Dorothee Pflueger

The genomic complexity of primary human prostate cancer

Prostate cancer is the second most common cause of male cancer deaths in the United States. However, the full range of prostate cancer genomic alterations is incompletely characterized. Here we present the complete sequence of seven primary human prostate cancers and their paired normal counterparts. Several tumours contained complex chains of balanced (that is, ‘copy-neutral’) rearrangements that occurred within or adjacent to known cancer genes. Rearrangement breakpoints were enriched near open chromatin, androgen receptor and ERG DNA binding sites in the setting of the ETS gene fusion TMPRSS2–ERG, but inversely correlated with these regions in tumours lacking ETS fusions. This observation suggests a link between chromatin or transcriptional regulation and the genesis of genomic aberrations. Three tumours contained rearrangements that disrupted CADM2, and four harboured events disrupting either PTEN (unbalanced events), a prostate tumour suppressor, or MAGI2 (balanced events), a PTEN interacting protein not previously implicated in prostate tumorigenesis. Thus, genomic rearrangements may arise from transcriptional or chromatin aberrancies and engage prostate tumorigenic mechanisms.

Discovery of non-ETS gene fusions in human prostate cancer using next-generation RNA sequencing

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date, little is known about the presence of non-ETS fusion events in prostate cancer. We used next-generation transcriptome sequencing (RNA-seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and four involving non-ETS genes such as CDKN1A (p21), CD9, and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency, but, interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement-prone phenotype.

FusionSeq: a modular framework for finding gene fusions by analyzing paired-end RNA-sequencing data.

We have developed FusionSeq to identify fusion transcripts from paired-end RNA-sequencing. FusionSeq includes filters to remove spurious candidate fusions with artifacts, such as misalignment or random pairing of transcript fragments, and it ranks candidates according to several statistics. It also has a module to identify exact sequences at breakpoint junctions. FusionSeq detected known and novel fusions in a specially sequenced calibration data set, including eight cancers with and without known rearrangements.

Distinct Genomic Aberrations Associated With ERG Rearranged Prostate Cancer

Emerging molecular and clinical data suggest that ETS fusion prostate cancer represents a distinct molecular subclass, driven most commonly by a hormonally regulated promoter and characterized by an aggressive natural history. The study of the genomic landscape of prostate cancer in the light of ETS fusion events is required to understand the foundation of this molecularly and clinically distinct subtype. We performed genome‐wide profiling of 49 primary prostate cancers and identified 20 recurrent chromosomal copy number aberrations, mainly occurring as genomic losses. Co‐occurring events included losses at 19q13.32 and 1p22.1. We discovered three genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in nonrearranged prostate cancer is accompanied by gene expression deregulation in an independent dataset and by protein deregulation of MYO6. To analyze copy number alterations within the ETS genes, we performed a comprehensive analysis of all 27 ETS genes and of the 3 Mbp genomic area between ERG and TMPRSS2 (21q) with an unprecedented resolution (30 bp). We demonstrate that high‐resolution tiling arrays can be used to pin‐point breakpoints leading to fusion events. This study provides further support to define a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.

Characterization of KRAS Rearrangements in Metastatic Prostate Cancer

UNLABELLED Using an integrative genomics approach called amplification breakpoint ranking and assembly analysis, we nominated KRAS as a gene fusion with the ubiquitin-conjugating enzyme UBE2L3 in the DU145 cell line, originally derived from prostate cancer metastasis to the brain. Interestingly, analysis of tissues revealed that 2 of 62 metastatic prostate cancers harbored aberrations at the KRAS locus. In DU145 cells, UBE2L3-KRAS produces a fusion protein, a specific knockdown of which attenuates cell invasion and xenograft growth. Ectopic expression of the UBE2L3-KRAS fusion protein exhibits transforming activity in NIH 3T3 fibroblasts and RWPE prostate epithelial cells in vitro and in vivo. In NIH 3T3 cells, UBE2L3-KRAS attenuates MEK/ERK signaling, commonly engaged by oncogenic mutant KRAS, and instead signals via AKT and p38 mitogen-activated protein kinase (MAPK) pathways. This is the first report of a gene fusion involving the Ras family, suggesting that this aberration may drive metastatic progression in a rare subset of prostate cancers. SIGNIFICANCE This is the first description of an oncogenic gene fusion of KRAS, one of the most studied proto-oncogenes. KRAS rearrangement may represent the driving mutation in a rare subset of metastatic prostate cancers, emphasizing the importance of RAS-RAF-MAPK signaling in this disease.

Testing mutual exclusivity of ETS rearranged prostate cancer

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5′ fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements.

Functional characterization of BC039389-GATM and KLK4-KRSP1 chimeric read-through transcripts which are up-regulated in renal cell cancer

BackgroundChimeric read-through RNAs are transcripts originating from two directly adjacent genes (<10 kb) on the same DNA strand. Although they are found in next-generation whole transcriptome sequencing (RNA-Seq) data on a regular basis, investigating them further has usually been refrained from. Therefore, their expression patterns or functions in general, and in oncogenesis in particular, are poorly understood.ResultsWe used paired-end RNA-Seq and a specifically designed computational data analysis pipeline (FusionSeq) to nominate read-through events in a small discovery set of renal cell carcinomas (RCC) and confirmed them in a larger validation cohort.324 read-through events were called overall; 22/27 (81%) selected nominees passed validation with conventional PCR and were sequenced at the junction region. We frequently identified various isoforms of a given read-through event. 2/22 read-throughs were up-regulated: BC039389-GATM was higher expressed in RCC compared to benign adjacent kidney; KLK4-KRSP1 was expressed in 46/169 (27%) RCCs, but rarely in normal tissue. KLK4-KRSP1 expression was associated with worse clinical outcome in the patient cohort. In cell lines, both read-throughs influenced molecular mechanisms (i.e. target gene expression or migration/invasion) in a way that counteracted the effect of the respective parent transcript GATM or KLK4.ConclusionsOur data suggests that the up-regulation of read-through RNA chimeras in tumors is not random but causes regulatory effects on cellular mechanisms and may impact patient survival.

