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1.  The Role of SPINK1 in ETS Rearrangement Negative Prostate Cancers 
Cancer cell  2008;13(6):519-528.
Summary
ETS gene fusions have been characterized in a majority of prostate cancers, however the key molecular alterations in ETS negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers.
doi:10.1016/j.ccr.2008.04.016
PMCID: PMC2732022  PMID: 18538735
2.  Targeted Next-generation Sequencing of Advanced Prostate Cancer Identifies Potential Therapeutic Targets and Disease Heterogeneity 
European urology  2012;63(5):920-926.
Background
Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue.
Objective
To demonstrate the feasibility of a novel next-generation sequencing (NGS) based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa.
Design, setting, and participants
FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression.
Intervention
At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform.
Outcome measurements and statistical analysis
A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations.
Results and limitations
We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected.
Conclusions
This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.
doi:10.1016/j.eururo.2012.08.053
PMCID: PMC3615043  PMID: 22981675
Next-generation sequencing; Castration-resistant prostate cancer; Prostate cancer genome
3.  Characterization of Transcriptional Changes in ERG Rearrangement-Positive Prostate Cancer Identifies the Regulation of Metabolic Sensors Such as Neuropeptide Y 
PLoS ONE  2013;8(2):e55207.
ERG gene rearrangements are found in about one half of all prostate cancers. Functional analyses do not fully explain the selective pressure causing ERG rearrangement during the development of prostate cancer. To identify transcriptional changes in prostate cancer, including tumors with ERG gene rearrangements, we performed a meta-analysis on published gene expression data followed by validations on mRNA and protein levels as well as first functional investigations. Eight expression studies (n = 561) on human prostate tissues were included in the meta-analysis. Transcriptional changes between prostate cancer and non-cancerous prostate, as well as ERG rearrangement-positive (ERG+) and ERG rearrangement-negative (ERG−) prostate cancer, were analyzed. Detailed results can be accessed through an online database. We validated our meta-analysis using data from our own independent microarray study (n = 57). 84% and 49% (fold-change>2 and >1.5, respectively) of all transcriptional changes between ERG+ and ERG− prostate cancer determined by meta-analysis were verified in the validation study. Selected targets were confirmed by immunohistochemistry: NPY and PLA2G7 (up-regulated in ERG+ cancers), and AZGP1 and TFF3 (down-regulated in ERG+ cancers). First functional investigations for one of the most prominent ERG rearrangement-associated genes - neuropeptide Y (NPY) - revealed increased glucose uptake in vitro indicating the potential role of NPY in regulating cellular metabolism. In summary, we found robust population-independent transcriptional changes in prostate cancer and first signs of ERG rearrangements inducing metabolic changes in cancer cells by activating major metabolic signaling molecules like NPY. Our study indicates that metabolic changes possibly contribute to the selective pressure favoring ERG rearrangements in prostate cancer.
doi:10.1371/journal.pone.0055207
PMCID: PMC3563644  PMID: 23390522
4.  Distinct Genomic Aberrations Associated With ERG Rearranged Prostate Cancer 
Genes, chromosomes & cancer  2009;48(4):366-380.
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 3 genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in non- rearranged 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 3Mbp 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 defining a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.
doi:10.1002/gcc.20647
PMCID: PMC2674964  PMID: 19156837
ETS genes; prostate cancer; gain; loss
5.  Prevalence of TMPRSS2-ERG and SLC45A3-ERG gene fusions in a large prostatectomy cohort 
The majority of prostate cancers harbor recurrent gene fusions between the hormone-regulated TMPRSS2 and members of the ETS family of transcription factors, most commonly ERG. Prostate cancer with ERG rearrangements represent a distinct subclass of tumor based on studies reporting associations with histomorphologic features, characteristic somatic copy number alterations, and gene expression signatures. The current study describes the frequency of ERG rearrangement prostate cancer and three 5 prime (5') gene fusion partners (i.e., TMPRSS2, SLC45A3 and NDRG1) in a large prostatectomy cohort.
ERG gene rearrangements and mechanism of rearrangement, as well as rearrangements of TMPRSS2, SLC45A3, and NDRG1 were assessed using fluorescence in-situ hybridization (FISH) on prostate cancer samples from 614 patients treated by radical prostatectomy. ERG rearrangement occurred in 53% of the 540 assessable cases. TMPRSS2 and SLC45A3 were the only 5' partner in 78% and 6% of these ERG rearranged cases, respectively. Interestingly, 11% of the ERG rearranged cases demonstrated concurrent TMPRSS2 and SLC45A3 rearrangements. TMPRSS2 or SLC45A3 rearrangements could not be identified for 5% of the ERG rearranged cases. From these remaining cases we identified one case with NDRG1 rearrangement. We did not observe any associations with pathologic parameters or clinical outcome.
