Prognostic and Predictive Potential of CCL5 Expression in Muscle-Invasive Bladder Cancer Patients.

Bladder cancer (BC) is the 12th most commonly diagnosed cancer worldwide. Although there are several well-established molecular and immunological classifications, prognostic and predictive markers for tumor cells and immune cells are still needed. Using a tissue microarray, we analyzed the expression of the chemokine CC motif ligand 5 (CCL5) by immunohistochemistry (IHC) in 175 muscle-invasive BC samples. The application of a single cutoff for the staining status of tumor cells (TCs; positive vs. negative) and immune cells (ICs; positive vs. negative) revealed 75 patients (42.9%) and 123 patients (70.3%) with CCL5-positive TCs or ICs, respectively. IHC results were associated with prognostic and predictive data. Multivariate Cox regression analysis revealed that positive CCL5 staining in TCs was associated with significantly shorter disease-specific survival (DSS; RR = 1.51; p = 0.047), but CCL5-negative ICs were associated with significantly shorter overall survival (OS; RR = 1.66; p = 0.005), DSS (RR = 2.02; p = 0.001) and recurrence-free survival (RFS; RR = 1.94; p = 0.002). Adjuvant chemotherapy was favorable for patients with CCL5-negative ICs for OS (RR = 0.30; p = 0.006), DSS (RR = 0.36; p = 0.022) and RFS (RR = 0.41; p = 0.046) but not for patients with CCL5-positive ICs, except in the subgroup of N1 + N2 patients, where it was associated with better OS. We suggest that CCL5 expression can be a prognostic and predictive marker for muscle-invasive bladder cancer patients.

A deep-learning workflow to predict upper tract urothelial carcinoma protein-based subtypes from H&E slides supporting the prioritization of patients for molecular testing.

Upper tract urothelial carcinoma (UTUC) is a rare and aggressive, yet understudied, urothelial carcinoma (UC). The more frequent UC of the bladder comprises several molecular subtypes, associated with different targeted therapies and overlapping with protein-based subtypes. However, if and how these findings extend to UTUC remains unclear. Artificial intelligence-based approaches could help elucidate UTUC's biology and extend access to targeted treatments to a wider patient audience. Here, UTUC protein-based subtypes were identified, and a deep-learning (DL) workflow was developed to predict them directly from routine histopathological H&E slides. Protein-based subtypes in a retrospective cohort of 163 invasive tumors were assigned by hierarchical clustering of the immunohistochemical expression of three luminal (FOXA1, GATA3, and CK20) and three basal (CD44, CK5, and CK14) markers. Cluster analysis identified distinctive luminal (N = 80) and basal (N = 42) subtypes. The luminal subtype mostly included pushing, papillary tumors, whereas the basal subtype diffusely infiltrating, non-papillary tumors. DL model building relied on a transfer-learning approach by fine-tuning a pre-trained ResNet50. Classification performance was measured via three-fold repeated cross-validation. A mean area under the receiver operating characteristic curve of 0.83 (95% CI: 0.67-0.99), 0.8 (95% CI: 0.62-0.99), and 0.81 (95% CI: 0.65-0.96) was reached in the three repetitions. High-confidence DL-based predicted subtypes showed significant associations (p < 0.001) with morphological features, i.e. tumor type, histological subtypes, and infiltration type. Furthermore, a significant association was found with programmed cell death ligand 1 (PD-L1) combined positive score (p < 0.001) and FGFR3 mutational status (p = 0.002), with high-confidence basal predictions containing a higher proportion of PD-L1 positive samples and high-confidence luminal predictions a higher proportion of FGFR3-mutated samples. Testing of the DL model on an independent cohort highlighted the importance to accommodate histological subtypes. Taken together, our DL workflow can predict protein-based UTUC subtypes, associated with the presence of targetable alterations, directly from H&E slides.

NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers.

BACKGROUND: The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization. METHODS: Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions. RESULTS: Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus. CONCLUSIONS: We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

Associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 with molecular subtypes, PD-L1 expression, and FGFR3 mutational status in two advanced urothelial bladder cancer cohorts.

