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Based on gene-expression profiles, prostate tumors can be subdivided into subtypes with different aggressiveness and response to treatment. We investigated if similar clinically relevant subgroups can be identified simply by the combination of two immunohistochemistry markers: one for tumor cell differentiation (prostate specific antigen, PSA) and one for proliferation (Ki67). This was analyzed in men with prostate cancer diagnosed at transurethral resection of the prostate 1975-1991 (n = 331) where the majority was managed by watchful waiting. Ki67 and PSA immunoreactivity was related to outcome and to tumor characteristics previously associated with prognosis. Increased Ki67 and decreased PSA were associated with poor outcome, and they provided independent prognostic information from Gleason score. A combinatory score for PSA and Ki67 immunoreactivity was produced using the median PSA and Ki67 levels as cut-off (for Ki67 the upper quartile was also evaluated) for differentiation into subgroups. Patients with PSA low/Ki67 high tumors showed higher Gleason score, more advanced tumor stage, and higher risk of prostate cancer death compared to other patients. Their tumor epithelial cells were often ERG positive and expressed higher levels of ErbB2, phosphorylated epidermal growth factor receptor (pEGF-R) and protein kinase B (pAkt), and their tumor stroma showed a reactive response with type 2 macrophage infiltration, high density of blood vessels and hyaluronic acid, and with reduced levels of caveolin-1, androgen receptors, and mast cells. In contrast, men with PSA high/Ki67 low tumors were characterized by low Gleason score, and the most favorable outcome amongst PSA/Ki67-defined subgroups. Men with PSA low/Ki67 low tumors showed clinical and tumor characteristics intermediate of the two groups above. A combinatory PSA/Ki67 immunoreactivity score identifies subgroups of prostate cancers with different epithelial and stroma phenotypes and highly different outcome but the clinical usefulness of this approach needs to be validated in other cohorts.

Increasing evidence indicates calcium-binding S100 protein involvement in inflammation and tumor progression. In this prospective study, we evaluated the mRNA levels of two members of this family, S100A9 and S100A12, in peripheral blood mononuclear cells (PBMCs) in a cohort of 121 prostate cancer patients using RT-PCR. Furthermore, monocyte count was determined by flow cytometry. By stratifying patients into different risk groups, according to TNM stage, Gleason score and PSA concentration at diagnosis, expression of S100A9 and S100A12 was found to be significantly higher in patients with metastases compared to patients without clinically detectable metastases. In line with this, we observed that the protein levels of S100A9 and S100A12 in plasma were higher in patients with advanced disease. Importantly, in patients with metastases at diagnosis, high monocyte count and high levels of S100A9 and S100A12 were significantly associated with short progression free survival (PFS) after androgen deprivation therapy (ADT). High monocyte count and S100A9 levels were also associated with short cancer-specific survival, with monocyte count providing independent prognostic information. These findings indicate that circulating levels of monocytes, as well as S100A9 and S100A12, could be biomarkers for metastatic prostate cancer associated with particularly poor prognosis.

Purpose: Bone is the most predominant site of distant metastasis in prostate cancer, and patients have limited therapeutic options at this stage. Experimental Design: We performed a system-wide quantitative proteomic analysis of bone metastatic prostate tumors from 22 patients operated to relieve spinal cord compression. At the time of surgery, most patients had relapsed after androgen-deprivation therapy, while 5 were previously untreated. An extended cohort of prostate cancer bone metastases (n = 65) was used for immunohistochemical validation. Results: On average, 5,067 proteins were identified and quantified per tumor. Compared with primary tumors (n = 26), bone metastases were more heterogeneous and showed increased levels of proteins involved in cell-cycle progression, DNA damage response, RNA processing, and fatty acid β-oxidation; and reduced levels of proteins were related to cell adhesion and carbohydrate metabolism. Within bone metastases, we identified two phenotypic subgroups: BM1, expressing higher levels of AR canonical targets, and mitochondrial and Golgi apparatus resident proteins; and BM2, with increased expression of proliferation and DNA repair–related proteins. The two subgroups, validated by the inverse correlation between MCM3 and prostate specific antigen immunoreactivity, were related to disease prognosis, suggesting that this molecular heterogeneity should be considered when developing personalized therapies. Conclusions: This work is the first system-wide quantitative characterization of the proteome of prostate cancer bone metastases and a valuable resource for understanding the etiology of prostate cancer progression. Clin Cancer Res; 24(21); 5433–44. ©2018 AACR.

Prostate cancer (PC) remains a leading cause of cancer-related deaths among men worldwide, despite continuously improved treatment strategies. Patients with metastatic disease are treated by androgen deprivation therapy (ADT) that with time results in the development of castration-resistant prostate cancer (CRPC) usually established as metastases within bone tissue. The androgen receptor (AR) transcription factor is the main driver of CRPC development and of acquired resistance to drugs given for treatment of CRPC, while a minority of patients have CRPC that is non-AR driven. Molecular mechanisms behind epithelial AR reactivation in CRPC include AR gene amplification and overexpression, AR mutations, expression of constitutively active AR variants, intra-tumoural and adrenal androgen synthesis and promiscuous AR activation by other factors. This review will summarize AR alterations of clinical relevance for patients with CRPC, with focus on constitutively active AR variants, their possible association with AR amplification and structural rearrangements as well as their ability to predict patient resistance to AR targeting drugs. The review will also discuss AR signalling in the tumour microenvironment and its possible relevance for metastatic growth and therapy.

Clinical management of the prostate needs improved prognostic tests and treatment strategies. Because proteins are the ultimate effectors of most cellular reactions, are targets for drug actions and constitute potential biomarkers; a quantitative systemic overview of the proteome changes occurring during prostate cancer (PCa) initiation and progression can result in clinically relevant discoveries.To study cellular processes altered in PCa using system-wide quantitative analysis of changes in protein expression in clinical samples and to identify prognostic biomarkers for disease aggressiveness.Mass spectrometry was used for genome-scale quantitative proteomic profiling of 28 prostate tumors (Gleason score 6-9) and neighboring nonmalignant tissue in eight cases, obtained from formalin-fixed paraffin-embedded prostatectomy samples. Two independent cohorts of PCa patients (summing 752 cases) managed by expectancy were used for immunohistochemical evaluation of proneuropeptide-Y (pro-NPY) as a prognostic biomarker.Over 9000 proteins were identified as expressed in the human prostate. Tumor tissue exhibited elevated expression of proteins involved in multiple anabolic processes including fatty acid and protein synthesis, ribosomal biogenesis and protein secretion but no overt evidence of increased proliferation was observed. Tumors also showed increased levels of mitochondrial proteins, which was associated with elevated oxidative phosphorylation capacity measured in situ. Molecular analysis indicated that some of the proteins overexpressed in tumors, such as carnitine palmitoyltransferase 2 (CPT2, fatty acid transporter), coatomer protein complex, subunit alpha (COPA, vesicle secretion), and mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2, protein kinase) regulate the proliferation of PCa cells. Additionally, pro-NPY was found overexpressed in PCa (5-fold, p0.05), but largely absent in other solid tumor types. Pro-NPY expression, alone or in combination with the ERG status of the tumor, was associated with an increased risk of PCa specific mortality, especially in patients with Gleason score ≤ 7 tumors.This study represents the first system-wide quantitative analysis of proteome changes associated to localized prostate cancer and as such constitutes a valuable resource for understanding the complex metabolic changes occurring in this disease. We also demonstrated that pro-NPY, a protein that showed differential expression between high and low risk tumors in our proteomic analysis, is also a PCa specific prognostic biomarker associated with increased risk for disease specific death in patients carrying low risk tumors.The identification of proteins whose expression change in prostate cancer provides novel mechanistic information related to the disease etiology. We hope that future studies will prove the value of this proteome dataset for development of novel therapies and biomarkers.

