Exploring vasculogenesis in the normal human kidney and clear cell renal cell carcinoma: insights from development to tumor progression and biomarkers for therapy response.

Vasculogenesis, which refers to the development of blood vessels from precursor cells, is a process that occurs predominantly during early embryonic life. It plays a crucial role in the establishment of the primitive vascular network. Vasculogenesis diminishes throughout the fetal vascular remodeling process, giving way to angiogenesis, which becomes the predominant mechanism after birth. At first, the development of the kidney's blood vessels depends on vasculogenesis, and then both vasculogenesis and angiogenesis happen simultaneously. Both processes are necessary for the normal development of the renal vasculature. Although the kidneys are highly vascularized, our understanding of normal kidney vasculogenesis is still incomplete. This lack of knowledge may explain the limited data available on the role of vasculogenesis in the progression and spread of renal cancers. In other types of cancer, researchers have well documented the phenomenon of tumor vasculogenesis. However, there is currently limited and fragmented information about the occurrence of clear-cell renal cell carcinomas (cc-RCC). In this article, we provide a comprehensive review of the current understanding of normal kidney vasculogenesis and vasculogenic pathways in clear cell renal cell carcinoma (cc-RCC). We specifically focus on cellular precursors, growth factors, and the influence of the normal and tumor environments on these processes. It will carefully look at how tumor vasculogenesis might affect the growth and metastasis of clear cell renal cell carcinoma (cc-RCC), as well as how it might affect the effectiveness of drugs and the development of therapy resistance.

Evaluating Established Roles, Future Perspectives and Methodological Heterogeneity for Wilms' Tumor 1 (WT1) Antigen Detection in Adult Renal Cell Carcinoma, Using a Novel N-Terminus Targeted Antibody (Clone WT49).

Renal cell carcinoma (RCC) is arguably the deadliest form of genitourinary malignancy and is nowadays viewed as a heterogeneous series of cancers, with the same origin but fundamentally different metabolisms and clinical behaviors. Immunohistochemistry (IHC) is increasingly necessary for RCC subtyping and definitive diagnosis. WT1 is a complex gene involved in carcinogenesis. To address reporting heterogeneity and WT1 IHC standardization, we used a recent N-terminus targeted monoclonal antibody (clone WT49) to evaluate WT1 protein expression in 56 adult RCC (aRCC) cases. This is the largest WT1 IHC investigation focusing exclusively on aRCCs and the first report on clone WT49 staining in aRCCs. We found seven (12.5%) positive cases, all clear cell RCCs, showing exclusively nuclear staining for WT1. We did not disregard cytoplasmic staining in any of the negative cases. Extratumoral fibroblasts, connecting tubules and intratumoral endothelial cells showed the same exclusively nuclear WT1 staining pattern. We reviewed WT1 expression patterns in aRCCs and the possible explanatory underlying metabolomics. For now, WT1 protein expression in aRCCs is insufficiently investigated, with significant discrepancies in the little data reported. Emerging WT1-targeted RCC immunotherapy will require adequate case selection and sustained efforts to standardize the quantification of tumor-associated antigens for aRCC and its many subtypes.

Heterogeneity of Platelet Derived Growth Factor Pathway Gene Expression Profile Defines Three Distinct Subgroups of Renal Cell Carcinomas.

