Loss of MTAP expression by immunohistochemistry is a surrogate marker for homozygous 9p21.3 deletion in urothelial carcinoma.

Homozygous deletion of the chromosomal region 9p21.3 is common in urothelial carcinoma (UC) and leads to loss of several genes, including CDKN2A and MTAP, resulting in loss of MTAP protein expression. Here, we aimed at exploring the diagnostic potential of MTAP immunohistochemistry (IHC) as a surrogate marker for homozygous 9p21.3 deletion (9p21 HD) in UC. MTAP status was determined by IHC on 27 UC tissue specimens with known 9p21.3 status as defined by fluorescence in situ hybridization (FISH) in matched cytological specimens, by IHC and FISH on a tissue microarray (TMA) containing 359 UC at different stages, and by IHC on 729 consecutive UC from routine practice. Moreover, we analyzed a longitudinal series of matched specimens from 38 patients with MTAP negative recurrent UC. MTAP loss by IHC was found in all 17 patients with 9p21 HD and in 2/8 cases without 9p21 HD. In the TMA, MTAP loss was more common in metastases (53%) than in muscle-invasive (33%) and non-muscle-invasive UC (29%) (p=0.03). In the consecutive series, 164/729 (22%) cases showed loss of MTAP expression. In 41 of these 164 cases (25%), loss of MTAP expression was heterogenous. We also discovered loss of MTAP expression in flat urothelium adjacent to MTAP negative low-grade UC, suggesting true flat low-grade neoplasia that could not be diagnosed by morphology alone. Longitudinal analysis of recurrences showed persistent negative MTAP status over time in 37/38 (97%) patients. MTAP IHC can serve as a surrogate marker for 9p21 HD in UC and as a diagnostic tool to differentiate reactive urothelium from urothelial neoplasia. It also provides a unique opportunity to study clinicopathological associations and the heterogeneity of 9p21 HD across the whole spectrum of UC manifestations.

Exploring the intratumoral heterogeneity of DNA ploidy in prostate cancer.

BACKGROUND: Prostate cancer is morphologically and molecularly heterogeneous. Genomic heterogeneity might be mirrored by variability in DNA ploidy. Aneuploidy is a hallmark of genomic instability and associated with tumor aggressiveness. Little attention has been paid to the biological significance of the diploid tumor cell population that often coexists with aneuploid populations. Here, we investigated the role of DNA ploidy in tumor heterogeneity and clonal evolution. METHODS: Three radical prostatectomy specimens with intratumoral heterogeneity based on nuclear features on H&E were selected. DNA content of each subpopulation was determined by DNA image cytometry and silver in situ hybridization (SISH). Genomic evolution was inferred from array comparative genomic hybridization (aCGH). Additionally, immunohistochemistry was used to examine the stemness-associated marker ALDH1A1. RESULTS: Nuclear morphology reliably predicted DNA ploidy status in all three cases. In one case, aCGH analysis revealed several shared deletions and one amplification in both the diploid and the aneuploid population, suggesting that these populations could be related. In the other two cases, a statement about relatedness was not possible. Furthermore, ALDH1A1 was expressed in 2/3 cases and exclusively observed in their diploid populations. CONCLUSIONS: In this proof-of-concept study, we demonstrate the feasibility to predict the DNA ploidy status of distinct populations within one tumor by H&E morphology. Future studies are needed to further investigate the clonal relationship between the diploid and the aneuploid subpopulation and test the hypothesis that the aneuploid population is derived from the diploid one. Finally, our analyses pointed to an enrichment of the stemness-associated marker ALDH1A1 in diploid populations, which warrants further investigation in future studies.

Cell-Free DNA Genomic Profiling and Its Clinical Implementation in Advanced Prostate Cancer.

