piRNAs and PIWI Proteins as Diagnostic and Prognostic Markers of Genitourinary Cancers.

piRNAs (PIWI-interacting RNAs) are small non-coding RNAs capable of regulation of transposon and gene expression. piRNAs utilise multiple mechanisms to affect gene expression, which makes them potentially more powerful regulators than microRNAs. The mechanisms by which piRNAs regulate transposon and gene expression include DNA methylation, histone modifications, and mRNA degradation. Genitourinary cancers (GC) are a large group of neoplasms that differ by their incidence, clinical course, biology, and prognosis for patients. Regardless of the GC type, metastatic disease remains a key therapeutic challenge, largely affecting patients' survival rates. Recent studies indicate that piRNAs could serve as potentially useful biomarkers allowing for early cancer detection and therapeutic interventions at the stage of non-advanced tumour, improving patient's outcomes. Furthermore, studies in prostate cancer show that piRNAs contribute to cancer progression by affecting key oncogenic pathways such as PI3K/AKT. Here, we discuss recent findings on biogenesis, mechanisms of action and the role of piRNAs and the associated PIWI proteins in GC. We also present tools that may be useful for studies on the functioning of piRNAs in cancers.

Ciprofloxacin and Levofloxacin as Potential Drugs in Genitourinary Cancer Treatment-The Effect of Dose-Response on 2D and 3D Cell Cultures.

INTRODUCTION: Introducing new drugs for clinical application is a very difficult, long, drawn-out, and costly process, which is why drug repositioning is increasingly gaining in importance. The aim of this study was to analyze the cytotoxic properties of ciprofloxacin and levofloxacin on bladder and prostate cell lines in vitro. METHODS: Bladder and prostate cancer cell lines together with their non-malignant counterparts were used in this study. In order to evaluate the cytotoxic effect of both drugs on tested cell lines, MTT assay, real-time cell growth analysis, apoptosis detection, cell cycle changes, molecular analysis, and 3D cultures were examined. RESULTS: Both fluoroquinolones exhibited a toxic effect on all of the tested cell lines. In the case of non-malignant cell lines, the cytotoxic effect was weaker, which was especially pronounced in the bladder cell line. A comparison of both fluoroquinolones showed the advantage of ciprofloxacin (lower doses of drug caused a stronger cytotoxic effect). Both fluoroquinolones led to an increase in late apoptotic cells and an inhibition of cell cycle mainly in the S phase. Molecular analysis showed changes in BAX, BCL2, TP53, and CDKN1 expression in tested cell lines following incubation with ciprofloxacin and levofloxacin. The downregulation of topoisomerase II genes (TOP2A and TOP2B) was noticed. Three-dimensional (3D) cell culture analysis confirmed the higher cytotoxic effect of tested fluoroquinolone against cancer cell lines. CONCLUSIONS: Our results suggest that both ciprofloxacin and levofloxacin may have great potential, especially in the supportive therapy of bladder cancer treatment. Taking into account the low costs of such therapy, fluoroquinolones seem to be ideal candidates for repositioning into bladder cancer therapeutics.

Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers.

Breast cancers and cancers of the genitourinary tract are the most common malignancies among men and women and are still characterized by high mortality rates. In order to improve the outcomes, early diagnosis is crucial, ideally by applying non-invasive and specific biomarkers. A key role in this field is played by extracellular vesicles (EVs), lipid bilayer-delimited structures shed from the surface of almost all cell types, including cancer cells. Subcellular structures contained in EVs such as nucleic acids, proteins, and lipids can be isolated and exploited as biomarkers, since they directly stem from parental cells. Furthermore, it is becoming even more evident that different body fluids can also serve as sources of EVs for diagnostic purposes. In this review, EV isolation and characterization methods are described. Moreover, the potential contribution of EV cargo for diagnostic discovery purposes is described for each tumor.

Sonic Hedgehog signaling pathway in gynecological and genitourinary cancer (Review).

Cancers of the urinary tract, as well as those of the female and male reproductive systems, account for a large percentage of malignancies worldwide. Mortality is frequently affected by late diagnosis or therapeutic difficulties. The Sonic Hedgehog (SHH) pathway is an evolutionary conserved molecular cascade, which is mainly associated with the development of the central nervous system in fetal life. The present review aimed to provide an in­depth summary of the SHH signaling pathway, including the characterization of its major components, the mechanism of its upstream regulation and non­canonical activation, as well as its interactions with other cellular pathways. In addition, the three possible mechanisms of the cellular SHH cascade in cancer tissue are discussed. The aim of the present review was to summarize significant findings with regards to the expression of the SHH pathway components in kidney, bladder, ovarian, cervical and prostate cancer. Reports associated with common deficits and de­regulations of the SHH pathway were summarized, despite the differences in molecular and histological patterns among these malignancies. However, currently, neither are SHH pathway elements included in panels of prognostic/therapeutic molecular patterns in any of the discussed cancers, nor have the drugs targeting SMO or GLIs been approved for therapy. The findings of the present review may support future studies on the treatment of and/or molecular targets for gynecological and genitourinary cancers.

