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Prostate cancer remains a leading cause of cancer-related mortality in men, with advanced stages posing significant treatment challenges due to high morbidity and mortality. Among genetic alterations, TP53 mutations are among the most prevalent in cancers and are strongly associated with poor clinical outcomes and therapeutic resistance. This review investigates the role of TP53 mutations in prostate cancer progression, prognosis, and therapeutic development. A comprehensive analysis of preclinical and clinical studies was conducted to elucidate the molecular mechanisms, clinical implications, and potential therapeutic approaches associated with TP53 alterations in prostate cancer. TP53 mutations are highly prevalent in advanced stages, contributing to genomic instability, aggressive tumor phenotypes, and resistance to standard treatments. Emerging evidence supports the utility of liquid biopsy techniques, such as circulating tumor DNA analysis, for detecting TP53 mutations, providing prognostic value and facilitating early intervention strategies. Novel therapeutic approaches targeting TP53 have shown promise in preclinical settings, but their clinical efficacy requires further validation. Overall, TP53 mutations represent a critical biomarker for disease progression and therapeutic response in prostate cancer. Advances in detection methods and targeted therapies hold significant potential to improve outcomes for patients with TP53-mutated prostate cancer. Further research is essential to integrate TP53-based strategies into routine clinical practice.

Background: Immune checkpoint inhibitors (ICIs) are an important therapeutic pillar in metastatic urothelial carcinoma (mUC). The occurrence of immune-related adverse events (irAEs) appears to be associated with improved outcomes in observational studies. However, these associations are likely affected by immortal time bias and do not represent causal effects. The aim of this study was to assess the effect of irAEs on outcomes while correcting for immortal time bias, using target trial emulation (TTE)., Methods: TTE was contrasted to adjusted naïve and time-updated Cox models. We performed a multi-institutional retrospective study involving mUC patients under ICI. The primary objective was to assess the impact of irAEs on progression-free survival (PFS) and overall survival (OS). Secondary endpoints included the influence of irAEs on objective response rates (ORRs) to ICI and the influence of systemic corticosteroids on outcomes., Results: Among 335 patients (median age: 69 yrs), 69.6% received ICI in the second line or further lines. During a median follow-up of 21.1 months, 122 (36.4%) patients developed irAEs of any grade (grade ≥ 3: 14.9%). Hazard ratios (HRs) for PFS ranged from 0.37 for naïve adjusted Cox model to 0.88 (95% confidence interval (CI), 0.59-1.30) with time-updated covariates, and from 0.41 to 1.10 (95% CI, 0.69-1.75) for OS. TTE accounting for immortal time bias yielded a HR of 1.02 (95% CI, 0.72-1.44) for PFS, and 0.90 (95% CI, 0.62-1.30) for OS. In contrast to the naïve Cox model (HR = 2.26, 95% CI 1.26-4.05), the presence of irAEs was no longer a predictive factor for improved ORR in time-updated Cox models (HR = 1.27, 95% CI 0.68-2.36) and TTE (HR = 1.43, 95% CI 0.89-2.29). In patients with irAEs, systemic corticosteroids did not negatively impact survival., Conclusion: Using TTE, we were able to show that the occurrence of irAEs is no longer associated with better survival or improved response rates to ICI in mUC patients, in contrast to the naïve analysis. These findings demonstrate that TTE is a suitable formal framework to avoid immortal time bias in studies with time-dependent non-interventional exposures., Competing Interests: Declarations. Conflict of interests: The authors have no relevant financial or nonfinancial interests to disclose. Ethical approval: The study was approved by the local ethical committee of the Medical University of Innsbruck (study number AN2014-0121; 336/4.3). Consent to participate: Informed consent was obtained from all individual participants included in the study., (© 2024. The Author(s).)

Background: Electronic patient-reported outcomes (ePROs) have been shown to enhance healthcare quality by improving patient symptom management or quality of life (QoL). However, ePROs data for urothelial cancer (UC) patients receiving systemic therapies are scarce, and the application of ePROs in this patient cohort may need specific setups. This study tested the feasibility of ePROs for UC patients receiving systemic therapies in the outpatient clinic of a tertiary care center., Patients and Methods: From January 2022 to April 2023, 30 UC patients receiving systemic cancer therapies received ePROs based on the Common Terminology Criteria for Adverse Events (CTCAE) and European Organization for Research and Treatment of Cancer Core Quality of Life questionnaires (EORTC QLQ-30) to report their symptoms and QoL during systemic therapy, in total, 125 questions for every therapy cycle. The proportion of patients adherent to the ePROs was assessed to evaluate feasibility, with a preset threshold of 50%. At least half of all treatment cycles with a minimum of two consecutive ePROs (corresponding to two successive therapy cycles) had to be completed to be counted as adherent, and a maximum of six successive therapy cycles was followed by ePROs. Descriptive statistics were calculated for clinical and demographic patient characteristics. T-test and chi-square-test analyses were performed to study the association between ePROs adherence and clinical or demographic factors. The digital process was closely monitored for procedural impediments that could occur., Results: 21 (70%) of the included 30 patients adhered to the provided ePROs, significantly higher than the predetermined threshold of 50%. Adherence remained above 70% until the end of the observation period. A significant negative effect of immigration background on ePROs compliance was observed (p = 0.006). No other variables were significantly associated with ePROs compliance., Conclusions: In this study, ePROs were a feasible method to assess symptoms and QoL during the systemic cancer therapy of UC patients at our center. The compliance of patients with immigration backgrounds was the most significant barrier to using ePROs in this setting. However, the study is limited by the exclusion of patients without email access and the lack of assessment of physician compliance with the ePROs data, which may affect the generalizability and implementation of the findings., Competing Interests: Declarations. Ethics approval and consent to participate: The local ethics committee approved the study (EK-Nr: 1972/2021, Ethics Committee of the Medical University of Vienna). Informed consent was obtained from all individual participants included in the study. Consent for publication: Not applicable. Competing interests: SFS reports honoraria for lectures/consulting boards for Astellas, Astra Zeneca, Bayer, BMS, Cepheid, Ferring, Ipsen, Janssen, Lilly, MSD, Olympus, Pfizer, Pierre Fabre, Richard Wolf Roche, Sanochemia, Sanofi, Takeda, Urogen; patents: method to determine prognosis after therapy for prostate cancer granted 2002-09-06, methods to determine prognosis after therapy for bladder cancer granted 2003-06-19, prognostic methods for patients with prostatic disease granted 2004-08-05 and soluble Fas urinary marker for the detection of bladder transitional cell carcinoma granted 2010-07-20. The other authors have no competing interests to declare that are relevant to the content of this article. The other authors have no competing interests to declare that are relevant to the content of this article., (© 2024. The Author(s).)