One in every ten men diagnosed with prostate cancer will have metastases at some point, causing the high mortality of prostate cancer. Only earlier diagnosis has the chance to save those lives. However, correct therapy stratification may prolong the life of the patients without severely reducing the quality of life, thereby possibly turning a deadly disease into a chronic disease. For this to happen, we need predictive biomarkers that can be employed to longitudinally follow the disease in order to identify vulnerabilities and to monitor acquired resistance for personalized medicine. Using the platelet-enriched fraction of a blood sample, we here show that we can use the ability of platelets to sequester material during circulation to provide information derived from the tumor. This information could be used for early detection of prostate cancer. By using the platelet test in combination with PSA measurements, we may be able to rule out a majority of the false negatives associated with the PSA test. Furthermore, the platelet platform can be used in synergy with other biomarker sources such as circulating tumor DNA or protein biomarkers, providing the possibility to predict and monitor outcome of abiraterone therapy, and providing alternatives to the nonfunctional therapy by guiding therapy switching, in metastatic castration-resistant patients. Citation Format: Lee-Ann Tjon-Kon-Fat, Linda Köhn, Myron Best, Marie Lundholm, Thomas Wurdinger, Anders Widmark, Camilla Thellenberg-Karlsson, Pernilla Wikström, Jonas Nilsson. Tumor-educated platelets for early prostate cancer diagnosis, and therapy stratification in patients with metastasized castration resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B08.

Background: Novel therapies for castration resistant prostate cancer (CRPC) have been introduced in the clinic with possibilities for individualized treatment plans. Best practice of those expensive drugs requires predictive biomarker monitoring. This study used circulating biomarker analysis to follow cancer-derived transcripts implicated in therapy resistance. Method: The isolated platelet population of blood samples and digital-PCR were used to identify selected biomarker transcripts in patients with CRPC prior chemo- or androgen synthesis inhibiting therapy. Results: Fifty patients received either docetaxel (n = 24) or abiraterone (n = 26) therapy, with therapy response rates of 54% and 48%, respectively. Transcripts for the PC-associated biomarkers kallikrein-related peptidase-2 and -3 (KLK2, KLK3), folate hydrolase 1 (FOLH1), and neuropeptide-Y (NPY) were uniquely present within the platelet fraction of cancer patients and not detected in healthy controls (n = 15). In the abiraterone treated cohort, the biomarkers provided information on therapy outcome, demonstrating an association between detectable biomarkers and short progression free survival (PFS) (FOLH1, P < 0.01; KLK3, P < 0.05; and NPY, P < 0.05). Patients with biomarker-negative platelets had the best outcome, while FOLH1 (P < 0.05) and NPY (P = 0.05) biomarkers provided independent predictive information in a multivariate analysis regarding PFS. KLK2 (P < 0.01), KLK3 (P < 0.001), and FOLH1 (P < 0.05) biomarkers were associated with short overall survival (OS). Combining three biomarkers in a panel (KLK3, FOLH1, and NPY) made it possible to separate long-term responders from short-term responders with 87% sensitivity and 82% specificity. Conclusion: Analyzing tumor-derived biomarkers in platelets of CRPC patients enabled prediction of the outcome after abiraterone therapy with higher accuracy than baseline serum PSA or PSA response.

Reply to Isabel Heidegger, Renate Pichler, and Andreas Pircher's Letter to the Editor re : Erik Bovinder Ylitalo, Elin Thysell, Emma Jernberg, et al. Subgroups of Castration-resistant Prostate Cancer Bone Metastases Defined Through an Inverse Relationship Between Androgen Receptor Activity and Immune Response. Eur Urol 2017;71:776-87

To elucidate if the density of inflammatory cells expressing S100A9 in malignant and surrounding non-malignant prostate tissues is a prognostic marker for outcome in prostate cancer patients.Tissue was obtained from 358 men diagnosed with prostate cancer at transurethral resection of the prostate due to obstructive voiding problems, of which 260 were then followed with watchful waiting. Tissue microarrays of both malignant and non-malignant tissues were stained with an antibody against S100A9. The number of stained inflammatory cells was scored and related to clinical outcome and histopathological parameters of known prognostic value.A high number of inflammatory cells expressing S100A9 in both malignant and surrounding non-malignant tissues were associated with significantly shorter cancer-specific survival. This association remained significant when Gleason score and local tumour stage were analysed together with S100A9 in a Cox regression model. Low number of S100A9 positive cells in non-malignant stroma was correlated to significantly longer cancer-specific survival also in patients with Gleason score 8-10 tumours. S100A9 positive cells in tumour stroma were correlated with Gleason score, hyaluronan, platelet-derived growth factor receptor beta (PDGFR-β), and androgen receptor (inverse correlation) in tumour stroma. S100A9 positive cells in non-malignant stroma correlated with androgen receptor in this tissue compartment (inverse correlation).A high number of S100A9 positive inflammatory cells in tumour stroma and in non-malignant stroma were associated with shorter cancer-specific survival in prostate cancer patients.