Most men with prostate cancer (PCa), despite potentially curable localized disease at initial diagnosis, progress to metastatic disease. Despite numerous treatment options, choosing the optimal treatment for individual patients remains challenging. Biomarkers guiding treatment sequences in an advanced setting are lacking. To estimate the diagnostic potential of liquid biopsies in guiding personalized treatment of PCa, we evaluated the utility of a custom-targeted next-generation sequencing (NGS) panel based on the AmpliSeq HD Technology. Ultra-deep sequencing on plasma circulating free DNA (cfDNA) samples of 40 metastatic castration-resistant PCa (mCRPC) and 28 metastatic hormone-naive PCa (mCSPC) was performed. CfDNA somatic mutations were detected in 48/68 (71%) patients. Of those 68 patients, 42 had matched tumor and cfDNA samples. In 21/42 (50%) patients, mutations from the primary tumor tissue were detected in the plasma cfDNA. In 7/42 (17%) patients, mutations found in the primary tumor were not detected in the cfDNA. Mutations from primary tumors were detected in all tested mCRPC patients (17/17), but only in 4/11 with mCSPC. AR amplifications were detected in 12/39 (31%) mCRPC patients. These results indicate that our targeted NGS approach has high sensitivity and specificity for detecting clinically relevant mutations in PCa.

Somatic Features of Response and Relapse in Non-muscle-invasive Bladder Cancer Treated with Bacillus Calmette-Guérin Immunotherapy.

BACKGROUND: High-risk non-muscle-invasive bladder cancer (NMIBC) is treated with bacillus Calmette-Guérin (BCG), but relapse is common. Improvement of patient outcomes requires better understanding of links between BCG resistance and genomic driver alterations. OBJECTIVE: To validate the prognostic impact of common genomic alterations in NMIBC pretreatment and define somatic changes present in post-BCG relapses. DESIGN, SETTING, AND PARTICIPANTS: We retrieved tumour tissues and outcomes for 90 patients with BCG-naive NMIBC initiating BCG monotherapy. Post-BCG tissue was available from 34 patients. All tissues underwent targeted sequencing of tumour and matched normal. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations between clinical outcomes and genomics were determined using Cox proportional hazard models. RESULTS AND LIMITATIONS: Of the patients, 58% were relapse free at data cut-off, 24% had NMIBC recurrence, and 18% experienced muscle-invasive progression. The risk of relapse was associated with ARID1A mutation (hazard ratio [HR] = 2.00; p = 0.04) and CCNE1 amplification (HR = 2.61; p = 0.02). Pre- and post-BCG tumours shared truncal driver alterations, with mutations in TERT and chromatin remodelling genes particularly conserved. However, shifts in somatic profiles were common and clinically relevant alterations in FGFR3, PIK3CA, TSC1, and TP53 were temporally variable, despite apparent clonal prevalence at one time point. Limitations include the difficulty of resolving the relative impact of BCG therapy versus surgery on genomics at relapse and biopsy bias. CONCLUSIONS: Somatic hypermutation and alterations in CCNE1 and ARID1A should be incorporated into future models predicting NMIBC BCG outcomes. Changes in tumour genomics over time highlight the importance of recent biopsy when considering targeted therapies, and suggest that relapse after BCG is due to persisting and evolving precursor populations. PATIENT SUMMARY: Changes in key cancer genes can predict bladder cancer relapse after treatment with bacillus Calmette-Guérin. Relapses after treatment can be driven by large-scale genetic changes within the cancer. These genetic changes help us understand how superficial bladder cancer can progress to be treatment resistant.

High Inter- and Intratumoral Variability of Ki67 Labeling Index in Newly Diagnosed Prostate Cancer with High Gleason Scores.

BACKGROUND: The majority of studies investigating the role of Ki67 labeling index (LI) in prostate carcinoma (PC) focused on localized PC treated radically, where Ki67 LI is regarded as a prognostic marker. The relevance of Ki67 in advanced PC remains largely unexplored. While Gleason score is still one of the best indicators of clinical outcomes in PC, differences in progression-free survival and overall survival in patients with high Gleason scores suggest that additional factors are involved in tumor progression. Understanding the underlying mechanisms could help to optimize treatment strategies for an individual patient. Here, we aimed to determine the inter- and intratumoral distribution of Ki67 LI in patients with PC with high Gleason scores and to correlate Ki67 LI with the status of ERG, PTEN, and Bcl-2. METHODS: Immunohistochemistry for Ki67, ERG, PTEN, and Bcl-2 was performed on core needle biopsies from 112 patients with newly diagnosed PC Gleason score 8, 9, and 10. RESULTS: Using a cutoff of ≥10%, 17/112 cases (15%) had a homogeneously low and 95/112 cases (85%) a high Ki67 LI. 41% of cases showed intratumoral heterogeneity containing areas with low and high proliferation. There was no association between Ki67 LI and ERG, PTEN, or Bcl-2 status. CONCLUSIONS: Our data demonstrate major inter- and intratumoral variability of Ki67 LI in high-grade PC with a surprisingly low Ki67 LI in a subset of cases. Further studies are necessary to explore the molecular basis and potential clinical implications of a paradoxically low proliferation rate in high-grade PC.