AIMS: Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody-drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN-4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. METHODS AND RESULTS: The TCGA BLCA (n = 405) and the CCC-EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN-4/NECTIN-4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN-4/NECTIN-4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN-4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H-score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN-4 expression was reduced in neuroendocrine-like and/or protein-based double-negative tumours. TROP2- and NECTIN-4-negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD-L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN-4 expression. CONCLUSIONS: TACSTD2/TROP2 and NECTIN-4/NECTIN-4 are widely expressed in aUC, independent of FGFR3 alterations or PD-L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(-like) and sarcomatoid aUC.

Proposal for a Novel Histological Scoring System as a Potential Grading Approach for Muscle-invasive Urothelial Bladder Cancer Correlating with Disease Aggressiveness and Patient Outcomes.

BACKGROUND: Grading of muscle-invasive bladder cancer (MIBC) according to the current World Health Organization (WHO) criteria is controversial due to its limited prognostic value. All MIBC cases except a tiny minority are of high grade. OBJECTIVE: To develop a prognostic histological scoring system for MIBC integrating histomorphological phenotype, stromal tumor-infiltrating lymphocytes (sTILs), tumor budding, and growth and spreading patterns. DESIGN, SETTING, AND PARTICIPANTS: Tissue specimens and clinical data of 484 patients receiving cystectomy and lymphadenectomy with curative intent with or without adjuvant chemotherapy. Histomorphological phenotypes, sTILs, tumor budding, and growth and spreading patterns were evaluated and categorized into four grade groups (GGs). GGs were correlated with molecular subtypes, immune infiltration, immune checkpoint expression, extracellular matrix (ECM) remodeling, and epithelial-mesenchymal transition (EMT) activity. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: GGs were associated with overall (OS), disease-specific (DSS), and progression-free (PFS) survival in univariable and multivariable analyses. Association with biological features was analyzed with descriptive statistics. RESULTS AND LIMITATIONS: Integration of two histomorphological tumor groups, three sTILs groups, three tumor budding groups, and four growth/spread patterns yielded four novel GGs that had high significance in the univariable survival analysis (OS, DSS, and PFS). GGs were confirmed as independent prognostic predictors with the greatest effect in the multivariable Cox regression analysis. Correlation with molecular data showed a gradual transition from basal to luminal subtypes from GG1 to GG4; a gradual decrease in survival, immune infiltration, and immune checkpoint activity; and a gradual increase in ECM remodeling and EMT activity. CONCLUSIONS: We propose a novel, prognostically relevant, and biologically based scoring system for MIBC in cystectomies applicable to routine pathological sections. PATIENT SUMMARY: We developed a novel approach to assess the aggressiveness of advanced bladder cancer, which allows improved risk stratification compared with the method currently proposed by the World Health Organization.

Risk Factor Analysis for Long-Term Graft Survival Following Pediatric Kidney Transplantation: The Importance of Pretransplantation Time on Dialysis and Donor/Recipient Age Difference.

Recognizing risk factors that may negatively affect long-term graft survival following pediatric kidney transplantation is a key element in the decision-making process during organ allocation. We retrospectively reassessed all cases of pediatric kidney transplantation performed in our center in the last 20 years with the aim of determining baseline characteristics that could be identified as prognostic risk factors for long-term graft survival. Between 2001 and 2020, a total of 91 kidney transplantations in children under the age of 18 years were undertaken in our center. Early graft failure was observed in six of the 91 patients (7%). The median follow-up of the remaining 85 children was 100 months, and the overall kidney graft survival rates at 5, 10, 15 and 20 years were 85.2%, 71.4%, 46.0% and 30.6%, respectively. Small children with a body surface area of <1 m2 were significantly associated with better long-term graft survival outcomes, while adolescents aged more than twelve years showed poorer graft survival rates than younger children. Body surface area of the recipient of ≥1 m2, pretransplantation duration of the recipient on dialysis ≥18 months, hemodialysis prior to transplantation and donor/recipient age difference of ≥25 years were significantly associated with poorer long-term graft survival.

