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Jan Trapman - One of the best experts on this subject based on the ideXlab platform.
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two unique novel prostate specific and androgen regulated fusion partners of ETV4 in prostate cancer
2008Co-Authors: Karin G. Hermans, Guido Jenster, Anke A Bressers, Hetty A Van Der Korput, Natasja F Dits, Jan TrapmanAbstract:Recently, fusion of ERG to the androgen-regulated, prostate-specific TMPRSS2 gene has been identified as the most frequent genetic alteration in prostate cancer. At low frequency, TMPRSS2-ETV1 and TMPRSS2-ETV4 fusion genes have been described. In this study, we report two novel ETV4 fusion genes in prostate cancer: KLK2-ETV4 and CANT1-ETV4. Both gene fusions have important unique aspects. KLK2 is a well-established androgen-induced and prostate-specific gene. Fusion of KLK2 to ETV4 results in the generation of an additional ETV4 exon, denoted exon 4a. This novel exon delivers an ATG for the longest open reading frame, in this way avoiding translation start in KLK2 exon 1. Although wild-type CANT1 has two alternative first exons (exons 1 and 1a), only exon 1a was detected in CANT1-ETV4 fusion transcripts. We show that CANT1 transcripts starting at exon 1a have an androgen-induced and prostate-specific expression pattern, whereas CANT1 transcripts starting at exon 1 are not prostate specific. So, the two novel ETV4 fusion partners possess as predominant common characteristics androgen-induction and prostate-specific expression.
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tmprss2 erg fusion by translocation or interstitial deletion is highly relevant in androgen dependent prostate cancer but is bypassed in late stage androgen receptor negative prostate cancer
2006Co-Authors: Karin G. Hermans, Ronald Van Marion, Herman Van Dekken, Guido Jenster, Wytske M Van Weerden, Jan TrapmanAbstract:Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1 , or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4 , and FLI1 , in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG , linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)–negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease. (Cancer Res 2006; 66(22): 10658-63)
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tmprss2 erg fusion by translocation or interstitial deletion is highly relevant in androgen dependent prostate cancer but is bypassed in late stage androgen receptor negative prostate cancer
2006Co-Authors: Karin G. Hermans, Guido Jenster, Ronald Van Marion, Herman Van Dekken, Wytske M Van Weerden, Jan TrapmanAbstract:Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1, or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4, and FLI1, in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG, linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)-negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease.
Scott A Tomlins - One of the best experts on this subject based on the ideXlab platform.
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novel rna hybridization method for the in situ detection of etv1 ETV4 and etv5 gene fusions in prostate cancer
2014Co-Authors: Lakshmi P Kunju, Shannon Carskadon, Scott A Tomlins, Javed Siddiqui, Arul M Chinnaiyan, Nallasivam PalanisamyAbstract:: The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.
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abstract 4216 novel rnain situhybridization method for the detection of etv1 ETV4 and etv5 rearrangements in prostate cancer
2013Co-Authors: Lakshmi P Kunju, Shannon Carskadon, Ritu Bhalla, Scott A Tomlins, Javed Siddiqui, Arul M Chinnaiyan, Nallasivam PalanisamyAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer (PCa) remains the most commonly diagnosed cancer in American men with an estimated incidence of 214,740 new cases and accounting for 28,170 deaths in 2012. The genetic basis of 50-60% of PCa is attributable to rearrangements in ETS genes (ERG, ETV1, ETV4, ETV5), BRAF, RAF1 and overexpression of SPINK1. The discovery and validation of reliable diagnostic methods are warranted to detect these molecular rearrangements. ETS gene rearrangements are typically detected by FISH and PCR methods. Recently, monoclonal antibodies against ERG have been developed which detect the truncated ERG protein and are strongly correlated with ERG rearrangement as detected by FISH. However, due to the lack of specific antibodies for ETV1, ETV4 and ETV5 genes, in situ detection of these markers is not feasible. We have developed a novel RNA in situ hybridization (RNA-ISH) based assay for in situ detection of ETV1, ETV4, and ETV5 in formalin fixed paraffin embedded (FFPE) tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using