BACKGROUND/AIM: We previously described four different vascular patterns (reticular, diffuse, fasciculate, and trabecular) in renal cell carcinoma (RCC) suggesting an early and heterogeneous acquisition of perivascular cells most probably due to a particular PDGF pathway gene expression profile. The aim of the study was to study PDGF pathway gene expression profiles, separately for each vascular pattern. MATERIALS AND METHODS: TaqMan assay for the PDGF pathway was performed on twelve cases of ccRCC previously evaluated by histopathology, immunohistochemistry, and RNAscope. Gene expression profile was correlated with grade, invasion, vascular patterns, and VEGF. RESULTS: PIK3C3 and SLC9A3 genes were overexpressed in all vascular patterns, but they were significantly correlated with high VEGF mRNA in the reticular and diffuse pattern. STAT1, JAK2, SHC2, SRF and CHUK (IKK) were exclusively overexpressed in cases with diffuse vascular pattern. SLC9A3, CHUK and STAT3 were overexpressed in G2 tumors. CONCLUSION: Three ccRCC subgroups were defined: 1) PIK3C3 (VSP34)/SLC9A3 which may be proper for anti PIK3C3 inhibitors; 2) VEGFhigh subgroup where association of anti VEGF may be a benefit and 3) JAK2/STAT1 subgroup, potentially being eligible for anti JAK/STAT therapy associated with IKK inhibitors.

The Mutually Mediated Chloride Intracellular Channel Protein 1 (CLIC1) Relationship between Malignant Cells and Tumor Blood Vessel Endothelium Exhibits a Significant Impact on Tumor Angiogenesis, Progression, and Metastasis in Clear Cell Renal Cell Carcinoma (ccRCC).

Background: Overexpression of chloride intracellular channel protein 1 (CLIC1) in tumor cells has been confirmed, but it has received less attention in the tumor blood vessel endothelium. Aim: The assessment of CLIC1 expression in ccRCC tumor blood vessels and its relationship with TNM parameters and tumor cell CLIC1 expression. Methods: CLIC1 immunostaining in ccRCC was evaluated in 50 cases in both malignant cells and tumor blood vessels (CLIC1 microvessel density-CLIC1-MVD) and was correlated with TNM staging parameters. Results: CLIC1-MVD was observed in approximately 65% of cases, and CLIC1 co-localization in both tumor and endothelial cells was observed in 59% of cases. ccRCC was classified into four groups (Classes 0−3) based on the percentage of positive tumor cells, with each group including sub-groups defined by CLIC1 expression in the endothelium. Class 3 (60−100% positive tumor cells) had the highest CLIC1-MVD, with an impact on T and M parameters (p value = 0.007 for T, and p value = 0.006 for M). For cases with CLIC1 intracellular translocation, there was a strong correlation between CLIC1-MVD and M (p value < 0.001). Conclusions: Co-expression of ccRCC tumor and endothelial cells promotes tumor progression and metastasis and should be investigated further as a potential therapeutic target for ccRCC and other human malignancies.

Anti-chloride Intracellular Channel Protein 1 (CLIC1) Antibodies Induce Tumour Necrosis and Angiogenesis Inhibition on In Vivo Experimental Models of Human Renal Cancer.

BACKGROUND/AIM: Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models. MATERIALS AND METHODS: Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques. RESULTS: RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy. CONCLUSION: Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models.

Tumour-associated Angiogenesis and Intermediate Blood Vessels in Renal Cell Carcinoma.

Background/Aim: Renal cell carcinoma is strongly vascularized, and formation of new blood vessels is a complex and multi-step process. In this study, we analysed the subtypes of intermediate blood vessels, as shown by double immunohistochemistry. Materials and Methods: Tumour-associated blood vessels were identified by double immunostaining based on CD34 and smooth muscle cell actin. Blood vessels were classified both quantitatively and qualitatively based on the expression of the aforementioned two markers. The main criteria to sub-classify intermediate blood vessels was the presence, distribution, and arrangement of perivascular cells. Results: We described three subtypes of intermediate blood vessels found particularly in the tumour area: Subtype 1 lacked perivascular cells, subtype 2 showed scattered pericytes attached to the vascular wall, and subtype 3 showed a continuous layer of perivascular cells on one side. Conclusion: We describe for the first time three subtypes of renal cell carcinoma-associated intermediate blood vessels, which could be important in prognosis and as potential targets for anti-vascular therapy.

Does sleep deprivation alter virtual reality-based robotic surgical skills?

