Tumor heterogeneity, aggressiveness, and immune cell composition in a novel syngeneic PSA-targeted Pten knockout mouse prostate cancer (MuCaP) model.

BACKGROUND: Prostate cancer is recognized as a heterogeneous disease demanding appropriate preclinical models that reflect tumor complexity. Previously, we established the PSA-Cre;PtenLoxP/LoxP genetic engineered mouse model (GEMM) for prostate cancer reflecting the various stages of tumor development. Prostate tumors in this Pten KO model slowly develop, requiring more than 10 months. In order to enhance its practical utility, we established a syngeneic panel of cell lines derived from PSA-Cre targeted Pten KO tumors, designated the mouse prostate cancer (MuCap) model. METHODS: Four different MuCaP epithelial cell lines were established from three independent primary Pten KO mouse prostate tumors. Tumorigenic capacity of the MuCaP cell lines was determined by subcutaneous inoculation of these cell lines in immunocompetent mice. Response to PI3K-targeted therapy was validated in ex vivo tissue slices of the established MuCaP tumors. RESULTS: The MuCaP cell lines were all tumorigenic in immunocompetent mice after subcutaneous inoculation. Interestingly, these syngrafted tumors represented different tumor growth rates and morphologies. Treatment with the specific PI3K inhibitor GDC0941 resulted in responses very similar between syngeneic MuCaP and primary Pten KO prostate tumors. Finally, immunoprofiling of the different syngeneic MuCaP tumors demonstrated differential numbers of tumor infiltrating lymphocytes and distinct immune gene profiles with expression of CD8, INFy, and PD1 being inversely related to tumor aggressiveness. CONCLUSIONS: Collectively, we present here a well-defined MuCaP platform of in vitro and in vivo mouse prostate cancer models that may support preclinical assessment of (immune)-therapies for prostate cancer.

Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras-driven lung tumours.

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision-cut slices from Kras-driven non-small-cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen-activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short-term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3-kinase-mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology-associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A mononucleotide repeat in PRRT2 is an important, frequent target of mismatch repair deficiency in cancer.

The DNA mismatch repair (MMR) system corrects DNA replication mismatches thereby contributing to the maintenance of genomic stability. MMR deficiency has been observed in prostate cancer but its impact on the genomic landscape of these tumours is not known. In order to identify MMR associated mutations in prostate cancer we have performed whole genome sequencing of the MMR deficient PC346C prostate cancer cell line. We detected a total of 1196 mutations in PC346C which was 1.5-fold higher compared to a MMR proficient prostate cancer sample (G089). Of all different mutation classes, frameshifts in mononucleotide repeat (MNR) sequences were significantly enriched in the PC346C sample. As a result, a selection of genes with frameshift mutations in MNR was further assessed regarding its mutational status in a comprehensive panel of prostate, ovarian, endometrial and colorectal cancer cell lines. We identified PRRT2 and DAB2IP to be frequently mutated in MMR deficient cell lines, colorectal and endometrial cancer patient samples. Further characterization of PRRT2 revealed an important role of this gene in cancer biology. Both normal prostate cell lines and a colorectal cancer cell line showed increased proliferation, migration and invasion when expressing the mutated form of PRRT2 (ΔPRRT2). The wild-type PRRT2 (PRRT2wt) had an inhibitory effect in proliferation, consistent with the low expression level of PRRT2 in cancer versus normal prostate samples.

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice.

Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m) developed at older age (>10m) into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC), adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK), and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7-8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1) and tumor class 2 (TC2). TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma/intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor model, histopathological, molecular and biological heterogeneity occurred during later stages of tumor development.

From Nf1 to Sdhb knockout: Successes and failures in the quest for animal models of pheochromocytoma.