Background and Objective: The role of genetic variants in response to systemic therapy in muscle-invasive bladder cancer (MIBC) is still elusive. We assessed variations in genes involved in DNA damage repair (DDR) before and after cisplatin-based neoadjuvant chemotherapy (NAC) and correlation of alteration patterns with DNA damage and response to therapy., Methods: Matched tissue from 46 patients with MIBC was investigated via Ion Torrent-based next-generation sequencing using a self-designed panel of 30 DDR genes. Phosphorylation of γ-histone 2A.X (H2AX) was analyzed via immunohistochemistry to evaluate DNA damage. Genetic variants were analyzed along with clinical data and quantitative phospho-H2AX data using the Kaplan-Meier method, Cox regression analysis, and factor analysis of mixed data., Key Findings and Limitations: Twenty-five patients (54%) had a response (

Introduction: Socioeconomic disparities have been linked to delayed prostate cancer diagnosis and poorer outcomes in various countries. This study aims to evaluate the socioeconomic disparities in prostate cancer diagnostics in Vienna, Austria, by examining initial prostate-specific antigen values and age at diagnosis across different districts and nationalities., Methods: This retrospective study included 1356 prostate cancer patients treated at the Medical University of Vienna between 2012 and 2022. Influence of residential districts and nationalities of the patients on the initial prostate-specific antigen (iPSA) value and on the age at diagnosis were analyzed. Patient data, including iPSA values, residential districts, and nationalities, were retrieved from the hospital's internal documentation system. The information on average income of residential districts was obtained from the City of Vienna's municipality data. Nationalities were grouped into EU and non-EU categories. Statistical analyses, including linear regression and t‑tests, were performed to examine the relationship between iPSA values, age at diagnosis, and socioeconomic variables. Linear regression was used to analyze the relationship between district income and both iPSA values and age at diagnosis., Results: The study found no significant differences in iPSA values and age at diagnosis between patients from higher income and lower income districts. Additionally, there were no significant differences among individual districts or between EU and non-EU nationals., Conclusion: The findings suggest that the Austrian healthcare system provides equitable access to prostate cancer diagnostics across different socioeconomic groups., (© 2024. The Author(s).)

Background and Objective: There are limited data on real-world outcomes for patients with advanced or metastatic urothelial cancer (mUC) since immune checkpoint inhibitors (ICIs) became available. Our objective was to analyze outcomes for patients with mUC since ICIs became available., Methods: We performed a retrospective analysis of 131 patients with mUC attending the outpatient clinic of a single tertiary care center who received systemic therapy between June 2017 and July 2021 with follow-up up to December 2022. Summary and descriptive statistics were calculated for categorical and continuous variables. The Kaplan-Meier method was applied to calculate survival, and a Cox proportional-hazards model was used to explore associations between clinical variables and outcomes., Key Findings and Limitations: The median patient age was 68 yr (range 35-90). The first systemic therapy administered was platinum-based in 79% of cases and ICI-based in 21%. Some 61% of the cohort received a second systemic treatment, with 75% of these an ICI. Median overall survival for the entire cohort was 24 mo (interquartile range 9-35). Patients on ICI therapy for ≥6 mo had median overall survival of 59 mo (95% confidence interval 39 mo-not reached). Metastatic sites on initiation of ICI therapy and C-reactive protein kinetics were prognostic in patients receiving ICIs. Limitations include the retrospective design and inherent selection bias., Conclusions and Clinical Implications: More than 60% of patients with mUC received second-line treatment, and 75% of these received an ICI. Patients staying on immunotherapy for more than 6 mo have substantially better outcomes in comparison to patients with less time on immunotherapy and historical cohorts., Patient Summary: We looked at the lines of therapy and outcomes for patients with advanced or metastatic cancer of the urinary tract, starting from when immunotherapy drugs called immune checkpoint inhibitors (ICIs) became available. We found that 60% of patients have received second-line therapy, which is a double the rate in comparison to historical groups of patients. Patients with long-term ICI therapy (>6 months) had significantly better outcomes, with a median survival of more than 3 years., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation. However, metabolic stabilization by phosphorothioate (PS), the only backbone chemistry used clinically, may be insufficient for targeting extrahepatic tissues. To improve oligonucleotide stabilization, we report the discovery, synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5'-C and 5'-OH of a nucleoside. exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone, provides stabilization against 3' and 5' exonucleases and is tolerated at multiple oligonucleotide positions. A combined exNA-PS backbone enhances resistance to 3' exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone, improving tissue exposure, tissue accumulation and efficacy in mice, both systemically and in the brain. The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues, both with siRNA and with other oligonucleotides such as CRISPR guide RNA, antisense oligonucleotides, mRNA and tRNA., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo . With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo . Administered intraventricularly, di-siRNA SOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders., Competing Interests: Competing interests: AK is a co-founder, on the scientific advisory board, and holds equities of Atalanta Therapeutics; AK is a founder of Comanche Pharmaceuticals, and on the scientific advisory board of Aldena Therapeutics, AlltRNA, Prime Medicine, and EVOX Therapeutics; Select authors hold patents or on patent applications relating to the divalent siRNA, SOD1-targeting oligonucleotides, and the methods described in this report. NH received compensation for other services from Myrobalan Inc. and General Dynamics during the conduct of this study, unrelated to the present work.