PD-L1 expression in anogenital and oropharyngeal squamous cell carcinomas associated with different clinicopathological features, HPV status and prognosis: a meta-analysis.

BACKGROUND: Little research has been done on clinicopathological characteristics and human papillomavirus (HPV) status of anogenital and oropharyngeal squamous cell carcinomas (SCC) with a strong expression of programmed death ligand 1 (PD-L1) in tumor cells. Therefore, we conducted this meta-analysis. METHODS: We performed a comprehensive research in PubMed, Embase and Cochrane databases up to 30 September 2020. The effect size was hazard ratio (HR) with 95% confidence interval (CI) for overall survival (OS), cancer-specific survival (CSS), disease-free survival (DFS). The pooled odds ratio (OR) with 95% CI were used to assess the association between PD-L1 expression and clinicopathological features along with HPV status. RESULTS: A total of 2003 cases (944 anogenital and 1059 oropharynx SCC patients) were included. High PD-L1 expression in anogenital SCC cases were associated with advanced age (OR = 1.63, 95% CI: 1.04-2.58) and HPV negativity (OR = 0.47, 95% CI: 0.31-0.71). Besides, PD-L1 positive anogenital SCC cases held a significantly declined OS (HR = 2.18, 95% CI: 1.37-3.47) and CSS (HR = 2.45, 95% CI: 1.30-4.65). For oropharynx SCC, PD-L1 was more frequent in younger and HPV positive patients (OR = 0.60, 95% CI: 0.37-0.98; OR = 3.01, 95% CI: 1.78-5.09) and PD-L1 expression was relevant to better OS and DFS (HR = 0.76, 95% CI: 0.60-0.97; HR = 0.50, 95% CI: 0.33-0.75). CONCLUSIONS: The meta-analysis demonstrated that in anogenital SCC, PD-L1 positivity had to do with a worse outcome, which might attribute to advanced age, higher tumor grade, lymph node metastasis and HPV negativity, while in oropharynx cancer, PD-L1 expression was related to better prognosis for the reason that PD-L1 was less frequent in the aged and negative HPV status.

The role of long non-coding RNA MIAT in cancers.

Long non-coding RNAs (lncRNAs), a kind of non-coding single-strand RNAs, play an important role as carcinogenic genes or tumor suppressors in the development of human cancer. Myocardial infarction-associated transcript (MIAT) was first identified as a lncRNA in 2006 and originally isolated as a candidate gene for myocardial infarction. Later, it was reported that MIAT exhibits regulatory effects on the human cell cycle. Since its discovery, MIAT has also been identified as a carcinogenic regulator in many malignant tumors. High expression of MIAT is related to the clinicopathological characteristics of cancer patients. It can also regulate cell proliferation, invasion, metastasis, and anti-apoptosis through a variety of mechanisms. Therefore, MIAT is considered a potential biomarker and therapeutic target in cancer. In this review, we summarize the biological function, mechanism, and potential clinical significance of MIAT during tumorigenesis.

The International Society of Urological Pathology Consultation on Molecular Pathology of Urogenital Cancer.

The 2019 Consultation Conference on the molecular pathology of urogenital cancers was organized by the International Society of Urological Pathology (ISUP) to have an understanding of the current use of molecular-genetic markers and to make recommendations on their application in prostate, bladder, renal, testicular, and penile cancer. This brief introductory article describes the organization of this conference and provides its rationale and main findings.

PD1/PD-L1 Axis in Uro-oncology.

The immune system is important to control tumor development and progression in humans. However, tumor cells and cells of the tumor microenvironment can induce immune escape mechanisms including activation of immune checkpoints such as PD-1/PD-L1. Based on this knowledge, new immune therapies, including PD-1 and PD-L1 inhibition, have been developed and are already recommended as a standard treatment in metastatic bladder and kidney cancer patients. In addition to its role as a therapeutic target, PD-L1 seems to be a prognostic parameter although data are controversial. Only little is known about signaling pathways inducing PD-L1 expression in tumor cells on one hand and about its functional role for tumor cells itself. However, the understanding of the complex biological function of PD-L1 will improve therapeutic options in urological malignancies. This review is giving an overview of the current knowledge concerning the PD-1/PD-L1 axis in urological tumors including bladder, kidney, prostate, testicular and penile cancer.