Prostate cancer patient-derived organoids: detailed outcome from a prospective cohort of 81 clinical specimens.

Patient-derived organoids (PDOs) represent promising preclinical models in various tumor types. In the context of prostate cancer (PCa), however, their establishment has been hampered by poor success rates, which impedes their broad use for translational research applications. Along with the necessity to improve culture conditions, there is a need to identify factors influencing outcomes and to determine how to assess success versus failure in organoid generation. In the present study, we report our unbiased efforts to generate PDOs from a cohort of 81 PCa specimens with diverse pathological and clinical features. We comprehensively analyzed histological features of each enrolled sample (Gleason score, tumor content, proliferation index) and correlated them with organoid growth patterns. We identified improved culture conditions favoring the generation of PCa organoids, yet no specific intrinsic tumor feature was broadly associated with sustained organoid growth. In addition, we performed phenotypic and molecular characterization of tumor-organoid pairs using immunohistochemistry, immunofluorescence, fluorescence in situ hybridization, and targeted sequencing. Morphological and immunohistochemical profiles of whole organoids altogether provided a fast readout to identify the most promising ones. Notably, primary samples were associated with an initial take-rate of 83% (n = 60/72) in culture, with maintenance of cancer cells displaying common PCa alterations, such as PTEN loss and ERG overexpression. These cancer organoids were, however, progressively overgrown by organoids with a benign-like phenotype. Finally, out of nine metastasis samples, we generated a novel organoid model derived from a hormone-naïve lung metastasis, which displays alterations in the PI3K/Akt and Wnt/ß-catenin pathways and responds to androgen deprivation. Taken together, our comprehensive study explores determinants of outcome and highlights the opportunities and challenges associated with the establishment of stable tumor organoid lines derived from PCa patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Patterns of stemness-associated markers in the development of castration-resistant prostate cancer.

BACKGROUND: Putative castration-resistant (CR) stem-like cells (CRSC) have been identified based on their ability to initiate and drive prostate cancer (PCa) recurrence following castration in vivo. Yet the relevance of these CRSC in the course of the human disease and particularly for the transition from hormone-naive (HN) to castration-resistance is unclear. In this study, we aimed at deciphering the significance of CRSC markers in PCa progression. METHODS: We constructed a tissue microarray comprising 112 matched HN and CR tissue specimens derived from 55 PCa patients. Expression of eight stemness-associated markers (ALDH1A1, ALDH1A3, ALDH3A1, BMI1, NANOG, NKX3.1, OCT4, SOX2) was assessed by immunohistochemistry and scored as a percentage of positive tumor cells. For each marker, the resulting scores were statistically analyzed and compared to pathological and clinical data associated with the samples. Unsupervised clustering analysis was performed to stratify patients according to the expression of the eight CRSC markers. Publicly-available transcriptional datasets comprising HN and CR PCa samples were interrogated to assess the expression of the factors in silico. RESULTS: Immunohistochemical assessment of paired samples revealed atypical patterns of expression and intra- and intertumor heterogeneity for a subset of CRSC markers. While the expression of particular CRSC markers was dynamic over time in some patients, none of the markers showed significant changes in expression upon the development of castration resistance (CR vs HN). Using unsupervised clustering approaches, we identified phenotypic subtypes based on the expression of specific stem-associated markers. In particular, we found (a) patterns of mutual exclusivity for ALDH1A1 and ALDH1A3 expression, which was also observed at the transcriptomic level in publicly-available PCa datasets, and (b) a phenotypic cluster associated with more aggressive features. Finally, by comparing HN and CR matched samples, we identified phenotypic cluster switches (ie, change of phenotypic cluster between the HN and CR state), that may be associated with clinical and predictive relevance. CONCLUSIONS: Our findings indicate stemness-associated patterns that are associated with the development of castration-resistance. These results pave the way toward a deeper understanding of the relevance of CRSC markers in PCa progression and resistance to androgen-deprivation therapy.