commercially available RNA-ISH probes. In order to assess the feasibility of RNA-ISH method for the reliable detection of ETS rearrangement, we compared RNA-ISH with ERG immunohistochemistry (IHC) using previously validated ERG positive cases. Of the 12 ERG IHC-positive cases, RNA-ISH was positive in all samples and no false positives were detected in the 70 ERG-negative cases, indicating a 100% concordance between IHC and RNA-ISH. Thus, we established that the RNA-ISH method is comparable to IHC for evaluation of ETS gene rearrangement in PCa We then tested ETV1 ([NM_004956][1]), ETV4 (NM_001079675), and ETV5 ([NM_004454][2]) probes on 10 known positive (ETV1-5; ETV4-4; ETV5-1) and 319 unknown cases (localized PCa N=265; Metastatic PCa N=54). All 10 known positive cases were confirmed positive by RNA-ISH. Of the 319 cases, 11 (3.4%) ETV1and 3 (1%) ETV4 positive cases were identified. No non-specific staining was observed. Positive cases with tissue available for evaluation were validated with break-apart FISH probes and found to be confirmatory in all cases. No non-specific staining was observed in rearrangement negative cases. In majority (20/24) of the positive cases the RNA-ISH staining pattern was homogeneous; however, 3 ETV1-positive cases and 1 ETV4-positive case showed heterogeneous staining pattern. ERG IHC was performed and we found that the regions negative for either ETV1 or ETV4 were positive for ERG. Thus we identified a subset of prostate cancer showing dual ETS rearrangement in two independent tumor foci. This is the first time dual ETS rearrangement in prostate cancer has been demonstrated in situ. We showed that RNA-ISH is a viable alternative method for in situ detection of ETS gene rearrangements in prostate cancer. Citation Format: Lakshmi P. Kunju, Shannon Carskadon, Ritu Bhalla, Javed Siddiqui, Scott A. Tomlins, Arul M. Chinnaiyan, Nallasivam Palanisamy. Novel RNA In situ hybridization method for the detection of ETV1, ETV4 and ETV5 rearrangements in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2013-4216 [1]: /lookup/external-ref?link_type=GEN&access_num=NM_004956&atom=%2Fcanres%2F73%2F8_Supplement%2F4216.atom [2]: /lookup/external-ref?link_type=GEN&access_num=NM_004454&atom=%2Fcanres%2F73%2F8_Supplement%2F4216.atom
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abstract 2780 xenografts of human prostate cancer a genetic profile analysis
2013Co-Authors: Nallasivam Palanisamy, Ritu Bhalla, Scott A Tomlins, Lakshmi P Kunju, Jun Yang, Xinhai Wan, John C Araujo, Eleni Efstathiou, Louis L Pisters, Arul M ChinnaiyanAbstract:Prostate cancer (PCa) is the second leading cause of cancer-related death in the US. Recent clinical trials have shown responses in a subpopulation of patients; thus we need methods to identify likely responders. The genetic basis of PCa is understood to the extent that patients can be classified based on underlying molecular aberrations: 50-60% of PCas have rearrangements in ERG, ETV1, ETV4, ETV5, BRAF, and RAF1 and overexpression of SPINK1 and AR. PCas with PTEN deletion along with ERG have altered clinical behavior. We developed a strategy to establish PCa xenografts with tissue taken directly from men and implanted subcutaneously in SCID mice. After its growth, the tumor is harvested and sequentially passaged over 4 or 5 mice. We have established 62 PCa xenografts since the program9s inception. These xenografts, which are often developed while the donor PCa patient is alive, have proven valuable for testing drugs and have led to initiation of a promising clinical study (ClinicalTrials.gov: NCT00831792). In the study reported here we systematically characterized 51/62 xenografts for the presence of known PCa markers by immunohistochemistry and fluorescence in situ hybridization. The PCa xenografts were derived from PCas in the prostate or direct extensions to adjacent organs (21) or from metastases to bone (4), lymph node (3), liver (6), thyroid (1), testis (1), adrenal gland (2), brain (3), and unusual sites (skin, chest wall, soft tissue) (4) or ascites (3), and pleural effusions (3). 