Establishment of primary prostate epithelial and tumorigenic cell lines using a non-viral immortalization approach.

BACKGROUND: Research on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells. METHODS: Radical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells. RESULTS: Initial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent. CONCLUSION: Whereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.

Evaluation of Gas 6 as a Prognostic Marker in Papillary Renal Cell Carcinoma.

INTRODUCTION: Growth arrest-specific protein 6 (Gas 6) is a ligand that plays a role in proliferation and migration of cells. For several tumor entities, high levels of Gas 6 are associated with poorer survival. We examined the prognostic role of Gas 6 in renal cell carcinoma (RCC), especially in papillary RCC (pRCC), which is still unclear. METHODS: The patients' sample collection is a joint collaboration of the PANZAR consortium. Patients' medical history and tumor specimens were collected from n = 240 and n = 128 patients with type 1 and 2 pRCC, respectively. Expression of Gas 6 was determined by immunohistochemistry. RESULTS: In total, Gas 6 staining was evaluable in 180 of 240 type 1 and 110 of 128 type 2 pRCC cases. Kaplan-Meier analysis disclosed no significant difference in 5-year overall survival for all pRCC nor either subtype. Also, Gas+ and Gas- groups did not significantly differ in any tumor or patient characteristics. CONCLUSION: Gas 6 was not found to be an independent prognostic marker in pRCC. Future studies are warranted to determine if Gas 6 plays a role as prognostic marker or therapeutic target in pRCC.

The prognostic potential of alkaline phosphatase and lactic acid dehydrogenase in bmCRPC patients without significant PSA response under enzalutamide.

BACKGROUND: In patients with bone metastatic castration-resistant prostate cancer (bmCRPC) on systemic treatment, it is difficult to differentiate between continuous rise of prostate specific antigen (PSA) representing progression, and PSA-surge, which is followed by clinical response or stable disease. The purpose of this study was to evaluate the prognostic value of dynamic changes of alkaline phosphatase (ALP) and lactic acid dehydrogenase (LDH) levels as a predictor of clinical efficacy or therapeutic resistance of patients who do not show a sufficient initial PSA decline of ≥50% from baseline during early therapy with Enzalutamide. METHODS: Forty-eight men with bmCRPC on Enzalutamide 07/2010-09/2019 with initially rising PSA were analyzed. We monitored PSA, LDH and ALP at week 0, 2, 4, and every 4 weeks thereafter and analyzed the correlation between ALP rising at 12 weeks with or without LDH-normalization and the association with survival. For this we used Kaplan Meier analysis and uni- and multivariate cox-regression models. RESULTS: In Kaplan-Meier analysis, ALP rising at 12 weeks with or without LDH-normalization was associated with significantly worse median progression-free survival (PFS) of 3 months vs. 5 months (Log rank P = 0.02) and 3 months vs. 5 months (P = 0.01), respectively and overall survival (OS) with 8 months vs. 15 months (P = 0.02) and 8 months vs. 17 months (P < 0.01). In univariate analysis of PFS, ALP rising at 12 weeks alone, ALP rising at 12 weeks without LDH-normalization and application of Enzalutamide after chemotherapy showed a statistically significant association towards shorter PFS (hazard ratio (HR): 0.51, P = 0.04; HR: 0.48, P = 0.03; HR: 0.48, P = 0.03). Worse OS was significantly associated with ALP rising at 12 weeks alone, ALP rising at 12 weeks without LDH-normalization, and application of Enzalutamide after chemotherapy (HR: 0.47, P = 0.02; HR: 0.36, P < 0.01; HR: 0.31, P < 0.01). In multivariate analysis only the application of Enzalutamide after chemotherapy remained an independent prognostic factor for worse OS (HR: 0.36, P = 0.01). CONCLUSIONS: Dynamic changes of ALP (non-rise) and LDH (normalization) under therapy with Enzalutamide may be associated with clinical benefit, better PFS, and OS in patients with bmCRPC who do not show a PSA decline.

The Prognostic Impact of PD-L2 in Papillary Renal-Cell Carcinoma.

INTRODUCTION: Programmed death-1 ligand (PD-L1) has been often studied in different types of renal-cell carcinoma (RCC). For example, in clear-cell renal carcinoma it is well established that programmed death-1 receptor and PD-L1 are important prognostic markers. In contrast, the role of programmed death-2 ligand (PD-L2) as prognostic marker remains unclear. The aim of this study was to evaluate if PD-L2 expression could play a role as a prognostic marker for papillary RCC (pRCC). METHODS: The patients' sample collection was a joint collaboration of the PANZAR consortium. Patients' medical history and tumor specimens were collected from n = 240 and n = 128 patients with type 1 and 2 pRCC, respectively. Expression of PD-L2 was determined by immunohistochemistry. In total, PD-L2 staining was evaluable in 185 of 240 type 1 and 99 of 128 type 2 pRCC cases. RESULTS: PD-L2 staining was positive in 67 (36.2%) of type 1 and in 31 (31.3%) of type 2 pRCC specimens. The prevalence of PD-L2+ cells was significantly higher in high-grade type 1 tumors (p = 0.019) and in type 2 patients with metastasis (p = 0.002). Kaplan-Meier analysis disclosed significant differences in 5-year overall survival (OS) for patients with PD-L2- compared to PD-L2+ in pRCC type 1 of 88.4% compared to 73.6% (p = 0.039) and type 2 of 78.8% compared to 39.1% % (p < 0.001). However, multivariate analysis did not identify the presence of PD-L2+ cells neither in type 1 nor type 2 pRCC as an independent predictor of poor OS. DISCUSSION/CONCLUSION: PD-L2 expression did not qualify as an independent prognostic marker in pRCC. Future studies will have to determine whether anti-PD-L2-targeted treatment may play a role in pRCC and expression can potentially serve as a predictive marker for these therapeutic approaches.

Serum Hepcidin and GDF-15 levels as prognostic markers in urothelial carcinoma of the upper urinary tract and renal cell carcinoma.

BACKGROUND: Cancer is a life-threatening disease that causes every fourth death. It is often hard to determine the time point of progression. Therefore, biomarkers for cancer entities that indicate disease progression or aggressiveness and thereby guide therapeutic decisions are required. Unfortunately, reliable biomarkers are rare. In this study, the potential of serum hepcidin and serum GDF-15 as biomarkers that correlate with patient's survival in the two entities upper urinary tract urothelial carcinomas (UUTUC) and renal cell carcinoma (RCC) were analyzed. METHODS: In this retrospective study n = 38 patients suffering from UUTUC, n = 94 patients suffering from RCC and n = 21 patients without infections or cancer, all hospitalized at the University Hospital Muenster, were included. Serum samples of patients were retrospectively analyzed. Serum hepcidin and GDF-15 levels were measured and correlated to aggressiveness and progression of the disease as well as patient's outcome. RESULTS: For both entities, UUTUC and RCC, serum hepcidin levels as well as serum GDF-15 levels were increased compared to sera of controls. High serum hepcidin and GDF-15 levels were associated with metastases and cancer relapse. Also, in both entities, the overall survival was decreased in patients with increased serum hepcidin and GDF-15 levels. Hence, high serum hepcidin and GDF-15 levels correlated with patient's outcome. CONCLUSION: To conclude, the data of this study show a correlation of high serum hepcidin and GDF-15 levels with aggressiveness and progression of the disease and demonstrate potential prognostic properties of serum hepcidin and GDF-15 levels. The data support the further assessment of serum hepcidin and GDF-15 as prognostic markers in RCC and UUTUC.

Performance comparison of two androgen receptor splice variant 7 (AR-V7) detection methods.

OBJECTIVES: To compare the performance of two established androgen receptor splice variant 7 (AR-V7) mRNA detection systems, as paradoxical responses to next-generation androgen-deprivation therapy in AR-V7 mRNA-positive circulating tumour cells (CTC) of patients with castration-resistant prostate cancer (CRPC) could be related to false-positive classification using detection systems with different sensitivities. MATERIALS AND METHODS: We compared the performance of two established mRNA-based AR-V7 detection technologies using either SYBR Green or TaqMan chemistries. We assessed in vitro performance using eight genitourinary cancer cell lines and serial dilutions in three AR-V7-positive prostate cancer cell lines, as well as in 32 blood samples from patients with CRPC. RESULTS: Both assays performed identically in the cell lines and serial dilutions showed identical diagnostic thresholds. Performance comparison in 32 clinical patient samples showed perfect concordance between the assays. In particular, both assays determined AR-V7 mRNA-positive CTCs in three patients with unexpected responses to next-generation anti-androgen therapy. Thus, technical differences between the assays can be excluded as the underlying reason for the unexpected responses to next-generation anti-androgen therapy in a subset of AR-V7 patients. CONCLUSIONS: Irrespective of the method used, patients with AR-V7 mRNA-positive CRPC should not be systematically precluded from an otherwise safe treatment option.

Molecularly-driven precision medicine for advanced bladder cancer.

Urothelial carcinoma of the bladder is a heterogeneous disease with many challenges for clinicians and patients alike. However, for the most part of the last three decades, treatment and outcomes for patients with this disease have not changed a lot. With recent advances in immunooncology leading to the approval of multiple agents for the metastatic setting, the treatment landscape started to change. With the emergent data from landmark multi-institutional sequencing projects as well as molecular data from recent trials, our understanding of the underlying disease biology, response patterns as well as definition of molecular subtypes has evolved tremendously. This review aims to summarize the currently available concepts of mutational profiles and molecular subtypes as well as their implications for management of urothelial carcinoma.

177Lu-PSMA-617 radioligand therapy and outcome in patients with metastasized castration-resistant prostate cancer.

PURPOSE: Radioligand therapies targeting prostate-specific membrane antigen (PSMA) have been established for the treatment of metastasized castration-resistant prostate cancer (mCRPC) in the last decade and show promising response rates and a favourable toxicity profile. The aim of this study was to evaluate the overall survival (OS) and to identify parameters predicting outcome in mCRPC patients treated with 177Lu-PSMA-617. METHODS: Between December 2014 and January 2017, 59 consecutive patients (median age 72 years; interquartile range, (IQR, 66-76 years) with mCRPC, who had been treated with at least one next-generation antihormonal drug as well as chemotherapy, were included in this study. Biochemical response was evaluated using Prostate Cancer Working Group 3 (PCWG3) criteria. Survival was evaluated using Kaplan-Meier estimates and Cox regression proportional hazards model. Toxicity was assessed using Common Toxicity Criteria for Adverse Events (CTCAE). The study was approved by the local ethics committee. RESULTS: The 59 patients were treated with a total of 159 cycles (median 3 cycles, range 1-7) of 177Lu-PSMA-617 (median dose 6.11 GBq, IQR 5.9-6.3 GBq). The median follow-up was 24 weeks (IQR 15-36 weeks). Follow-up data for at least 12 weeks (PCWG3) were available in 76% (45) of the patients. For outcome results data from all patients treated with at least one cycle were analysed. A decline in prostate-specific antigen (PSA) of ≥50% occurred in 53%, and a decline in PSA of any amount in 91% of patients. The estimated median OS was 32 weeks. An initial alkaline phosphatase (ALP) level <220 U/L and a PSA decline after the first cycle were associated with a longer OS (56 vs. 28 weeks, p < 0.01, and 56 vs. 29 weeks, p = 0.04, respectively). The median estimated PSA progression-free survival (PPFS) was 18 weeks. Only ALP level <220 U/L was significantly associated with a longer PPFS (41 vs. 18 weeks, p < 0.01). CONCLUSIONS: A PSA decline after the first cycle of 177Lu-PSMA-617 and an initial ALP level <220 U/L were predictors of a longer OS in patients with end-stage mCRPC. An ALP level <220 U/L was additionally associated with a longer PPFS.

The Role of the Neutrophil to Lymphocyte Ratio for Survival Outcomes in Patients with Metastatic Castration-Resistant Prostate Cancer Treated with Abiraterone.

The purpose of this study was to examine the prognostic capability of baseline neutrophil-to-lymphocyte-ratio (NLR) and NLR-change under Abiraterone in metastatic castration-resistant prostate cancer patients. The impact of baseline NLR and change after eight weeks of treatment on progression-free survival (PFS) and overall survival (OS) was analyzed using Kaplan-Meier-estimates and Cox-regression. 79 men with baseline NLR <5 and 17 with NLR >5 were analyzed. In baseline analysis of PFS NLR >5 was associated with non-significantly shorter median PFS (five versus 10 months) (HR: 1.6 (95%CI:0.9-2.8); p = 0.11). After multivariate adjustment (MVA), ECOG > 0-1, baseline LDH>upper limit of normal (UNL) and presence of visceral metastases were independent prognosticators. For OS, NLR >5 was associated with shorter survival (seven versus 19 months) (HR: 2.3 (95%CI:1.3-4.0); p < 0.01). In MVA, ECOG > 0-1 and baseline LDH > UNL remained independent prognosticators. After 8 weeks of Abiraterone NLR-change to <5 prognosticated worse PFS (five versus 12 months) (HR: 4.1 (95%CI:1.1-15.8); p = 0.04). MVA showed a trend towards worse PFS for NLR-change to <5 (p = 0.11). NLR-change to <5 led to non-significant shorter median OS (seven versus 16 months) (HR: 2.3 (95%CI:0.7-7.1); p = 0.15). MVA showed non-significant difference for OS. We concluded baseline NLR <5 is associated with improved survival. In contrast, in patients with baseline NLR >5, NLR-change to <5 after eight weeks of Abiraterone was associated with worse survival and should be interpreted carefully.

Dynamic changes of alkaline phosphatase are strongly associated with PSA-decline and predict best clinical benefit earlier than PSA-changes under therapy with abiraterone acetate in bone metastatic castration resistant prostate cancer.

BACKGROUND: Significant progress in treatment of metastatic castration resistant prostate cancer (mCRPC) has been made. Biomarkers to tailor therapy are scarce. To facilitate decision-making we evaluated dynamic changes of alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and prostate specific antigen (PSA) under therapy with Abiraterone. METHODS: Men with bone mCRPC (bmCRPC) on Abiraterone 12/2009-01/2014 were analyzed. Dynamic ALP-, LDH- and PSA-changes were analyzed as predictors of best clinical benefit and overall survival (OS) with logistic-regression, Cox-regression and Kaplan-Meier-analysis. RESULTS: Thirty-nine pre- and 45 post-chemotherapy patients with a median follow up of 14.0 months were analyzed. ALP-Bouncing can be observed very early during therapy with Abiraterone. ALP-Bouncing is defined as rapidly rising ALP-levels independent of baseline ALP during the first 2-4 weeks of Abiraterone-therapy with subsequent equally marked decline to pretreatment levels or better within 8 weeks of therapy, preceding potentially delayed PSA-decline. In univariate analysis failure of PSA-reduction ≥ 50% and failure of ALP-Bouncing were the strongest predictors of progressive disease (p = 0.003 and 0.021). Rising ALP at 12 weeks, no PSA-reduction ≥ 50% and no ALP-Bouncing were strongest predictors of poor OS, (all p < 0.001). Kaplan-Meier-analysis showed worse OS for rising ALP at 12 weeks, no PSA-reduction ≥ 50% and no ALP-Bouncing (p < 0.001). In subgroup-analysis of oligosymptomatic patients all parameters remained significant predictors of poor OS, with no PSA-reduction ≥ 50% and rising ALP at 12 weeks being the strongest (p < 0.001). In multivariate analysis PSA-reduction ≥ 50% remained an independent predictor of OS for the whole cohort and for the oligosymptomatic subgroup (both p = 0.014). No patient with ALP-Bouncing had PD for best clinical benefit. Patients with rising ALP at 12 weeks had no further benefit of Abiraterone. CONCLUSIONS: Dynamic changes of ALP, LDH and PSA during Abiraterone-therapy are associated with best clinical benefit and OS in bmCRPC. ALP-Bouncing occurring earlier than PSA-changes as well as prior to equivocal imaging results and rising ALP at 12 weeks under Abiraterone may help to decide whether to discontinue Abiraterone. An external validation of these findings on a prospective cohort is planned.

Early Prediction of Therapy Response to Abiraterone Acetate Using PSA Subforms in Patients with Castration Resistant Prostate Cancer.

The purpose of this study was to evaluate the prognostic ability of early changes of total prostate specific antigen (tPSA), free PSA (fPSA), [-2]proPSA and the Prostate Health Index (PHI) following initiation of Abiraterone-therapy in men with castration resistant prostate cancer (mCRPC). In 25 patients, PSA-subforms were analyzed before and at 8-12 weeks under therapy as prognosticators of progression-free-survival (PFS) and overall survival (OS). Comparing patients with a PFS < vs. ≥12 months by using Mann-Whitney-Wilcoxon Tests, the relative-median-change of tPSA (-0.1% vs. -86.8%; p = 0.02), fPSA (12.1% vs. -55.3%; p = 0.03) and [-2]proPSA (8.1% vs. -59.3%; p = 0.05) differed significantly. For men with ≤ vs. >15 months of OS there was a non-significant trend for a difference in the relative-median-change of fPSA (17.0% vs. -46.3%; p = 0.06). In Kaplan-Meier analyses, declining fPSA and [-2]proPSA were associated with a longer median PFS (13 months, 95% confidence interval (CI): 9.6-16.4 vs. 10 months, 95% CI: 3.5-16.5; p = 0.11), respectively. Correspondingly, decreasing fPSA and [-2]proPSA values indicated an OS of 32 months (95% CI: not reached (NR)) compared to 21 months in men with rising values (95% CI: 7.7-34.3; p = 0.14), respectively. We concluded that the addition of fPSA- and [-2]proPSA-changes to tPSA-information might be further studied as potential markers of early Abiraterone response in mCRPC patients.

Positive surgical margins in nephron-sparing surgery: risk factors and therapeutic consequences.

The increased use of nephron-sparing surgery to treat localized renal cell carcinoma (RCC) lends weight to the question of the value of microscopically positive surgical margins (PSM) in cases with a tumor bed macroscopically free of residual tumor. The aim of this article is to highlight the data available on risk factors for PSM, their clinical relevance, and possible therapeutic consequences. For this purpose, publications on the incidence and relevance of PSM after partial nephrectomy from the last 15 years were examined and evaluated. We summarize that PSM are generally rare, regardless of the surgical procedure, and are seen more often in connection with an imperative indication for nephron-sparing surgery as well as a central tumor location. Most studies describe that PSM lead to a moderate increase in the rate of local relapses, but no study has thus far been able to demonstrate an association with shorter tumor-specific overall survival. Intraoperative frozen section analysis had no positive influence on the risk of definite PSM in most trials. Therefore, we conclude that PSM should definitely be avoided. However, in cases with a macroscopically tumor-free intraoperative resection bed, they should lead to close surveillance of the affected kidney and not to immediate (re)intervention.

["Pressure kills kidneys": protection of the upper urinary tract through neuro-urological management]. / "Pressure kills kidneys": Schutz des oberen Harntrakts durch neuro-urologisches Management.

Low compliance is not an independent disease but a severe manifestation resulting from various disorders of the storage and excretory function of the bladder. Reduced distensibility of the detrusor muscle can lead to a pressure load on the upper urinary tract but also to urge-dominant LUTS and urinary tract infections. Neurogenic causes, especially spinal cord injury/spina bifida but also iatrogenic damage or subvesical obstruction, can contribute to the development. This article is intended to provide information about the epidemiology and complex pathogenetic mechanisms as well as the necessary diagnostic steps. Various medical, interventional and surgical treatment strategies are evaluated on the basis of current scientific findings, thus supporting the reader in the care of this complex patient group.

Preoperative serum C- reactive protein: a prognostic marker in patients with upper urinary tract urothelial carcinoma.

BACKGROUND: To analyse the prognostic significance of preoperative C-reactive protein (CRP) serum level in patients with upper urinary tract urothelial carcinoma (UUT-UC). METHODS: We evaluated 158 UUT-UC patients who had undergone surgery in the University Hospital of Hannover (MHH). 143 (89.4%) suffered from cancer in the renal pelvis, 13 (8.1%) patients presented with tumour located in the ureter. A preoperative CRP value was available for 115 patients. The mean (median) follow-up for these patients was 28.3 (15.1) months. RESULTS: The median (mean) CRP value of all evaluable patients was 10.0 (40.7) mg/l. The CRP-level, stratified into two subgroups (CRP ≤5 vs. >5 mg/l), correlated significantly with muscle invasive tumour stage (36.4 vs. 78.9%; p<0.001), the risk of presenting nodal disease (4.5 vs. 26.8%; p=0.002) and distant metastasis (2.3 vs. 16.9%; p<0.016). The Kaplan-Meier 5-year cancer specific survival (CSS) rates were 54.2 and 26.4% for patients with preoperative CRP levels ≤ and >5 mg/l, respectively (p<0.006). Next to age and the presence of metastasis, multivariate analysis also identified CRP as a continuous variable as an independent prognosticator for CSS. CONCLUSIONS: A high preoperative serum CRP level is associated with locally advanced and metastatic disease in patients with UUT-UC. Its routine use could allow better risk stratification and risk-adjusted follow-up of UUT-UC patients.

Immunohistochemical expression of TROP­2 (TACSTD2) on the urothelial carcinoma of the urinary bladder and other types of cancer.

In metastatic or locally advanced urothelial carcinoma (UC), therapeutic options have been limited to chemotherapy and immune checkpoint inhibitors. Novel targets and drugs such as antibody drug conjugates have been developed, and enfortumab vedotin targeting nectin-4 and sacituzumab govitecan (SG) targeting trophoblast cell surface antigen 2 (TROP-2), the protein product of the TACSTD2 gene, have been approved. The expression of TROP-2 was investigated within UC and other types of carcinomas, and within the tissue of different healthy organs to understand treatment responses and toxicities. The expression of TROP-2 in the tissues of 42 patients with UC, 13 patients with other types of cancer and in the normal tissues of 11 patients was retrospectively analyzed. Immunohistochemical staining of the TROP-2 protein was performed on a BenchMark ULTRA IHC/ISH System (Roche Tissue Diagnostics; Roche Diagnostics, Ltd.) according to accredited staining protocols in a routine immunohistochemistry accredited and certified facility of the laboratory of immunohistochemistry at the Institute of Pathology (Gerhard-Domagk Institute)- University Hospital Muenster (UKM)-Muenster-Germany]. Different expression levels of TROP-2 were observed, and the highest expression rate of TROP-2 was observed in UC, independent of the tumor stage. However, normal urothelial cells had similar expression levels. Except for ductal carcinoma in situ, the expression of TROP-2 was reduced in other types of cancer and in the healthy tissues from other organs, including pancreas, gall bladder, colon and prostate. Given the treatment response based on the expression level of TROP-2, SG would be effective in almost all cases of UC. However, it would also have an effect on the normal urothelium.

Co-expression and clinical utility of AR-FL and AR splice variants AR-V3, AR-V7 and AR-V9 in prostate cancer.

BACKGROUND: Androgen receptor (AR) splice variants (AR-Vs) have been discussed as a biomarker in prostate cancer (PC). However, some reports question the predictive property of AR-Vs. From a mechanistic perspective, the connection between AR full length (AR-FL) and AR-Vs is not fully understood. Here, we aimed to investigate the dependence of AR-FL and AR-V expression levels on AR gene activity. Additionally, we intended to comprehensively analyze presence of AR-FL and three clinically relevant AR-Vs (AR-V3, AR-V7 and AR-V9) in different stages of disease, especially with respect to clinical utility in PC patients undergoing AR targeted agent (ARTA) treatment. METHODS: AR-FL and AR-V levels were analyzed in PC and non-PC cell lines upon artificial increase of AR pre-mRNA using either drug treatment or AR gene activation. Furthermore, expression of AR-FL and AR-Vs was determined in PC specimen at distinct stages of disease (primary (n = 10) and metastatic tissues (n = 20), liquid biopsy samples (n = 422), mCRPC liquid biopsy samples of n = 96 patients starting novel treatment). Finally, baseline AR-FL and AR-V status was correlated with clinical outcome in a defined cohort of n = 65 mCRPC patients undergoing ARTA treatment. RESULTS: We revealed rising levels of AR-FL accompanied with appearance and increase of AR-Vs in dependence of elevated AR pre-mRNA levels. We also noticed increase in AR-FL and AR-V levels throughout disease progression. AR-V expression was always associated with high AR-FL levels without any sample being solely AR-V positive. In patients undergoing ARTA treatment, AR-FL did show prognostic, yet not predictive validity. Additionally, we observed a substantial clinical response to ARTA treatment even in AR-V positive patients. Accordingly, multivariate analysis did not demonstrate independent significance of AR-Vs in neither predictive nor prognostic clinical utility. CONCLUSION: We demonstrate a correlation between AR-FL and AR-V expression during PC progression; with AR-V expression being a side-effect of elevated AR pre-mRNA levels. Clinically, AR-V positivity relies on high levels of AR-FL, making cells still vulnerable to ARTA treatment, as demonstrated by AR-FL and AR-V positive patients responding to ARTA treatment. Thus, AR-FL and AR-V might be considered as a prognostic, yet not predictive biomarker in mCRPC patients.

Localization of FOXP3-positive cells in renal cell carcinoma.

Regulatory T cells (Treg cells), which are lymphocyte subsets capable of suppressing immune responses, appear to play a crucial role in maintaining immune homeostasis and mediating peripheral tolerance. However, Treg cells also accumulate in cancer patients and have been implicated in tumor immune escape. The forkhead box P3 (FOXP3) transcription factor is currently regarded as the most specific and reliable marker for Treg cells in men. We investigated the frequency and characterized the distribution of FOXP3(+) cells in renal cell carcinoma (RCC) patients, focusing on the tumor microenvironment. FOXP3 expression was assessed in kidney tissue samples from 32 RCC patients by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Both conventional and quantitative RT-PCR disclosed higher FOXP3 expression levels in RCC than in adjacent normal renal tissue. Immunohistochemical staining of FOXP3-expressing cells confirmed the accumulation of FOXP3(+) cells in tumor tissue, particularly at the border between malignant and adjacent benign kidney tissues. Our findings indicate that Treg cells accumulate at the tumor invasion zone and could thus be part of an immune escape mechanism of RCC that promotes disease progression.

GSTP1 CpG island hypermethylation for DNA-based detection of occult tumor cells in surgical margins after radical prostatectomy.

OBJECTIVE: The risk of local recurrence after radical prostatectomy (RP) is considerably dependent on local tumor stage. To improve local staging, the aim of this study was to assess the feasibility of quantitative methylation-specific PCR (Q-MSP) for the identification of promoter hypermethylation of the detoxifying glutathione-S-transferase P1 gene (GSTP1) to detect occult prostate cancer (PCa) cells in the prostatic fossa after RP. METHODS: A total of 39 consecutive patients with clinically organ-confined PCa underwent RP. After gland excision, biopsies were obtained from eight defined areas of the prostatic fossa and bisected for both histopathological and molecular analyses. Results were related to clinicopathological data including tumor stage, Gleason score, prostate-specific antigen (PSA), and biochemical recurrence. RESULTS: Of 39 patients, 11 with PCa had at least one positive molecular margin status indicated by GSTP1 methylation. These included 5 of 17 (29.4%) with organ-confined and 6 of 22 (27.3%) with advanced (≥pT3 and/or pN+) PCa. GSTP1 methylation in surgical margins strongly correlated with histopathological R-status (P = 0.022) and preoperative PSA (P = 0.01) whereas no association with tumor stage (pT2 vs pT3), grade (Gleason score <7 vs ≥7), and lymph node status was found. No patient experienced biochemical relapse. CONCLUSIONS: GSTP1 hypermethylation detected by Q-MSP in prostatic fossa biopsies after RP is well suited for the detection of occult tumor cells in surgical margins. However, the limited number of patients and the short-term follow-up does not allow definite conclusions on the prognostic value of GSTP1 in surgical margins.

The "Aging Males' Symptoms" Scale (AMS): predictive value for lowered circulating androgens.

BACKGROUND: Symptoms of the "male climacteric" are often at least in part referred to an age-dependent decline of serum androgen levels. Therefore, we evaluated the relationship of climacteric symptoms as assessed by the "Aging Males' Symptoms" (AMS) Questionnaire with circulating androgen levels. METHODS: 146 ambulatory men (age, 27-85 years) were surveyed with the AMS Questionnaire and sampled for serum values of total testosterone (tT) and sexual hormone binding globulin (SHBG). Free testosterone (fT) was calculated from tT and SHBG. A total AMS score ≥37 was considered pathological; the lower limits for tT and fT were set to 8 nmol/l and 180 pmol/l, respectively. RESULTS: A significant deficit in tT and fT was shown in 25 (17.1%) and 34 (24.5%) men, respectively; the AMS Questionnaire showed pathological results for 66 (45.2%) men. In predicting a tT deficit, the AMS Questionnaire rendered a sensitivity of 76% and a specificity of 61.6%, only. However, multiple regression analysis revealed a significant correlation of lowered tT with a pathological somatovegetative and psychological AMS subscore (p = 0.042 and p = 0.01) and a correlation of lowered fT with a pathological sexual subscore (p = 0.039). CONCLUSION: In predicting hypogonadism the AMS Questionnaire in total did not render a sufficient diagnostic efficiency.

Expression of nectin-4 in papillary renal cell carcinoma.

BACKGROUND: Nectin-4 contributes to tumor proliferation, lymphangiogenesis and angiogenesis in malignant tumors and is an emerging target in tumor therapy. In renal cell carcinoma (RCC) VEGF-directed tyrosine kinase inhibitors and checkpoint inhibitors are currently treatments of choice. Enfortumab vedotin-ejf (EV) is an antibody drug conjugate that targets Nectin-4. The aim of our study was to investigate the expression of Nectin-4 in a large cohort of papillary RCC specimens. PATIENTS AND METHODS: Specimens were derived from the PANZAR consortium (Erlangen, Heidelberg, Herne, Homburg, Mainz, Mannheim, Marburg, Muenster, LMU Munich, TU Munich, and Regensburg). Clinical data and tissue samples from n = 190 and n = 107 patients with type 1 and 2 pRCC, respectively, were available. Expression of Nectin-4 was determined by immunohistochemistry (IHC). RESULTS: In total, Nectin-4 staining was moderately or strongly positive in of 92 (48.4%) of type 1 and 39 (36.4%) type 2 of pRCC cases. No associations between Nectin-4 expression and age at diagnosis, gender, grading, and TNM stage was found. 5 year overall survival rate was not statistically different in patients with Nectin-4 negative versus Nectin-4 positive tumors for the overall cohort and the pRCC type 2 subgroup, but higher in patient with Nectin-4 positive pRCC type 1 tumors compared to Nectin-4 negative tumors (81.3% vs. 67.8%, p = 0.042). CONCLUSION: Nectin-4 could not be confirmed as a prognostic marker in pRCC in general. Due to its high abundance on pRCC specimens Nectin-4 is an interesting target for therapeutical approaches e.g. with EV. Clinical trials are warranted to elucidate its role in the pRCC treatment landscape.