Fatty acid amide hydrolase (FAAH) is responsible for the hydrolysis of the endogenous cannabinoid (CB) receptor ligand anandamide. Here we have investigated whether the expression levels of FAAH and CB1 receptors influence the prognostic value of markers of angiogenesis in prostate cancer. Data from a cohort of 419 patients who were diagnosed with prostate cancer at transurethral resection for lower urinary tract symptoms, of whom approximately 2/3 had been followed by expectancy, were used. Scores for the angiogenesis markers endoglin and von Willebrand factor (vWf), the endocannabinoid markers fatty acid amide hydrolase (FAAH) and cannabinoid CB1 receptors and the cell proliferation marker Ki-67 were available in the database. For the cases followed by expectancy, the prognostic value of endoglin was dependent upon the tumour epithelial FAAH immunoreactivity (FAAH-IR) and CB1IR scores, and the non-malignant epithelial FAAH-IR scores, but not the non-malignant CB1IR scores or the tumour blood vessel FAAH-IR scores. This dependency upon the tumour epithelial FAAH-IR or CB1IR scores was less apparent for vWf, and was not seen for Ki-67. Using an endoglin cut-off value of 10 positively stained vessels per core and a median split of tumour FAAH-IR, four groups could be generated, with 15year of disease-specific survival (%) of 68±7 (low endoglin, low FAAH), 45±11 (high endoglin, low FAAH), 77±6 (low endoglin, high FAAH) and 21±10 (high endoglin, high FAAH). Thus, the cases with high endoglin and high FAAH scores have the poorest rate of disease-specific survival. At diagnosis, the number of cases with tumour stages 1a-1b relative to stages 2-4 was sensitive to the endoglin score in a manner dependent upon the tumour FAAH-IR. It is concluded that the prognostic value of endoglin as a marker of neovascularisation in prostate cancer can be influenced by the expression level of markers of the endocannabinoid system. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Prostate cancer (PCa) is the most common cancer in men. PCa is strongly age associated; low death rates in surveillance cohorts call into question the widespread use of surgery, which leads to overtreatment and a reduction in quality of life. There is a great need to increase the understanding of tumor characteristics in the context of disease progression.To perform the first multigenome investigation of PCa through analysis of both autosomal and mitochondrial DNA, and to integrate exome sequencing data, and RNA sequencing and copy-number alteration (CNA) data to investigate how various different tumor characteristics, commonly analyzed separately, are interconnected.Exome sequencing was applied to 64 tumor samples from 55 PCa patients with varying stage and grade. Integrated analysis was performed on a core set of 50 tumors from which exome sequencing, CNA, and RNA sequencing data were available.Genes, mutated at a significantly higher rate relative to a genomic background, were identified. In addition, mitochondrial and autosomal mutation rates were correlated to CNAs and proliferation, assessed as a cell cycle gene expression signature.Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for.The mitochondrial genome displays an elevated mutation rate compared to the autosomal chromosomes. By integrated analysis, we demonstrated that different tumor characteristics are interconnected, providing an increased understanding of PCa etiology.

Prostate cancer (PCa) patients with bone metastases are primarily treated with androgen deprivation therapy (ADT). Less pronounced ADT effects are seen in metastases than in primary tumors. To test if acute effects of ADT was enhanced by concurrent inhibition of pro-survival insulin-like growth factor 1 (IGF-1), rats were inoculated with Dunning R3327-G tumor cells into the tibial bone marrow cavity and established tumors were treated with castration in combination with IGF-1 receptor (IGF-1R) inhibitor NVP-AEW541, or by each treatment alone. Dunning R3327-G cells were stimulated by androgens and IGF-1 in vitro. In rat tibia, Dunning R3327-G cells induced bone remodeling, identified through increased immunoreactivity of osteoblast and osteoclast markers. Tumor cells occasionally grew outside the tibia, and proliferation and apoptotic rates a few days after treatment were evaluated by scoring BrdU- and caspase-3-positive tumor cells inside and outside the bone marrow cavity, separately. Apoptosis was significantly induced outside, but unaffected inside, the tibial bone by either castration or NVP-AEW541, and the maximum increase (2.7-fold) was obtained by the combined treatment. Proliferation was significantly reduced by NVP-AEW541, independently of growth site, although the maximum decrease (24%) was observed when NVP-AEW541 was combined with castration. Tumor cell IGF-1R immunoreactivity was evaluated in clinical PCa bone metastases (n = 61), and positive staining was observed in most cases (74%). In conclusion, IGF-1R inhibition may be evaluated in combination with ADT in patients with metastatic PCa, or in combination with therapies for the subsequent development of castration-resistant disease, although diverse responses could be anticipated depending on metastasis site. Electronic supplementary material The online version of this article (doi:10.1007/s10585-017-9848-8) contains supplementary material, which is available to authorized users.

The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment.Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software.AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion.AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc.

Accumulating data indicates that tumor-derived extracellular vesicles (EVs) are responsible for tumor-promoting effects. However, if tumor EVs also prepare the tumor-bearing organ for subsequent tumor growth and if this effect is different in low and high malignant tumors is not thoroughly explored. Here we used orthotopic rat Dunning R-3327 prostate tumors to compare the role of EVs from fast growing and metastatic MatLyLu (MLL) tumors with EVs from more indolent and non-metastatic Dunning G (G) tumors. Prostate tissue pre-conditioned with MLL-EVs in vivo facilitated G tumor establishment compared to G-EVs. MLL-EVs increased prostate epithelial proliferation and macrophage infiltration into the prostate compared to G-EVs. Both types of EVs increased macrophage endocytosis and the mRNA expression of genes associated with M2 polarization in vitro, with MLL-EVs giving the most pronounced effects. MLL-EVs also altered the mRNA expression of growth factors and cytokines in primary rat prostate fibroblasts compared to G-EVs, suggesting fibroblast activation. Our findings propose that EVs from metastatic tumors have the ability to prime the prostate tissue and enhance tumor growth to a higher extent than EVs from non-metastatic tumors. Identifying these differences could lead to novel therapeutic targets and potential prognostic markers for prostate cancer.

Background: Novel therapies for men with castration-resistant prostate cancer (CRPC) are needed, particularly for cancers not driven by androgen receptor (AR) activation. Objectives: To identify molecular subgroups of PC bone metastases of relevance for therapy. Design, setting, and participants: Fresh-frozen bone metastasis samples from men with CRPC (n = 40), treatment-naïve PC (n = 8), or other malignancies (n = 12) were characterized using whole-genome expression profiling, multivariate principal component analysis (PCA), and functional enrichment analysis. Expression profiles were verified by reverse transcription–polymerase chain reaction (RT-PCR) in an extended set of bone metastases (n = 77) and compared to levels in malignant and adjacent benign prostate tissue from patients with localized disease (n = 12). Selected proteins were evaluated using immunohistochemistry. A cohort of PC patients (n = 284) diagnosed at transurethral resection with long follow-up was used for prognostic evaluation. Results and limitations: The majority of CRPC bone metastases (80%) was defined as AR-driven based on PCA analysis and high expression of the AR, AR co-regulators (FOXA1, HOXB13), and AR-regulated genes (KLK2, KLK3, NKX3.1, STEAP2, TMPRSS2); 20% were non–AR-driven. Functional enrichment analysis indicated high metabolic activity and low immune responses in AR-driven metastases. Accordingly, infiltration of CD3+ and CD68+ cells was lower in AR-driven than in non–AR-driven metastases, and tumor cell HLA class I ABC immunoreactivity was inversely correlated with nuclear AR immunoreactivity. RT-PCR analysis showed low MHC class I expression (HLA-A, TAP1, and PSMB9 mRNA) in PC bone metastases compared to benign and malignant prostate tissue and bone metastases of other origins. In primary PC, low HLA class I ABC immunoreactivity was associated with high Gleason score, bone metastasis, and short cancer-specific survival. Limitations include the limited number of patients studied and the single metastasis sample studied per patient. Conclusions: Most CRPC bone metastases show high AR and metabolic activities and low immune responses. A subgroup instead shows low AR and metabolic activities, but high immune responses. Targeted therapy for these groups should be explored. Patient summary: We studied heterogeneities at a molecular level in bone metastasis samples obtained from men with castration-resistant prostate cancer. We found differences of possible importance for therapy selection in individual patients.