81% of xenografts derived from prostatic adenocarcinomas were AR positive (27/33); 16 were small-cell, poorly differentiated neuroendocrine carcinomas or ductal adenocarcinomas and did not express AR. One sarcomatoid and 1 ductal adenocarcinoma expressed AR; 77% of evaluable tumors had a deletion in PTEN (31/40); 48% of AR-positive tumors expressed recurrent gene fusions (eg, ERG, ETV1, ETV5) (13/27). Together, these results in this cohort_AR and recurrent gene fusion expression and PTEN deletion_nicely correlate with findings in human PCa. We next assessed whether PCa xenografts maintained histopathologic and molecular fidelity with the human tumor of origin in selected cases (n=16). Histopathologic pattern and recurrent gene fusion expression were the same in the paired human and mouse tissue. The AR and PTEN status were the same in most paired human and mouse samples. In 4 cases, AR expression was lost or PTEN deleted in the PCa xenograft, suggesting that selection for more aggressive genotypes may occur during xenograft development and that PCa xenografts develop by selecting cells’ drivers of cancer progression. In conclusion, we have developed a protocol for xenograft development that has fidelity with human PCa. This approach has provided a repository of clinically annotated samples that can be linked prospectively to clinical progression/response to therapy and thus will help identify therapy responders. Citation Format: Nallasivam Palanisamy, Jun Yang, Xinhai Wan, John C. Araujo, Eleni Efstathiou, Louis Pisters, Ritu Bhalla, Scott Tomlins, Lakshmi P. Kunju, Arul Chinnaiyan, Christopher J. Logothetis, Patricia Troncoso, Nora M. Navone. Xenografts of human prostate cancer - a genetic profile analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2013-2780
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characterization of tmprss2 ets gene aberrations in androgen independent metastatic prostate cancer
2008Co-Authors: Scott A Tomlins, Anjana Menon, Mark A Rubin, Rajal B Shah, Xuhong Cao, Kenneth J Pienta, Arul M ChinnaiyanAbstract:Recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factor family members ERG, ETV1, and ETV4 have been identified as a critical event in prostate cancer development. In this study, we characterized the prevalence and diversity of these rearrangements in hormone-refractory metastatic prostate cancer. We used a fluorescence in situ hybridization (FISH) split probe strategy to comprehensively evaluate TMPRSS2-ETS aberrations across 97 nonosseous metastatic sites of prostate cancer from 30 rapid autopsies of men who died of androgen-independent disease. Tissue microarrays were constructed representing multiple metastatic sites from each patient, and split signal FISH probes for TMPRSS2, ERG, ETV1, and ETV4 were used to assess for TMPRSS2-ETS rearrangements. In patients exhibiting these aberrations, multiple sites from an individual case harbored the same gene fusion molecular subtype suggesting clonal expansion of disease. The most common prostate cancer gene fusion, TMPRSS2-ERG, can be generated by the mechanism of interstitial deletion (Edel) about 39% to 60% of the time in clinically localized disease. Interestingly, we observed that all of the androgen-independent metastatic prostate cancer sites harboring TMPRSS2-ERG were associated with Edel. These findings suggest that TMPRSS2-ERG with Edel is an aggressive and, in this study, uniformly lethal molecular subtype of prostate cancer associated with androgen-independent disease.
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characterization of tmprss2 etv5 and slc45a3 etv5 gene fusions in prostate cancer
2008Co-Authors: Beth E Helgeson, Scott A Tomlins, Bharathi Laxman, Xuhong Cao, Qi Cao, Nameeta Shah, John R Prensner, Nirmish Singla, James E Montie, Sooryanarayana VaramballyAbstract:Recurrent gene fusions involving oncogenic ETS transcription factors (including ERG, ETV1, and ETV4) have been identified in a large fraction of prostate cancers. The most common fusions contain the 5′ untranslated region of TMPRSS2 fused to ERG. Recently, we identified additional 5′ partners in ETV1 fusions, including TMPRSS2, SLC45A3, HERV-K_22q11.23, C15ORF21, and HNRPA2B1. Here, we identify ETV5 as the fourth ETS family member involved in recurrent gene rearrangements in prostate cancer. Characterization of two cases with ETV5 outlier expression by RNA ligase–mediated rapid amplification of cDNA ends identified one case with a TMPRSS2:ETV5 fusion and one case with a SLC45A3:ETV5 fusion. We confirmed the presence of these fusions by quantitative PCR and fluorescence in situ hybridization. In vitro recapitulation of ETV5 overexpression induced invasion in RWPE cells, a benign immortalized prostatic epithelial cell line. Expression profiling and an integrative molecular concepts analysis of RWPE-ETV5 cells also revealed the induction of an invasive transcriptional program, consistent with ERG and ETV1 overexpression in RWPE cells, emphasizing the functional redundancy of ETS rearrangements. Together, our results suggest that the family of 5′ partners previously identified in ETV1 gene fusions can fuse with other ETS family members, suggesting numerous rare gene fusion permutations in prostate cancer. [Cancer Res 2008;68(1):73–80]
Barbara J. Graves - One of the best experts on this subject based on the ideXlab platform.
