correlated with rearrangement of the ETV1 chromosomal region. Moreover, we identified a novel, N-truncated ETV1 isoform with the same activity as full-length ETV1. Results and Discussion Previously, we reported ETV1 overexpression in eight out of 84 prostate cancer samples. In four cases this was caused by a gene fusion, in the other four a full-length ETV1 transcript was overexpressed [10]. In the present study we investigated overexpression of ETV1 by quantitative reverse transcriptase reaction (QPCR) in a novel cohort of 66 prostate cancers. In six RNAs ETV1 overexpression was detected. Samples G51, G59, G233, G270 and G268 were derived from primary tumors and G210 was derived from a recurrent tumor. The six samples were further studied by QPCR with primer pairs at the 59-end of ETV1 mRNA (exon 1F and exon 4R) and at the 39-end of the mRNA (exon 11F and exon 12R) (Figure 1A). A high exon 11–12 to exon 1–4 ratio is indicative for a fusion gene; a 1:1 ratio indicated overexpression of full-length ETV1 mRNA. Based on these criteria, tumors G51, G59, G233 and G270 expressed full-length ETV1 whereas G210 and G268 expressed a fusion transcript (see also control PC374 that expresses TMPRSS2-ETV1). HNRPA2B1- ETV1 was found as the fusion transcript in sample G210 (data not shown); the fusion transcript in G268 has not been identified as yet. These two samples were not investigated further in this study. In the next experiments we focused on the elucidation of the mechanism of overexpression of full-length ETV1. Recently, it has been shown that in two prostate cancer cell lines overexpressing full-length ETV1, LNCaP and MDA PCa2b, ETV1 is translocated [8]. We now addressed the question whether in clinical samples translocation of the complete ETV1 gene might occur. To detect genomic rearrangements tissue slides of all four prostate cancer samples with full-length ETV1 overexpression were analyzed by break-apart interphase FISH with two labeled BAC probes, one BAC recognized ETV1 and the second one the flanking gene DGKB (Figure 1B). The series was supplemented with two samples harboring full-length ETV1 overexpression from our previous study, G89 and G308 [10], for which frozen tissues of sufficient morphological quality were available. Figure 1B shows the results of the FISH experiments. Interestingly, we found split signals in all samples except for G59, indicating frequent ETV1 rearrangements in prostate cancers that overexpress full-length ETV1. Absence of a split signal in G59 suggests absence of translocation, although we cannot exclude a breakpoint outside the investigated region. Its observed high frequency indicates genomic rearrangement as an important mechanism of full-length ETV1 overexpression in clinical prostate cancer. Obviously, the chromosomal region to which ETV1 is translocated can contribute to elucidation of the mechanism of ETV1 overexpression. We were able to identify more details of the rearrangements in samples G270 and G89. It has been shown in LNCaP cells that ETV1 is translocated to 14q13.3-q21.1. The whole gene is integrated in the last intron of MIPOL1. In MDA PCa2b, ETV1 is translocated to the same region, although the precise position is unknown [8]. Moreover, we previously described insertion of truncated ETV1 in the intron of a two exon gene encoding a ncRNA, denoted EST14, giving rise to an EST14-ETV1 fusion transcript that contains ETV1 exon 5–12 sequences (sample G342 in ref. 10; Figure 2A). Importantly, EST14 maps directly adjacent to MIPOL1 on 14q. Like most ETV1 fusion partners, EST14 is an androgen-regulated prostate-specific gene. To investigate whether the same chromosomal region was involved in full-length ETV1 translocation in our novel cohort, interphase FISH was performed with the ETV1 BAC (Figure 1B) in combination with ETV1 Translocations in Prostate Cancer a MIPOL1 BAC (Figure 1C). A merging yellow signal was detected in sample G270 (Figure 1C) but in none of the other tumors. These data indicate that although there seems a preference for chromosome 14q13.3-21.1, other genomic regions will also contribute to rearrangement and overexpression of full-length ETV1. Additional information of ETV1 rearrangement in G270 came from 59-RACE of tumor cDNA (data not shown). Remarkably, we did not only detect as expected the full-length ETV1 transcript but also a fusion transcript (Figure 2A). Such a result was not found for any of the other tumors overexpressing full-length ETV1. Sequencing showed that the fusion transcript in G270 was composed of ETV1 exon 1–12 preceded by the first exon of EST14 (Figure 2A). Scanning the EST14 intron and ETV1 flanking region by long-range PCR and sequencing mapped the break- points in G270 ,1.9 Kbp upstream of ETV1 exon 1 and ,5 Kbp downstream of EST14 exon 1. The breakpoint in EST14 is only 180 bp apart from the breakpoint in G342 (Figure 2B and ref. 10). Further information of ETV1 rearrangement was also collected for sample G89. Previously, a xenograft propagated on male nude mice had been generated from this tumor (PC135). Like tumor G89, PC135 overexpressed full-length ETV1 [3]. The availability of the xenograft allowed the preparation of metaphase chromo- some spreads. In multicolor FISH a complex chromosomal rearrangement pattern was found (data not shown). Individual chromosome paints were used to validate the multicolor FISH data. Painting of chromosome 7 indicated the presence of multiple chromosome 7 fragments (Figure 3). Hybridization with an ETV1 BAC identified the presence of three gene copies: two in apparently normal chromosomes 7 and one in a complex marker chromosome. Follow-up experiments showed that the marker chromosome contained fragments of chromosomes 4, 7 and 10, as first indicated by multicolor FISH (Figure 3). The ETV1 BAC hybridized at the junction of the chromosome 7 and the chromosome 4 fragment, strongly suggesting that the 4;7 translocation was instrumental in overexpression of ETV1. The precise positions of the breakpoints in 4 and 7 remain to be determined. Our data predict that multiple chromosomal regions are involved in overexpression of full-length ETV1. At least one of these regions is on chromosome 14 and a second one on chromosome 4. The chromosome 14 region is also involved in ETV1 gene fusion. Deep sequencing technology could be instrumental in identification of other ETV1 rearrangements. Detailed characterization of the full-length ETV1 transcripts in the various tumors by 59-RACE and sequencing showed that not only ETV1 exon 1- exon 12 transcripts were present but also various other full-length ETV1 transcripts, resulting from alterna- tive promoter usage. These transcripts were denoted as ETV1, ETV1-1a, ETV1-1b and ETV1-1c. Figure 4A and Supplementary Figure S1 show the positions of the different first exons in the gene and indicate the various ATG start codons. Of both ETV1-1a and ETV1-1b two splice variants were found (data not shown). QPCR experiments using transcript-specific primers on RNA from all six clinical prostate cancer samples that overexpressed ETV1 showed that the level of expression of the different transcripts was variable in the various tumors (see Figure S2). ETV1 was hardly expressed in control benign prostate hyperplasia sample G277. Figure 4B schematically represents the predicted composition of the various ETV1 protein isoforms that will be produced. Note that ETV1-1c is by far the shortest, lacking the N-terminal 60 amino acids, including the major part of the conserved acidic TAD. In dETV1 that is expressed by most fusion genes, the N-termimal 131 amino acids are absent. ETV1, ETV1-1b1 and -1b2 were of similar size, as shown in Western blots of lysates from transfected HEK293T cells (Figure 4B). PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16332