Background and purpose The criteria for selecting patients who may benefit from surgery of spinal cord compression in metastatic prostate cancer are poorly defined. We therefore studied patients operated for metastatic spinal cord compression in order to evaluate outcome of surgery and to find predictors of survival. Patients and methods We reviewed the records of 54 consecutive patients with metastatic prostate cancer who were operated for spinal cord compression at Umeå University Hospital. The indication for surgery was neurological deficit due to spinal cord compression. 41 patients had hormone-refractory cancer and 13 patients had previously untreated, hormone-naïve prostate cancer. 29 patients were operated with posterior decompression only, and in 25 patients posterior decompression and stabilization was performed. Results Preoperatively, 6/54 of patients were able to walk. 1 month after surgery, 33 patients were walking, 15 were non-ambulatory, and 6 had died. Mortality rate was 11% at 1 month, 41% at 6 months, and 59% at 1 year. In the hormone-naïve group, 8/13 patients were still alive with a median postoperative follow-up of 26 months. In the hormone-refractory group, median survival was 5 months. Patients with hormone-refractory disease and Karnofsky performance status (KPS) of ≤ 60% had median survival of 2.5 months, whereas those with KPS of 70% and KPS of ≥ 80% had a median survival of 7 months and 18 months, respectively (p < 0.001). Visceral metastases were present in 12/41 patients with hormone-refractory tumor at the time of spinal surgery, and their median survival was 4 months—as compared to 10 months in patients without visceral metastases (p = 0.003). Complications within 30 days of surgery occurred in 19/54 patients. Interpretation Our results indicate that patients with hormone-naive disease, and those with hormone-refractory disease with good performance status and lacking visceral metastases, may be helped by surgery for metastatic spinal cord compression.

A molecular classification system with clinical impact has been lacking for prostate cancer until You and colleagues published “Integrated Classification of Prostate Cancer Reveals a Novel Luminal Subtype with Poor Prognosis” ([1][1]). Gene expression profiles were used to stratify localized

Hypoxia-inducible factor (HIF)-2alpha is upregulated in hypoxia or by inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. In a number of malignancies, increased HIF-2alpha expression may indicate worse prognosis. The aim of this study was to evaluate the prognostic information of HIF-2alpha mRNA expression in renal cell carcinoma (RCC).HIF-2alpha mRNA was quantified by real time polymerase chain reaction (rt-PCR) in tumour tissue samples from 202 patients. Samples from 50 corresponding kidney cortex tissue were analysed as controls. mRNA levels were evaluated in relation to tumour cell type, TNM stage, nuclear grade and disease specific survival.The levels of HIF-2alpha mRNA were significantly higher in 168 clear cell (c)RCC than in 23 papillary (p)RCC (p0.001) or 11 chromophobe (ch)RCC (p0.006). Among cRCC there was an inverse correlation between HIF-2alpha mRNA levels and TNM stage I and II-IV tumours (p=0.01), and nuclear grade (p = 0.006). After a median follow-up time of 99 months (range 34-247), 106 patients had died of RCC. No correlation of HIF-2alpha mRNA to survival was observed. A multivariate analysis of prognostic factors in cRCC showed that TNM stage alone was an independent predictor of prognosis; HIF-2alpha mRNA levels did not add further prognostic information.The results demonstrated that HIF-2alpha mRNA levels were higher in cRCC compared to pRCC and chRCC. Furthermore, HIF-2alpha mRNA levels were inversely related to TNM stage and nuclear grade in cRCC.

Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1). To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RTPCR showed that the main site of HO-1 synthesis was HO-1(+) macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1(+) macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1(+) macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1(+) macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1(+) macrophages may have an important role in prostate cancer.

In the present study we have investigated whether Caveolin-1 expression in non-malignant and malignant prostate tissue is a potential prognostic marker for outcome in prostate cancer patients managed by watchful waiting. Caveolin-1 was measured in prostate tissues obtained through transurethral resection of the prostate from 395 patients diagnosed with prostate cancer. The majority of the patients (n = 298) were followed by watchful waiting after diagnosis. Tissue microarrays constructed from malignant and non-malignant prostate tissue were stained with an antibody against Caveolin-1. The staining pattern was scored and related to clinicopathologic parameters and outcome. Microdissection and qRT-PCR analysis of Cav-1 was done of the prostate stroma from non-malignant tissue and stroma from Gleason 3 and 4 tumors. Cav-1 RNA expression was highest in non-malignant tissue and decreased during cancer progression. High expression of Caveolin-1 in tumor stroma was associated with significantly longer cancer specific survival in prostate cancer patients. This association remained significant when Gleason score and local tumor stage were combined with Caveolin-1 in a Cox regression model. High stromal Caveolin-1 immunoreactivity in prostate tumors is associated with a favourable prognosis in prostate cancer patients managed by watchful waiting. Caveolin-1 could possibly become a useful prognostic marker for prostate cancer patients that are potential candidates for active surveillance.

The epidermal growth factor receptor (EGFR) mediates regulatory signals in the normal prostate, but the functional importance of this is unclear.Adult male rats were castrated, or castrated + treated with gefitinib (Iressa, ZD1839), an EGFR tyrosine kinase inhibitor, for 3 days. Seven-day castrated rats were treated with testosterone, or testosterone + gefitinib, for 3 days.Both castration alone and testosterone treatment in castrated animals increased the mRNA and protein levels of EGFR and phospho-EGFR in the ventral prostate. Inhibition of EGFR during castration and during testosterone-stimulated prostate growth resulted in a decrease in total epithelial weight, epithelial cell proliferation, endothelial cell proliferation, and increased epithelial cell apoptosis.This study suggests that increased EGFR signaling during castration mediates stimulatory effects balancing castration-induced prostate regression, and that EGFR signaling is a necessary component in testosterone-stimulated prostate growth.

Castration-induced involution of the normal prostate is caused by primary effects in the prostate stroma and vasculature, but if this is the case also in tumors is unknown.Androgen-independent AT-1 prostate tumor cells were therefore injected into the ventral prostate (VP) in Copenhagen rats. Seven days later when the growing tumor was surrounded by normal VP tissue the rats were castrated and the effect examined 3 and 7 days later.Castration reduced vascular density in the surrounding VP tissue and this was accompanied by tumor cell hypoxia, apoptosis, and temporarily retarded tumor growth. Castration-induced VP tissue regression occurred more rapidly in the contra-lateral than in the tumor-bearing lobe.Androgen-independent tumor cell respond to castration when growing in an androgen-dependent environment. The presence of a tumor influences the castration response in the surrounding normal tissue. The microenvironment determines how prostate epithelial cells respond to castration.