Application and Pitfalls of Immunohistochemistry in Diagnosis of Challenging Genitourinary Cases.

CONTEXT.­: Immunohistochemistry (IHC) has become increasingly important in the evaluation of pathologic conditions in the genitourinary (GU) organs. In addition to careful evaluation of hematoxylin-eosin sections and generation of a differential diagnosis, choosing the optimal panel of IHC markers becomes even more important when the biopsy material is very limited. The following summary of our experience supplemented with relevant literature review exemplifies how to use IHC to facilitate pathologic diagnosis in the GU system. OBJECTIVE.­: To describe our experience with the most common immunohistochemical markers used in GU pathology. DATA SOURCES.­: Institutional experience and literature search comprise our data sources. CONCLUSIONS.­: Application of IHC provides enormous benefits to the interpretation of GU pathologic conditions, including benign and malignant lesions. However, both insufficient and excessive types of use of IHC, as well as incorrect interpretations in common and rare GU conditions, could present pitfalls in diagnosis.

Status of Programmed Death Ligand 1 (PD-L1) by Immunohistochemistry and Scoring Algorithms.

The detection of the Programmed Death Ligand 1 (PD-L1) protein by immunohistochemistry is currently the only approved test predictive of response to drugs targeting the PD1/PDL1 axis. The role of this test is debated since several reagents have been used as companion diagnostics for different drugs on diverse immunostaining platforms. In addition, different scoring systems for PD-L1 immunohistochemistry have been applied in the registration studies regarding single drugs. This review deals with the various issues that are related to the immunohistochemical test for PD-L1. We discuss currently unsolved problems such as the advantages and the flaws of PD-L1 immunohistochemistry; the choice of the best reagents and the best scoring system. Finally, we review the current experiences on the role of immunohistochemistry for PD-L1 in clinical trials with immune checkpoint inhibitors.

Practical Molecular Testing in a Clinical Genitourinary Service.

CONTEXT.­: Molecular testing is increasingly playing a key role in the diagnosis, prognosis, and treatment of neoplasms of the genitourinary system. OBJECTIVE.­: To provide a general overview of the clinically relevant molecular tests available for neoplasms of the genitourinary tract. DATA SOURCES.­: Relevant medical literature indexed on PubMed. CONCLUSIONS.­: Understanding of the molecular oncology of genitourinary neoplasms is rapidly advancing, and the pathologist must be aware of the practical implications of molecular testing. While many genomic abnormalities are not yet clinically relevant, there is an increasing library of ancillary tests that may guide diagnosis, prognosis, and/or treatment of many neoplasms. Recurrent genomic abnormalities have been identified in many types of renal cell carcinoma, and some types of renal cell carcinoma are specifically defined by the molecular abnormality. Two major routes of developing urothelial carcinoma have been molecularly described. Recurrent translocations involving ETS family genes are found in approximately half of prostate cancer cases. Testicular germ cell tumors typically harbor i(12p). Penile neoplasms are often high-risk human papillomavirus-driven cancers. Nonetheless, even as genitourinary neoplasms are increasingly better understood at the molecular level, further research with eventual clinical validation is needed for optimal diagnosis, prognosis, and treatment of aggressive malignancies in the genitourinary tract.

TGF-ß and microRNA Interplay in Genitourinary Cancers.

Genitourinary cancers (GCs) include a large group of different types of tumors localizing to the kidney, bladder, prostate, testis, and penis. Despite highly divergent molecular patterns, most GCs share commonly disturbed signaling pathways that involve the activity of TGF-ß (transforming growth factor beta). TGF-ß is a pleiotropic cytokine that regulates key cancer-related molecular and cellular processes, including proliferation, migration, invasion, apoptosis, and chemoresistance. The understanding of the mechanisms of TGF-ß actions in cancer is hindered by the "TGF-ß paradox" in which early stages of cancerogenic process are suppressed by TGF-ß while advanced stages are stimulated by its activity. A growing body of evidence suggests that these paradoxical TGF-ß actions could result from the interplay with microRNAs: Short, non-coding RNAs that regulate gene expression by binding to target transcripts and inducing mRNA degradation or inhibition of translation. Here, we discuss the current knowledge of TGF-ß signaling in GCs. Importantly, TGF-ß signaling and microRNA-mediated regulation of gene expression often act in complicated feedback circuits that involve other crucial regulators of cancer progression (e.g., androgen receptor). Furthermore, recently published in vitro and in vivo studies clearly indicate that the interplay between microRNAs and the TGF-ß signaling pathway offers new potential treatment options for GC patients.

