Downstaging and Survival Associated with Neoadjuvant Immunotherapy Before Radical Cystectomy for Muscle-invasive Bladder Cancer.

BACKGROUND: Neoadjuvant cisplatin-containing chemotherapy before radical cystectomy is the standard of care for patients with localized muscle-invasive bladder cancer (MIBC). However, a large proportion of patients are ineligible for cisplatin. Single-arm phase 2 neoadjuvant immunotherapy trials have reported promising tumor response rates, but interpretation is limited owing to lack of a comparator arm. OBJECTIVE: To compare rates of pathologic downstaging and overall survival between patients receiving neoadjuvant immunotherapy (NAI), neoadjuvant chemotherapy (NAC), or no neoadjuvant therapy (NNAT). DESIGN, SETTING, AND PARTICIPANTS: We identified 18 483 patients in the National Cancer Data Base who were diagnosed with clinically localized MIBC and underwent radical cystectomy from 2014 to 2019. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Nearest-neighbor propensity-score caliper matching was used to create three demographically similar and equally sized cohorts stratified by NAT receipt. Logistic regression was used to examine the association of treatment received with pathologic downstaging to pT0N0 and pT < 2N0. Cox proportional-hazards regression was used to assess the association of treatment received with overall survival (OS). RESULTS AND LIMITATIONS: Propensity score matching yielded three equally sized cohorts without significant differences in baseline characteristics (n = 840). The NAI group had a higher rate of pathologic downstaging to pT0N0 than the NNAT group and a similar rate to the NAC group (NNAT 6.7% vs NAC 26.4%, odds ratio 5.0, 95% confidence interval [CI] 2.9-8.3; NAI 22.5%, odds ratio 4.0, 95% CI 2.4-7.1). The NAI group had better OS than the NNAT group and similar OS to the NAC group (NAC: hazard ratio 0.62, 95% CI 0.42-0.92; NAI: hazard ratio 0.68, 95% CI 0.46-0.97, with NNAT as the reference). The primary limitation is selection bias from confounding by clinical indication. CONCLUSIONS: NAI is a promising alternative to NAC for patients with clinically localized MIBC, as evidenced by similar pathologic downstaging rates and OS benefits in comparison to no NAT. Phase 3 trials should be conducted to test the noninferiority of NAI to NAC. PATIENT SUMMARY: We compared outcomes for patients with muscle-invasive bladder cancer according to whether they received chemotherapy, immunotherapy, or no medical therapy before surgical removal of their bladder. We found that preoperative immunotherapy improved patient survival and regression of the cancer stage in comparison to no medical therapy, similar to the outcomes seen with preoperative chemotherapy. Randomized clinical trials are needed to confirm these findings.

Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment.

Background: Immunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naïve and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates. Results: 21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naïve tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin αV+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. In CPTAC, collagen IV protein was more abundant in advanced stages of disease. Conclusions: On spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naïve tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC.

Predicting Limited Survival Following Inguinal Lymph Node Dissection in Penile Cancer: Should We Revisit the Goals of Care?

OBJECTIVE: Patients with advanced penile squamous cell cancer have a poor prognosis and can benefit from early palliative care consultation. We built a model to identify those patients most likely to benefit. METHODS: Patients with penile squamous cell cancer undergoing inguinal lymph node dissection were identified from the National Cancer Database (NCDB) and a multi-institutional international dataset (INT). A multivariable Cox proportional hazards model for overall survival (OS) was developed using the NCDB and applied to the INT dataset. Parameters were used to make receiver operating characteristic (ROC) curves. ROC-related criteria were optimized to identify a predictive probability cut point and dichotomize patients from INT into risk groups for limited OS of <6 and <12 months. RESULTS: NCDB had 860 deaths; 105 (5%) at 6 months and 296 (15%) at 12 months. INT had 257 deaths; 56 (8%) at 6 months and 124 (18%) at 12 months. Limited OS was associated with older age, greater T and N stage, and fewer lymph nodes removed. Optimized ROC criteria using the OS <6 months curve best dichotomized INT patients into high-risk group with median OS of 24 months (95% CI 18-34) and low-risk group with median OS of 174 months (95% CI 120-NE). CONCLUSION: We developed a simple model that could be used as a screening tool for early palliative care referral.