This study was designed to test whether early castration-induced short-term cellular changes in primary prostate tumours could predict clinical outcome in advanced disease.Biopsies from 83 patients obtained before and within two weeks after surgical castration were investigated. Tumour epithelial cell apoptosis, proliferation, and prostate specific antigen (PSA) levels were quantified using immunohistochemistry, laser capture micro-dissection, and real time RT-PCR. Cellular effects were related to changes in serum PSA levels and clinical outcome.Decreased proliferation and PSA mRNA levels, and increased apoptosis were observed in most tumours. These early cellular responses were not correlated to each other and did not predict serum PSA response or cancer-specific survival. A nadir PSA level below 1 ng/ml predicted a longer cancer-specific survival after castration therapy.Castration therapy causes primary tumour regression in most patients with advanced prostate cancer, but these primary tumour effects are not predictive for systemic disease control. Studies of early changes in metastases during hormonal therapy will probably give more predictive information for clinical outcome than further studies in primary tumours.

Castration rapidly reduces stroma insulin-like growth factor (IGF)-1 synthesis and action in mouse prostate epithelium. We explore if similar changes are of importance for castration-induced prostate regression in humans.Epithelial and surrounding stroma cells were micro-dissected from patient biopsies obtained before and shortly after castration. IGF-1 mRNA levels were quantified by RT-PCR and related to epithelial apoptosis and IGF-1, IGF-1 receptor, and androgen receptor (AR) immunoreactivity.IGF-1 mRNA was principally produced in the stroma and IGF-R1 in the epithelium. Stroma IGF-1 mRNA levels were significantly decreased after castration in non-malignant but not malignant tissue. Lack of stroma IGF-1 reduction after castration was associated with low stroma AR expression before therapy. Reduction of IGF-1 mRNA levels in the tumor stroma and/or epithelium was associated with epithelial apoptosis after therapy.Low AR expression and maintained stroma IGF-1 synthesis may result in limited tumor cell death after castration therapy.

To investigate the prognostic value of potential markers in localized, Gleason score 6 and 7 prostate cancer (PC).From a consecutive series of men with PC diagnosed at transurethral resection (1975-1990), specimens from 132 patients without metastases, with Gleason score (GS) 6 (n=80) or 7 (n=52) tumors followed with watchful waiting were examined. The fraction of resected prostate tissue containing cancer, the micro-vessel density (v.d.) when stained for endoglin or von Willebrand factor (vWf), and the percentage of Ki-67 labeled tumor cells were measured using immunohistochemistry.High levels of vWf v.d., endoglin v.d., and percent cancer of the TURP specimen were significantly associated with short cancer-specific survival in Kaplan-Meier analysis of all patients with GS 6 and 7 tumors. Interestingly, a combined estimate of percent cancer and vWF v.d. could be used to identify a large subset (50%) of GS 6 tumors with only a 2.5% risk of PC death within 15 years. None of the tested markers gave independent prognostic information for the GS 7 tumors.Estimates of tumor size and vascular density may identify a large proportion of non-aggressive GS 6, but not GS 7, tumors.

A rye bran diet has been suggested to retard prostate cancer growth and increase tumor cell apoptosis in animal models and in men with prostate cancer. Transgenic mice designed to develop prostate cancer (transgenic adenocarcinoma of the mouse prostate, TRAMP) were therefore treated with a rye bran diet. TRAMP mice were fed a rye bran (n = 13) or a control diet (n = 15) from 4 to 24 wk of age. Tumor histology, cell proliferation, and apoptosis were accessed using stereological and immunohistochemical techniques. In the dorsolateral prostate lobe in TRAMP mice, epithelial cell apoptosis was increased by 31%, and the volume fraction of epithelial cells was reduced by 20% (P0.05) in the rye bran-treated group compared with the control diet group. There were, however, no significant changes detected in lobe weights or tumor grade. In conclusion, a rye bran diet increases epithelial apoptosis and reduces the epithelial cell fraction in TRAMP tumors.

The involution of the rat ventral prostate gland after castration could be caused by primary changes in the vasculature. To explore the mechanisms, we studied the effects of castration and testosterone treatment on the vasculature in the ventral prostate in adult rats and mice. Androgen receptor expression, vascular morphology, and the expression of angiopoietin (ang) 1 and 2 and their receptor tie 2 were examined 1, 3, and 7 d after castration and after testosterone treatment of castrated animals using stereological methods, immunohistochemistry, laser capture microdissection, and Western blotting. One day after castration, the percentage of blood vessels covered with smooth muscle actin, endothelial cell proliferation, and vascular volume had decreased, whereas endothelial cell apoptosis had increased. Simultaneously, ang 1 and tie 2 protein levels decreased. Nuclear expression of androgen receptor was observed not only in glandular and stroma smooth muscle cells but also in the mural cells of prostate arteries and veins and was markedly down-regulated already 1 d after castration. Testosterone administration of castrated mice and rats reversed all the observed effects. At the mRNA level, tie 2 was exclusively, but ang 1 predominantly, expressed in the stroma, compared with the epithelial compartment. Local delivery of soluble tie 2 during testosterone-stimulated growth, inhibited vascular maturation and increased vascular volume and leukocyte infiltration compared with controls. We conclude that androgens may regulate the prostate vasculature by direct effects on mural vascular cells and by influencing the secretion of the angiopoietins, in above all, the stroma cells.

Angiopoietin 2 expression is related to histological grade, vascular density, metastases, and outcome in prostate cancer.

Pigment epithelium-derived factor, a potent angiogenesis inhibitor in the eye, is also expressed in the prostate. Prostate size and angiogenesis is increased in pigment epithelium-derived factor knockout mice, and pigment epithelium-derived factor is down-regulated in some prostate cancers. To investigate whether pigment epithelium-derived factor expression correlates with tumor progression, we examined 5 Dunning rat prostate sublines with different growth rates, differentiation, androgen dependence, vascular density, and metastatic ability and 26 human prostate cancers of Gleason score 8–10 obtained from patients at transurethral resection selected to represent two groups, with and without metastases at diagnosis. By Western blot, real-time quantitative reverse transcription-PCR, and immunostaining, pigment epithelium-derived factor was detected in highly differentiated, nonmetastatic, androgen-sensitive Dunning tumors and in the anaplastic, androgen insensitive but nonmetastatic Dunning tumors. In contrast, the metastatic Dunning tumor sublines showed very low pigment epithelium-derived factor expression levels. In human cancer tissues, by immunohistochemistry and real-time quantitative reverse transcription-PCR, patients without metastases at diagnosis had higher tumor pigment epithelium-derived factor levels than tumors from patients with metastases at diagnosis. In both the rat model and in the human tumors, the proliferation index and vascular count, as determined by Ki-67 staining and endoglin and/or factor VIII-related antigen staining, inversely correlated with pigment epithelium-derived factor mRNA levels. These observations indicate that loss of pigment epithelium-derived factor expression could be associated with the progression toward a metastatic phenotype in prostate cancer.