[Individualized precision medicine]. / Individualisierte Präzisionsmedizin.

Spectacular advances have been made in personalized medicine , which has rapidly revolutionized our traditional understanding of disease diagnosis and treatment. Molecular testing of tissue and liquid samples using next generation sequencing has developed into a key technology in this scenario. It can be used for both the determination of biomarkers for diagnostic, prognostic and predictive purposes, as well as the possible improvement of treatment outcome through the use of targeted therapies and the avoidance of therapies in the event of special resistance situations. In addition to drugs that have already been approved, which among other things intervene in cellular DNA repair, many new drugs have been developed and are in clinical testing. Furthermore, new possibilities in molecular imaging have dramatically expanded our understanding of tumor spread and created new approaches for targeted therapies.

Frequency of microsatellite instability (MSI) in upper tract urothelial carcinoma: comparison of the Bethesda panel and the Idylla MSI assay in a consecutively collected, multi-institutional cohort.

AIMS: Upper tract urothelial carcinoma (UTUC) is a rare malignancy with a poor prognosis which occurs sporadically or in few cases results from a genetic disorder called Lynch syndrome. Recently, examination of microsatellite instability (MSI) has gained importance as a biomarker: MSI tumours are associated with a better response to immunomodulative therapies. Limited data are known about the prevalence of MSI in UTUC. New detection methods using the fully automated Idylla MSI Assay facilitate analysis of increased patient numbers. METHODS: We investigated the frequency of MSI in a multi-institutional cohort of 243 consecutively collected UTUC samples using standard methodology (Bethesda panel), along with immunohistochemistry of mismatch repair (MMR) proteins. The same tumour cohort was retested using the Idylla MSI Assay by Biocartis. RESULTS: Using standard methodology, 230/243 tumours were detected as microsatellite stable (MSS), 4/243 tumours as MSI and 9/243 samples as invalid. In comparison, the Idylla MSI Assay identified four additional tumours as MSS, equalling 234/243 tumours; 4/243 were classified as MSI and only 5/243 cases as invalid. At the immunohistochemical level, MSI results were supported in all available cases with a loss in MMR proteins. The overall concordance between the standard and the Idylla MSI Assay was 98.35%. Time to result differed between 3 hours for Idylla MSI Assay and 2 days with the standard methodology. CONCLUSION: Our data indicate a low incidence rate of MSI tumours in patients with UTUC. Furthermore, our findings highlight that Idylla MSI Assay can be applied as an alternative method of MSI analysis for UTUC.

Experimental in vitro, ex vivo and in vivo models in prostate cancer research.

Androgen deprivation therapy has a central role in the treatment of advanced prostate cancer, often causing initial tumour remission before increasing independence from signal transduction mechanisms of the androgen receptor and then eventual disease progression. Novel treatment approaches are urgently needed, but only a fraction of promising drug candidates from the laboratory will eventually reach clinical approval, highlighting the demand for critical assessment of current preclinical models. Such models include standard, genetically modified and patient-derived cell lines, spheroid and organoid culture models, scaffold and hydrogel cultures, tissue slices, tumour xenograft models, patient-derived xenograft and circulating tumour cell eXplant models as well as transgenic and knockout mouse models. These models need to account for inter-patient and intra-patient heterogeneity, the acquisition of primary or secondary resistance, the interaction of tumour cells with their microenvironment, which make crucial contributions to tumour progression and resistance, as well as the effects of the 3D tissue network on drug penetration, bioavailability and efficacy.

Spatial Immunephenotypes of Distant Metastases but not Matched Primary Urothelial Carcinomas Predict Response to Immune Checkpoint Inhibition.

BACKGROUND: The value of programmed cell death ligand-1 (PD-L1) to predict durable responses to immune checkpoint inhibitors (ICIs) in metastatic urothelial carcinoma (mUC) is inconsistent. We hypothesize that the use of archived primary tumor material (PRIM) for PD-L1 testing in clinical trials not properly reflecting the metastatic disease status (MET) contributes to this clinical issue. OBJECTIVE: To analyze the predictive and prognostic value of PD-L1, spatial immunephenotypes, and major histocompatibility complex class I (MHC-I) determined in patient-matched PRIM/MET. DESIGN, SETTING, AND PARTICIPANTS: PD-L1, spatial immunephenotypes, and MHC-I were examined in 154 mUC patients with at least one available pretreatment MET (138 patient-matched PRIM/MET pairs). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: PD-L1, spatial immunephenotype, and MHC-I status of (patient-matched PRIM and) pretreatment MET were correlated with chemotherapy and ICI response and outcomes. RESULTS AND LIMITATIONS: Discordance rates in patient-matched PRIM/MET were 25/30%, 36%, and 49% for PD-L1 (CPS10/IC5%), immunephenotypes, and MHC-I (loss vs preserved), respectively. Correlations with chemotherapy and ICI responses were observed for immunephenotypes and MHC-I status determined in MET (not for PD-L1 alone), but not in PRIM. In case of ICIs, patients with cytotoxic tumor immune microenvironment (TIME) showed durable responses with disease control rates of 90% and a hazard ratio for disease progression/death of 0.05 (95% confidence interval: 0.01-0.65) versus patients with immunedepleted MET (disease control rate 29%). MET MHC-I status added an incremental value to predict durable ICI responses. Limitations include the partly retrospective design and the lack of MET multisampling on individual patient level. CONCLUSIONS: The TIME is subject to substantial dynamics during metastatic evolution. MET immunephenotypes and MHC-I statuses show promising potential to predict chemotherapy and durable ICI responses, while the PRIM TIME does not. Thus, future clinical trials should rather rely on pretreatment MET biopsies reflecting the current immunological disease state than on PRIM. PATIENT SUMMARY: Prediction of chemotherapy and responses to immune checkpoint inhibitors might be possible using representative pretreatment metastatic biopsies.

Clean Intermittent Catheterization in Children under 12 Years Does Not Have a Negative Impact on Long-Term Graft Survival following Pediatric Kidney Transplantation.

BACKGROUND: Congenital anomalies of the kidneys and urinary tract (CAKUTs) are one of the most prevalent primary causes of end-stage renal disease (ESRD) in young children, and approximately one-third of these children present with lower urinary tract dysfunction (LUTD). Many children with LUTD require therapy with clean intermittent catheterization (CIC). CIC commonly leads to bacteriuria, and considerations have arisen regarding whether CIC in immunosuppressed children is safe or whether repeated febrile urinary tract infections (UTIs) may lead to the deterioration of kidney graft function. MATERIAL AND METHODS: We retrospectively reviewed all cases of primary kidney transplantation performed in our center between 2001 and 2020 in recipients aged less than twelve years. The number of episodes of febrile UTIs as well as the long-term kidney graft survival of children undergoing CIC were compared to those of children with urological causes of ESRD not undergoing CIC, as well as to those of children with nonurological causes of ESRD. RESULTS: Following successful kidney transplantation in 41 children, CIC was needed in 8 of these patients. These 8 children undergoing CIC had significantly more episodes of febrile UTIs than did the 18 children with a nonurological cause of ESRD (p = 0.04) but not the 15 children with a urological cause of ESRD who did not need to undergo CIC (p = 0.19). Despite being associated with a higher rate of febrile UTIs, CIC was not identified as a risk factor for long-term kidney graft survival, and long-term graft survival did not significantly differ between the three groups at a median follow-up of 124 months. CONCLUSIONS: Our study demonstrates that, under regular medical care, CIC following pediatric transplantation is safe and is not associated with a higher rate of long-term graft loss.

Cell-Free DNA Sequencing Reveals Gene Variants in DNA Damage Repair Genes Associated with Prognosis of Prostate Cancer Patients.

In the present study, we further analyzed the data obtained in our previous study, where we investigated the cell-free DNA (cfDNA) of 34 progressive prostate cancer patients via targeted sequencing. Here, we studied the occurrence and prognostic impact of sequence variants according to their clinical pathological significance (CPS) or their functional impact (FI) in 23 DNA damage repair (DDR) genes with a focus on the ATM serine/threonine kinase gene (ATM). All patients had at least one DDR gene with a CPS or FI variant. Kaplan-Meier analysis indicated that the group with a higher number of CPS variants in DDR genes had a shorter time to treatment change (TTC) compared to the group with a lower number of CPS variants (p = 0.038). Analysis of each DDR gene revealed that CPS variants in the ATM gene and FI variants in the nibrin (NBN) gene showed a shorter TTC (p = 0.034 and p = 0.042). In addition, patients with CPS variants in the ATM gene had shorter overall survival (OS; p = 0.022) and disease-specific survival (DSS; p = 0.010) than patients without these variants. Interestingly, patients with CPS variants in seven DDR genes possessed a better OS (p = 0.008) and DSS (p = 0.009), and patients with FI variants in four DDR genes showed a better OS (p = 0.007) and DSS (p = 0.008). Together, these findings demonstrated that the analysis of cfDNA for gene variants in DDR genes provides prognostic information that may be helpful for future temporal and targeted treatment decisions for advanced PCa patients.

Reduced Recurrence Rates Are Associated with Photodynamic Diagnostics Compared to White Light after Extended Transurethral Resection of Bladder Tumors.

One pillar in treating non-muscle-invasive bladder cancer (NMIBC) is the complete and high-quality transurethral resection of the primary tumor (TURBT). However, even after a high-quality primary resection, the residual tumor risk is considerable, thus requiring a re-TURBT. Resections performed with the aid of a photodynamic diagnostics report improved recurrence-free survival rates and increased detection rates of carcinoma in situ (CIS). This monocentric retrospective study reports on patients treated with an extended TURBT procedure using conventional white-light cystoscopy or photodynamic diagnostics (PDD). Only patients undergoing a TURBT resection for their primary tumor were included in the statistical analysis. Recurrence-free survival and overall survival were the clinical endpoints. Mann−Whitney U tests and chi-squared tests were used for descriptive intergroup comparisons. The associations with overall survival and recurrence-free survival were determined by univariate and multivariate analyses. The test results were considered significant when p was < 0.05. In comparison to conventional white-light cystoscopy, PDD increased the detection rates of CIS (p = 0.004) and tumor multifocality (p = 0.005) and led to reduced residual tumor incidence at the primary resection site (p < 0.001). Likewise, tumor recurrence rates were reduced in the PDD cohort (p < 0.001). Patient age and the presence of residual tumor at the primary resection site were identified as independent predictors of overall survival. For recurrence-free survival, only the PDD resection method was an independent predictor (HR = 0.43; p < 0.001). In summary, we demonstrated that the utilization of PDD techniques was associated with improved detection rates of CIS and multifocal tumors and with reduced recurrence rates. The extended resection protocol allowed us to determine that PDD resections lead to a reduced residual tumor rate at the initial resection site. This residual tumor state at the resection site, determined by extended TURBT, became an independent predictor of long-term survival. On the other hand, the PDD technique was confirmed as the only independent predictor of recurrence-free survival.

Combined miR-486 and GP88 (Progranulin) Serum Levels Are Suggested as Supportive Biomarkers for Therapy Decision in Elderly Prostate Cancer Patients.

