Oncogene (2015) 34, 568–577 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Next-generation sequencing reveals novel rare fusion events with functional implication in prostate cancer I Teles Alves1, T Hartjes2, E McClellan3,4, S Hiltemann2,RBo¨ ttcher2, N Dits2, MR Temanni5, B Janssen6, W van Workum6, P van der Spek3, A Stubbs3, A de Klein7, B Eussen7, J Trapman8 and G Jenster2 Gene fusions, mainly between TMPRSS2 and ERG, are frequent early genomic rearrangements in prostate cancer (PCa). In order to discover novel genomic fusion events, we applied whole-genome paired-end sequencing to identify structural alterations present in a primary PCa patient (G089) and in a PCa cell line (PC346C). Overall, we identified over 3800 genomic rearrangements in each of the two samples as compared with the reference genome. Correcting these structural variations for polymorphisms using whole-genome sequences of 46 normal samples, the numbers of cancer-related rearrangements were 674 and 387 for G089 and PC346C, respectively. From these, 192 in G089 and 106 in PC346C affected gene structures. Exclusion of small intronic deletions left 33 intergenic breaks in G089 and 14 in PC346C. Out of these, 12 and 9 reassembled genes with the same orientation, capable of generating a feasible fusion transcript. Using PCR we validated all the reliable predicted gene fusions. Two gene fusions were in-frame: MPP5–FAM71D in PC346C and ARHGEF3–C8ORF38 in G089. Downregulation of FAM71D and MPP5–FAM71D transcripts in PC346C cells decreased proliferation; however, no effect was observed in the RWPE-1-immortalized normal prostate epithelial cells. Together, our data showed that gene rearrangements frequently occur in PCa genomes but result in a limited number of fusion transcripts. Most of these fusion transcripts do not encode in-frame fusion proteins. The unique in-frame MPP5–FAM71D fusion product is important for proliferation of PC346C cells. Oncogene (2015) 34, 568–577; doi:10.1038/onc.2013.591; published online 3 February 2014 Keywords: FAM71D; gene fusions; next-generation sequencing; prostate cancer; MPP5 INTRODUCTION approach uses only 1–2% of the genomic sequences as a Prostate cancer (PCa) is one of the most frequently diagnosed template through capture and enrichment before the sequencing cancers and a major cause of death in men in countries with a process.15,16 This allows for higher sequence coverage at the western lifestyle.1 Throughout the past two decades, several expense of a few disadvantages such as the uneven capture genetic events have been revealed that are important in efficiency and the absence of unknown or yet to be annotated development and progression of PCa.2 The predominant genetic exons.17 Instead of a focused exome-sequencing approach, more abnormalities identified so far include the forming of ETS-fusion challenging whole-genome sequencing provides the most genes,3 loss of phosphatase and tensin homolog (PTEN) tumor complete view of genomic changes.18 A range of sequencing suppressor gene,4 amplification of AR and amplification of the technologies is now available and more are becoming available MYC oncogene.5 Most of these studies used comparative genomic soon that provide different approaches for library construction, hybridization/SNP (single nucleotide length polymorphism) arrays clone separation and amplification, and nucleotide detection.19–22 and performed genome-wide copy number variation analysis The technology utilized in this study from Complete Genomics Inc. as a start to identify specific genetic alterations.6,7 The use of (Mountain View, CA, USA) makes use of array technology to gene expression arrays was important in the detection of genes separate amplified DNA template organized into single-strand with aberrant expression patterns.8 The integration of these two coils, known as DNA nanoballs. Nucleotide read-out is based on a different sets of data, copy number variation and gene expression ligation protocol.23 reinforced and expanded this panel of genetic alterations.9,10 In PCa genomics the use of NGS has allowed significant Next-generation sequencing (NGS) techniques emerging over progress in cataloging systematically all the DNA changes present the last few years proved to be a major breakthrough in in cancer.24 The use of RNA-seq has produced major insight documenting novel genetic changes resulting in a better under- into the identification and expression of novel long noncoding standing of cancer cell biology.11,12 Both RNA and DNA can be RNAs and novel gene fusions in PCa.25,26 Exome sequencing of used as templates for NGS methods and it is possible to analyze PCa samples was utilized for the detection of novel small either paired-end, mate pair, as short or long sequence reads mutations.27–30 In the same way, a mutational landscape of depending on the platform applied.13,14 An exome-sequencing patients with