BACKGROUND: Androgen ablation is the standard treatment for advanced prostate cancer but the short-term cellular effects are largely unknown. METHODS: Sextant prostate biopsies were taken from 77 p ...

Characterization of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) as a model to study effects of castration therapy.

Vascular endothelial cell growth factor (VEGF-A) is synthesized in the testis but its role and regulation in this organ have not been examined. VEGF and its receptors (VEGF-R) were quantified using reverse transcription-polymerase chain reaction and Western blot. VEGF, VEGF-R1, and VEGF-R2 mRNAs and VEGF protein were increased after treatment with 50 IU hCG. Injection of 100 ng human recombinant VEGF 165 into the testis caused an increase in endothelial cell proliferation, but only a moderate increase in testicular interstitial fluid volume. In contrast with systemic hCG treatment, local VEGF injection did not increase the permeability to intravenously injected colloidal carbon particles. However, if VEGF was given locally in the testes of animals pretreated with hCG 4 or 8 h earlier, VEGF acted in synergy with hCG to increase vascular carbon leakage by forming interendothelial cell gaps. Testicular blood flow was unaffected by local VEGF 165 injection. Treatment with a specific VEGF-R2 tyrosine kinase inhibitor blocked the hCG-induced increase in endothelial cell proliferation but did not affect the hCG-induced accumulation of polymorphonuclear leukocytes in testicular blood vessels or the increase in the testicular interstitial space. The present study demonstrated that testicular VEGF secretion is increased by hormonal stimulation of Leydig cells and that VEGF, through effects mediated via VEGF-R2, regulates endothelial cell proliferation in the rat testis. VEGF does not appear to regulate testicular blood flow and it is not involved in inducing the hCG-induced inflammation-like response in the testicular microvasculature. The permeability-increasing effect of VEGF is low in the testis under basal conditions but is apparently up-regulated by hCG treatment.

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.

BACKGROUND Endoglin, a receptor for some of the members of the transforming growth factor-β (TGF-β) family, is expressed on proliferating endothelial cells and has been suggested as a marker of ongoing angiogenesis. In this study, endoglin was evaluated as a prognostic factor for prostate cancer progression. METHODS Immunohistochemical staining of endoglin was examined in 72 cases of prostate cancer and compared with immunohistochemical staining of the pan-endothelial marker von Willebrand factor (vWf), clinicopathological factors, and cancer-specific survival. Micro-vessels were measured in the most vascularized fields. Double staining with antibodies against smooth muscle actin and endoglin or vWf, respectively, was performed in order to evaluate vessel maturation. RESULTS Endoglin-stained tumor vessels were generally small and only 19% also stained with actin. Endoglin was a better prognostic marker than vWf. The median survival times were shorter for patients with tumor vascular count (vc) above median than for patients with vc below median (4 vs. 12 years, P = 0.0007, and 5 vs. 10 years, P = 0.018, for endoglin and vWf, respectively). Endoglin vc was associated with Gleason score (P = 0.001), local tumor stage (P = 0.0006), metastasis (P = 0.01), tumor cell immunoreactivity for TGF-β1 (P = 0.0003), and tumor cell proliferation index (rs = 0.319, P = 0.02). Endoglin, in contrast to vWf, vc was prognostic for survival in the subgroup of patients with Gleason score 5, 6, and 7 tumors. CONCLUSIONS Endoglin marks principally small, probably newly formed tumor vessels in zthe prostate, and is a promising prognostic marker for prostate cancer patients. Prostate 51: 268–275, 2002. © 2002 Wiley-Liss, Inc.

e583 Background: Novel therapies for castration resistant prostate cancer (CRPC) have been introduced in the clinic with possibilities for individualized treatment plans. Best practice of those expensive drugs requires predictive biomarker monitoring. This study used circulating biomarker analysis to follow cancer-derived transcripts implicated in therapy resistance. Methods: The isolated platelet population of blood samples and QRT-PCR were used to identify selected biomarker transcripts in patients with CRPC prior chemo- or androgen synthesis directed therapies. The association between biomarker status in platelets (positive vs. negative) and therapy response, progression-free survival (PFS) and overall survival (OS) was examined. Results: Fifty patients received either docetaxel (n = 24) or abiraterone (n = 26) therapy, with therapy response rates of 54% and 48%, respectively. Transcripts for the PC-associated biomarkers kallikrein-related peptidase-3 ( KLK3), androgen receptor splice-variant 7 (ARV7), folate hydrolase 1 (FOLH1), and neuropeptide-Y ( NPY) were present within the platelet fraction. Analyzing biomarkers in the chemotherapy group did not add information about PFS, but FOLH1 was associated with short OS (p = 0.00015). In the abiraterone treated cohort, detectable FOLH1 (p = 0.009), KLK3 (p = 0.018), and NPY (p = 0.028) were all associated with short PFS. Patients with biomarker-negative platelets had the best outcome, while FOLH1 and NPY provided independent predictive information regarding PFS and KLK3 (p = 0.001) and FOLH1 (p = 0.002) were associated with short OS. Conclusions: Analyzing tumor-derived biomarkers in platelets of CRPC patients enabled prediction of the outcome after abiraterone therapy with high accuracy. Platelet-based analysis of FOLH1, NPY, and KLK3 may be used for treatment stratification of patients scheduled for treatment with abiraterone.

BACKGROUND Inhibitory effects of gonadotropin-releasing hormone (GnRH) analogs on prostate cancer cell proliferation, both in vivo and in vitro, indicate the presence of specific binding sites for GnRH on prostate cancer cells. To investigate this issue further, we examined the expression of GnRH receptor (GnRH-R) mRNA and protein in human prostate biopsies as well as in other extrapituitary tissues. METHODS The relative quantity of GnRH-R mRNA was determined by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in human prostate biopsies. Extrapituitary GnRH-R levels were determined by a semiquantitative PCR reaction. RESULTS Using PCR, a relatively high expression level of GnRH-R mRNA was found in prostate tumor tissue followed by normal prostate, thymus, and kidney expression levels. The levels showed by heart, brain, placenta, lung, liver, skeletal muscle, pancreas, colon, ovary, small intestine, spleen, and testis were low but detectable, whereas peripheral blood leukocyte showed no demonstrable product. GnRH-R immunoreactivity was localized in both luminal and basal epithelial cells in benign and malignant prostate tissue, and GnRH-R were also observed in intraprostatic lymphocytes. The relative GnRH-R mRNA levels in prostate biopsies from 16 patients showed a wide range of individual differences, but these differences were not related to histological grade. Castration therapy did not significantly influence GnRH-R mRNA expression in normal and malignant prostate tissue. CONCLUSIONS These results suggest that epithelial cells and infiltrating lymphocytes are targets for GnRH action in the human prostate. Comparative data show relatively high GnRH-R expression in human prostate tissue compared to other human tissues. Prostate 47:276–284, 2001. © 2001 Wiley-Liss, Inc.