Our study aimed to assess the applicability of miR-486 in combination with soluble GP88 protein as a diagnostic and/or predictive biomarker for prostate cancer (PCa) patients. miR-486 and GP88 levels in serum samples from 136 patients undergoing MRI-guided biopsy of the prostate were assessed by qRT−PCR and ELISA, respectively. Of these, 86 patients received a histologically confirmed diagnosis of PCa. Neither marker showed an association with the diagnosis of cancer. PCa patients were separated based on (i) treatment into patients with active surveillance or patients with any type of curative treatment and (ii) age into elderly (>68 years) patients and younger patients (≤68 years). In elderly patients (N = 41) with the intention of curative treatment at optimized cut-off values, significantly higher GP88 levels (p = 0.018) and lower miR-486 levels (p = 0.014) were observed. The total PSA level and ISUP biopsy grade were used in a baseline model for predicting definitive therapy. The baseline model exhibited an area under the curve (AUC) of 0.783 (p = 0.005). The addition of the serum biomarkers miR-486 and GP88 to the baseline model yielded an improved model with an AUC of 0.808 (p = 0.002). Altogether, combined miR-486 and GP88 serum levels are associated with and are therefore suggested as supportive biomarkers for therapy decisions, particularly in elderly PCa patients.

The Human Leukocyte Antigen G as an Immune Escape Mechanism and Novel Therapeutic Target in Urological Tumors.

The non-classical human leukocyte antigen G (HLA-G) is a potent regulatory protein involved in the induction of immunological tolerance. This is based on the binding of membrane-bound as well as soluble HLA-G to inhibitory receptors expressed on various immune effector cells, in particular NK cells and T cells, leading to their attenuated functions. Despite its restricted expression on immune-privileged tissues under physiological conditions, HLA-G expression has been frequently detected in solid and hematopoietic malignancies including urological cancers, such as renal cell and urothelial bladder carcinoma and has been associated with progression of urological cancers and poor outcome of patients: HLA-G expression protects tumor cells from anti-tumor immunity upon interaction with its inhibitory receptors by modulating both the phenotype and function of immune cells leading to immune evasion. This review will discuss the expression, regulation, functional and clinical relevance of HLA-G expression in urological tumors as well as its use as a putative biomarker and/or potential therapeutic target for the treatment of renal cell carcinoma as well as urothelial bladder cancer.

Utility of stromal tumor infiltrating lymphocyte scoring (sTILs) for risk stratification of patients with muscle-invasive urothelial bladder cancer after radical cystectomy.

BACKGROUND: Multi-omics analyses of muscle-invasive bladder cancer (MIBC) demonstrated that specific patterns of tumor infiltrating lymphocytes (TILs) associates with improved outcomes in patients treated with radical cystectomy. However, methodologies for simple and robust quantification of TILs, especially for daily practice purposes, are lacking. Thus, we investigated the feasibility of stromal TIL scoring on hematoxylin/eosin stained (HE) slides in MIBC. MATERIALS AND METHODS: sTILs were scored on HE whole slides of 241 MIBC patients treated with radical cystectomy and adjuvant chemotherapy. Median infiltration of 10% was used as objective cut-off. Additionally, immunohistochemistry was performed on spatially organized tissue microarrays to quantify key immune cell populations objectively for correlational analyses with sTIL scoring results (CD3+/Pan-T-cells, CD8+/cytotoxic T-Cells, CD56+/NK-cells, CD68+/macrophages). sTILs amounts were correlated with clinicopathological features, recurrence-free (RFS), disease-specific (DSS), and overall survival (OS). RESULTS: sTIL amounts correlated moderately to strongly with quantitatively estimated amounts of pan-T-cells (r = 0.73, P <0.0001), cytotoxic T-cells (r = 0.73, P <0.0001), NK-cells (r = 0.68, P <0.0001), macrophages (r = 0.55, P <0.0001) and with pan-cytotoxic immune infiltration (r = 0.78, P <0.0001), thus reflecting overall infiltration with key immune cell populations. sTIL infiltration ≥10% was associated with significantly higher 5-year OS (45.5% vs. 19.8%), DSS (56.6% vs. 25.6%) and RFS (56.2% vs. 18.9%; P <0.0001 for all three comparisons) rates, and lower pT-stage (P = 0.015), lower pN-stage (P = 0.028), lower rates of lymphovascular invasion (P = 0.0003) and blood vessel invasion (P = 0.01) when compared to sTIL infiltration of <10%. Multivariable regressions models confirmed sTILs as strongest independent predictor for improved outcomes following radical cystectomy. CONCLUSIONS: HE based sTIL scoring is a reliable tool to assess MIBC inflammation status and to stratify the survival of MIBC patients undergoing radical cystectomy. sTIL amount is an independent predictor for improved survival, and might be an useful, routinely applicable tool to identify patients benefiting from perioperative platinum-based chemotherapy and checkpoint inhibitor therapy. However, external validation of our data is required.