INTRODUCTION: Robotic surgery is widely used in many surgical specialities, and there has been no study to assess the impact of sleep deprivation on the complex environment of robotic surgery. AIM: To compare specific metrics of selected robotic simulator exercises on sleep-deprived and non-sleep-deprived surgical residents. MATERIAL AND METHODS: We enrolled 20 volunteers, residents in surgery, evaluated before and after an 18-hour overnight shift, regarding their results on virtual robotic surgery simulator - the sleep deprivation (SD) group. As a control group, the same subjects were evaluated 5-7 days after the post-shift evaluation, without having a shift overnight and at least 7 h of sleep the previous night - the non-sleep-deprivation (nSD) group. RESULTS: A statistically significant difference between the pre-shift and post-shift overall results for all exercises in the SD group and no statistical differences for the nSD group were observed. As the difficulty of the exercises increased, statistical differences were observed on specific metrics for all exercises between the pre-shift and post-shift as well as between the post-shift and the morning after a normal sleep period overnight. In a subgroup analysis, the overall results revealed a stronger statistical difference between pre-shift and post-shift for residents with more intense sleep deprivation (< 3 h of sleep vs. > 3 h of sleep). CONCLUSIONS: Sleep deprivation leads to impairment of surgical skills assessed by robotic virtual simulator. The more complex and skill demanding the exercise, the higher the difference between sleep deprived and non-deprived residents.

The impact of tiredness on virtual reality robotic surgical skills.

INTRODUCTION: The effect of tiredness has been proved for the surgeons' musculature performing laparoscopic or robotic procedures (physical stress). Mental stress after robotic surgery has been reported as well. It is still unclear how much the surgical skills are altered and which types of skills are more affected at the final steps of long, complex robotic surgical procedures. AIM: To evaluate to what extent the surgeon's skills are influenced by long procedures, using the objective assessment of different surgical skills by a virtual reality robotic simulator. MATERIAL AND METHODS: Fifteen surgeons were asked to perform a continuous 4 h virtual robotic surgical simulator training session. At the beginning of simulator training and at the end of each of the 4 h of training, three exercises of increasing difficulty were selected to be performed in order to assess the surgeons' skills. RESULTS: There were statistically significant differences between the initial and final overall scores for all the three exercises, the final outcomes being inferior. The specific metrics for each exercise slightly improved within 1 h from the beginning and thereafter decreased to a statistically significantly inferior value. CONCLUSIONS: The specific metrics on the virtual reality robotic surgical simulator were altered after a 4-hour console training period. Further larger and more complex studies are necessary to evaluate the translation from the simulator to real-life robotic surgery.

Urinary Microbiota-Are We Ready for Prime Time? A Literature Review of Study Methods' Critical Steps in Avoiding Contamination and Minimizing Biased Results.

Within the last few years, there have been an increased number of clinical studies involving urinary microbiota. Low-biomass microbiome sequencing (e.g., urine, lung, placenta, blood) is easily biased by contamination or cross-contamination. So far, a few critical steps, from sampling urine to processing and analyzing, have been described (e.g., urine collection modality, sample volume size, snap freezing, negative controls usage, laboratory risks for contamination assessment, contamination of negative results reporting, exploration and discussion of the impact of contamination for the final results, etc.) We performed a literature search (Pubmed, Scopus and Embase) and reviewed the published articles related to urinary microbiome, evaluating how the aforementioned critical steps to obtain unbiased, reliable results have been taken or have been reported. We identified different urinary microbiome evaluation protocols, with non-homogenous reporting systems, which can make gathering results into consistent data for similar topics difficult and further burden the already so complex emerging field of urinary microbiome. We concluded that to ease the progress in this field, a joint approach from researchers, authors and publishers would be necessary in order to create mandatory reporting systems which would allow to recognize pitfalls and avoid compromising a promising field of research.

Ethical, legal and clinical aspects of live surgery in urology - contemporary issues and a glimpse of the future.