Immunocytochemistry for ARID1A as a potential biomarker in urine cytology of bladder cancer.

BACKGROUND: Mutations of AT-rich interactive domain 1 (ARID1A) have been associated with a worse outcome after intravesical treatment with bacille Calmette-Guérin in patients with non-muscle-invasive bladder cancer (NMIBC). Loss of ARID1A protein expression in urine cytology may serve as an indication of an ARID1A mutation. Therefore, the authors examined the expression of ARID1A in urine cytology and histological specimens of bladder cancer for correlation with ARID1A mutational status. METHODS: The authors constructed a tissue microarray containing samples from 164 tissue samples from 150 patients with NMIBC and 100 tissue samples from 81 patients with muscle-invasive bladder cancer. A second cohort consisted of archived cytological specimens and matched tissue sections from 62 patients with high-grade NMIBC. The authors established immunohistochemistry and immunocytochemistry (ICC) protocols, respectively, for the analysis of ARID1A protein expression in histological and cytological specimens. Confirmatory next-generation sequencing (NGS) was performed on tumor specimens using a targeted NGS panel containing all exonic regions of ARID1A. RESULTS: The prevalence of ARID1A loss of expression on the tissue microarray was 3.6% in NMIBC (6 of 164 tissue samples) and 10% in muscle-invasive bladder cancer (10 of 100 tissue samples) (P = .059). Loss of ARID1A expression in cytology was concordantly immunohistochemistry negative in 6 of 8 matched tissue specimens. NGS confirmed an ARID1A mutation on all 6 histology samples with loss of ARID1A expression. When NGS demonstrated an absence of ARID1A mutation, histology was concordantly positive (16 of 16 cases). CONCLUSIONS: The authors have suggest ARID1A ICC as a promising surrogate marker for ARID1A mutational status in patients with urothelial carcinoma. Pitfalls in ICC scoring include benign umbrella cells that often are negative for ARID1A. Further prospective studies are needed to determine the clinical relevance of ARID1A ICC in urinary cytology.

Tissue microarray analysis delineate potential prognostic role of Annexin A7 in prostate cancer progression.

BACKGROUND: Annexin A7 (ANXA7) is a member of the multifunctional calcium or phospholipid-binding annexin gene family. While low levels of ANXA7 are associated with aggressive types of cancer, the clinical impact of ANXA7 in prostate cancer remains unclear. Tissue microarrays (TMA) have revealed several new molecular markers in human tumors. Herein, we have identified the prognostic impact of ANXA7 in a prostate cancer using a tissue microarray containing 637 different specimens. METHODS: The patients were diagnosed with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival data (median 5.9 years) were available. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), syndecan-1 (CD-138) and ANXA7 were analyzed by immunohistochemistry. RESULTS: A bimodal distribution of ANXA7 was observed. Tumors expressing either high or no ANXA7 were found to be associated with poor prognosis. However, ANXA7 at an optimal level, in between high and no ANXA7 expression, had a better prognosis. This correlated with low Ki67, Bcl-2, p53 and high syndecan-1 which are known predictors of early recurrence. At Gleason grade 3, ANXA7 is an independent predictor of poor overall survival with a p-value of 0.003. Neoadjuvant hormonal therapy, which is known to be associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, was found to be associated with under-expression of ANXA7. CONCLUSIONS: The results of this TMA study identified ANXA7 as a new prognostic factor and indicates a bimodal correlation to tumor progression.

Delineation of human prostate cancer evolution identifies chromothripsis as a polyclonal event and FKBP4 as a potential driver of castration resistance.