Genitourinary Tumors: Update on Molecular Biomarkers for Diagnosis, Prognosis and Prediction of Response to Therapy.

BACKGROUND: Research of biomarkers in genitourinary tumors goes along with the development of complex emerging techniques ranging from next generation sequencing platforms, applied to archival pathology specimens, cytological samples, liquid biopsies, and to patient-derived tumor models. METHODS: This contribution is an update on molecular biomarkers for diagnosis, prognosis and prediction of response to therapy in genitourinary tumors. The following major topics are dealt with: Immunological biomarkers, including the microbiome, and their potential role and caveats in renal cell carcinoma, bladder and prostate cancers and testicular germ cell tumors; Tissue biomarkers for imaging and therapy, with emphasis on Prostate-specific membrane antigen in prostate cancer; Liquid biomarkers in prostate cancer, including circulating tumor cell isolation and characterization in renal cell carcinoma, bladder cancer with emphasis on biomarkers detectable in the urine and testicular germ cell tumors; and Biomarkers and economic sustainability. CONCLUSION: The identification of effective biomarkers has become a major focus in cancer research, mainly due to the necessity of selecting potentially responsive patients in order to improve their outcomes, as well as to reduce the toxicity and costs related to ineffective treatments.

Metabolomic Heterogeneity of Urogenital Tract Cancers Analyzed by Complementary Chromatographic Techniques Coupled with Mass Spectrometry.

BACKGROUND: In regard to urogenital tract cancer studies, an estimated 340,650 new cases and 58,360 deaths from genital system cancer and about 141,140 new cases and 29330 deaths from urinary system were projected to occur in the United States in 2012. The main drawbacks of currently available diagnostic tests constitute the low specificity, costliness and quite high invasiveness. OBJECTIVE: The main goal of this pilot study was to determine and compare urine metabolic fingerprints in urogenital tract cancer patients and healthy controls. METHOD: A comparative analysis of the metabolic profile of urine from 30 patients with cancer of the genitourinary system (bladder (n=10), kidney (n=10) and prostate (n=10)) and 30 healthy volunteers as a control group was provided by LC-TOF/MS and GCQqQ/ MS. The data analysis was performed by the use of U-Mann Whitney test or Student's t-test, principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). RESULTS: As a result, 33, 43, and 22 compounds were identified as statistically significant in bladder, prostate and kidney cancer, respectively, compared to healthy groups. CONCLUSION: Diverse compounds such as purine, sugars, amino acids, nucleosides, organic acids which play a role in purine metabolism, in tricarboxylic acid cycle, in amino acid metabolism or in gut microbiota metabolism were identified. Only two metabolites namely glucocaffeic acid and lactic acid were found to be in common in studied three types of cancer.

New plasma preparation approach to enrich metabolome coverage in untargeted metabolomics: plasma protein bound hydrophobic metabolite release with proteinase K.

Plasma untargeted metabolomics is a common method for evaluation of the mechanisms underlying human pathologies and identification of novel biomarkers. The plasma proteins provide the environment for transport of hydrophobic metabolites. The current sample preparation protocol relies on the immediate precipitation of proteins and thus leads to co-precipitation of a significant fraction of hydrophobic metabolites. Here we present a new simple procedure that overcomes the co-precipitation problem and improves metabolome coverage. Introducing an additional step preceding the protein precipitation, namely limited digestion with proteinase K, allows release of associated metabolites through the relaxation of the native proteins tertiary structure. The modified protocol allows clear detection of hydrophobic metabolites including fatty acids and phospholipids. Considering the potential involvement of the hydrophobic metabolites in human cardiovascular and cancer diseases, the method may constitute a novel approach in plasma untargeted metabolomics.

Blocking PD-1/PD-L1 in Genitourinary Malignancies: To Immunity and Beyond.