Abstract 3925: Characterization of complex chromosomal aberrations in prostate cancer from whole genome sequencing

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prostate cancer is the second most common cause of male cancer deaths in the United States, accounting for 200,000 new cases and 32,000 deaths per year. Chromosomal rearrangements comprise a major mechanism driving prostate carcinogenesis. For example, recurrent gene fusions that render ETS transcription factors under the control of androgen-responsive promoters are present in the majority of prostate cancers. Other types of somatic alterations, such as base substitutions, small insertions/deletions, and chromosomal copy number alterations, have also been described, yet the full repertoire of genomic alterations that underlie primary human prostate cancer remains incompletely characterized. We present here the most comprehensive genome sequencing effort in prostate cancer reported to date. We have characterized the complete genomes of 7 primary prostate cancers and patient-matched normal samples using massively parallel sequencing technology. We observed a mean mutation frequency of 0.9 per megabase, consistent with what has been reported for other tumor types. However, our results indicate that translocations and other chromosomal rearrangements are far more common than expected, with a median of 90 per prostate cancer genome. Several tumors contained chains of balanced rearrangements involving multiple loci associated with known cancer genes. We observed a striking and unexpected relationship between rearrangement breakpoints and chromatin structure, which differed for tumors harboring the ETS gene fusion TMPRSS2-ERG and tumors lacking ETS fusions. We also observed an enrichment of point mutations near rearrangement breakpoints. Three of seven tumors contained rearrangements that disrupted CADM2, a nectin-like member of the immunoglobulin-like cell adhesion molecules; recurrent CADM2 rearrangements were also detected in an independent cohort by fluorescent in situ hybridization (FISH). Four tumors harbored rearrangements disrupting either PTEN, a prostate tumor suppressor, or MAGI2, a PTEN interacting protein not previously implicated in prostate cancer. Together, these results illuminate potential avenues for target discovery and reveal the potential of complex rearrangements to engage prostate tumorigenic mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3925. doi:10.1158/1538-7445.AM2011-3925

Abstract 1139: Complete characterization of prostate cancer genomes by massively parallel sequencing

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Prostate cancer is the most common type of cancer diagnosed among men in the United States, accounting for 200,000 new cases and 27,000 deaths per year. Prior genetic studies have shown that chromosomal rearrangements comprise a major mechanism of oncogene activation in prostate cancer. For example, androgen-regulated gene fusions involving ETS family transcription factors are present in the majority of prostate cancers, yet the full repertoire of genomic alterations driving prostate carcinogenesis and progression remains unknown. Toward this end, recent technological advances have made it possible to characterize the full complement of somatic mutations in a single tumor through whole genome sequencing. We are using massively parallel sequencing technology to characterize the complete genomes of several primary prostate adenocarcinomas at >30x coverage. All samples are high-grade primary tumors (Gleason grade 7 to 9) and include cases with and without known ETS family translocations. For each tumor, we are also obtaining >30x sequence coverage of matched normal DNA from blood of these same patients in order to determine the somatic component of the overall variation we observe. Our results indicate that translocations and other chromosomal rearrangements occur frequently in prostate cancer, at a rate of >100 per genome. Further, we have discovered many nonsynonymous sequence mutations (point mutations and indels) in each tumor, some of which may represent novel candidate drivers of tumor progression. The overall rate of somatic point mutations is approximately 1 per Megabase. Integrated analysis of all genomes reveals both recurrent and private alterations. Together, these results illuminate potential avenues for target discovery and demonstrate the unparalleled value in performing complete genome sequencing in this malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1139.

Abstract 2743: Accelerating the exploration of novel gene fusion events in prostate cancer

Gene fusions are regarded as cancer defining and often proven causative for cancer development. Up to 50% of prostate cancers harbor recurrent gene fusions, most often the TMPRSS2-ERG fusion. Other ETS gene fusions have been described involving ETV1, ETV4, and ETV5. The remaining prostate cancers are considered gene fusion negative based on fluorescence in-situ hybridization (FISH) or RT-PCR for known fusion specific transcripts. Paired-end RNA-sequencing is a novel approach to systematically interrogate for fusion transcripts in a global and un-biased manner. Herein, we exploit FusionSeq, a computational tool specifically developed to nominate chimeric transcripts from paired-end RNA-seq data, with the goal of identifying new gene fusions. Using FusionSeq we identified the androgen-induced genes FKBP5 and KLK2 as two novel 5′ fusion partners for the ETS genes ERG and ETV1, respectively. General transcriptional chimerism (e.g. read-through transcripts) without evidence of underlying genomic rearrangements is not restricted to prostate cancer but also occurs in the patient9s matched benign prostate tissue, providing evidence for an extended complexity of the cellular transcriptome and supporting the notion that a gene9s definition might have to be considered flexible. Finally, we report the identification and the validation of two novel gene fusions: i) the tumor suppressor and cell cycle regulator CDKN1A (p21 WAF , CIP1) is fused to CD9; besides down-regulating p21 gene expression, the CDKN1A-CD9 fusion also alters the CD9 protein. ii) by fusing IKBKB (IKK2, IκB kinase beta subunit) to TNPO1 (transportin 1), IKBKB gene expression increases over other prostate cancers, suggesting a pronounced activation of the NFκB complex. Our results show that a small subset of prostate cancers may harbor private gene fusions involving tumor suppressors and regulators of pathways implicated in cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2743.