Understanding the evolutionary mechanisms and genomic events leading to castration-resistant (CR) prostate cancer (PC) is key to improve the outcome of this otherwise deadly disease. Here, we delineated the tumour history of seven patients progressing to castration resistance by analysing matched prostate cancer tissues before and after castration. We performed genomic profiling of DNA content-based flow-sorted populations in order to define the different evolutionary patterns. In one patient, we discovered that a catastrophic genomic event, known as chromothripsis, resulted in multiple CRPC tumour populations with distinct, potentially advantageous copy number aberrations, including an amplification of FK506 binding protein 4 (FKBP4, also known as FKBP52), a protein enhancing the transcriptional activity of androgen receptor signalling. Analysis of FKBP4 protein expression in more than 500 prostate cancer samples revealed increased expression in CRPC in comparison to hormone-naïve (HN) PC. Moreover, elevated FKBP4 expression was associated with poor survival of patients with HNPC. We propose FKBP4 amplification and overexpression as a selective advantage in the process of tumour evolution and as a potential mechanism associated with the development of CRPC. Furthermore, FKBP4 interaction with androgen receptor may provide a potential therapeutic target in PC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MED15 overexpression in prostate cancer arises during androgen deprivation therapy via PI3K/mTOR signaling.

Androgen deprivation therapy (ADT) is the main therapeutic option for advanced prostate cancer (PCa). After initial regression, most tumors develop into castration-resistant PCa (CRPC). Previously, we found the Mediator complex subunit MED15 to be overexpressed in CRPC and to correlate with clinical outcome. Therefore, we investigated whether MED15 is implicated in the signaling changes taking place during progression to CRPC. Immunohistochemistry (IHC) for MED15 on matched samples from the same patients before and after ADT reveals significantly increased MED15 expression after ADT in 72%. A validation cohort comprising samples before and after therapy confirmed our observations. Protein analysis for pAKT and pSMAD3 shows that MED15 correlates with PI3K and TGFß activities, respectively, and that hyper-activation of both pathways simultaneously correlates with highest levels of MED15. We further show that MED15 protein expression increases in LNCaP cells under androgen deprivation, and via EGF mediated PI3K activation. PI3K/mTOR and TGFß-receptor inhibition results in decreased MED15 expression. MED15 knockdown reduces LNCaP cell viability and induces apoptosis during androgen deprivation, while cell cycle is not affected. Collectively, MED15 overexpression arises during ADT via hyper-activation of PI3K/mTOR signaling, thus MED15 may serve as a predictive marker for response to PI3K/mTOR inhibitors. Furthermore, MED15 is potentially a therapeutic target for the treatment of CRPC.

Primary synovial cell sarcoma of the kidney: case report and review of the literature.

Synovial cell sarcoma (SCS) of the kidney is a rare tumor entity with a poor prognosis. Morphologic and immunohistochemical characteristics may overlap with other more common neoplasms of the kidney. Therefore, the diagnosis of primary renal SCS not only requires the exclusion of similar tumor types, but also a confirmation of SYT-SSX gene fusion using molecular techniques. The treatment comprises radical surgery, and, depending on age and health status, adjuvant chemotherapy in selected patients. Here, we present an elderly SCS patient in whom straightforward radical surgical treatment resulted in a sustained complete remission; it allowed us to perform a literature survey focusing on current diagnostic tools for SCS.

Early development of human lymphomas in a prostate cancer xenograft program using triple knock-out immunocompromised mice.

BACKGROUND: There is an urgent need for preclinical models of prostate cancer; however, clinically relevant patient-derived prostate cancer xenografts (PDXs) are demanding to establish. METHODS: Sixty-seven patients who were undergoing palliative transurethral surgery or radical prostatectomy for histologically confirmed, clinically relevant prostate cancer were included in the study. Fresh prostate cancer tissue was identified by frozen analysis in 48 patients. The cancer tissue was transplanted subcutaneously and under the renal capsule of NSG and NOG mice supplemented with human testosterone. All growing PDXs were evaluated by histology and immunohistochemistry. RESULTS: Early assessment of the animals at least three months after transplantation included 27/48 (56.3%) eligible PDX cohorts. PDX growth was detected in 10/27 (37%) mouse cohorts. Eight of the ten PDXs were identified as human donor derived lymphomas, including seven Epstein Barr virus (EBV)-positive diffuse large B-cell lymphomas and one EBV-negative peripheral T-cell lymphoma. One sample consisted of benign prostatic tissue, and one sample comprised a benign epithelial cyst. Prostate cancer was not detected in any of the samples. CONCLUSIONS: Tumors that arise within the first three months after prostate cancer xenografting may represent patient-derived EBV-positive lymphomas in up to 80% of the early growing PDXs when using triple knockout NSG immunocompromised mice. Therefore, lymphoma should be excluded in prostate cancer xenografts that do not resemble typical prostatic adenocarcinoma.