Purpose: Determining the frequency and distribution of pathogenic germline variants (PGVs) in Austrian prostate cancer (PCa) patients and to assess the accuracy of different clinical risk scores to correctly predict PGVs., Methods: This cross-sectional study included 313 men with advanced PCa. A comprehensive personal and family history was obtained based on predefined questionnaires. Germline DNA sequencing was performed between 2019 and 2021 irrespective of family history, metastatic or castration status or age at diagnosis. Clinical risk scores for hereditary cancer syndromes were evaluated and a PCa-specific score was developed to assess the presence of PGVs., Results: PGV presence was associated with metastasis (p = 0.047) and castration resistance (p = 0.011), but not with personal cancer history or with relatives with any type of cancer. Clinical risk scores (Manchester score, PREMM5 score, Amsterdam II criteria or Johns Hopkins criteria) showed low sensitivities (3.3-20%) for assessing the probability of PGV presence. A score specifically designed for PCa patients stratifying patients into low- or high-risk regarding PGV probability, correctly classified all PGV carriers as high-risk, whereas a third of PCa patients without PGVs was classified as low risk of the presence of PGVs., Conclusion: Application of common clinical risk scores based on family history are not suitable to identify PCa patients with high PGV probabilities. A PCa-specific score stratified PCa patients into low- or high-risk of PGV presence with sufficient accuracy, and germline DNA sequencing may be omitted in patients with a low score. Further studies are needed to evaluate the score., (© 2023. The Author(s).)

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

Survival outcomes after radical cystectomy (RC) for bladder cancer (BCa) have not improved in recent decades; nevertheless, RC remains the standard treatment for patients with localized muscle-invasive BCa. Identification of the patients most likely to benefit from RC only versus a combination with systemic therapy versus systemic therapy first/only and bladder-sparing is needed. This systematic review and meta-analysis pools the data from published studies on blood-based biomarkers to help prognosticate disease recurrence after RC. A literature search on PubMed and Scopus was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Articles published before November 2022 were screened for eligibility. A meta-analysis was performed on studies investigating the association of the neutrophil-to-lymphocyte ratio (NLR), the only biomarker with sufficient data, with recurrence-free survival. The systematic review identified 33 studies, and 7 articles were included in the meta-analysis. Our results demonstrated a statistically significant correlation between elevated NLR and an increased risk of disease recurrence (HR 1.26; 95% CI 1.09, 1.45; p = 0.002) after RC. The systematic review identified various other inflammatory biomarkers, such as interleukin-6 or the albumin-to-globulin ratio, which have been reported to have a prognostic impact on recurrence after RC. Besides that, the nutritional status, factors of angiogenesis and circulating tumor cells, and DNA seem to be promising tools for the prognostication of recurrence after RC. Due to the high heterogeneity between the studies and the different cut-off values of biomarkers, prospective and validation trials with larger sample sizes and standardized cut-off values should be conducted to strengthen the approach in using biomarkers as a tool for risk stratification in clinical decision-making for patients with localized muscle-invasive BCa.

We summarise the available data for and assess the prognostic value of circulating tumour DNA (ctDNA) in patients treated with systemic therapy for urothelial carcinoma (UC). Studies were deemed eligible if they reported on oncologic outcomes for patients with UC treated with systemic therapy according to the baseline ctDNA profile (before starting systemic therapy) and/or changes over the course of therapy. Five studies met the eligibility criteria. We found a strong association between high baseline ctDNA levels and worse disease-free survival (DFS; hazard ratio [HR] 3.53, 95% confidence interval [CI] 2.58-4.84) and overall survival (OS; HR 2.99, 95% CI 2.17-4.13). Patients with a decline in ctDNA level after immunotherapy had better DFS (HR 0.25, 95% CI 0.13-0.49) and OS (HR 0.10, 95% CI 0.03-0.42) in comparison to patients without a ctDNA decline. Conversely, an increase in ctDNA levels after immunotherapy was associated with worse survival outcomes. Patients with UC who exhibited a decrease in ctDNA levels during systemic therapy had better survival outcomes compared to those with stable or increasing ctDNA levels. PATIENT SUMMARY: Measurement of tumour DNA in blood may help in identifying patients with cancer of the urinary tract who are unlikely to respond to chemotherapy or immunotherapy. This could serve as a biomarker for monitoring cancer treatment., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Small interfering RNAs are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS, enabling development of CNS-targeted therapeutics. Many genetically-defined neurodegenerative disorders are dominant, favoring selective silencing of the mutant allele. In some cases, successfully targeting the mutant allele requires targeting single nucleotide polymorphism (SNP) heterozygosities. Here, we use Huntington's disease (HD) as a model. The optimized compound exhibits selective silencing of mutant huntingtin protein in patient-derived cells and throughout the HD mouse brain, demonstrating SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. Targeting a disease-causing allele using RNAi-based therapies could be helpful in a range of dominant CNS disorders where maintaining wild-type expression is essential., (© 2022. The Author(s).)