TGF-beta1 is an important regulator of the normal and malignant prostate. In the non-malignant prostate, TGF-beta1 stimulates cell differentiation, inhibits epithelial cell proliferation, and induces epithelial cell death. TGF-beta1 is secreted into semen where it is an important immunosuppressive factor. Prostate cancer cells express high levels of TGF-beta1, which seems to enhance prostate cancer growth and metastasis by stimulating angiogenesis and by inhibiting immune responses directed against tumour cells. Prostate cancer cells frequently lose their TGF-beta receptors and acquire resistance to the anti-proliferative and pro-apoptotic effects of TGF-beta1. Accordingly, high expression of TGF-beta1 and loss of TGF-beta receptor expression have been associated with a particularly bad prognosis in human prostate cancer patients. TGF-beta1 also seems to be a mediator of castration-induced apoptosis in androgen dependent normal and malignant prostate epithelial cells. The ability of some prostate tumours to avoid castration-induced apoptosis may not, however, be simply due to loss of TGF-beta receptor type I or type II expression in the tumour cells. It may also be related to an inability of these cells to up-regulate TGF-beta receptor levels in response to castration or possibly due to defects downstream of the receptors. Short-term therapy-induced changes in the TGF-beta system in prostate tumours can probably be used to predict the long-term response to androgen ablation treatment. Further investigations into the TGF-beta system in the prostate are needed, however, to elucidate how alterations in this system affect the behaviour of prostate tumours, and whether this system can be manipulated for therapeutical purposes.

OBJECTIVE This study was carried out to explore the capability of partial least squares (PLS) analysis, a multivariate projection method, in the evaluation of investigative prognostic biomarkers. MATERIAL AND METHODS Using PLS analysis, survival and life expectancy were prognosticated in 53 patients with prostate cancer treated with transurethral resection. The age of the patient, stage and grade of the tumours, and immunoreactivity in the tumours for p53, Bcl-2, and TGF-beta1 and its receptors type I and type II (TGFbeta-RI and TGFalpha-RII), and markers for proliferation, vascular density, and macrophage density were included as prognostic factors in the analysis. RESULTS The prognostic impact of the factors, in decreasing order, was tumour grade, proliferation, stage, vascular density, TGFbeta-RI, metastasis, TGF-beta1, and TGFbeta-RII. Macrophage staining, p53, Bcl-2, and patient age did not contribute to the model. The model explained 62% of the total variance in survival, and the standard deviation of error of prediction was 29 months. CONCLUSIONS PLS analysis may become a useful tool for evaluation of putative prognostic factors.

Transforming growth factor-beta1 (TGF-beta1) inhibits epithelial cell proliferation in the normal prostate. Prostate tumours express high levels of TGF-beta1, and seem to acquire resistance to its anti-proliferative effects with tumour progression. In this study, TGFbeta variations with tumour progression were examined in the Dunning prostatic adenocarcinoma model. Expression of TGF-beta1 and TGFbeta receptor type I and type II (TGFbeta-RI and TGFbeta-RII) in rat dorsolateral prostate (DLP) and Dunning tumour sublines (PAP, AT-1, AT-2, AT-3 and MatLyLu) was examined in vitro and in vivo, using competitive reverse transcription-polymerase chain reaction (RT-PCR), Northern and Western blot, and immunohistochemistry. All tumours expressed elevated levels of TGF-beta1 and TGFbeta-RI mRNA, when compared with the DLP (Por = 0.05). All tumours except MatLyLu also expressed elevated levels of TGFbeta-RII mRNA (Por = 0.05). Interestingly, TGFbeta-RII protein levels were very low in the highly metastatic AT-3 and MatLyLu tumours in vivo, when compared with levels in the PAP, AT-1, and AT-2 tumours. This difference was not detected for the AT-1, AT-2, and AT-3 cells in vitro. Immunostaining of TGF-beta1, TGFbeta-RI, and TGFbeta-RII was localised principally in normal and tumour epithelial cells, and occasionally in smooth muscle cells. In conclusion, high expression of TGF-beta1 and TGFbeta-RI and low expression of TGFbeta-RII may contribute to tumour progression and metastasis in the Dunning prostatic adenocarcinoma model.

BACKGROUND Continuous administration of gonadotropin-releasing hormone (GnRH) agonists in prostate cancer patients results in involution of the tumors due to suppression of androgen production. In addition to the effect of GnRH at the hypothalamic-pituitary level, experiments in vitro on breast, ovary, and prostatic cells have shown an inhibition of cell proliferation, indicating the presence of local GnRH receptors (GnRH-R). The aim of the present study was to investigate the expression of GnRH-R mRNA in the normal rat ventral prostate (VP) and Dunning R3327 PAP adenocarcinoma and to evaluate the effects of castration on receptor mRNA expression. METHODS RNA was prepared from ovaries, pituitaries, VP, and Dunning tumors from both intact and castrated animals. GnRH-R mRNA levels were quantified by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS GnRH-R mRNA was detected in normal VP and Dunning tumors. Normal VP showed lower amounts of GnRH-R mRNA compared to Dunning tumors. An elevation of mRNA expression was observed 7 days after castration in Dunning tumors. CONCLUSIONS GnRH-R mRNA was found in both VP and Dunning tumors, indicating the presence of a local GnRH system. Normal VP showed lower amounts of GnRH-R mRNA when compared to malignant tissues. GnRH-R mRNA levels were elevated in Dunning tumors following castration. Prostate 39:101–107, 1999. © 1999 Wiley-Liss, Inc.

BACKGROUND. The mechanism behind castration-induced apoptosis in prostate cells is unknown, but data from other species suggest that transforming growth factor β1 (TGF-β1) may be involved. METHODS. By using quantitative RT-PCR and immunohistochemistry, expression of TGF-β1 and its receptors type I and II (RI and RII) was studied in normal and tumor areas of core biopsies taken before and 2-11 days after castration therapy. The TGF-β responses were related to changes in apoptotic index and to changes in serum prostate-specific antigen (PSA). RESULTS. In normal prostate tissue, apoptosis was generally increased by castration, and apoptosis was accompanied by an increase in TGF-β1 and RII mRNA levels (P < 0.05). In tumors, apoptosis was seen only in 44% of the cases and in these, but not in the others, TGF-β1, RI, and RII mRNA levels were increased (P < 0.05). In the patients showing a prognostically favorable PSA response (nadir PSA

BACKGROUND Prostate tumors express high levels of transforming growth factor-β1 (TGF-β1) and seem to acquire resistance to its antiproliferative effects with tumor progression. Moreover, TGF-β1 could be involved in tumor-promoting processes such as angiogenesis, cell migration, and immunosuppression. METHODS Immunoreactivity for TGF-β1 and its receptors type I and type II (TGFβ-RI and TGFβ-RII), tumor vascular count, and cell proliferation were studied in 73 cases of prostate cancer, diagnosed between 1975–1983 and followed with surveillance. RESULTS Patients with tumor overproduction of TGF-β1 had shorter median cancer-specific survival than patients with normal TGF-β1 immunoreactivity (5.0 vs. 10 years, P = 0.006). Furthermore, increased TGF-β1 staining was associated with tumor grade, high vascular counts, and metastasis (P = 0.02, 0.02, and 0.01, respectively). Patients with loss of tumor TGFβ-RII expression in combination with TGF-β1 overproduction showed particularly short survival (2.6 vs. 10 years, P = 0.0000), when compared to patients with normal immunoreactivity. CONCLUSIONS Overproduction of TGF-β1 and loss of TGFβ-RII expression are associated with poor clinical outcome in prostate cancer, and TGF-β1 may promote tumor progression by stimulating angiogenesis and metastasis. Prostate 37:19–29, 1998. © 1998 Wiley-Liss, Inc.

Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. Summary: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

In the normal prostate, transforming growth factor-beta 1 (TGF-beta 1) inhibits epithelial cell growth and is associated with apoptosis. The role of TGF-beta 1 in prostate cancer remains, however, unclear. In this work, the expression of TGF-beta receptor type I and II (TGF beta-RI and TGF beta-RII) in the Dunning R3327 PAP adenocarcinoma was studied, after castration and oestrogen treatment. Since castration induces apoptosis in the rat ventral prostate (VP) [21], but not in the Dunning R3327 PAP tumour [46], the TGF-beta receptor levels in the tumour were compared to the receptor levels in the VP. Methods used were competitive reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. In the VP, TGF beta-RI and TGF beta-RII expressions were increased after castration, indicating a negative regulation of TGF beta receptors by androgens. In the Dunning tumour, TGF beta-RI and TGF beta-RII levels were elevated and only TGF beta-RI showed a clear-cut increase after castration. The receptors were located in epithelial and smooth muscle cells in the VP and mainly in epithelial cells in the Dunning tumour. In conclusion, the elevated TGF beta receptor levels and the diminished androgen regulation of TGF beta-RII in the tumour distinguish the Dunning R3327 PAP tumour from the normal prostate and need to be further elucidated.

Prostate cancer is a common and multifocal disease but the diagnostic methods available are unsatisfactory. Most tumors present are of low malignant potential, whereas others are highly aggressive. At present, imaging cannot be used to guide tissue biopsies safely towards the most aggressive tumor present. To handle this problem multiple needle biopsies are taken. The biopsies often contain only normal prostate tissue, and even if the tumor is sampled it is not known whether a more aggressive cancer is present elsewhere in the organ. If changes in the normal tissue indicate the presence and nature of tumors, this information could be used to improve diagnostics and prognostics of prostate cancer.Current evidence that the tumor-adjacent morphologically normal prostate tissue is not completely normal is reviewed, and that this tissue, named tumor indicating normal tissue (TINT) by the authors, can be used to diagnose prostate cancer.The reader will understand that tumors need to affect their surroundings in order to grow and metastasize and that the normal prostate tissue is therefore tinted by the presence and nature of cancer and that this knowledge can be used to develop new diagnostic and prognostic markers.TINT changes could probably, when more rigorously defined and validated, be used to diagnose and prognosticate prostate cancer.

BACKGROUND: Intra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated with castration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables. METHODOLOGY/PRINCIPAL FINDINGS: Untreated, hormone-naïve (HN, n = 9) and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases. Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases. CONCLUSIONS/SIGNIFICANCE: Induced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis. Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2(nd) line androgen-deprivation therapy.

The aim of this study was to explore whether vascular density and tumour cell proliferation are related to the risk of prostate cancer death in patients managed by watchful waiting.From a consecutive series of men diagnosed with prostate cancer at transurethral resection in 1975-1990, tissue microarrays (TMAs) were constructed. A majority of men had no metastases at diagnosis and were followed by watchful waiting (n = 295). The TMAs were stained for Ki67, endoglin and factor VIII-related antigen (vWf).In univariate Cox analyses, increased Ki67 index, endoglin vascular density and vWf vascular density were associated with shorter cancer-specific survival. Ki67 index and endoglin vascular density added independent prognostic information to clinical stage, estimated tumour size and Gleason score (GS) in multivariate Cox analysis. In GS 6 tumours, high Ki67 index and high endoglin vascular density identified patients with poor outcome. After 15 years of follow-up not a single man out of 34 men with low staining for both markers (35% of all GS 6 tumours) had died of prostate cancer, in contrast to 15 prostate cancer deaths among the remaining 63 men with GS 6 tumours (65% cumulative risk of prostate cancer death). vWf vascular density in benign areas was a prognostic marker in GS 6 and 7 tumours.Men with GS 6 tumours with both low Ki67 index and endoglin vascular density staining scores have a low risk of progression. Additional studies are needed to test whether these two markers can be applied to core biopsies to select patients suitable for surveillance.

Background: Constitutively active androgen receptor variants (AR-V) lacking the ligand binding domain (LBD) may promote the development of castration-resistant prostate cancer (CRPC). The expression of AR-Vs in the clinically most important metastatic site, the bone, has, however, not been well documented. Our aim was therefore to compare levels of AR-Vs in hormone-naive (HN) and CRPC bone metastases in comparison to primary PC and non-malignant prostate tissue, as well as in relation to AR protein expression, whole-genome transcription profiles and patient survival. Methodology/Principal Findings: Hormone-naive (n=10) and CRPC bone metastases samples (n=30) were obtained from 40 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomized men. Levels of full length AR (ARfl) and AR-Vs termed AR-V1, AR-V7, and AR-V567es mRNA were measured with RT-PCR and whole-genome transcription profiles with an Illumina Beadchip array. Protein levels were examined by Western blotting and immunohistochemistry. Transcripts for ARfl, AR-V1, and AR-V7 were detected in most primary tumors and metastases, and levels were significantly increased in CRPC bone metastases. The AR-V567es transcript was detected in 23% of the CRPC bone metastases only. A sub-group of CRPC bone metastases expressed LBD-truncated AR proteins at levels comparable to the ARfl. Detectable AR-V567es and/or AR-V7 mRNA in the upper quartile, seen in 1/3 of all CRPC bone metastases, was associated with a high nuclear AR immunostaining score, disturbed cell cycle regulation and short survival. Conclusions/Significance: Expression of AR-Vs is increased in CRPC compared to HN bone metastases and associated with a particularly poor prognosis. Further studies are needed to test if patients expressing such AR-Vs in their bone metastases benefit more from drugs acting on or down-stream of these AR-Vs than from therapies inhibiting androgen synthesis. Citation: Hornberg E, Ylitalo EB, Crnalic S, Antti H, Stattin P, et al. (2011) Expression of Androgen Receptor Splice Variants in Prostate Cancer Bone Metastases is