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens.

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis.

MED12 overexpression is a frequent event in castration-resistant prostate cancer.

In a recent effort to unravel the molecular basis of prostate cancer (PCa), Barbieri and colleagues using whole-exome sequencing identified a novel recurrently mutated gene, MED12, in 5.4% of primary PCa. MED12, encoding a subunit of the Mediator complex, is a transducer of Wnt/ß-catenin signaling, linked to modulation of hedgehog signaling and to the regulation of transforming growth factor beta (TGFß)-receptor signaling. Therefore, these studies prompted us to investigate the relevance of MED12 in PCa. Expression of MED12, SMAD3 phosphorylation, and proliferation markers was assessed by immunohistochemistry on tissue microarrays from 633 patients. siRNA-mediated knockdown of MED12 was carried out on PCa cell lines followed by cellular proliferation assays, cell cycle analysis, apoptosis assays, and treatments with recombinant TGFß3. We found nuclear overexpression of MED12 in 40% (28/70) of distant metastatic castration-resistant prostate cancer (CRPC(MET)) and 21% (19/90) of local-recurrent CRPC (CRPC(LOC)) in comparison with frequencies of less than 11% in androgen-sensitive PCa, and no overexpression in benign prostatic tissues. MED12 expression was significantly correlated with high proliferative activity in PCa tissues, whereas knockdown of MED12 decreased proliferation, reduced G1- to S-phase transition, and increased the expression of the cell cycle inhibitor p27. TGFß signaling activation associates with MED12 nuclear overexpression in tissues and results in a strong increase in MED12 nuclear expression in cell lines. Furthermore, MED12 knockdown reduced the expression of the TGFß target gene vimentin. Our findings show that MED12 nuclear overexpression is a frequent event in CRPC in comparison with androgen-sensitive PCa and is directly implicated in TGFß signaling.

MED15, encoding a subunit of the mediator complex, is overexpressed at high frequency in castration-resistant prostate cancer.

The mediator complex is an evolutionary conserved key regulator of transcription of protein-coding genes and an integrative hub for diverse signaling pathways. In this study, we investigated whether the mediator subunit MED15 is implicated in castration-resistant prostate cancer (CRPC). MED15 expression and copy number/rearrangement status were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively on 718 prostate cancer (PCa) specimens and sequenced by Sanger on a subset. Furthermore, SMAD3 phosphorylation, androgen receptor (AR) and proliferation markers were evaluated by IHC. In PCa cells, siRNA/shRNA knockdown of MED15 was followed by proliferation assays with/without dihydrotestosterone (DHT), and treatments with recombinant TGF-ß3. Our results show that MED15 is overexpressed in 76% of distant metastatic CRPC (CRPC(MET) ) and 70% of local-recurrent CRPC (CRPC(LOC) ), in contrast to low frequencies in androgen-sensitive PCa, and no expression in benign prostatic tissue. Furthermore, MED15 overexpression correlates with worse clinical outcome thus defining a highly lethal phenotype. Moreover, TGF-ß signaling activation associates with MED15 overexpression in PCa tissues, and leads to increased expression of MED15 in PCa cells. MED15 knockdown effects phosphorylation and shuttling of p-SMAD3 to the nucleus as well as TGF-ß-enhanced proliferation. In PCa tissues, MED15 overexpression associates with AR overexpression/amplification and correlates with high proliferative activity. MED15 knockdown decreases both androgen-dependent and -independent proliferation in PCa cells. Taken together, these findings implicate MED15 in CRPC, and as MED15 is evolutionary conserved, it is likely to emerge as a lethal phenotype in other therapeutic-resistant diseases, and not restricted to our disease model.