This is the first study to describe the frequency of SLC45A3-ERG fusions in a large clinical cohort. Most studies have assumed that all ERG rearrangement prostate cancers harbor TMPRSS2-ERG fusions. This is also the first study to report concurrent TMPRSS2 and SLC45A3 rearrangements in the same tumor focus suggesting additional complexity that had not been previously appreciated. This study has important clinical implications for the development of diagnostic assays to detect ETS rearrangement prostate cancer. Incorporation of these less common ERG rearrangement prostate cancer fusion assays could further increase the sensitivity of these PCR-based approaches.
doi:10.1038/modpathol.2009.193
PMCID: PMC2848699  PMID: 20118910
Prostate cancer; ETS rearrangements; prevalence
6.  Chromosome rearrangement associated inactivation of tumour suppressor genes in prostate cancer 
Prostate cancer, the most common male cancer in Western countries, is commonly detected with complex chromosomal rearrangements. Following the discovery of the recurrent TMPRSS2:ETS fusions in prostate cancer and EML4:ALK in non-small-cell lung cancer, it is now accepted that fusion genes not only are the hallmark of haematological malignancies and sarcomas, but also play an important role in epithelial cell carcinogenesis. However, previous studies aiming to identify fusion genes in prostate cancer were mainly focused on expression changes and fusion transcripts. To investigate the genes recurrently affected by the chromosome breakpoints in prostate cancer, we analysed Affymetrix array 6.0 and 500K SNP microarray data from 77 prostate cancer samples. While the two genes most frequently affected by genomic breakpoints were, as expected, ERG and TMPRSS2, surprisingly more known tumour suppressor genes (TSGs) than known oncogenes were identified at recurrent chromosome breakpoints. Certain well-characterised TSGs, including p53, PTEN, BRCA1 and BRCA2 are recurrently truncated as a result of chromosome rearrangements in prostate cancer. Interestingly, many of the genes residing at recurrent breakpoint sites have not yet been implicated in prostate carcinogenesis such as HOOK3, PPP2R2A and TCBA1. We have confirmed the generally reduced expression of selected genes in clinical samples using quantitative RT-PCR analysis. Subsequently, we further investigated the genes associated with the t(4:6) translocation in LNCaP cells and reveal the genomic fusion of SNX9 and putative TSG UNC5C, which led to the reduced expression of both genes. This study reveals another common mechanism that leads to the inactivation of TSGs in prostate cancer and the identification of multiple TSGs inactivated by chromosome rearrangements will lead to new direction of research for the molecular basis of prostate carcinogenesis.
PMCID: PMC3189822  PMID: 21994901
prostate cancer; chromosome rearrangements; chromosome breakpoints; tumour suppressor gene; oncogene; SNP array; FISH; QRT-PCR
7.  mCOPA: analysis of heterogeneous features in cancer expression data 
Background
Cancer outlier profile analysis (COPA) has proven to be an effective approach to analyzing cancer expression data, leading to the discovery of the TMPRSS2 and ETS family gene fusion events in prostate cancer. However, the original COPA algorithm did not identify down-regulated outliers, and the currently available R package implementing the method is similarly restricted to the analysis of over-expressed outliers. Here we present a modified outlier detection method, mCOPA, which contains refinements to the outlier-detection algorithm, identifies both over- and under-expressed outliers, is freely available, and can be applied to any expression dataset.
Results
We compare our method to other feature-selection approaches, and demonstrate that mCOPA frequently selects more-informative features than do differential expression or variance-based feature selection approaches, and is able to recover observed clinical subtypes more consistently. We demonstrate the application of mCOPA to prostate cancer expression data, and explore the use of outliers in clustering, pathway analysis, and the identification of tumour suppressors. We analyse the under-expressed outliers to identify known and novel prostate cancer tumour suppressor genes, validating these against data in Oncomine and the Cancer Gene Index. We also demonstrate how a combination of outlier analysis and pathway analysis can identify molecular mechanisms disrupted in individual tumours.
Conclusions
We demonstrate that mCOPA offers advantages, compared to differential expression or variance, in selecting outlier features, and that the features so selected are better able to assign samples to clinically annotated subtypes. Further, we show that the biology explored by outlier analysis differs from that uncovered in differential expression or variance analysis. mCOPA is an important new tool for the exploration of cancer datasets and the discovery of new cancer subtypes, and can be combined with pathway and functional analysis approaches to discover mechanisms underpinning heterogeneity in cancers.