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ETV4 is necessary for estrogen signaling and growth in endometrial cancer cells
2020Co-Authors: Adriana C Rodriguez, Kathleen A Clark, Jeffery M Vahrenkamp, Kristofer C Berrett, Katrin P Guillen, Sandra D Scherer, Chiehhsiang Yang, Bryan E Welm, Margit M Janatamsbury, Barbara J. GravesAbstract:Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression; however, the molecular mechanisms underlying ERs regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways; ETV4 loss impacts chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture where hollow organoids were formed and in vivo in the context of estrogen dependent growth. Our results show that ETV4 plays a necessary role in estrogen signaling in endometrial cancer cells.
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ETV4 and ap1 transcription factors form multivalent interactions with three sites on the med25 activator interacting domain
2017Co-Authors: Simon L Currie, Jedediah J Doane, Lawrence P. Mcintosh, Kathryn S Evans, Niraja Bhachech, Bethany J Madison, Desmond K W Lau, Jack J Skalicky, Kathleen A Clark, Barbara J. GravesAbstract:The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. Within the ETS family, high-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker or no detectable binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-binding sequences were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.
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structured and disordered regions cooperatively mediate dna binding autoinhibition of ets factors etv1 ETV4 and etv5
2017Co-Authors: Simon L Currie, Jedediah J Doane, Frank G Whitby, Barbara J. Graves, Mark Okon, Lawrence P. McintoshAbstract:: Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein-protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.
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the ets gene ETV4 is required for anchorage independent growth and a cell proliferation gene expression program in pc3 prostate cells
2010Co-Authors: Peter C Hollenhorst, Litty Paul, Mary W Ferris, Barbara J. GravesAbstract:Chromosomal abnormalities that give rise to elevated expression levels of the ETS genes ETV1, ETV4, ETV5, or ERG are prevalent in prostate cancer, but the function of these transcription factors in carcinogenesis is not clear. Previous work in cell lines implicates ERG, ETV1, and ETV5 as regulators of invasive growth but not transformation. Here, we show that the PC3 prostate cancer cell line provides a model system to study the overexpression of ETV4. Migration assays, anchorage-independent growth assays, and microarray analysis indicate that high ETV4 expression contributes to both transformation and cellular motility in PC3 cells. ETV4 directly bound the 5′ and 3′ MYC enhancers and modulated expression of both MYC and other cell proliferation genes, demonstrating a potential role in cell growth control. Despite this novel role for ETV4 in anchorage-independent growth, ETV4 overexpression in normal prostate-derived RWPE-1 cells showed effects similar to ETV1 overexpression: increased cellular motility and an upregulation of genes encoding extracellular proteins as well as ones important for development, inflammation, and wound healing. Because ETV1 and ETV4 have similar roles when introduced to the same cellular background, we suggest that the requirement of high ETV4 expression for maintenance of the anchorage-independent growth in PC3 cells is due to a specific characteristic of this cell line rather than a function of ETV4 that is distinct from the other oncogenic ETS genes. Thus, the function of ETS genes in prostate cancer may differ based on other genetic alterations in a tumor.
Arul M Chinnaiyan - One of the best experts on this subject based on the ideXlab platform.
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novel rna hybridization method for the in situ detection of etv1 ETV4 and etv5 gene fusions in prostate cancer
2014Co-Authors: Lakshmi P Kunju, Shannon Carskadon, Scott A Tomlins, Javed Siddiqui, Arul M Chinnaiyan, Nallasivam PalanisamyAbstract:: The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.