Characterization and clinical relevance of ALDHbright populations in prostate cancer.

PURPOSE: High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN: We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. RESULTS: ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS: Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

Estrogen receptor ß expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naive prostate cancer and are elevated in castration-resistant disease.

Patients with advanced prostate cancer (PC) are usually treated with androgen withdrawal. While this therapy is initially effective, nearly all PCs become refractory to it. As hormone receptors play a crucial role in this process, we constructed a tissue microarray consisting of PC samples from 107 hormone-naïve (HN) and 101 castration-resistant (CR) PC patients and analyzed the androgen receptor (AR) gene copy number and the protein expression profiles of AR, Serin210-phosphorylated AR (pAR(210)), estrogen receptor (ER)ß, ERα and the proliferation marker Ki67. The amplification of the AR gene was virtually restricted to CR PC and was significantly associated with increased AR protein expression (P<0.0001) and higher tumor cell proliferation (P=0.001). Strong AR expression was observed in a subgroup of HN PC patients with an adverse prognosis. In contrast, the absence of AR expression in CR PC was significantly associated with a poor overall survival. While pAR(210) was predominantly found in CR PC patients (P<0.0001), pAR(210) positivity was observed in a subgroup of HN PC patients with a poor survival (P<0.05). Epithelial ERα expression was restricted to CR PC cells (9%). ERß protein expression was found in 38% of both HN and CR PCs, but was elevated in matched CR PC specimens. Similar to pAR(210), the presence of ERß in HN patients was significantly associated with an adverse prognosis (P<0.005). Our results strongly suggest a major role for pAR(210) and ERß in HN PC. The expression of these markers might be directly involved in CR tumor growth.

ERG rearrangement in local recurrences compared to distant metastases of castration-resistant prostate cancer.

Castration-resistant prostate cancer is the second most common cause of cancer death and results in a median survival of less than 2 years. In prostate cancer, fusions between TMPRSS2 and ERG are common. The ERG rearrangement prevalence in local recurrent castration-resistant prostate cancer compared to distant metastatic prostate cancer is unknown. We investigated the frequency of ERG rearrangement in local recurrent castration-resistant prostate cancer compared to distant metastatic prostate cancer, and assessed for associations between androgen receptor (AR) amplification and ERG rearrangement status. Samples from 134 patients diagnosed with prostate cancer (84 local recurrent castration resistant prostate cancer, 55 distant metastatic prostate cancer) were assessed for their ERG rearrangement and AR amplification status by fluorescence in situ hybridization. Statistical analysis was performed using the χ (2) test. We found that the ERG rearrangement occurs at a significantly lower frequency in distant metastatic prostate cancer (25 %) than in local recurrent castration-resistant prostate cancer (45 %). The AR amplification frequencies were 45 and 35 % in local recurrent castration-resistant prostate cancer and distant metastatic prostate cancer, respectively. The ERG rearrangement occurred at a lower frequency in distant metastatic prostate cancer compared to local recurrent castration-resistant prostate cancer.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth.

BACKGROUND: We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA). METHODS: The effect of NRBP1 expression on tumor cell growth was analyzed by using RNAi. NRBP1 protein expression was evaluated on two TMAs containing prostate samples from more than 1,000 patients. Associations with clinico-pathological features, the proliferation marker Ki67 and survival data were analyzed. RESULTS: RNAi mediated silencing of NRBP1 expression in prostate cancer cell lines resulted in reduced cell growth (P < 0.05). TMA analysis revealed NRBP1 protein expression in benign prostate hyperplasia in 6% as compared to 60% in both, high-grade intraepithelial neoplasia and prostate cancer samples. Strong NRBP1 protein expression was restricted to prostate cancer and correlated with higher expression of the proliferation marker Ki67 (P < 0.05). Further, patients with strong NRBP1 protein expression showed poor clinical outcomes (P < 0.05). Analysis of matched localized cancer tissues before and after castration revealed that post-therapy-related repression of NRBP1 expression was significantly associated with better overall survival. CONCLUSIONS: We demonstrate that expression of NRBP1 is up-regulated during the progression of prostate cancer and that high NRBP1 expression is linked with poor prognosis and enhanced tumor cell growth.