doi:10.1186/2043-9113-2-22
PMCID: PMC3553066  PMID: 23216803
Cancer; Outliers; Expression data; Expression profile; Cluster; Subtype; Heterogeneous; Bioinformatics; Percentile; Feature selection
8.  ERG–TMPRSS2 rearrangement is shared by concurrent prostatic adenocarcinoma and prostatic small cell carcinoma and absent in small cell carcinoma of the urinary bladder: evidence supporting monoclonal origin 
Prostatic carcinoma is a heterogeneous disease with frequent multifocality and variability in morphology. Particularly, prostatic small cell carcinoma is a rare variant with aggressive behavior. Distinction between small cell carcinoma of the prostate and urinary bladder may be challenging, especially in small biopsy specimens without associated prostatic adenocarcinoma or urothelial carcinoma. Recently, gene fusions between ETS genes, particularly ETS-related gene (ERG), and transmembrane protease, serine 2 (TMPRSS2) have been identified as a frequent event in prostate cancer. Thus, molecular methods may be helpful in determining the primary site of small cell carcinoma. Thirty cases of prostatic small cell carcinoma from the authors’ archives were studied, among which 13 had concurrent prostatic adenocarcinoma. Tricolor fluorescence in situ hybridization (FISH) was performed on formalin-fixed paraffin-embedded tissue sections with a probe cocktail for 3′/5′ ERG and TMPRSS2. Cases of small cell carcinoma of the bladder and conventional prostatic adenocarcinoma (25 each) were also tested as controls. ERG gene alterations were found only in prostate malignancies and not in benign prostatic tissue or bladder small cell carcinoma. TMPRSS2–ERG gene fusion was found in 47% (14/30) of prostatic small cell carcinoma. Of cases with concurrent prostatic adenocarcinoma, 85% (11/13) had identical findings in both components. In 20% of rearranged cases, the ERG abnormality was associated with 5′ ERG deletion. In 17% (5/30) of cases, gain of the 21q22 locus was present. Two cases showed discordant aberrations in the small cell carcinoma and adenocarcinoma, one with deletion of 5′ ERG and one with gain of chromosome 21q, both in only the adenocarcinoma component. Small cell carcinoma of the prostate demonstrates TMPRSS2–ERG rearrangement with comparable frequency to prostatic adenocarcinoma. In cases with concurrent adenocarcinoma and small cell carcinoma, the majority showed identical abnormalities in both components, indicating a likely common clonal origin. Discordant alterations were present in rare cases, suggesting that acquisition of additional genetic changes in multifocal tumors may be responsible for disease progression to a more aggressive phenotype. TMPRSS2–ERG fusion is absent in bladder small cell carcinoma, supporting the utility of FISH in distinguishing prostate from bladder primary tumors and identifying metastatic small cell carcinoma of unknown origin.
doi:10.1038/modpathol.2011.56
PMCID: PMC3441178  PMID: 21499238
ERG–TMPRSS2 rearrangement; fluorescence in situ hybridization; histogenesis; morphology; prostate; small cell carcinoma; urothelium
9.  Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing 
Genome Medicine  2013;5(4):30.
Background
Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively.
Methods
We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC).
Results
The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone.
Conclusions
The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'.
doi:10.1186/gm434
PMCID: PMC3707016  PMID: 23561577
10.  Novel 5′ Fusion Partners of ETV1 and ETV4 in Prostate Cancer12 
Neoplasia (New York, N.Y.)  2013;15(7):720-726.
Gene fusions involving the erythroblast transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, ETV5, and FLI1 are a common feature of prostate carcinomas (PCas). The most common upstream fusion partner described is the androgen-regulated prostate-specific gene TMPRSS2, most frequently with ERG, but additional 5′ fusion partners have been described. We performed 5′ rapid amplification of cDNA ends in 18 PCas with ETV1, ETV4, or ETV5 outlier expression to identify the 5′ fusion partners. We also evaluated the exon-level expression profile of these ETS genes in 14 cases. We identified and confirmed by fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction the two novel chimeric genes OR51E2-ETV1 and UBTF-ETV4 in two PCas. OR51E2 encodes a G-protein-coupled receptor that is overexpressed in PCas, whereas UBTF is a ubiquitously expressed gene encoding an HMG-box DNA-binding protein involved in ribosome biogenesis. We additionally describe two novel gene fusion combinations of previously described genes, namely, SLC45A3-ETV4 and HERVK17-ETV4. Finally, we found one PCa with TMPRSS2-ETV1, one with C15orf21-ETV1, one with EST14-ETV1, and two with 14q133-q21.1-ETV1. In nine PCas (eight ETV1 and one ETV5), exhibiting ETS outlier expression and genomic rearrangement detected by FISH, no 5′ fusion partner was found. Our findings contribute significantly to characterize the heterogeneous group of ETS gene fusions and indicate that all genes described as 5′ fusion partners with one ETS gene can most likely be rearranged with any of the other ETS genes involved in prostate carcinogenesis.
PMCID: PMC3689235  PMID: 23814484
11.  Comparison of doses and NTCP to risk organs with enhanced inspiration gating and free breathing for left-sided breast cancer radiotherapy using the AAA algorithm 
Background
The purpose of this study was to investigate the potential dose reduction to the heart, left anterior descending (LAD) coronary artery and the ipsilateral lung for patients treated with tangential and locoregional radiotherapy for left-sided breast cancer with enhanced inspiration gating (EIG) compared to free breathing (FB) using the AAA algorithm. The radiobiological implication of such dose sparing was also investigated.
Methods
Thirty-two patients, who received tangential or locoregional adjuvant radiotherapy with EIG for left-sided breast cancer, were retrospectively enrolled in this study. Each patient was CT-scanned during FB and EIG. Similar treatment plans, with comparable target coverage, were created in the two CT-sets using the AAA algorithm. Further, the probability of radiation induced cardiac mortality and pneumonitis were calculated using NTCP models.