Background: The heterogeneity of bladder cancers (BCs) is a major challenge for the development of novel therapies. However, given the high rates of recurrence and/or treatment failure, the identification of effective therapeutic strategies is an urgent clinical need., Objective: We aimed to establish a model system for drug identification/repurposing in order to identify novel therapies for the treatment of BC., Design, Setting, and Participants: A collection of commercially available BC cell lines (n = 32) was comprehensively characterized. A panel of 23 cell lines, representing a broad spectrum of BC, was selected to perform a high-throughput drug screen., Outcome Measurements and Statistical Analysis: Positive hits were defined as compounds giving >50% inhibition in at least one BC cell line., Results and Limitations: Amongst >1700 tested chemical compounds, a total of 471 substances exhibited antineoplastic effects. Clofarabine, an antimetabolite drug used as third-line treatment for childhood acute lymphoblastic leukaemia, was amongst the limited number of drugs with inhibitory effects on cell lines of all intrinsic subtypes. We, thus, reassessed the substance and confirmed its inhibitory effects on commercially available cell lines and patient-derived cell cultures representing various disease stages, intrinsic subtypes, and histologic variants. To verify these effects in vivo, a patient-derived cell xenograft model for urothelial carcinoma (UC) was used. Well-tolerated doses of clofarabine induced complete remission in all treated animals (n = 12) suffering from both early- and late-stage disease. We further took advantage of another patient-derived cell xenograft model originating from the rare disease entity sarcomatoid carcinoma (SaC). Similarly to UC xenograft mice, clofarabine induced subcomplete to complete tumour remissions in all treated animals (n = 8)., Conclusions: The potent effects of clofarabine in vitro and in vivo suggest that our findings may be of high clinical relevance. Clinical trials are needed to assess the value of clofarabine in improving BC patient care., Patient Summary: We used commercially available cell lines for the identification of novel drugs for the treatment of bladder cancer. We confirmed the effects of one of these drugs, clofarabine, in patient-derived cell lines and two different mouse models, thereby demonstrating a potential clinical relevance of this substance in bladder cancer treatment., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose of Review: To highlight the latest changes in prostate cancer (PCa), urothelial carcinoma, upper tract urothelial carcinoma (UTUC) and renal cell carcinoma (RCC) diagnosis and the impact of genetics in this field., Recent Findings: Breast cancer1/2 mutations start to play a major role in PCa treatment with regard to personalized medicine. In urothelial carcinoma an overlap between histological pathological and molecular findings exists, fibroblast growth factor receptor alteration are starting to play a major role, programmed death-ligand 1 although problematic is still important in the treatment setting. UTUC is rare, but genetically different from urothelial carcinoma. In the development of RCC, different genetic pathways such as Von Hippel-Lindau, but also tuberous sclerosis 1/2 and others play a major role in tumor development., Summary: Over the last years, genetics has become increasingly important role in the diagnosis and the treatment of patients with urological malignancies. The upcoming 5th edition (1) of the WHO still considers conventional surgical pathology as the diagnostic gold standard, but molecular pathology is gaining importance not only for diagnosis, but also in personalized treatment, of prostate, kidney cancer and urothelial carcinomas. Therefore, a close collaboration between surgical urology, pathology and oncology departments is mandatory. In this review, we will discuss the latest evolutions in PCa, urothelial carcinoma, upper urinary tract carcinomas and RCC s in the field of genetics in urology., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Histone deacetylases (HDACs) target acetylated lysine residues in histone and non-histone proteins. HDACs are implicated in the regulation of genomic stability, cell cycle, cell death and differentiation and thus critically involved in tumorigenesis. Further, HDACs regulate T-cell development and HDAC inhibitors (HDACis) have been approved for clinical use in some T-cell malignancies. Still, the exact targets and mechanisms of HDAC inhibition in cancer are understudied. We isolated tumor cell lines from a transgenic mouse model of anaplastic large cell lymphoma (ALCL), a rare T-cell lymphoma, and abrogated HDAC activity by treatment with the HDACis Vorinostat and Entinostat or Cre-mediated deletion of Hdac1 . Changes in overall protein expression as well as histone and protein acetylation were measured following Hdac1 deletion or pharmacological inhibition using label-free liquid chromatography mass spectrometry (LC-MS/MS). We found changes in overall protein abundance and increased acetylation of histones and non-histone proteins, many of which were newly discovered and associated with major metabolic and DNA damage pathways. For non-histone acetylation, we mapped a total of 1204 acetylated peptides corresponding to 603 proteins, including chromatin modifying proteins and transcription factors. Hyperacetylated proteins were involved in processes such as transcription, RNA metabolism and DNA damage repair (DDR). The DDR pathway was majorly affected by hyperacetylation following HDAC inhibition. This included acetylation of H2AX, PARP1 and previously unrecognized acetylation sites in TP53BP1. Our data provide a comprehensive view of the targets of HDAC inhibition in malignant T cells with general applicability and could have translational impact for the treatment of ALCL with HDACis alone or in combination therapies., Competing Interests: The authors declare no conflict of interest.

Squamous cell carcinoma of the penis (PSC) is a rare disease with limited information on the molecular events leading to malignant transformation. In a third of PSC cases, presence of human papilloma virus (HPV) is found. The APOBEC3 family of proteins is known to play a significant role in defense against HPV infection, but their role in PSC is largely unknown. In this study, we aim to assess mRNA expression levels of APOBEC3 family members in HPV+ and HPV- PSC to get insight into their association with clinicopathological features and to evaluate their prognostic impact. Expression levels of six APOBEC3 family members in tissue from 50 patients with PSC were determined by RT-PCR and correlated with clinical and histopathological features. Lower expression of APOBEC3A, APOBEC3B, and APOBEC3C was observed in advanced PSC stages. Except for APOBEC3D, HPV+ samples showed higher expression of APOBEC3s compared to HPV- samples. In univariate analyses, APOBEC3A and APOBEC3C expression tended to be associated with disease-free survival and APOBEC3A expression with overall survival; however, multivariable analyses failed to confirm these associations with outcome. More extensive external validation and functional laboratory studies are needed to evaluate further their role in PSC development and progression., (© 2022. The Author(s).)

