (class N). (b) Signals from rearranged ERG gene with retention of separated red (3′) and green (5′) probes (class Esplit). (c) Example of a ‘1Edel’ cancer: the rearrangement is associated with deletion of sequences 5′ to ERG (green) with retention of a single red 3′-ERG signal. (d and e) Examples of 2+Edel cancers: the rearrangement is associated with deletion of sequences 5′ to ERG (green) with retention of two or more red 3′-ERG signals. Bottom: Map of the ERG and TMPRSS2 genes showing the position of the BACs and fosmids used as probes in FISH assays. Probe I: 1:RP11-95G19, 2:RP11-720N21, 3:CTD-2511E13; probe II 4:RP11-372O18, 5:RP11-115E14, 6:RP11-729O4; probe III, 7:three pooled fosmids (G248P89444D12, G248P800876A1, G248P8239C5), 8:RP11-35C4, 9:RP11-282I20; probe IV, 10:RP11-114H1, 11:RP11-662D5. Probes I and II correspond, respectively, to sequences immediately 5′ (green) and 3′ (red) to the ERG gene. Probes III and IV correspond, respectively, to sequences immediately 5′ (green) and 3′ (red) to the TMPRSS2 gene.

Figure 2

Distribution of the pattern of ERG gene alterations in human prostate cancer. Each cancer is scored for three classes of FISH signals: normal unrearranged ERG loci (adjacent red and green signals); separate red signals that correspond to sequences 3′ to a rearranged ERG locus and separate green signals that correspond to sequences 5′ to a rearranged ERG locus. For example, ‘2,1,0’ means that the cancer contains two normal alleles, one separate red 3′-ERG signal and no green 5′-ERG signals. Cancers with only normal signals are designated as class N. When the ERG locus is split to form both separate 3′-ERG (red) and 5′-ERG (green) signals, the cancer is designated as class Esplit. Cancers containing separate red 3′ to ERG signals, but lacking separate green signals are designated ‘Edel’ cancers.

Figure 3

FISH detection of TMPRSS2 and ERG gene status. (left-hand side): the status of the ERG gene was examined using the ERG ‘break-apart’ assay using probes that detected sequences immediately 5′ (green, probe II) and 3′ (red, probe I) to ERG. (Righthand side): the same representative cancer cell was re-hybridized with FISH probes that detected sequences 5′ to TMPRSS2 (green, probe III) and 3′ to ERG (red, probe I). The principle of detection is in each case shown at the top and the precise origin of the three separate FISH probes used in these studies (probes I–III) can be seen in Figure 1. Nuclei shown in this figure were from cancers with the following ERG status: class N (a), class Esplit (b) and class Edel (c). RT–PCR studies carried out as described previously (Clark et al., 2007) confirmed the presence of TMPRSS2–ERG fusion transcripts only in cancers that contained rearranged ERG (result not shown). A comparison of the left- and right-hand panels in this figure shows that rearranged 3′-ERG always remains linked to 5′-TMPRSS2, and that the sequences examined by FISH located between the TMPRSS2 and ERG loci (probe II) are either missing (class Edel cancers) or positioned elsewhere in the nucleus (class Esplit cancers).

Figure 4

Kaplan–Meier analysis comparing prostate cancer outcomes for different categories of ERG gene alteration. (a and b) A comparison of class N, class Esplit and class Edel cancers. (c and d) Outcome stratified according to the copy number of 3′-ERG FISH signal in Edel cancers. The Kaplan–Meier curves compare class N cancer with Edel cancer containing a single copy of 3′-ERG sequences (1Edel), and with Edel cancer that contain two or more copies of 3′-ERG sequences (2+Edel). a and c are cause-specific survival. b and d are overall survival.

Figure 5

FISH analysis of the TMPRSS2 and ERG loci in 2+Edel cancers. The ‘left’ and ‘middle’ panels in this figure are the same as the ‘left’ and ‘right panels’ in Figure 3 showing, respectively, results from the ERG locus ‘break-apart’ assay (left), and from the FISH detection of sequences 5′ to TMPRSS2 and 3′ to ERG (middle). Additionally in the right-hand panel, the status of the TMPRSS2 gene has been examined using a TMPRSS2 ‘break-apart’ assay by re-hybridizing the same representative cancer cell with FISH probes that detected sequences immediately 5′ (green, probe III) and 3′ (red, probe IV) to TMPRSS2. The principle of detection is in each case shown at the top and the precise origin of the four separate FISH probes used in these studies (probes I–IV) can be seen in Figure 1. The nuclei shown in this figure were from a class N cancer (a) and a class 2+Edel cancer. (b) RT–PCR studies carried out as described previously (Clark et al., 2007) confirmed the presence of TMPRSS2–ERG fusion transcripts only in the 2+Edel cancer (result not shown). These analyses demonstrate that cancers with a rearranged ERG locus also have a rearranged TMPRSS2 locus, (ii) the rearranged 5′-TMPRSS2 and 3′-ERG sequences remain joined together and (iii) the rearrangement and joining of 5′-TMPRSS2 and 3′-ERG is accompanied by deletion of intervening sequences corresponding to both probes II and IV.

Figure 6

(a and b) Kaplan–Meier analysis stratifying prostate cancer outcomes according to copy number of 3′-ERG FISH signal: (a) class Esplit cancers, cause-specific survival; (b) class Esplit cancers, overall survival. (c and d) Kaplan–Meier analysis comparing prostate cancers outcomes depending on ERG ploidy status: (c) cause-specific survival and (d) overall survival.