Results
For tangential treatment, the median V25Gy for the heart and LAD was decreased for EIG from 2.2% to 0.2% and 40.2% to 0.1% (p < 0.001), respectively, whereas there was no significant difference in V20Gy for the ipsilateral lung (p = 0.109). For locoregional treatment, the median V25Gy for the heart and LAD was decreased for EIG from 3.3% to 0.2% and 51.4% to 5.1% (p < 0.001), respectively, and the median ipsilateral lung V20Gy decreased from 27.0% for FB to 21.5% (p = 0.020) for EIG. The median excess cardiac mortality probability decreased from 0.49% for FB to 0.02% for EIG (p < 0.001) for tangential treatment and from 0.75% to 0.02% (p < 0.001) for locoregional treatment. There was no significant difference in risk of radiation pneumonitis for tangential treatment (p = 0.179) whereas it decreased for locoregional treatment from 6.82% for FB to 3.17% for EIG (p = 0.004).
Conclusions
In this study the AAA algorithm was used for dose calculation to the heart, LAD and left lung when comparing the EIG and FB techniques for tangential and locoregional radiotherapy of breast cancer patients. The results support the dose and NTCP reductions reported in previous studies where dose calculations were performed using the pencil beam algorithm.
doi:10.1186/s13014-015-0375-y
PMCID: PMC4465142  PMID: 25884950
12.  Molecular characterisation of ERG, ETV1 and PTEN gene loci identifies patients at low and high risk of death from prostate cancer 
British Journal of Cancer  2010;102(4):678-684.
Background:
The discovery of ERG/ETV1 gene rearrangements and PTEN gene loss warrants investigation in a mechanism-based prognostic classification of prostate cancer (PCa). The study objective was to evaluate the potential clinical significance and natural history of different disease categories by combining ERG/ETV1 gene rearrangements and PTEN gene loss status.
Methods:
We utilised fluorescence in situ hybridisation (FISH) assays to detect PTEN gene loss and ERG/ETV1 gene rearrangements in 308 conservatively managed PCa patients with survival outcome data.
Results:
ERG/ETV1 gene rearrangements alone and PTEN gene loss alone both failed to show a link to survival in multivariate analyses. However, there was a strong interaction between ERG/ETV1 gene rearrangements and PTEN gene loss (P<0.001). The largest subgroup of patients (54%), lacking both PTEN gene loss and ERG/ETV1 gene rearrangements comprised a ‘good prognosis' population exhibiting favourable cancer-specific survival (85.5% alive at 11 years). The presence of PTEN gene loss in the absence of ERG/ETV1 gene rearrangements identified a patient population (6%) with poorer cancer-specific survival that was highly significant (HR=4.87, P<0.001 in multivariate analysis, 13.7% survival at 11 years) when compared with the ‘good prognosis' group. ERG/ETV1 gene rearrangements and PTEN gene loss status should now prospectively be incorporated into a predictive model to establish whether predictive performance is improved.
Conclusions:
Our data suggest that FISH studies of PTEN gene loss and ERG/ETV1 gene rearrangements could be pursued for patient stratification, selection and hypothesis-generating subgroup analyses in future PCa clinical trials and potentially in patient management.
doi:10.1038/sj.bjc.6605554
PMCID: PMC2837564  PMID: 20104229
ERG/ETV1 gene rearrangements; fluorescence in situ hybridisation; PTEN gene loss; prostate cancer; survival
13.  The Mutational Landscape of Lethal Castrate Resistant Prostate Cancer 
Nature  2012;487(7406):239-243.
Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.
doi:10.1038/nature11125
PMCID: PMC3396711  PMID: 22722839
14.  Characterization of ETS Gene Aberrations in Select Histologic Variants of Prostate Carcinoma 
Histologic variants of prostate carcinoma account for 5-10% of the disease and are typically seen in association with conventional acinar carcinoma. These variants often differ from the latter in clinical, immunophenotypic, and biologic potential. Recently, recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factors ERG, ETV1, ETV4 or ETV5 have been identified in a majority of conventional prostate carcinomas. However, the frequency and significance of this critical molecular event is unknown in the histologic variants of prostate carcinoma. Here, we used break-apart fluorescence in situ hybridization to assess TMPRSS2 and ETS aberrations in a series of select histologic variants: foamy gland carcinoma (N=17), ductal adenocarcinoma (N=18), mucinous carcinoma (N=18), and small cell carcinoma (N=7). A histologic variation of acinar adenocarcinoma, demonstrating glomeruloid morphology (N=9), was also investigated. Overall, 55% of histologic variant or variation morphologies demonstrated ETS aberrations (ERG in 54% and ETV1 in 1%). TMPRSS2:ERG fusion was identified in 83% (15/18), 71% (5/7), 50% (9/18), 33% (3/9) and 29% (5/17) of mucinous, small cell, ductal, glomeruloid, and foamy gland prostate carcinomas, respectively. Previously, we reported that 100% of androgen-independent metastatic prostate carcinomas harboring TMPRSS2:ERG gene fusion were associated with interstitial deletion (Edel). Interestingly, ERG rearrangement in small cell carcinomas occurred exclusively through EDel, supporting the notion that TMPRSS2:ERG with Edel is an aggressive molecular subtype. SPINK-1, a biomarker expressed exclusively in a subset of ETS negative prostate carcinomas, was expressed in 6% of ETS negative histologic variants, specifically in ductal adenocarcinoma. Notably, 88% (43/49) variant morphologies in this cohort showed concordance of TMPRSS2:ERG fusion with associated conventional acinar type, suggesting that variant morphology is clonally related to the latter. Overall, our data provides insight into the origin, molecular mechanism and phenotypic association of ETS fusions in histologic variants of prostate carcinoma.
doi:10.1038/modpathol.2009.79
PMCID: PMC2760291  PMID: 19465903
ETS; Rearrangement; Histologic variant; Prostate carcinoma; Fluorescence in situ hybridization
15.  deFuse: An Algorithm for Gene Fusion Discovery in Tumor RNA-Seq Data 
PLoS Computational Biology  2011;7(5):e1001138.