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors characterized by a high frequency of hereditary forms. Based on transcriptome classification, PPGL can be classified in two different clusters. Cluster 1 tumors are caused by mutations in SDHx, VHL and FH genes and are characterized by a pseudohypoxic signature. Cluster 2 PPGL carry mutations in RET, NF1, MAX or TMEM127 genes and display an activation of the MAPK and mTOR signaling pathways. Many genetically engineered and allografted mouse models have been generated these past 30 years to investigate the mechanisms of PPGL tumorigenesis and test new therapeutic strategies. Among them, only Cluster 2-related models have been successful while no Cluster 1-related knockout mouse was so far reported to develop a PPGL. In this review, we present an overview of existing, successful or not, PPGL models, and a description of our own experience on the quest of Sdhb knockout mouse models of PPGL.

Discriminating somatic and germline mutations in tumor DNA samples without matching normals.

Tumor analyses commonly employ a correction with a matched normal (MN), a sample from healthy tissue of the same individual, in order to distinguish germline mutations from somatic mutations. Since the majority of variants found in an individual are thought to be common within the population, we constructed a set of 931 samples from healthy, unrelated individuals, originating from two different sequencing platforms, to serve as a virtual normal (VN) in the absence of such an associated normal sample. Our approach removed (1) >96% of the germline variants also removed by the MN sample and (2) a large number (2%-8%) of additional variants not corrected for by the associated normal. The combination of the VN with the MN improved the correction for polymorphisms significantly, with up to ∼30% compared with MN and ∼15% compared with VN only. We determined the number of unrelated genomes needed in order to correct at least as efficiently as the MN is about 200 for structural variations (SVs) and about 400 for single-nucleotide variants (SNVs) and indels. In addition, we propose that the removal of common variants with purely position-based methods is inaccurate and incurs additional false-positive somatic variants, and more sophisticated algorithms, which are capable of leveraging information about the area surrounding variants, are needed for optimal accuracy. Our VN correction method can be used to analyze any list of variants, regardless of sequencing platform of origin. This VN methodology is available for use on our public Galaxy server.

High Efficacy of Combination Therapy Using PI3K/AKT Inhibitors with Androgen Deprivation in Prostate Cancer Preclinical Models.

BACKGROUND: The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway is frequently activated during prostate cancer (PCa) progression through loss or mutation of the phosphatase and tensin homolog (PTEN) gene. Following the androgen receptor (AR) pathway, it is the second major driver of PCa growth. OBJECTIVE: To assess efficacy of novel PI3K/AKT-targeted therapies in PCa models, as a single agent and in combination with androgen deprivation. DESIGN, SETTING, AND PARTICIPANTS: Twelve human PCa cell lines were tested in vitro for sensitivity to the AKT inhibitor AZD5363 and the PI3K beta/delta inhibitor AZD8186. The combination of AZD5363 and AZD8186 with castration was evaluated in vivo in PTEN-negative versus PTEN-positive patient-derived xenografts. Tumors and plasma were collected for biomarker analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: In vitro growth inhibition was determined by methylthiazolyldiphenyl-tetrazolium bromide assay. In vivo efficacy was monitored by caliper measurements of subcutaneous tumor volume. PI3K/AKT and AR pathway activity was analyzed by Western blot, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. RESULTS AND LIMITATIONS: AZD5363 and AZD8186 inhibited in vitro growth of 10 of 12 and 7 of 12 PCa cell lines, respectively, with increased sensitivity under androgen depletion. In vivo, AZD5363 and AZD8186 as single agents significantly inhibited growth of PTEN-negative PC346C xenografts compared to placebo by 60% and 66%, respectively. Importantly, combination of either agent with castration resulted in long-lasting tumor regression, which persisted after treatment cessation. Expression of AR-target genes kallikrein-related peptidase 3 (KLK3, also known as PSA); transmembrane protease, serine 2 (TMPRSS2); and FK506 binding protein 5 (FKBP5) was upregulated after PI3K/AKT inhibition. Neither compound inhibited tumor growth in the PTEN-positive PC310 model. CONCLUSIONS: Combination with hormonal therapy improved efficacy of PI3K/AKT-targeted agents in PTEN-negative PCa models. Upregulation of AR-target genes upon PI3K/AKT inhibition suggests a compensatory crosstalk between the PI3K-AR pathways. These data strongly advocate for further clinical evaluation. PATIENT SUMMARY: Inactivation of the PTEN gene is a common event promoting prostate cancer (PCa) progression. This preclinical study illustrates the potent anticancer activity of novel PTEN-targeted drugs on PCa models, particularly in combination with hormonal therapy.

ETS fusion genes in prostate cancer.

Prostate cancer is very common in elderly men in developed countries. Unravelling the molecular and biological processes that contribute to tumor development and progressive growth, including its heterogeneity, is a challenging task. The fusion of the genes ERG and TMPRSS2 is the most frequent genomic alteration in prostate cancer. ERG is an oncogene that encodes a member of the family of ETS transcription factors. At lower frequency, other members of this gene family are also rearranged and overexpressed in prostate cancer. TMPRSS2 is an androgen-regulated gene that is preferentially expressed in the prostate. Most of the less frequent ETS fusion partners are also androgen-regulated and prostate-specific. During the last few years, novel concepts of the process of gene fusion have emerged, and initial experimental results explaining the function of the ETS genes ERG and ETV1 in prostate cancer have been published. In this review, we focus on the most relevant ETS gene fusions and summarize the current knowledge of the role of ETS transcription factors in prostate cancer. Finally, we discuss the clinical relevance of TMRPSS2-ERG and other ETS gene fusions in prostate cancer.

iFUSE: integrated fusion gene explorer.

UNLABELLED: We present iFUSE (integrated fusion gene explorer), an online visualization tool that provides a fast and informative view of structural variation data and prioritizes those breaks likely representing fusion genes. This application uses calculated break points to determine fusion genes based on the latest annotation for genomic sequence information, and where relevant the structural variation (SV) events are annotated with predicted RNA and protein sequences. iFUSE takes as input a Complete Genomics (CG) junction file, a FusionMap fusion detection report file or a file already analysed and annotated by the iFUSE application on a previous occasion. RESULTS: We demonstrate the use of iFUSE with case studies from tumour-normal SV detection derived from Complete Genomics whole-genome sequencing results. AVAILABILITY: iFUSE is available as a web service at http://ifuse.erasmusmc.nl.

Gene fusions by chromothripsis of chromosome 5q in the VCaP prostate cancer cell line.

The VCaP cell line is widely used in prostate cancer research as it is a unique model to study castrate resistant disease expressing high levels of the wild type androgen receptor and the TMPRSS2-ERG fusion transcript. Using next generation sequencing, we assembled the structural variations in VCaP genomic DNA and observed a massive number of genomic rearrangements along the q arm of chromosome 5, characteristic of chromothripsis. Chromothripsis is a recently recognized phenomenon characterized by extensive chromosomal shattering in a single catastrophic event, mainly detected in cancer cells. Various structural events identified on chromosome 5q of VCaP resulted in gene fusions. Out of the 18 gene fusion candidates tested, 15 were confirmed on genomic level. In our set of gene fusions, only rarely we observe microhomology flanking the breakpoints. On RNA level, only five transcripts were detected and NDUFAF2-MAST4 was the only resulting in an in-frame fusion transcript. Our data indicate that although a marker of genomic instability, chromothripsis might lead to only a limited number of functionally relevant fusion genes.

A multi-parameter imaging assay identifies different stages of ligand-induced androgen receptor activation.

Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands.

A 36-gene signature predicts clinical progression in a subgroup of ERG-positive prostate cancers.

BACKGROUND: The molecular basis of the clinical heterogeneity of prostate cancer (PCa) is not well understood. OBJECTIVE: The purpose of our study was to identify and characterize genes in a clinically relevant gene expression signature in a subgroup of primary PCa positive for transmembrane protease, serine 2 (TMPRSS2)-v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG). DESIGN, SETTING, AND PARTICIPANTS: We studied gene expression profiles by unsupervised hierarchical clustering in 48 primary PCas from patients with a long clinical follow-up. Results were correlated with clinical outcome and validated in an independent patient cohort. Selected genes from a defined classifier were tested in vitro for biologic properties. INTERVENTION: Initial treatment of primary tumors was radical prostatectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations between clinical and histopathologic variables were evaluated by the Pearson χ(2) test, Mann-Whitney U test, or Kruskal-Wallis test, where appropriate. The log-rank test or Breslow method was used for statistical analysis of Kaplan-Meier survival curves. RESULTS AND LIMITATIONS: Most tumors that overexpressed ERG clustered separately from other primary PCas. No differences in any clinical end points between ERG-positive and ERG-negative cancers were detected. Importantly, within the ERG-positive samples, two subgroups were identified, which differed significantly in prostate-specific antigen recurrence-free survival, and cancer-specific and overall survival. From our findings, we defined a gene expression classifier of 36 genes. In a second, completely independent tumor set, the classifier also distinguished ERG-positive subgroups with different clinical outcome. In both patient cohorts, the classifier was not predictive in ERG-negative tumors. Biologic processes regulated by genes in the classifier included cell adhesion and bone remodeling. Tumor growth factor-ß signaling was indicated as the main differing signaling pathway between the two ERG subgroups. In vitro biologic assays of two selected genes from the classifier (inhibin, beta A [INHBA] and cadherin 11, type 2, OB-cadherin (osteoblast) [CDH11]) supported a functional role in PCa progression. Possible multifocality and limited number of PCa samples can be limitations of the study. CONCLUSIONS: The classifier identified can contribute to prediction of tumor progression in ERG-positive primary prostate tumors and might be instrumental in therapy decisions.

Identification of TDRD1 as a direct target gene of ERG in primary prostate cancer.

Molecular classification of ERG-rearranged prostate cancer clarifies the role of TMPRSS2-ERG in the development and progression of prostate cancer. The objective of our study was to identify direct ERG target genes in ERG-rearranged prostate cancer. Two independent cohorts of primary prostate cancer (Cohort A, n=48; Cohort B, n=31), a cohort of late-stage prostate cancer (n=51) and expression array data of a cohort of primary prostate tumors from a different institute (n=128) were analyzed for expression of genes that were coexpressed with ERG overexpression. By genome-wide expression analysis and Q-RT-PCR it was shown that the gene Tudor domain containing 1 (TDRD1) was by far the strongest correlated gene with ERG overexpression in both Cohort A and B. Expression array analysis of the patient cohort from a different institute showed a large overlap in genes that were positively correlated with ERG overexpression, including TDRD1. In late-stage prostate cancer, TDRD1 was also coexpressed with ERG overexpression, although a proportion of ERG-negative late-stage samples expressed TDRD1. TDRD1 expression was not associated with ETV1 overexpression. In the prostate cancer cell line VCaP, downregulation of ERG by shRNA lead to a lower expression level of TDRD1 and resulted in a decreased activity of the TDRD1 promoter. By mutation analysis we identified a functional ERG binding site in the TDRD1 promoter. Our findings show TDRD1 as the first identified upregulated direct ERG target gene that is strongly associated with ERG overexpression in primary prostate cancer.

Trp53 inactivation leads to earlier phaeochromocytoma formation in pten knockout mice.

Phaeochromocytomas (PCCs) are benign neuroendocrine tumours of the adrenal medulla. Approximately 10% of PCC patients develop metastases, but this frequency is much higher in specific subtypes of patients. The reliable diagnosis of malignant PCC can only be made after identification of a metastasis. To study the effect of Trp53 inactivation on PCC pathogenesis in Pten KO mice, we investigated the adrenals of a large cohort of mice with conditional monoallelic and biallelic inactivation of Trp53 and Pten. The adrenal weights were determined for all mice, and in a proportion of these mice, immunohistochemistry for tyrosine hydroxylase and dopamine ß-hydroxylase was performed on the adrenals and corresponding lungs. Finally, comparative genomic hybridization (CGH) was performed. The histological and immunohistochemical results confirmed that the adrenal tumours were PCCs. Inactivation of one or both alleles of Trp53 resulted in earlier tumour occurrence in the Pten(loxP/loxP) mice as well as in the Pten(loxP/+) mice. In addition, lung metastases were found in up to 67% of mice. The CGH results showed that the most frequent genomic alterations were loss of chromosome 19 (86%) and gain of chromosome 15 (71%). In this study, we have shown that Pten/Trp53 KO mice showed metastatic PCC at high frequency and primary tumours occurred at younger ages in mice with Trp53 inactivation. Therefore, the present model appears to be a suitable model that might allow the preclinical study of new therapeutics for these tumours.

Stepwise androgen receptor dimerization.

Androgen-regulated gene expression is a highly coordinated dynamic process mediated by androgen receptor (AR) ligand binding and DNA binding, and by specific AR protein-protein interactions. The latter include DNA-binding domain (D-box) interactions in AR homodimers, and the interaction of the FQNLF motif in the AR N-terminal domain and the coactivator groove in the ligand-binding domain (N/C interaction). We have studied these interactions in AR homodimerization using quantitative imaging techniques. We found that the initial cytoplasmic intramolecular AR N/C interaction after ligand binding is followed by a D-box-dimerization-dependent transition to intermolecular N/C interaction in a proportion of nuclear ARs. The consecutive steps leading to homodimerization are initiated prior to DNA binding. Our data indicate the presence of nuclear pools of both AR homodimers and monomers. On the basis of AR-regulated reporter assays we propose specificity in regulation of gene expression by AR homodimers and monomers mediated by AR domain interactions. Moreover, our findings elucidate important steps in the spatiotemporal organization of AR intra- and inter-molecular interactions.

ERG immunohistochemistry is not predictive for PSA recurrence, local recurrence or overall survival after radical prostatectomy for prostate cancer.

In prostate cancer genomic rearrangements involving genes encoding ETS transcription factors are commonly present, with androgen-regulated transmembrane protease, serine 2 (TMPRSS2)-v-ets erythroblastosis virus E26 oncogen homologue (ERG) gene fusion occurring in 40-70%. Studies on the predictive value of ERG rearrangement as detected by in-situ hybridization or polymerase chain reaction have resulted in varying outcomes. The objective of this study was to correlate immunohistochemical ERG protein expression with clinico-pathological parameters at radical prostatectomy specimens, and to determine its predictive value for postoperative disease recurrence and progression in a prostate cancer screening cohort. Since androgen receptor is downregulated by ERG in cell lines, we also compared the expression of respective proteins. We selected 481 participants from the European Randomized Study of Screening for Prostate Cancer treated by radical prostatectomy for prostate adenocarcinoma. A tissue microarray was constructed containing representative cores of all prostate cancer specimens as well as 22 xenografts and seven cell lines. Immunohistochemical expression of ERG and androgen receptor was correlated with prostate-specific antigen (PSA), Gleason sum, pT-stage, surgical margins, biochemical recurrence, local recurrence, overall death and disease-specific death. ERG expression was detected in 284 patients (65%). Expression occurred significantly more frequent in patients with PSA ≤10 ng/ml (P=0.024). There was no significant association between ERG and Gleason sum, pT-stage or surgical margin status. PSA (P=0.011), Gleason sum (P=0.003), pT-stage (P=0.001) and surgical margin status (P<0.001) all had independent value for postoperative biochemical recurrence, while positive surgical margin (P=0.021) was the only independent predictor for local recurrence. ERG protein expression did not have prognostic value for the clinical end points in uni- and multivariate analyses. A positive correlation existed between ERG and androgen receptor expression in single tissue cores (P<0.001). In conclusion, immunohistochemical ERG expression has no predictive value for prostate cancer recurrence or progression after radical prostatectomy. Increasing ERG levels are associated with the upregulation of androgen receptor expression in clinical specimens.

Androgen receptor coregulators: recruitment via the coactivator binding groove.

Androgens are key regulators of male sexual differentiation and essential for development and maintenance of male reproductive tissues. The androgens testosterone and dihydrotestosterone mediate their effect by binding to, and activation of the androgen receptor (AR). Upon activation, the AR is able to recognize specific DNA sequences in gene promoters and enhancers from where it recruits coregulators to orchestrate chromatin remodeling and transcription regulation. The number of proteins that bind to the AR has surpassed 200 and many of them enhance (coactivator) or repress (corepressor) its transactivating capacity. For most of these coregulators, their AR binding interface and their exact mode of action still needs to be elucidated, but for some of the more classical coactivators and corepressors, we gained insight in their working mechanisms. Of particular interest are specific sequences (LxxLL and FxxLF-like motifs) in a subset of coactivators that interact with the AR via a coactivator binding groove in the ligand-binding domain. As compared to other steroid receptors, the conformation of the AR coactivator binding pocket is unique and preferentially binds FxxLF-like motifs. This predisposition is expected to contribute to the regulation of specific sets of target genes via recruitment of selected coregulators. This review provides an overview of these (inter)actions with a focus on the unique characteristics of the AR coactivator binding groove.

Androgen regulation of ETS gene fusion transcripts in prostate cancer.

Fusion between androgen-regulated TMPRSS2 and ETS transcription factor gene ERG is the most frequent genetic alteration that occurs in 40-70% of prostate cancers. Not only ERG but also other ETS transcription factor genes are involved in gene fusions. ETV1, ETV4, and ETV5 have all several fusion partners. One common feature shared by the majority of these partners is androgen-regulated expression. Despite its high frequency, the biological and molecular effects of ETS gene fusion in prostate cancer development and progression are unknown. In this chapter quantitative polymerase chain reaction (Q-PCR) is used for detection and further studying the incidence and properties of these fusion transcripts. The focus is on the expression of TMPRSS2-ERG transcripts in clinical prostate samples. Androgen regulation of TMPRSS2 is measured in commonly used LNCaP prostate cancer cells grown with and without the synthetic androgen R1881. Furthermore, combining Q-PCR with 5' RLM-RACE and sequencing are described for the identification of novel ETS fusion partners.

Immunohistochemical ETS-related gene detection in a Japanese prostate cancer cohort: diagnostic use in Japanese prostate cancer patients.

Chromosomal rearrangements that result in high expression levels of the ETS-related gene (ERG) present in approximately 50% of prostate cancer (PCa) patients, making this one of the most common oncogenic alterations in PCa. However, ERG overexpression at the protein level has not been rigorously evaluated in Japanese PCa patients. In this study, we evaluated ERG expression using antibody-based detection in 230 prostate specimens in a Japanese PCa cohort. Overall, we identified 20.1% ERG-positive PCa cases. ERG was not detected in benign glands. The specificity of ERG staining for detecting PCa was almost 100%; all of the ERG-positive samples were also diagnosed as PCa. The expression level of the ERG protein correlated with clinicopathological variables, including grade (P= 0.038), stage (P= 0.005), and metastatic status (P= 0.014). No correlation was observed with age (P= 0.196) or with preoperative prostate-specific antigen level (P= 0.322). Although the frequency of ERG-positive cases in Japanese PCa patients (20.1%) was lower than that reported in a PCa cohort in Western countries (approximately 50%), our study demonstrates that the clinical utility of ERG detection at the protein level can serve as an ancillary tool for diagnosing PCa in the Japanese population.