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abstract 4216 novel rnain situhybridization method for the detection of etv1 ETV4 and etv5 rearrangements in prostate cancer
2013Co-Authors: Lakshmi P Kunju, Shannon Carskadon, Ritu Bhalla, Scott A Tomlins, Javed Siddiqui, Arul M Chinnaiyan, Nallasivam PalanisamyAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer (PCa) remains the most commonly diagnosed cancer in American men with an estimated incidence of 214,740 new cases and accounting for 28,170 deaths in 2012. The genetic basis of 50-60% of PCa is attributable to rearrangements in ETS genes (ERG, ETV1, ETV4, ETV5), BRAF, RAF1 and overexpression of SPINK1. The discovery and validation of reliable diagnostic methods are warranted to detect these molecular rearrangements. ETS gene rearrangements are typically detected by FISH and PCR methods. Recently, monoclonal antibodies against ERG have been developed which detect the truncated ERG protein and are strongly correlated with ERG rearrangement as detected by FISH. However, due to the lack of specific antibodies for ETV1, ETV4 and ETV5 genes, in situ detection of these markers is not feasible. We have developed a novel RNA in situ hybridization (RNA-ISH) based assay for in situ detection of ETV1, ETV4, and ETV5 in formalin fixed paraffin embedded (FFPE) tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using commercially available RNA-ISH probes. In order to assess the feasibility of RNA-ISH method for the reliable detection of ETS rearrangement, we compared RNA-ISH with ERG immunohistochemistry (IHC) using previously validated ERG positive cases. Of the 12 ERG IHC-positive cases, RNA-ISH was positive in all samples and no false positives were detected in the 70 ERG-negative cases, indicating a 100% concordance between IHC and RNA-ISH. Thus, we established that the RNA-ISH method is comparable to IHC for evaluation of ETS gene rearrangement in PCa We then tested ETV1 ([NM_004956][1]), ETV4 (NM_001079675), and ETV5 ([NM_004454][2]) probes on 10 known positive (ETV1-5; ETV4-4; ETV5-1) and 319 unknown cases (localized PCa N=265; Metastatic PCa N=54). All 10 known positive cases were confirmed positive by RNA-ISH. Of the 319 cases, 11 (3.4%) ETV1and 3 (1%) ETV4 positive cases were identified. No non-specific staining was observed. Positive cases with tissue available for evaluation were validated with break-apart FISH probes and found to be confirmatory in all cases. No non-specific staining was observed in rearrangement negative cases. In majority (20/24) of the positive cases the RNA-ISH staining pattern was homogeneous; however, 3 ETV1-positive cases and 1 ETV4-positive case showed heterogeneous staining pattern. ERG IHC was performed and we found that the regions negative for either ETV1 or ETV4 were positive for ERG. Thus we identified a subset of prostate cancer showing dual ETS rearrangement in two independent tumor foci. This is the first time dual ETS rearrangement in prostate cancer has been demonstrated in situ. We showed that RNA-ISH is a viable alternative method for in situ detection of ETS gene rearrangements in prostate cancer. Citation Format: Lakshmi P. Kunju, Shannon Carskadon, Ritu Bhalla, Javed Siddiqui, Scott A. Tomlins, Arul M. Chinnaiyan, Nallasivam Palanisamy. Novel RNA In situ hybridization method for the detection of ETV1, ETV4 and ETV5 rearrangements in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2013-4216 [1]: /lookup/external-ref?link_type=GEN&access_num=NM_004956&atom=%2Fcanres%2F73%2F8_Supplement%2F4216.atom [2]: /lookup/external-ref?link_type=GEN&access_num=NM_004454&atom=%2Fcanres%2F73%2F8_Supplement%2F4216.atom
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abstract 2780 xenografts of human prostate cancer a genetic profile analysis
2013Co-Authors: Nallasivam Palanisamy, Ritu Bhalla, Scott A Tomlins, Lakshmi P Kunju, Jun Yang, Xinhai Wan, John C Araujo, Eleni Efstathiou, Louis L Pisters, Arul M ChinnaiyanAbstract:Prostate cancer (PCa) is the second leading cause of cancer-related death in the US. Recent clinical trials have shown responses in a subpopulation of patients; thus we need methods to identify likely responders. The genetic basis of PCa is understood to the extent that patients can be classified based on underlying molecular aberrations: 50-60% of PCas have rearrangements in ERG, ETV1, ETV4, ETV5, BRAF, and RAF1 and overexpression of SPINK1 and AR. PCas with PTEN deletion along with ERG have altered clinical behavior. We developed a strategy to establish PCa xenografts with tissue taken directly from men and implanted subcutaneously in SCID mice. After its growth, the tumor is harvested and sequentially passaged over 4 or 5 mice. We have established 62 PCa xenografts since the program9s inception. These xenografts, which are often developed while the donor PCa patient is alive, have proven valuable for testing drugs and have led to initiation of a promising clinical study (ClinicalTrials.gov: NCT00831792). In the study reported here we systematically characterized 51/62 xenografts for the presence of known PCa markers by immunohistochemistry and fluorescence in situ hybridization. The PCa xenografts were derived from PCas in the prostate or direct extensions to adjacent organs (21) or from metastases to bone (4), lymph node (3), liver (6), thyroid (1), testis (1), adrenal gland (2), brain (3), and unusual sites (skin, chest wall, soft tissue) (4) or ascites (3), and pleural effusions (3). 