Purpose of Review: Due to the limited number of cases, there are no guidelines for basal cell carcinoma (BCC) of the prostate. This review combines an unpublished case report of a 55-year-old patient with BCC with an assessment of the latest literature., Recent Findings: BCC of the prostate has previously been described in only approximately 140 cases. We describe the diagnostic process, including the uropathological and DNA-sequencing results, which allowed us to start an experimental treatment with pemigatinib. BCC of the prostate is associated with an aggressive biological and clinical behavior, such as recurrence and metastasis. Several immunohistochemical stainings are available to differentiate BCC from adenocarcinoma of the prostate. Based on pathology and results from next-generation sequencing (NGS), patients can be offered targeted therapies., Summary: With the aid of histological work-up and immunostaining, prostatic BCC can be accurately diagnosed. Our patient underwent radical prostatectomy and staged extended lymphadenectomy due to lymph node recurrence. The patient subsequently developed progressive disease and was treated with the FGFR-inhibitor pemigatinib. The patient's liver metastasis significantly responded. The present case confirms the possibility of aggressive behavior of prostatic BCC and highlights the importance of a thorough uropathological and molecular biological analysis with a precision medicine strategy., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3' end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed., Competing Interests: B.M.D.C.G., M.R.H., and A.K. have filed a patent application for dynamic PK-modifying oligonucleotide anchors., (© 2022 The Authors.)

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplant, with no approved therapeutics available. Although the exact molecular mechanism of NASH progression is not well understood, a widely held hypothesis is that fat accumulation is the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long-term gene silencing with single subcutaneous administration. Here, we identified a hyper-functional, fully chemically stabilized GalNAc-conjugated small interfering RNA (siRNA) targeting DGAT2 (Dgat2-1473) that, upon injection, elicits up to 3 months of DGAT2 silencing (>80%-90%, p < 0.0001) in wild-type and NSG-PiZ "humanized" mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (>85%, p < 0.0001) without increased accumulation of diglycerides, resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat did not translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose of Review: The aim of this study was to give an overview of molecular alterations in upper tract urothelial carcinomas (UTUCs) and to discuss them in the context of current and prospective systemic therapies., Recent Findings: UTUCs not only share a similar molecular landscape with urothelial carcinoma of the bladder (UCB), but also have distinct molecular features that can have an impact on therapy selection. FGFR3 alterations occur with a significant higher frequency in UTUC, with up to 40% of tumours harbouring FGFR3 driver mutations compared with 20% in UCB. In addition, a substantial number of high-grade UTUC show an immune-depleted phenotype and a luminal papillary expression subtype, thus predisposing them for FGFR inhibitor treatment. Approximately 20% of UTUC tumours have acquired mutations in TP53 and demonstrate a significant degree of genomic instability, which makes them candidates for systemic chemotherapy or immunotherapy. Whereas microsatellite instability (MSI) is rare in sporadic UTUC, 5-10% of UTUC patients have germline mutations in DNA mismatch repair genes, which leads to high MSI with enriched neoantigen load and presumably better response rates to immunotherapy., Summary: Treatment decisions in UTUC should take molecular tumour characteristics into account. The currently most therapy-relevant molecular alterations in UTUC comprise FGFR3 mutational status and mutations in DNA mismatch repair genes with concomitant microsatellite instability., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The gene coding for histone methyltransferase KMT2D is found among the top mutated genes in upper tract urothelial carcinoma (UTUC); however, there is a lack of data regarding its association with clinicopathologic features as well as survival outcomes. Therefore, we aimed to investigate KMT2D expression, mutation patterns, and their utility as prognostic biomarkers in patients with UTUC. A single-center study was conducted on tumor specimens from 51 patients treated with radical nephroureterectomy (RNU). Analysis of KMT2D protein expression was performed using immunohistochemistry (IHC). Customized next-generation sequencing (NGS) was used to assess alterations in KMT2D exons. Cox regression was used to assess the relationship of KMT2D protein expression and mutational status with survival outcomes. KMT2D expression was increased in patients with a previous history of bladder cancer (25% vs. 0%, p = 0.02). The NGS analysis of KMT2D exons in 27 UTUC tumors revealed a significant association between pathogenic KMT2D variants and tumor location ( p = 0.02). Pathogenic KMT2D variants were predominantly found in patients with non-pelvic or multifocal tumors (60% vs. 14%), while the majority of patients with a pelvic tumor location (81% vs. 20%) did not harbor pathogenic KMT2D alterations. Both IHC and NGS analyses of KMT2D failed to detect a statistically significant association between KMT2D protein or KMT2D gene alteration status and clinical variables such as stage/grade of the disease or survival outcomes (all p > 0.05). KMT2D alterations and protein expression were associated with UTUC features such as multifocality, ureteral location, and previous bladder cancer. While KMT2D protein expression and KMT2D mutational status do not seem to have prognostic value in UTUC, they appear to add information to improve clinical decision-making regarding the type of therapy.