Beside dry and wet lab training, simulators, video tapes, fellowships and clinical visits, live surgery has gained popularity during the last years, being an attraction point at large scientific meetings and at postgraduate courses as well. This type of surgical training raises both ethical and legal issues. Thus, there are professional societies that have banned such meetings, mainly due to safety reasons for the patient. The current article aims to identify and to discuss ethical and legal issues related to the topic, advantages, disadvantages and weak points of this emerging challenge for modern medicine, trying to analyze the issues from all relevant points of view: those of the patient, the surgeon and the session attendant.

Does previous open surgical experience have any influence on robotic surgery simulation exercises?

INTRODUCTION: Within the last years, there has been a trend in many hospitals to switch their surgical activity from open/laparoscopic procedures to robotic surgery. Some open surgeons have been shifting their activity to robotic surgery. It is still unclear whether there is a transfer of open surgical skills to robotic ones. AIM: To evaluate whether such transfer of skills occurs and to identify which specific skills are more significantly transferred from the operative table to the console. MATERIAL AND METHODS: Twenty-five volunteers were included in the study, divided into 2 groups: group A (15 participants) - medical students (without any surgical experience in open, laparoscopic or robotic surgery); and group B (10 participants) - surgeons with exclusively open surgical experience, without any previous laparoscopic or robotic experience. Participants were asked to complete 3 robotic simulator console exercises structured from the easiest one (Peg Board) to the toughest one (Sponge Suture). Overall scores for each exercise as well as specific metrics were compared between the two groups. RESULTS: There were no significant differences between overall scores of the two groups for the easiest task. Overall scores were better for group B as the exercises got more complex. For the intermediate and high-difficulty level exercises, most of the specific metrics were better for group B, with the exception of the working master space item. CONCLUSIONS: Our results suggest that the open surgical skills transfer to robotic skills, at least for the very beginning of the training process.

The Involvement of PDGF-B/PDGFRß Axis in the Resistance to Antiangiogenic and Antivascular Therapy in Renal Cancer.

BACKGROUND/AIM: Studies developed in the field of platelet-derived growth factors/platelet-derived growth factor receptors (PDGFs/PDGFRs) inhibition have focused on the therapeutic effects on tumor cells, neglecting their potential effects on tumor blood vessels. We herein propose a differential and critic assessment of platelet-derived growth factor B (PDGF-B) and platelet-derived growth factor receptor ß (PDGFRß) in renal cell carcinoma, correlated with the four main vascular patterns previously reported by our team. MATERIALS AND METHODS: PDGF-B and PDGFRß were evaluated on 50 archival paraffin embedded specimens related to vascular endothelial growth factor (VEGF), its inhibitory isoform VEGF165b and vascular patterns. RESULTS AND CONCLUSION: Our results support the involvement of VEGF165b in the phosphorylation of PDGFRß with an inhibitory effect on endothelial proliferation and migration. The simultaneous action of PDGF-B/PDGFRß and VEGF165b on the same type of receptor may explain the resistance to antiangiogenic therapy, which depends on the degree of modulation of PDGFRß phosphorylation.

Endostatin Effects on Tumor Cells and Vascular Network of Human Renal Cell Carcinoma Implanted on Chick Embryo Chorioallantoic Membrane.

Rare and inconsistent data are reported for chorioallantoic tumor models of renal cell carcinoma and none of them has used endostatin as an inhibitory agent of tumor development. We aimed to perform a comparative analysis of tumor cells and blood vessels from renal cell carcinoma on endostatin-treated and non-treated chorioallantoic membrane (CAM) implants by the assessment of endoglin, vascular endothelial growth factor (VEGF) and smooth muscle actin expression. Endostatin triple action on tumor, endothelial and perivascular cells was observed in the present study. Differential impact of endostatin treatment on intratumor and peritumor blood vessels was noticed on the VEGF expression and behaviour of tumor cells between clear cell and papillary components of RCC. Based on our findings, a high tumor heterogenity response to endostatin has been highlighted. Interplay between VEGF, endoglin and endostatin in RCC could support a combined targeted therapy to improve prognosis of patients with RCC and reduce therapy resistance often developed by monotherapy.