Gene fusions created by somatic genomic rearrangements are known to play an important role in the onset and development of some cancers, such as lymphomas and sarcomas. RNA-Seq (whole transcriptome shotgun sequencing) is proving to be a useful tool for the discovery of novel gene fusions in cancer transcriptomes. However, algorithmic methods for the discovery of gene fusions using RNA-Seq data remain underdeveloped. We have developed deFuse, a novel computational method for fusion discovery in tumor RNA-Seq data. Unlike existing methods that use only unique best-hit alignments and consider only fusion boundaries at the ends of known exons, deFuse considers all alignments and all possible locations for fusion boundaries. As a result, deFuse is able to identify fusion sequences with demonstrably better sensitivity than previous approaches. To increase the specificity of our approach, we curated a list of 60 true positive and 61 true negative fusion sequences (as confirmed by RT-PCR), and have trained an adaboost classifier on 11 novel features of the sequence data. The resulting classifier has an estimated value of 0.91 for the area under the ROC curve. We have used deFuse to discover gene fusions in 40 ovarian tumor samples, one ovarian cancer cell line, and three sarcoma samples. We report herein the first gene fusions discovered in ovarian cancer. We conclude that gene fusions are not infrequent events in ovarian cancer and that these events have the potential to substantially alter the expression patterns of the genes involved; gene fusions should therefore be considered in efforts to comprehensively characterize the mutational profiles of ovarian cancer transcriptomes.
Author Summary
Genome rearrangements and associated gene fusions are known to be important oncogenic events in some cancers. We have developed a novel computational method called deFuse for detecting gene fusions in RNA-Seq data and have applied it to the discovery of novel gene fusions in sarcoma and ovarian tumors. We assessed the accuracy of our method and found that deFuse produces substantially better sensitivity and specificity than two other published methods. We have also developed a set of 60 positive and 61 negative examples that will be useful for accurate identification of gene fusions in future RNA-Seq datasets. We have trained a classifier on 11 novel features of the 121 examples, and show that the classifier is able to accurately identify real gene fusions. The 45 gene fusions reported in this study represent the first ovarian cancer fusions reported, as well as novel sarcoma fusions. By examining the expression patterns of the affected genes, we find that many fusions are predicted to have functional consequences and thus merit experimental followup to determine their clinical relevance.
doi:10.1371/journal.pcbi.1001138
PMCID: PMC3098195  PMID: 21625565
16.  The Robustness of a Signaling Complex to Domain Rearrangements Facilitates Network Evolution 
PLoS Biology  2014;12(12):e1002012.
The broad tolerance of domain-rearranging mutations by a yeast signaling network suggests that signaling complexes have loose spatial constraints, making manipulation and perhaps evolution easier.
The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering.
Author Summary
Cells use complex protein interaction networks to sense and process external signals. Proteins involved in signaling are often composed of multiple functional units called domains. Because domains are modular, mutations that rearrange domains among proteins have the potential to result in the creation of novel proteins with altered functions. At an evolutionary timescale, domain rearrangements contribute to the functional diversification of signaling networks; at the shorter timescale of the life of an individual, domain rearrangements can impair cellular functions and lead to disease. Here, we investigated how domain-rearranging mutations alter the function of signaling networks, in particular when these mutations disrupt pre-existing proteins. We used as a model system the yeast mating signaling pathway, which shares many properties with more complex pathways active in human cells. Our results demonstrate that signaling networks are often robust to domain rearrangements that disrupt pre-existing genes. In addition, our experiments suggest a possible mechanism to explain this robustness: rather than being a rigid multi-protein machine, the yeast mating signaling complex is a dynamic ensemble with loose spatial constraints. Because of this, the changes in protein interaction partners caused by domain-rearrangement mutations can be accommodated without disrupting network function.
doi:10.1371/journal.pbio.1002012
PMCID: PMC4260825  PMID: 25490747
17.  ERG rearrangement is specific to prostate cancer and does not occur in any other common tumor 
Background
Identification of specific somatic gene alterations is crucial for the insight into the development, progression, and clinical behavior of individual cancer types. The recently discovered recurrent ERG rearrangement in prostate cancer (PCa) might represent a PCa specific alteration that has not been systematically assessed in tumors other than PCa. Aim of this study was to assess, whether the ERG rearrangement and the distinct deletion site between TMPRSS2 and ERG, both predominantly resulting in a TMPRSS2-ERG fusion, occurs in tumors other than PCa.
Design
We assessed 54 different tumor types (2942 samples in total) for their ERG rearrangement status by FISH. To calibrate, we analyzed 285 PCa samples for the ERG rearrangement frequency. Additionally, we interrogated a high-resolution SNP data set across 3131 cancer specimens (26 tumor types) for copy number alterations.