High expression of ERBB2 is an independent risk factor for reduced recurrence-free survival in patients with stage T1 non-muscle-invasive bladder cancer.

INTRODUCTION: Molecular markers associated with breast cancer are assumed to be associated with outcome in non-muscle-invasive bladder cancer (NMIBC). MATERIALS AND METHODS: We retrospectively investigated the association of the mRNA expression of estrogen receptor 1 (ESR1) and 2 (ESR2), progesterone receptor (PGR), MKI67, and HER2 (ERBB2) with recurrence-free (RFS), cancer-specific (CSS), and overall survival (OS) in 80 patients with stage T1 NMIBC. RESULTS: High expression of ESR2 (P = 0.003), ERBB2 (P < 0.001), and MKI67 (P = 0.029) was associated with shorter RFS. Only high ERBB2 was an independent prognostic factor for reduced RFS (HR = 2.98; P = 0.009). When sub stratifying the cohort, high ESR2 was associated with reduced RFS (P < 0.001), CSS (P = 0.037) and OS (P = 0.006) in patients without instillation therapy. High ESR2 was associated with reduced CSS (P = 0.018) and OS (P = 0.029) in females and with shorter RFS in both sexes (males: P = 0.035; females: P = 0.010). Patients with high ERBB2 showed reduced CSS (P = 0.011) and OS (P = 0.042) in females and reduced CSS (P = 0.012) in those without instillation, while RFS was significantly reduced irrespective of sex or instillation. CONCLUSION: High mRNA expression of ERBB2 is an independent predictor of reduced RFS in patients with stage T1 NMIBC. High ERBB2 and ESR2 are associated with reduced outcomes, especially in females and patients without instillation therapy.

Cell-Free DNA Variant Sequencing Using Plasma and AR-V7 Testing of Circulating Tumor Cells in Prostate Cancer Patients.

Prostate cancer (PCa) is the second most common malignant cancer and is a major cause of morbidity and mortality among men worldwide. There is still an urgent need for biomarkers applicable for diagnosis, prognosis, therapy prediction, or therapy monitoring in PCa. Liquid biopsies, including cell-free DNA (cfDNA) and circulating tumor cells (CTCs), are a valuable source for studying such biomarkers and are minimally invasive. In our study, we investigated the cfDNA of 34 progressive PCa patients, via targeted sequencing, for sequence variants and for the occurrence of CTCs, with a focus on androgen receptor splice variant 7 (AR-V7)-positive CTCs. The cfDNA content was associated with overall survival (OS; p = 0.014), disease-specific survival (DSS; p = 0.004), and time to treatment change (TTC; p = 0.001). Moreover, when considering all sequence variants grouped by their functional impact and allele frequency, a significant association with TTC (p = 0.017) was observed. When investigating only pathogenic or likely pathogenic gene variants, variants of the BRCA1 gene (p = 0.029) and the AR ligand-binding domain (p = 0.050) were associated with a shorter TTC. Likewise, the presence of CTCs was associated with a shorter TTC (p = 0.031). The presence of AR-V7-positive CTCs was associated with TTC (p < 0.001) in Kaplan-Meier analysis. Interestingly, all patients with AR-V7-positive CTCs also carried TP53 point mutations. Altogether, analysis of cfDNA and CTCs can provide complementary information that may support temporal and targeted treatment decisions and may elucidate the optimal choice within the variety of therapy options for advanced PCa patients.

Analysis of tripartite motif (TRIM) family gene expression in prostate cancer bone metastases.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFß-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFß signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.