Genitourinary malignancies represent a diverse biologic and immunologic landscape. Recently, checkpoint blockade has transformed the treatment paradigms for bladder and kidney cancer. However, continued progress will be essential in bladder and kidney cancer, given response to inhibition of the PD-1/PD-L1 (PD-1/PD-L1) axis remains variable and only a minority of patients respond. In contrast with the clinical trial results in bladder and kidney cancer, studies of anti-PD-1/PD-L1 therapy in prostate cancer have generally been disappointing. Nevertheless, an exciting array of studies is underway that are translating lessons learned from tumor biology into promising clinical trials. Here we highlight important features of the immune tumor microenvironment of bladder, kidney, and prostate cancer and review key completed and ongoing clinical trials of anti-PD-1/PD-L1 therapy in these tumor types.

The stem cell factor (SCF)/c-KIT system in carcinogenesis of reproductive tissues: What does the hormonal regulation tell us?

The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF) are expressed in several tissues of male and female reproductive tract, playing an important role in the regulation of basic biological processes. The activation of c-KIT by SCF controls, cell survival and death, cell differentiation and migration. Also, the SCF/c-KIT system has been implicated in carcinogenesis of reproductive tissues due to its altered expression pattern or overactivation in consequence of gain-of-functions mutations. Over the years, it has also been shown that hormones, the primary regulators of reproductive function and causative agents in the case of hormone-dependent cancers, are also able to control the SCF/c-KIT tissue levels. Therefore, it is liable to suppose that disturbed SCF/c-KIT expression driven by (de)regulated hormone actions can be a relevant step towards carcinogenesis. The present review describes the SCF and c-KIT expression in cancers of reproductive tissues, discussing the implications of the hormonal regulation of the SCF/c-KIT system in cancer development. Understanding the relationship between hormonal imbalance and the SCF/c-KIT expression and activity would be relevant in the context of novel therapeutic approaches in reproductive cancers.

[Endocrinology of cancer and age: early and late stages of ontogenesis].

Processes important for hormone-mediated carcinogenesis are present on different, even very early, ontogenesis stages. Early shifts in hormone-metabolic status often display opposite correlations with the risk of most common age-associated non-communicable pathologies (namely, hormone-dependent cancers and cardiovascular diseases). Additional known contradiction is the raise of reproductive system tumors incidence in the age associated with lower production of mitogenic hormones. Consequently, one should take into account production of steroids in target tissues themselves, recognize the importance of progenotoxic effect, which, apart from mitogenic function, is characteristic for estrogens and their derivatives, as well as the role of endocrine-genotoxic switchings forming so called basic triad, which is born under the influence of age-associated endocrine shifts and environmental factors. Aside from steroids-related system of increased cancer risk, attention should be paid to non-steroid ones (in particular insulin resistance- and inflammatory cytokines-associated), with their close connection to immune system functional state, low-grade chronic inflammation, obesity phenotype, and pro-/anti-inflammatory lipid factors ratio. In total, it confirms and importance of timely preventive interventions on both ontogenesis stages, early and late ones, which are often separated by several decades.

Targeting the Programmed Cell Death-1 Pathway in Genitourinary Tumors: Current Progress and Future Perspectives.

BACKGROUND: Immune checkpoint inhibitors have revolutionized the treatment of many malignancies with over a dozen new United States Food and Drug Administration (FDA) approvals in the past six years. Due to the combination of potent treatment success and potentially deadly adverse effects from immune checkpoint inhibitors, gathering prognostic and predictive information about FDA-indicated tumors is prudent. METHOD: PD-L1 expression is a poor prognostic factor and predictive of better responses from both PD-1 and PD-L1 inhibitors in a variety of tumor types including Renal Cell Carcinoma (RCC) and urothelial carcinoma. Each FDAapproved PD-1/PD-L1 drug is paired with a PD-L1 Immunohistochemistry (IHC) assay. The majority of PD-1/PDL1 inhibitor clinical trials use proprietary IHC antibodies with undefined validation data. Thus, there is need for improved knowledge and application of PD-1/PD-L1 IHC biomarkers. There is a wealth of recent publications using antibody clones to characterize tumor PD-1/PD-L1 expression profiles. RESULTS: PD-1 is expressed on lymphocytes. PD-L1 is expressed on both tumor cells and immune cells. IHC staining appears in membranous fashion. A cutoff of at least 5% tumor cell PD-L1 staining for positivity has worked for most studies. Caution should be observed when employing tissue microarray techniques. CONCLUSION: RCC has been the most studied of the genitourinary malignancies for PD-L1 expression. The atezolizumab- approved IHC assay is unique in that only immune cell staining is quantified for the use of this assay in urothelial carcinoma. With familiarity of the current FDA guidelines, published medical literature, and general immunohistochemical considerations, the use of immune checkpoint biomarkers can continue to flourish.