Results
None of the 54 different tumor types assessed by FISH harbored an ERG rearrangement, whereas the PCa samples revealed an ERG rearrangement in 31.2%–49.5%, depending on the cohort. Furthermore, within the 26 tumor types assessed for copy number alterations by SNP, the distinct deletion site between TMPRSS2 and ERG (21q22.2-3) was detectable exclusively in PCa.
Conclusion
Although Ewing's sarcoma and AML have known rearrangements rarely involving ERG, we hypothesize that the ERG rearrangement as well as the distinct deletion site on 21q22.2-3 between TMPRSS2 and ERG, are PCa specific genomic alterations. These observations provide further insight into the oncogenesis of PCa and might be critical for the development of ERG rearrangement assessment as a clinical tool.
doi:10.1038/modpathol.2010.87
PMCID: PMC3606550  PMID: 20473283
ERG rearrangement; prostate cancer; carcinoma
18.  The novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under stress 
Cell death & disease  2010;1(4):e32-.
We previously identified a novel estrogen-induced gene, EIG121, as being differentially regulated in endometrioid and nonendometrioid endometrial carcinoma. The function of EIG121 was unknown. Using a tetracycline-inducible system, we found that overexpression of EIG121, but not of LacZ, caused a profound suppression of cell growth. Subcellular fractionation and immunofluroscent labeling indicated that EIG121 was a transmembrane protein localized in the plasma membrane-late endosome–lysosome compartments. Deletion of the putative transmembrane domain abolished the membrane association. In cells overexpressing EIG121, cytoplasmic vacuoles accumulated after EIG121 induction, and the autophagosome marker LC3 translocated into punctuate, dot-like structures. Electron microscopy revealed that in cells overexpressing EIG121, autophagosomes were markedly increased. Overexpression of EIG121 also increased the cells containing acidic vesicles and induced lysosomal degradation of long-lived proteins. In MCF-7 cells, both EIG121 and LC3 were rapidly degraded by a lysosomal mechanism after starvation. Knockdown of EIG121 blocked starvation-induced LC3 degradation. By itself, knockdown of EIG121 did not affect cell survival. When combined with starvation or cytotoxic agents, EIG121 knockdown greatly increased apoptosis. Our results suggest that EIG121 is associated with the endosome–lysosome compartments and may have an important role in autophagy. Under unfavorable conditions such as starvation and exposure to cytotoxic agents, EIG121 may protect cells from cell death by upregulating the autophagy pathway.
doi:10.1038/cddis.2010.9
PMCID: PMC2976047  PMID: 21072319
EIG121; autophagy; lysosome; starvation; apoptosis; cell survival
19.  The novel estrogen-induced gene EIG121 regulates autophagy and promotes cell survival under stress 
Cell Death & Disease  2010;1(4):e32-.
We previously identified a novel estrogen-induced gene, EIG121, as being differentially regulated in endometrioid and nonendometrioid endometrial carcinoma. The function of EIG121 was unknown. Using a tetracycline-inducible system, we found that overexpression of EIG121, but not of LacZ, caused a profound suppression of cell growth. Subcellular fractionation and immunofluroscent labeling indicated that EIG121 was a transmembrane protein localized in the plasma membrane-late endosome–lysosome compartments. Deletion of the putative transmembrane domain abolished the membrane association. In cells overexpressing EIG121, cytoplasmic vacuoles accumulated after EIG121 induction, and the autophagosome marker LC3 translocated into punctuate, dot-like structures. Electron microscopy revealed that in cells overexpressing EIG121, autophagosomes were markedly increased. Overexpression of EIG121 also increased the cells containing acidic vesicles and induced lysosomal degradation of long-lived proteins. In MCF-7 cells, both EIG121 and LC3 were rapidly degraded by a lysosomal mechanism after starvation. Knockdown of EIG121 blocked starvation-induced LC3 degradation. By itself, knockdown of EIG121 did not affect cell survival. When combined with starvation or cytotoxic agents, EIG121 knockdown greatly increased apoptosis. Our results suggest that EIG121 is associated with the endosome–lysosome compartments and may have an important role in autophagy. Under unfavorable conditions such as starvation and exposure to cytotoxic agents, EIG121 may protect cells from cell death by upregulating the autophagy pathway.
doi:10.1038/cddis.2010.9
PMCID: PMC2976047  PMID: 21072319
EIG121; autophagy; lysosome; starvation; apoptosis; cell survival
20.  Exploration of the gene fusion landscape of glioblastoma using transcriptome sequencing and copy number data 
BMC Genomics  2013;14(1):818.
Background
RNA-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. Gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities.