81% of xenografts derived from prostatic adenocarcinomas were AR positive (27/33); 16 were small-cell, poorly differentiated neuroendocrine carcinomas or ductal adenocarcinomas and did not express AR. One sarcomatoid and 1 ductal adenocarcinoma expressed AR; 77% of evaluable tumors had a deletion in PTEN (31/40); 48% of AR-positive tumors expressed recurrent gene fusions (eg, ERG, ETV1, ETV5) (13/27). Together, these results in this cohort_AR and recurrent gene fusion expression and PTEN deletion_nicely correlate with findings in human PCa. We next assessed whether PCa xenografts maintained histopathologic and molecular fidelity with the human tumor of origin in selected cases (n=16). Histopathologic pattern and recurrent gene fusion expression were the same in the paired human and mouse tissue. The AR and PTEN status were the same in most paired human and mouse samples. In 4 cases, AR expression was lost or PTEN deleted in the PCa xenograft, suggesting that selection for more aggressive genotypes may occur during xenograft development and that PCa xenografts develop by selecting cells’ drivers of cancer progression. In conclusion, we have developed a protocol for xenograft development that has fidelity with human PCa. This approach has provided a repository of clinically annotated samples that can be linked prospectively to clinical progression/response to therapy and thus will help identify therapy responders. Citation Format: Nallasivam Palanisamy, Jun Yang, Xinhai Wan, John C. Araujo, Eleni Efstathiou, Louis Pisters, Ritu Bhalla, Scott Tomlins, Lakshmi P. Kunju, Arul Chinnaiyan, Christopher J. Logothetis, Patricia Troncoso, Nora M. Navone. Xenografts of human prostate cancer - a genetic profile analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2780. doi:10.1158/1538-7445.AM2013-2780
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ets gene aberrations in atypical cribriform lesions of the prostate implications for the distinction between intraductal carcinoma of the prostate and cribriform high grade prostatic intraepithelial neoplasia
2010Co-Authors: Khalid Suleman, Linda Sercia, Javed Siddiqui, Arul M Chinnaiyan, Nallasivam Palanisamy, Rajal B Shah, Cristina Magigalluzzi, Ming ZhouAbstract:Background: Atypical cribriform lesions (ACLs) of the prostate consist of cribriform glands lined with cytologically malignant cells with partial or complete basal cell lining. It may represent cribriform ‘‘high-grade prostatic intraepithelial neoplasia’’ (HGPIN) or ‘‘intraductal carcinoma of the prostate’’ (IDC-P), which is almost always associated with clinically aggressive prostate carcinoma (PCa). Distinction between these 2 lesions has profound clinical significance, especially on needle biopsies. However, there are lesions that do not fully satisfy the criteria for IDC-P yet are worse than typical HGPIN and are difficult to distinguish based on morphologic criteria alone. Methods: To better understand the biologic and molecular basis of distinction between cribriform HGPIN and IDC, we used break-apart fluorescence in-situ hybridization assay to assess ETS gene aberrations, a specific and commonest molecular alteration involving PCa, in a cohort of 16 isolated ACL, presumed to be an isolated cribriform HGPIN, and 45 carcinoma-associated ACL (ACL-PCa) on radical prostatectomy specimens, presumed to be spectrum of IDC-P. The latter was further divided into 2 groups: group A with marked nuclear atypia (nuclear size 6 � normal or larger) and/or comedonecrosis (n = 21) and group B that did not fulfill these criteria (n = 24). Results: Overall, ERG rearrangement was absent (0 of 16) in isolated cribriform HGPIN, whereas present in 75% (36 of 48) of IDC-P, of which 65% (23 of 36) were through deletion and 35% (13 of 36) through insertion. Notably, 17% (6 of 36) of the IDC-P showed duplication of ERG rearrangement in combination with deletion of 5 0 -ERG. Hundred percent (34 of 34) of the IDC-P showed concordance of ERG rearrangement status with adjacent invasive carcinoma. There was no difference between the 2 groups of IDC-P lesions regarding prevalence of ERG rearrangement (group A 79% vs. group B 74%) and EDel2+ (20% vs. 15%). No case with ETV1, ETV4, or ETV5 rearrangement was identified. Conclusions: Our molecular data suggest that isolated cribriform HGPIN and IDC-P are biologically distinct lesions. Majority of ACL-PCa most likely represent intraductal spread of PCa. There is a significant overlap between IDC-P and HGPIN at the lower grade morphologic spectrum. ERG break-apart fluorescence in-situ hybridization assay provides insight into understanding the molecular basis of cribriform HGPIN and IDC-P and has potential clinical implications in their distinction on needle biopsies.