Initial reports of a clinical response in patients treated with the radioligand [ 177 Lu]-PSMA-617 for castration-resistant prostate cancer (CRPC) are promising, despite known inter- and intrapatient heterogeneity. In metastatic CRPC, we examined the association of baseline immunohistochemical (IHC) expression of prostate-specific membrane antigen (PSMA) in a single lesion and responsiveness to [ 177 Lu]-PSMA-617 therapy, measured as the PSMA maximum standardized uptake value (SUV max ). Between 2015 and 2020, 19 patients with multiple metastases underwent single-lesion biopsy, [ 68 Ga]-PSMA positron emission tomography (PET) imaging, and treatment with [ 177 Lu]-PSMA-617. A monoclonal anti-PSMA antibody was used to semiquantitatively assess PSMA IHC in the biopsy specimen. Imaging evaluation of the biopsied single lesion and overall response was performed according to Positron Emission Tomography Response Criteria in Solid Tumors. The PSMA IHC histoscore correlated positively with pretreatment same-site PSMA SUV max ( r s  = 0.6). Nine patients had imaging after three cycles of [ 177 Lu]-PSMA-617 and were included in the lesion-specific analysis. Of these, five patients (55.6%) had an SUV max response at the biopsy site, but three experienced overall progression. The histoscore was unable to predict the lesion-specific change in SUV max (95% confidence interval [CI] -44.2 to 69.2) or PSA (95% CI-125.2 to 17.2). There was no correlation between single-lesion SUV max and overall progression ( r s  = 0.1) on [ 68 Ga]-PSMA PET imaging. Additional studies need to interrogate the clinical consequence of PSMA expression heterogeneity in metastases and the association with response to [ 177 Lu]-PSMA-671., Patient Summary: Treatment with a radioactive binding molecule called [ 177 Lu]-PSMA-617 for men with prostate cancer resistant to castration (CRPC) is showing promise. We investigated the association between the presence of PSMA protein in metastatic lesions at biopsy and response to [ 177 Lu]-PSMA-617 among men with metastatic CRPC. We found that assessment of PSMA presence at biopsy is not a reliable predictor of response to [ 177 Lu]-PSMA-617. Additional studies are needed to better determine which CRPC metastatic sites will respond to this therapy., (© 2021 The Authors.)

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3'-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3- and androgen-mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

Malignant transformation depends on genetic and epigenetic events that result in a burst of deregulated gene expression and chromatin changes. To dissect the sequence of events in this process, we used a T-cell-specific lymphoma model based on the human oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) translocation. We find that transformation of T cells shifts thymic cell populations to an undifferentiated immunophenotype, which occurs only after a period of latency, accompanied by induction of the MYC-NOTCH1 axis and deregulation of key epigenetic enzymes. We discover aberrant DNA methylation patterns, overlapping with regulatory regions, plus a high degree of epigenetic heterogeneity between individual tumors. In addition, ALK-positive tumors show a loss of associated methylation patterns of neighboring CpG sites. Notably, deletion of the maintenance DNA methyltransferase DNMT1 completely abrogates lymphomagenesis in this model, despite oncogenic signaling through NPM-ALK, suggesting that faithful maintenance of tumor-specific methylation through DNMT1 is essential for sustained proliferation and tumorigenesis., (© 2020 Redl et al.)

Small interfering RNAs (siRNAs) have the potential to treat a broad range of diseases. siRNAs need to be extensively chemically modified to improve their bioavailability, safety, and stability in vivo. However, chemical modifications variably impact target silencing for different siRNA sequences, making the activity of chemically modified siRNA difficult to predict. Here, we systematically evaluated the impact of 3' terminal modifications (2'-O-methyl versus 2'-fluoro) on guide strands of different length and showed that 3' terminal 2'-O-methyl modification negatively impacts activity for >60% of siRNA sequences tested but only in the context of 20- and not 19- or 21-nt-long guide strands. These results indicate that sequence, modification pattern, and structure may cooperatively affect target silencing. Interestingly, the introduction of an extra 2'-fluoro modification in the seed region at guide strand position 5, but not 7, may partially compensate for the negative impact of 3' terminal 2'-O-methyl modification. Molecular modeling analysis suggests that 2'-O-methyl modification may impair guide strand interactions within the PAZ domain of argonaute-2, which may affect target recognition and cleavage, specifically when guide strands are 20-nt long. Our findings emphasize the complex nature of modified RNA-protein interactions and contribute to design principles for chemically modified siRNAs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Bacillus Calmette-Guérin (BCG) immunotherapy, the standard adjuvant intravesical therapy for some intermediate and most high-risk non-muscle invasive bladder cancers (NMIBCs), suffers from a heterogenous response rate. Molecular markers to help guide responses are scarce and currently not used in the clinical setting., Methods: To identify novel biomarkers and pathways involved in response to BCG immunotherapy, we performed a genome-wide DNA methylation analysis of NMIBCs before BCG therapy. Genome-wide DNA methylation profiles of DNA isolated from tumors of 26 BCG responders and 27 failures were obtained using the Infinium MethylationEPIC BeadChip., Results: Distinct DNA methylation patterns were found by genome-wide analysis in the two groups. Differentially methylated CpG sites were predominantly located in gene promoters and gene bodies associated with bacterial invasion of epithelial cells, chemokine signaling, endocytosis, and focal adhesion. In total, 40 genomic regions with a significant difference in methylation between responders and failures were detected. The differential methylation state of six of these regions, localized in the promoters of the genes GPR158 , KLF8 , C12orf42 , WDR44 , FLT1, and CHST11, were internally validated by bisulfite-sequencing. GPR158 promoter hypermethylation was the best predictor of BCG failure with an AUC of 0.809 ( p -value < 0.001)., Conclusions: Tumors from BCG responders and BCG failures harbor distinct DNA methylation profiles. Differentially methylated DNA regions were detected in genes related to pathways involved in bacterial invasion of cells or focal adhesion. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts.

Context: While upper tract urothelial carcinoma (UTUC) share histological appearance with bladder cancer (BC), the former has differences in etiology and clinical phenotype consistent with characteristic molecular alterations., Objective: To systematically evaluate current genomic sequencing and proteomic data examining molecular alterations in UTUC., Evidence Acquisition: A systematic review using PubMed, Scopus, and Web of Science was performed in December 2019 according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement., Evidence Synthesis: A total of 46 publications were selected for inclusion in this report, including 13 studies assessing genome-wide alterations, 18 studies assessing gene expression or microRNA expression profiles, three studies assessing proteomics, one study assessing genome-wide DNA methylation, and 14 studies evaluating distinct pathway alteration patterns. Differences between sporadic and hereditary UTUC, and between UTUC and BC, as well as molecular profiles associated with exposure to aristolochic acid are highlighted. Molecular pathways relevant to UTUC biology, such as alterations in FGFR3, TP53, or microsatellite instability, are discussed. Our findings are limited by tumor and patient heterogeneity and different platforms used in the studies., Conclusions: Molecular events in UTUC and BC can be shared or distinct. Consequently, molecular subtypes differ according to location. Further work is needed to define the epigenomic and proteomic features of UTUC, and understand the mechanisms by which they shape the clinical behavior of UTUC., Patient Summary: We report the current data on the molecular alterations specific to upper tract urothelial carcinoma (UTUC), resulting from novel genomic and proteomic technologies. Although UTUC biology is comparable with that of bladder cancer, the rates and UTUC-enriched alterations support its uniqueness and the need for precision medicine strategies for this rare tumor type., (Copyright © 2020. Published by Elsevier B.V.)

Purpose of Review: The aim of this article is to give an overview of poly(ADP-ribose) polymerase inhibitors (PARPis) trials in prostate cancer and to discuss emerging approaches with potential future clinical implementation in both prostate and urothelial cancer., Recent Findings: PARPis are a class of drugs that can be applied for the treatment of homologous recombination repair (HRR)-deficient tumors. Tumors are potentially sensitive to PARPi harbor mutations in genes relevant for DNA damage repair, such as BRCA1/2 or ATM, which are present to a significant degree in metastatic prostate and urothelial cancer patients. Several PARPis have been successfully tested in clinical trials for HRR-deficient metastatic castration-resistant prostate cancer (mCRPC), and olaparib and rucaparib have recently received breakthrough approval in BRCA1/2 mutated mCRPC. Combination treatment of PARPis with androgen-receptor inhibitors or with checkpoint inhibitors and earlier frontline applications are currently being evaluated, and clinical trials enrolling bladder cancer (BCa) patients with HRR deficiency have recently been initiated., Summary: Approximately 10% of mCRPC patients and 34% of metastatic BCa patients have tumors with HRR deficiency and may benefit from PARPi treatment. Correct identification of these patients as well as determining the most adequate time point for drug administration will be key to successful clinical implementation.

Purpose of Review: The aim of the article to summarize recent changes of treatment options in metastatic renal cell carcinoma (mRCC) with a special emphasis on immune checkpoint inhibition., Recent Findings: The introduction of checkpoint inhibitor (CPI) therapy has led to a paradigm change in advanced renal cell carcinoma (RCC). Dual immune checkpoint inhibition or the combination of CPI and tyrosine kinase inhibitors (TKIs) was shown to improve survival when compared with the former standard of care sunitinib. Moreover, these novel strategies were shown to enable unprecedented rates of complete and durable responses, particularly with dual checkpoint inhibition. Although the treatment landscape has rapidly evolved, it remains unknown which combination is the best for the individual patient. Pivotal trials have used sunitinib as a comparator but no head to head comparisons have been conducted between novel agents so far. Moreover, no predictive biomarker has been identified yet to bring the best treatment to the individual patient., Summary: The aim of this review is to summarize the findings of CPI-based trials conducted in RCC and to discuss the future of mRCC treatment.

Purpose: To assess the prognostic significance of the nuclear receptor binding SET protein 2 (NSD2), a co-activator of the NFkB-pathway, on tumour progression in patients with advanced prostate cancer (PCa). Methods: We retrospectively assessed NSD2 expression in 53 patients with metastatic and castration-resistant PCa. Immunohistochemical staining for NSD2 was carried out on specimen obtained from palliative resection of the prostate. Univariable and multivariable analyses were performed to assess the association between NSD2 expression and PCa progression. Results: Of the 53 patients, 41 had castration-resistant PCa and 48 men had metastases at time of tissue acquisition. NSD2 expression was increased in tumour specimen from 42 patients (79.2%). In univariable Cox regression analyses, NSD2 expression was associated with PSA progression, progression on imaging and overall survival ( p  = 0.04, respectively). In multivariable analyses, NSD2 expression did not retain its association with these endpoints. Conclusions: NSD2 expression is abnormal in almost 80% of patients with advanced PCa. Expression levels of this epigenetic regulator are easily detected by immunohistochemistry while this biomarker exhibited prognostic value for PCa progression and death in univariable analysis. Further studies on NSD2 involvement in PCa proliferation, progression, metastasis and resistance mechanisms are needed.

Recent meta-analyses on checkpoint inhibitors in cancer report conflicting data regarding the association of patient gender with inhibitor efficacy. In advanced kidney cancer, checkpoint inhibitors have shown improved outcomes in first- and second-line settings compared with standard of care, but the role of patient gender on treatment outcome is unclear. We aimed to assess the efficacy of immunotherapy according to patient gender in advanced kidney cancer. We performed a systematic review and meta-analysis according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A literature search was performed using PubMed, Scopus, Web of Science, and The Cochrane Library to identify eligible studies published through February 16, 2019. Studies were included if they reported on the differential outcomes of male and female patients with metastatic kidney cancer receiving immunotherapy. Our outcomes of interest were overall survival (OS) or progression-free survival (PFS). Four randomized controlled trials comprising a total of 3664 patients (2715 males and 949 females) met our inclusion criteria. Both men and women with metastatic kidney cancer had an OS and PFS advantage with immunotherapy compared with standard-of-care, but no statistically significant difference between the genders was observed (OS hazard ratio [HR] for men, 0.69; 95% confidence interval [CI], 0.59-0.8; P = .40; HR for women, 0.62; 95% CI, 0.48-0.81; P = .13; PFS HR for men, 0.7; 95% CI, 0.59-0.82; P = .24; HR for women, 0.68; 95% CI, 0.52-0.90; P = .105). In patients with advanced kidney cancer receiving checkpoint inhibitors, there seems to be no association of patient gender with treatment outcome., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose of Review: Testicular germ cell tumours (TGCTs) exhibit, in contrast to other cancer types, a relatively low mutational burden. However, numerous epigenetic alterations have been shown to impact TGCT. In this review, we summarize the most relevant findings of the past 2 years., Recent Findings: Recent studies focused on the functions of microRNAs and the impact of aberrant DNA methylation. Moreover, several epigenetic drugs with antineoplastic effects in TGCTs were identified., Summary: Aberrant DNA methylation and differentially expressed microRNAs have an important effect on TGCT pathogenesis. Moreover, differential DNA methylation patterns were found to be specific for different TGCT subtypes. Various microRNAs, such as miR-371a-3p, were found to be highly sensitive and specific biomarkers for TGCT. The epigenetic drugs guadecitabine, animacroxam, and JQ1 showed promising effects on TGCT in preclinical in-vivo and in-vitro studies.

Introduction: The purpose of this study was to investigate the prevalence and prognostic value of the polymorphic variant (1245A>C), a single nucleotide polymorphism (SNP) of the HSD3B1 gene, in the tumors of patients with castration-resistant prostate cancer (CRPC)., Materials and Methods: We retrospectively evaluated 44 patients with CRPC who underwent palliative transurethral resection of the prostate. Genomic DNA was extracted from formalin-fixed and paraffin-embedded material, and 1245A>C SNP of the HSD3B1 gene was analyzed via Sanger sequencing. Cox regression analysis was used to assess the prognostic value of the respective SNP with time to progression as well as cancer-specific and overall survival in the subgroup of patients receiving second systemic treatment., Results: The SNP was present in 20 patients (51.2%) who received second line systemic treatment additionally to androgen deprivation, of which 16 (80%) patients were heterozygous and 4 (20%) were homozygous. Correlation analysis revealed no association of the SNP with any clinical characteristics at initiation of second-line systemic treatment. Moreover, the presence of the variant (1245A>C) of HSD3B1 was not associated with any survival endpoint., Conclusions: The variant allele 1245C of the HSD3B1 gene is present in approximately one-half of patients with CRPC; however, it is not associated with oncologic outcomes. These findings, however, need to be interpreted with caution as the sample size is small. Further research on biomarkers is needed to help tailor clinical decision making in prostate cancer, especially in the increasingly complex therapeutic landscape of CRPC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an "artificial exosome," in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose of Review: To give an overview of current options for conservative treatment of patients failing intravesical bacillus Calmette-Guerin (BCG) and to discuss emerging approaches with potential future clinical applications., Recent Findings: Radical cystectomy is the standard-of-care for patients failing BCG therapy. In patients unfit or unwilling to undergo surgery, salvage therapy options could be proposed with the aim to offer local cancer control and prevent progression to muscle-invasive disease. Salvage treatments have been conducted using intravesical chemotherapy regimens, chemoradiation or chemohyperthermia. Intravesical agents such as valrubicin, gemcitabine or docetaxel showed response rates varying between 16 and 40%, whereas combination treatments of gemcitabine with docetaxel or mitomycin reported response rates in up to 50% of all patients with durable responses in about one out of three patients. For chemohyperthermia, 2-year recurrence rates between 41 and 56% have been reported. Ongoing clinical trials are evaluating chemoradiation as well as novel approaches such as systemic immunotherapy, viral gene therapy, targeted therapy or vaccination strategies with promising preliminary outcomes., Summary: Salvage therapeutic bladder-sparing strategies for BCG failure such as intravesical chemotherapy or chemoradiation should currently only be considered in patients unfit for or refusing surgery. Innovative concepts such as chemohyperthermia, checkpoint inhibitors, targeted therapy or viral gene therapy could lead to major changes in clinical management of BCG failures in the future.

Context: The optimal treatment of urothelial bladder cancer (UBC) with micropapillary (MP) variant histology is not clear., Objective: To review the current literature on disease characteristics and treatment outcomes of MP UBC., Evidence Acquisition: A systematic search was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement and the Cochrane Handbook for Systematic Reviews of Interventions. The primary end points were recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS)., Evidence Synthesis: We identified 758 reports comprising a total of 3154 patients, of which 28 and 15 articles were selected for qualitative and quantitative analysis, respectively. In patients with T1 MP UBC, the 5-yr CSS rates for early radical cystectomy (RC) ranged from 81% to 100%, while they were between 60% and 85% for transurethral resection of the bladder and Bacillus Calmette-Guérin (BCG). In studies reporting on neoadjuvant chemotherapy (NAC), the rates of complete pathological response (ypT0) ranged from 11% to 55%. Nevertheless, the use of NAC did not improve RFS (hazard ratio [HR] 1.23, 95% confidence interval [CI] 0.52-2.93, p=0.6), CSS (HR 0.9, 95% CI 0.48-1.7, p=0.8), or OS (HR 1.35, 95% CI 0.98-1.86, p=0.1). Fifty-three percent (95% CI 43-63%) of patients who underwent RC alone had locally advanced disease (≥pT3), and 43% (95% CI 33-52%) were harbouring lymph node metastases. MP component at RC was not significantly associated with worse RFS (HR 1.25, 95% CI 0.88-1.78, p=0.2), CSS (HR 0.96, 95% CI 0.57-1.6, p=0.9), or OS (HR 1.20, 95% CI 0.88-1.62, p=0.3) when adjusted for pathological features., Conclusions: While MP UBC is associated with clinicopathological features of advanced disease, it is not associated with worse survival outcomes in patients undergoing RC. NAC results in pathological downstaging in a significant number of patients. Nevertheless, this does not translate into better survival outcomes. The optimal treatment of patients with cT1 remains controversial., Patient Summary: Our results suggest that micropapillary urothelial bladder cancer does not necessarily mandate different treatment algorithms. Nevertheless, each case should be discussed individually considering other clinicopathological factors., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia.

Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5'-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5'-(E)-vinylphosphonate and alternating 2'-fluoro and 2'-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at ∼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.