Results
In this study, we provide comprehensive view of gene fusion landscape in 185 glioblastoma multiforme patients from two independent cohorts. Fusions occur in approximately 30-50% of GBM patient samples. In the Ivy Center cohort of 24 patients, 33% of samples harbored fusions that were validated by qPCR and Sanger sequencing. We were able to identify high-confidence gene fusions from RNA-seq data in 53% of the samples in a TCGA cohort of 161 patients. We identified 13 cases (8%) with fusions retaining a tyrosine kinase domain in the TCGA cohort and one case in the Ivy Center cohort. Ours is the first study to describe recurrent fusions involving non-coding genes. Genomic locations 7p11 and 12q14-15 harbor majority of the fusions. Fusions on 7p11 are formed in focally amplified EGFR locus whereas 12q14-15 fusions are formed by complex genomic rearrangements. All the fusions detected in this study can be further visualized and analyzed using our website: http://ivygap.swedish.org/fusions.
Conclusions
Our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in GBM. The majority of the fusions are private fusions, and a minority of these recur with low frequency. A small subset of patients with fusions of receptor tyrosine kinases can benefit from existing FDA approved drugs and drugs available in various clinical trials. Due to the low frequency and rarity of clinically relevant fusions, RNA-seq of GBM patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-14-818) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-14-818
PMCID: PMC4046790  PMID: 24261984
Gene fusion; Glioblastoma; RNA-seq; EGFR fusions; NTRK1; ROS1; FGFR3-TACC3; PIK3C2B; Non-coding gene fusions
21.  Reconstitution of the ERG Gene Expression Network Reveals New Biomarkers and Therapeutic Targets in ERG Positive Prostate Tumors 
Journal of Cancer  2015;6(6):490-501.
Background: Despite a growing number of studies evaluating cancer of prostate (CaP) specific gene alterations, oncogenic activation of the ETS Related Gene (ERG) by gene fusions remains the most validated cancer gene alteration in CaP. Prevalent gene fusions have been described between the ERG gene and promoter upstream sequences of androgen-inducible genes, predominantly TMPRSS2 (transmembrane protease serine 2). Despite the extensive evaluations of ERG genomic rearrangements, fusion transcripts and the ERG oncoprotein, the prognostic value of ERG remains to be better understood. Using gene expression dataset from matched prostate tumor and normal epithelial cells from an 80 GeneChip experiment examining 40 tumors and their matching normal pairs in 40 patients with known ERG status, we conducted a cancer signaling-focused functional analysis of prostatic carcinoma representing moderate and aggressive cancers stratified by ERG expression.
Results: In the present study of matched pairs of laser capture microdissected normal epithelial cells and well-to-moderately differentiated tumor epithelial cells with known ERG gene expression status from 20 patients with localized prostate cancer, we have discovered novel ERG associated biochemical networks.
Conclusions: Using causal network reconstruction methods, we have identified three major signaling pathways related to MAPK/PI3K cascade that may indeed contribute synergistically to the ERG dependent tumor development. Moreover, the key components of these pathways have potential as biomarkers and therapeutic target for ERG positive prostate tumors.
doi:10.7150/jca.8213
PMCID: PMC4439933  PMID: 26000039
Prostate cancer; differentiation status; TMPRSS2-ERG fusion.
22.  A Targeted Next-Generation Sequencing Assay Detects a High Frequency of Therapeutically Targetable Alterations in Primary and Metastatic Breast Cancers: Implications for Clinical Practice 
The Oncologist  2014;19(5):453-458.
The aim of this study was to assess the frequency of potentially actionable genomic alterations in breast cancer that could be targeted with approved agents or investigational drugs in clinical trials using a next-generation sequencing-based genomic profiling assay. Systematic evaluation of the predictive value of these genomic alterations is critically important for further progress in this field.
Background.
The aim of this study was to assess the frequency of potentially actionable genomic alterations in breast cancer that could be targeted with approved agents or investigational drugs in clinical trials using a next-generation sequencing-based genomic profiling assay performed in a Clinical Laboratory Improvement Amendments-certified and College of American Pathologists-accredited commercial laboratory.
Methods.
Fifty-one breast cancers were analyzed, including primary tumor biopsies of 33 stage I–II and 18 stage IV cancers (13 soft tissue, 3 liver, and 2 bone metastases). We assessed 3,230 exons in 182 cancer-related genes and 37 introns in 14 genes often rearranged in cancer for base substitutions, indels, copy number alterations, and gene fusions. The average median sequencing depth was 1,154×.
Results.
We observed 158 genomic alterations in 55 genes in 48 of 51 (94%) tumors (mean 3.1, range 0–9). The average number of potentially therapeutically relevant alterations was similar in primary (1.6, range 0–4) and in heavily pretreated metastatic cancers (2.0, range 0–4) (p = .24). The most common actionable alterations were in PIK3CA (n = 9, phosphatidylinositol 3-kinase [PI3K]/mammalian target of rapamycin [mTOR] inhibitors), NF1 (n = 7, PI3K/mTOR/mitogen-activated protein kinase inhibitors), v-akt murine thymoma viral oncogene homolog 1-3 (n = 7, PI3K/mTOR/AKT inhibitors), BRCA1/2 (n = 6, poly[ADP-ribose] polymerase inhibitors), and CCND1,2 and CCNE (n = 8)/cycline dependent kinase (CDK)6 (n = 1) (CDK4/6 inhibitors), KIT (n = 1, imatinib/sunitinib), ALK (n = 1, crizotinib), FGFR1,2 (n = 5, fibroblast growth factor receptor inhibitors), and EGFR (n = 2, epidermal growth factor receptor inhibitors). Our sequencing assay also correctly identified all six cases with HER2 (ERBB2) amplification by fluorescence in situ hybridization when tumor content was adequate. In addition, two known activating HER2 mutations were identified, both in unamplified cases.
Conclusion.
Overall, 84% of cancers harbored at least one genomic alteration linked to potential treatment options. Systematic evaluation of the predictive value of these genomic alterations is critically important for further progress in this field.
doi:10.1634/theoncologist.2013-0377
PMCID: PMC4012963  PMID: 24710307
Next-generation sequencing; Precision medicine; Molecularly targeted therapy; Predictive markers
23.  A Novel 4-color Fluorescence in Situ Hybridization Assay for Detection of TMPRSS2 and ERG Rearrangements in Prostate Cancer 
Cancer genetics  2013;206(0):1-11.
Purpose
Since the identification of TMPRSS2/ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We hereby report a novel 4-color fluorescence in situ hybridization (FISH) assay that not only detects the typical TMPRSS2:ERG fusion but also alternative rearrangements of either the TMPRSS2 or ERG gene.
Experimental design
We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens including cell lines, primary prostate cancer, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients.
Results
When compared with RT-PCR or Gen-Probe method as the technical reference, the 4-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% Confidence Interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting TMPRSS2:ERG fusion. TMPRSS2:ERG fusion was detected at 41% and 43% in primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Alternative rearrangements other than the typical TMPRSS2:ERG fusion were confirmed by karyotype analysis and shown present in 7% primary cancer and 13% CRPC tumors. Successful karyotype analysis is reported for the first time on four of the xenograft samples, complementing the FISH results.
Conclusions
This 4-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer.
doi:10.1016/j.cancergen.2012.12.004
PMCID: PMC3632390  PMID: 23352841
Prostate cancer; TMPRSS2; ERG; FISH
24.  Breakpoint Analysis of Transcriptional and Genomic Profiles Uncovers Novel Gene Fusions Spanning Multiple Human Cancer Types 
PLoS Genetics  2013;9(4):e1003464.
Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.
Author Summary
Gene fusions represent an important class of cancer genes, created by rearrangements of the genome that bring together two different genes. Because they are unique to cancer cells, gene fusions are ideal diagnostic markers and therapeutic targets. While gene fusions were once thought restricted mainly to blood cancers, recent discoveries suggest they are more widespread. Here, we have developed an approach for mining DNA microarray data to detect the tell-tale signatures of gene fusions, as “breakpoints” occurring within the encoding DNA or expressed transcripts. We apply this approach to a large collection of nearly 1,000 human cancer specimens. From this analysis, we discover and verify twelve new gene fusions occurring in diverse cancer types. We verify that some of these rearrangements recur in other samples of the same cancer type (supporting a causal role) and that the cancers show dependency on the fusion for cancer cell growth. Notably, some of these fusions (e.g. CEP85L/ROS1 in angiosarcoma) represent the first for that cancer type and thus provide important new biological insight. Some are also good drug targets (including rearrangements of ROS1, RAF1, and CDK6 kinases), with clear implications for therapy.
doi:10.1371/journal.pgen.1003464
PMCID: PMC3636093  PMID: 23637631
25.  N-myc Downstream Regulated Gene 1 (NDRG1) Is Fused to ERG in Prostate Cancer12 
Neoplasia (New York, N.Y.)  2009;11(8):804-811.
A step toward the molecular classification of prostate cancer was the discovery of recurrent erythroblast transformation-specific rearrangements, most commonly fusing the androgen-regulated TMPRSS2 promoter to ERG. The TMPRSS2-ERG fusion is observed in around 90% of tumors that overexpress the oncogene ERG. The goal of the current study was to complete the characterization of these ERG-overexpressing prostate cancers. Using fluorescence in situ hybridization and reverse transcription-polymerase chain reaction assays, we screened 101 prostate cancers, identifying 34 cases (34%) with the TMPRSS2-ERG fusion. Seven cases demonstrated ERG rearrangement by fluorescence in situ hybridization without the presence of TMPRSS2-ERG fusion messenger RNA transcripts. Screening for known 5′ partners, we determined that three cases harbored the SLC45A3-ERG fusion. To discover novel 5′ partners in these ERG-overexpressing and ERG-rearranged cases, we used paired-end RNA sequencing. We first confirmed the utility of this approach by identifying the TMPRSS2-ERG fusion in a known positive prostate cancer case and then discovered a novel fusion involving the androgen-inducible tumor suppressor, NDRG1 (N-myc downstream regulated gene 1), and ERG in two cases. Unlike TMPRSS2-ERG and SCL45A3-ERG fusions, the NDRG1-ERG fusion is predicted to encode a chimeric protein. Like TMPRSS2, SCL45A3 and NDRG1 are inducible not only by androgen but also by estrogen. This study demonstrates that most ERG-overexpressing prostate cancers harbor hormonally regulated TMPRSS2-ERG, SLC45A3-ERG, or NDRG1-ERG fusions. Broader implications of this study support the use of RNA sequencing to discover novel cancer translocations.
PMCID: PMC2713587  PMID: 19649210

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