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characterization of tmprss2 ets gene aberrations in androgen independent metastatic prostate cancer
2008Co-Authors: Scott A Tomlins, Anjana Menon, Mark A Rubin, Rajal B Shah, Xuhong Cao, Kenneth J Pienta, Arul M ChinnaiyanAbstract:Recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factor family members ERG, ETV1, and ETV4 have been identified as a critical event in prostate cancer development. In this study, we characterized the prevalence and diversity of these rearrangements in hormone-refractory metastatic prostate cancer. We used a fluorescence in situ hybridization (FISH) split probe strategy to comprehensively evaluate TMPRSS2-ETS aberrations across 97 nonosseous metastatic sites of prostate cancer from 30 rapid autopsies of men who died of androgen-independent disease. Tissue microarrays were constructed representing multiple metastatic sites from each patient, and split signal FISH probes for TMPRSS2, ERG, ETV1, and ETV4 were used to assess for TMPRSS2-ETS rearrangements. In patients exhibiting these aberrations, multiple sites from an individual case harbored the same gene fusion molecular subtype suggesting clonal expansion of disease. The most common prostate cancer gene fusion, TMPRSS2-ERG, can be generated by the mechanism of interstitial deletion (Edel) about 39% to 60% of the time in clinically localized disease. Interestingly, we observed that all of the androgen-independent metastatic prostate cancer sites harboring TMPRSS2-ERG were associated with Edel. These findings suggest that TMPRSS2-ERG with Edel is an aggressive and, in this study, uniformly lethal molecular subtype of prostate cancer associated with androgen-independent disease.
Karin G. Hermans - One of the best experts on this subject based on the ideXlab platform.
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two unique novel prostate specific and androgen regulated fusion partners of ETV4 in prostate cancer
2008Co-Authors: Karin G. Hermans, Guido Jenster, Anke A Bressers, Hetty A Van Der Korput, Natasja F Dits, Jan TrapmanAbstract:Recently, fusion of ERG to the androgen-regulated, prostate-specific TMPRSS2 gene has been identified as the most frequent genetic alteration in prostate cancer. At low frequency, TMPRSS2-ETV1 and TMPRSS2-ETV4 fusion genes have been described. In this study, we report two novel ETV4 fusion genes in prostate cancer: KLK2-ETV4 and CANT1-ETV4. Both gene fusions have important unique aspects. KLK2 is a well-established androgen-induced and prostate-specific gene. Fusion of KLK2 to ETV4 results in the generation of an additional ETV4 exon, denoted exon 4a. This novel exon delivers an ATG for the longest open reading frame, in this way avoiding translation start in KLK2 exon 1. Although wild-type CANT1 has two alternative first exons (exons 1 and 1a), only exon 1a was detected in CANT1-ETV4 fusion transcripts. We show that CANT1 transcripts starting at exon 1a have an androgen-induced and prostate-specific expression pattern, whereas CANT1 transcripts starting at exon 1 are not prostate specific. So, the two novel ETV4 fusion partners possess as predominant common characteristics androgen-induction and prostate-specific expression.
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tmprss2 erg fusion by translocation or interstitial deletion is highly relevant in androgen dependent prostate cancer but is bypassed in late stage androgen receptor negative prostate cancer
2006Co-Authors: Karin G. Hermans, Ronald Van Marion, Herman Van Dekken, Guido Jenster, Wytske M Van Weerden, Jan TrapmanAbstract:Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1 , or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4 , and FLI1 , in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG , linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)–negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease. (Cancer Res 2006; 66(22): 10658-63)
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tmprss2 erg fusion by translocation or interstitial deletion is highly relevant in androgen dependent prostate cancer but is bypassed in late stage androgen receptor negative prostate cancer
2006Co-Authors: Karin G. Hermans, Guido Jenster, Ronald Van Marion, Herman Van Dekken, Wytske M Van Weerden, Jan TrapmanAbstract:Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1, or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4, and FLI1, in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG, linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)-negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease.