Joost L. Boormans

Antibody EPR3864 is specific for ERG genomic fusions in prostate cancer: implications for pathological practice

Genomic rearrangements involving genes encoding erythroblast transformation-specific transcription factors are commonly present in prostate cancer. The TMPRSS2–ERG gene fusion that leads to ERG overexpression occurs in ∼70% of prostate cancers. Implementation of fusion gene detection in pathological practice, however, has been hampered by the lack of reliable ERG antibodies. The objective of this study was first to compare ERG immunohistochemistry using the recently described antibody EPR3864 with ERG mRNA by quantitative PCR and, second, to investigate ERG immunohistochemistry in diagnostic prostate cancer needle biopsies. We analyzed 41 primary prostate adenocarcinomas obtained by radical prostatectomy and 83 consecutive prostate cancer needle biopsies. In the prostatectomy specimens, immunohistochemical ERG expression was highly concordant with the ERG mRNA overexpression (sensitivity 100% and specificity 85%). ERG overexpression was due to TMPRSS2–ERG gene fusion in all cases. ERG protein expression was identified in 51/83 adenocarcinomas (61%) on needle biopsies. ERG expression was more frequent in tumors infiltrating ≥2 needle biopsies (P<0.001) or occupying ≥50% of a single biopsy (P=0.018). Expression of ERG also occurred in 11/21 (52%) high-grade prostate intraepithelial neoplasia lesions. In 5/87 (6%) needle biopsies containing benign secretory glands, weak ERG staining was focally observed. In all of these cases, respective glands were adjacent to adenocarcinomas. In conclusion, immunohistochemistry for ERG strongly correlated with ERG mRNA overexpression and was specific for prostate cancer on needle biopsies. Therefore, ERG immunohistochemistry is an important adjunctive tool for pathophysiological studies on ERG gene fusions, and might support the pathological diagnosis of adenocarcinoma in a subset of prostate needle biopsies.

Truncated ETV1, Fused to Novel Tissue-Specific Genes, and Full-Length ETV1 in Prostate Cancer

In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1, an EST (EST14), and an endogenous retroviral repeat sequence (HERVK17). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized nontumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage-independent growth of these cells. PNT2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs, were up-regulated in both cell types. Integrin beta3 (ITGB3) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is proposed.

Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy

BACKGROUND An early report on the molecular subtyping of muscle-invasive bladder cancer (MIBC) by gene expression suggested that response to neoadjuvant chemotherapy (NAC) varies by subtype. OBJECTIVE To investigate the ability of molecular subtypes to predict pathological downstaging and survival after NAC. DESIGN, SETTING, AND PARTICIPANTS Whole transcriptome profiling was performed on pre-NAC transurethral resection specimens from 343 patients with MIBC. Samples were classified according to four published molecular subtyping methods. We developed a single-sample genomic subtyping classifier (GSC) to predict consensus subtypes (claudin-low, basal, luminal-infiltrated and luminal) with highest clinical impact in the context of NAC. Overall survival (OS) according to subtype was analyzed and compared with OS in 476 non-NAC cases (published datasets). INTERVENTION Gene expression analysis was used to assign subtypes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Receiver-operating characteristics were used to determine the accuracy of GSC. The effect of GSC on survival was estimated by Cox proportional hazard regression models. RESULTS AND LIMITATIONS The models generated subtype calls in expected ratios with high concordance across subtyping methods. GSC was able to predict four consensus molecular subtypes with high accuracy (73%), and clinical significance of the predicted consensus subtypes could be validated in independent NAC and non-NAC datasets. Luminal tumors had the best OS with and without NAC. Claudin-low tumors were associated with poor OS irrespective of treatment regimen. Basal tumors showed the most improvement in OS with NAC compared with surgery alone. The main limitations of our study are its retrospective design and comparison across datasets. CONCLUSIONS Molecular subtyping may have an impact on patient benefit to NAC. If validated in additional studies, our results suggest that patients with basal tumors should be prioritized for NAC. We discovered the first single-sample classifier to subtype MIBC, which may be suitable for integration into routine clinical practice. PATIENT SUMMARY Different molecular subtypes can be identified in muscle-invasive bladder cancer. Although cisplatin-based neoadjuvant chemotherapy improves patient outcomes, we identified that the benefit is highest in patients with basal tumors. Our newly discovered classifier can identify these molecular subtypes in a single patient and could be integrated into routine clinical practice after further validation.

Overexpression of Prostate-Specific TMPRSS2(exon 0)-ERG Fusion Transcripts Corresponds with Favorable Prognosis of Prostate Cancer

Purpose: To gain insight in the mechanism and clinical relevance of TMPRSS2-ERG expression in prostate cancer, we determined the specific characteristics of fusion transcripts starting at TMPRSS2 exon 1 and at a more upstream and less characterized exon 0. Experimental Design: We used quantitative PCR analysis to investigate expression of wild-type TMPRSS2(exon 0) and TMPRSS2(exon 1) and of ERG fusion transcripts. Expression was tested in normal tissue samples, in prostate cancer cell lines and xenografts, and in fresh-frozen clinical prostate cancer samples (primary tumors and recurrences). Expression in clinical samples was correlated with disease progression. Results: TMPRSS2(exon 0) and TMPRSS2(exon 1) transcripts were similarly androgen regulated in prostate cancer cell lines, but the expression levels of TMPRSS2(exon 1) were much higher. Comparison of expression in different tissues showed TMPRSS2(exon 0) expression to be much more prostate specific. In androgen receptor–positive prostate cancer xenografts, TMPRSS2(exon 1) transcripts were expressed at similar levels, but TMPRSS2(exon 0) transcripts were expressed at very variable levels. The same phenomenon was observed for TMPRSS2-ERG fusion transcripts. In clinical prostate cancers, the expression of TMPRSS2(exon 0)-ERG was even more variable. Expression of TMPRSS2(exon 0)-ERG transcripts was detected in 55% (24 of 44) of gene fusion–positive primary tumors but only in 15% (4 of 27) of gene fusion–positive recurrences and at much lower levels. Furthermore, in primary tumors, expression of TMPRSS2(exon 0)-ERG transcripts was an independent predictor of biochemical progression-free survival. Conclusion: The expression of TMPRSS2(exon 0)-ERG fusion transcripts in prostate cancer is associated with a less-aggressive biological behavior. (Clin Cancer Res 2009;15(20):6398–403)

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.

Expression of the Androgen-Regulated Fusion Gene TMPRSS2-ERG Does Not Predict Response to Endocrine Treatment in Hormone-Naïve, Node-Positive Prostate Cancer

BACKGROUND Fusion of the androgen-regulated gene transmembrane protease, serine 2, TMPRSS2, to the v-ets erythroblastosis virus E26 oncogene homolog (avian), ERG, of the erythroblast transformation-specific (ETS) family is the most common genetic alteration in prostate cancer (PCa). OBJECTIVE To determine whether expression of androgen-regulated TMPRSS2-ERG predicts response to endocrine treatment in hormone-naïve, node-positive PCa. DESIGN, SETTING, AND PARTICIPANTS Eighty-five patients with histologically confirmed, node-positive PCa who were without treatment at the moment of lymph node dissection were analysed. RNA was isolated from the paraffin-embedded lymph node metastases and complementary DNA (cDNA) was made. The quality of cDNA was tested by polymerase chain reaction (PCR) analysis of the expression of the housekeeping gene hydroxymethylbilane synthase, HMBS (formerly PBGD). TMPRSS2-ERG expression was analysed by PCR using a forward primer in TMPRSS2 exon 1 and a reverse primer in ERG exon 4. MEASUREMENTS The primary end point was time from start of endocrine therapy to the occurrence of three consecutive rises in prostate-specific antigen (PSA) that were at least 2 wk apart and resulted in two 50% increases over the PSA nadir. Secondary end points were time to PSA nadir after start of endocrine treatment and cancer-specific and overall survival. RESULTS AND LIMITATIONS TMPRSS2-ERG was expressed in 59% of the 71 patients who could be analysed. Median duration of response to endocrine therapy was 20.9 mo versus 24.1 mo for gene fusion-positive versus gene fusion-negative patients (95% confidence intervals: 18.6-23.1 vs 18.9-29.4, p=0.70). Furthermore, no significant differences were seen between the two groups for the secondary end points. CONCLUSIONS Expression of TMPRSS2-ERG is frequent in lymph node metastases of patients with untreated PCa; however, expression of this androgen-regulated fusion gene did not correspond with duration of response to endocrine therapy. Our results suggest that expression of TMPRSS2-ERG is not a candidate marker to select for metastatic PCa patients who will benefit more from endocrine treatment.

Percutaneous nephrolithotomy for treating renal calculi in children

There are various topics covered in the paediatric section this month. Another series of percutaneous nephrolithotomy is reported, as is the necessity or not to use the GFR in the follow‐up of children who have had a unilateral nephrectomy.

Histopathological characteristics of lymph node metastases predict cancer-specific survival in node-positive prostate cancer.

To correlate the histopathological characteristics of lymph node metastases in prostate cancer with cancer‐specific survival (CSS).

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.

Validation of a DNA Methylation-Mutation Urine Assay to Select Patients with Hematuria for Cystoscopy

Purpose: Only 3% to 28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer. Cystoscopy leads to high diagnostic costs and a high patient burden. Therefore, to improve the selection of patients for cystoscopy and reduce costs and over testing we aimed to validate a recently developed diagnostic urine assay. Materials and Methods: Included in study were 200 patients from a total of 3 European countries who underwent cystoscopy for hematuria, including 97 with bladder cancer and 103 with nonmalignant findings. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and HRAS, and methylation of OTX1, ONECUT2 and TWIST1. Logistic regression was used to analyze the association between predictor variables and bladder cancer. Results: Combining the methylation and mutation markers with age led to an AUC of 0.96 (95% CI 0.92–0.99) with 93% sensitivity and 86% specificity, and an optimism corrected AUC of 0.95. The AUC was higher for T1 or greater tumors compared to Ta tumors (0.99 vs 0.93). The AUC was also higher for high grade tumors compared to low grade tumors (1.00 vs 0.93). Overall negative predictive value was 99% based on the 5% to 10% prevalence of bladder cancer in patients with hematuria. This would lead to a 77% reduction in diagnostic cystoscopy. Conclusions: Analyzing hematuria patients for the risk of bladder cancer using novel molecular markers may lead to a reduction in diagnostic cystoscopy. Combining methylation analysis (OTX1, ONECUT2 and TWIST1) with mutation analysis (FGFR3, TERT and HRAS) and patient age resulted in a validated accurate prediction model.