castration-resistant PCa was described by Tomlins 1Department of Urology and Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands; 2Department of Urology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands; 3Department of Bioinformatics, Erasmus University Medical Center, Rotterdam, The Netherlands; 4Department of Mathematical and Computer Sciences, Metropolitan State University of Denver, Colorado, USA; 5CMNS-Institute for Advanced Computer Studies, University of Maryland, College Park, MD, USA; 6ServiceXS B.V., Leiden, The Netherlands; 7Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands and 8Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Correspondence: Dr G Jenster, Department of Urology, Josephine Nefkens Institute, Erasmus University Medical Center, Be 362a, P.O. Box 2040, Rotterdam 3000 CA, The Netherlands. E-mail: [email protected] Received 12 March 2013; revised 30 November 2013; accepted 18 December 2013; published online 3 February 2014 Novel rare fusion events with functional implication in prostate cancer I Teles Alves et al 569 et al.3 in 2012.31 In addition, deep RNA sequencing is also used to same orientation were identified (Table 2; Supplementary Tables identify transcription-induced chimeras associated with human S3–S5). prostate adenocarcinoma such as the novel TMEM79-SMG5.32,33 However, a full overview of all structural variations (SVs) and mutations can only be provided by whole-genome sequencing. Validation of fusion genes present in PC346C and G089 Many studies so far have applied NGS to obtain a more thorough For each possible gene fusion event in PC346C and G089, we perceptive of already known genetic alterations.34 The mechanism checked all the mapped reads, both the normal and the mispaired involving PTEN loss has been correlated with novel genetic reads (Supplementary Figure S3). We observed that a number of alterations in genes located in the vicinity of PTEN.24 Moreover, the fusion events were not supported by a convincing number of expression of constitutively active androgen receptor splice discordant mate pairs (Table 2). To evaluate whether the criterion variants in castration resistance PCa has been described.35 A few of three or more mispaired reads was justified, all 9 þ 11 fusion studies have been published making use of whole or focused events were validated using (RT)–PCR both at the DNA and RNA genome NGS technologies to discover and describe new genetic levels (Supplementary Figures S4 and S5, Supplementary Tables alterations in PCa. The study by Berger et al.24 in 2011 provided a S6–S9). Most of the fusion events with low mispaired read counts comprehensive approach towards both known and newly (o5) could not be confirmed, showing that the cutoff number of identified mutations and genomic rearrangements. Other studies three discordant mate pairs was set low. have focused on either a particular genomic region such as the For most gene fusions, the event was confirmed by sequencing 10q11.2 97-kb region comprising the MSMB gene or in the analysis on the RNA and/or DNA level. Out of these six PC346C fusion of all alterations present in a particular PCa sample.36,37 This was events, all were validated on the DNA level with only one that could the case for the new type of prostate adenocarcinoma identified not be verified at the transcript level (Supplementary Tables S10 that has a hybrid phenotype of both luminal and neuroendocrine and S11). The fusion in question, ITGA10–RBM8A, comprises the cells.37,38 downstream part of the 30-untranslated repeat sequence of RBM8A In this study, we applied whole-genome paired-end sequencing so it was not expected to be present within the fusion transcript. In in two PCa samples. Analysis of the PC346C PCa cell line and the G089, out of the nine reliable fusion events eight were validated at G089 primary PCa patient sample, which were selected based on the DNA level and five at the RNA level. The four fusions that were the absence of ETS-fusion genes,39 generated a wide panel of validated only at the DNA level were assessed for the expression new SV events. We focused on the detection of SVs to unravel levels of the involving genes using Affymetrix Exon-array analysis the events leading to fusion genes. The potential fusion genes (Affymetrix, Santa Clara, CA, USA) of the G089 PCa sample. In were validated and the genes involved were checked for general, we found a low expression pattern of these fusion genes. copy number alteration in other PCa samples. The in-frame The OBP2A–OBP2B fusion, comprising only three mispaired reads, fusion found in PC346C was assessed for possible functional was verified at the RNA level and involved two homologous genes. implications. The AATK–LAMA5 fusion was confirmed at the DNA level, although the number of discordant mate pairs was only five (Table 2). Next, we assessed which fusion events generated an in-frame fusion protein.