Geospatial characterization of immune cell distributions and dynamics across the microenvironment in clear cell renal cell carcinoma.

INTRODUCTION: In clear cell renal cell carcinoma (ccRCC), tumor-associated macrophage (TAM) induction of CD8+T cells into a terminally exhausted state has been implicated as a major mechanism of immunotherapy resistance, but a deeper biological understanding is necessary. METHODS: Primary ccRCC tumor samples were obtained from 97 patients between 2004 and 2018. Multiplex immunofluorescence using lymphoid and myeloid markers was performed in seven regions of interest per patient across three predefined zones, and geospatial analysis was performed using Ripley's K analysis, a methodology adapted from ecology. RESULTS: Clustering of CD163+M2 like TAMs into the stromal compartment at the tumor-stroma interface was associated with worse clinical stage (tumor/CD163+nK(75): stage I/II: 4.4 (IQR -0.5 to 5.1); stage III: 1.4 (IQR -0.3 to 3.5); stage IV: 0.6 (IQR -2.1 to 2.1); p=0.04 between stage I/II and stage IV), and worse overall survival (OS) and cancer-specific survival (CSS) (tumor/CD163+nK(75): median OS-hi=149 months, lo=86 months, false-discovery rate (FDR)-adj. Cox p<0.001; median CSS-hi=174 months, lo=85 months; FDR-adj. Cox p<0.001). An RNA-seq differential gene expression score was developed using this geospatial metric, and was externally validated in multiple independent cohorts of patients with ccRCC including: TCGA KIRC, and the IMmotion151, IMmotion150, and JAVELIN Renal 101 clinical trials. In addition, this CD163+ geospatial pattern was found to be associated with a higher TIM-3+ proportion of CD8+T cells, indicative of terminal exhaustion (tumor-core: 0.07 (IQR 0.04-0.14) vs 0.40 (IQR 0.15-0.66), p=0.05). CONCLUSIONS: Geospatial clustering of CD163+M2 like TAMs into the stromal compartment at the tumor-stromal interface was associated with poor clinical outcomes and CD8+T cell terminal exhaustion.

Selecting patients for magnetic resonance imaging cognitive versus ultrasound fusion biopsy of the prostate: A within-patient comparison.

Objectives: To compare overall agreement between magnetic resonance imaging (MRI)-ultrasound (US) fusion biopsy (FB) and MRI cognitive fusion biopsy (CB) of the prostate and determine which factors affect agreement for prostate cancer (PCa) who underwent both modalities in a prospective within-patient protocol. Patients and Methods: From August 2017 to January 2021, patients with at least one Prostate Imaging Reporting & Data System (PI-RADS) 3 or higher lesion on multiparametric MRI underwent transrectal FB and CB in a prospective within-patient protocol. CB was performed for each region of interest (ROI), followed by FB, followed by standard 12 core biopsy. Patients who were not on active surveillance were analysed. The primary endpoint was agreement for any PCa detection. McNemar's test and kappa statistic were used to analyse agreement. Chi-square test, Fisher's exact test and Wilcoxon rank sum test were used to analyse disagreement across clinical and MRI spatial variables. A multivariable generalized mixed-effect model was used to compare the interaction between select variables and fusion modality. Statistics were performed using SAS and R. Results: Ninety patients and 98 lesions were included in the analysis. There was moderate agreement between FB and CB (k = 0.715). McNemar's test was insignificant (p = 0.285). Anterior location was the only variable associated with a significant variation in agreement, which was 70% for anterior lesions versus 89.7% for non-anterior lesions (p = 0.035). Discordance did not vary significantly across other variables. In a mixed-effect model, the interaction between anterior location and use of FB was insignificant (p = 0.411). Conclusion: In a within-patient protocol of patients not on active surveillance, FB and CB performed similarly for PCa detection and with moderate agreement. Anterior location was associated with significantly higher disagreement, whereas other patient and lesion characteristics were not. Additional studies are needed to determine optimal biopsy technique for sampling anterior ROI.

Proteogenomic, Epigenetic, and Clinical Implications of Recurrent Aberrant Splice Variants in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Alternative mRNA splicing can be dysregulated in cancer, resulting in the generation of aberrant splice variants (SVs). Given the paucity of actionable genomic mutations in clear cell renal cell carcinoma (ccRCC), aberrant SVs may be an avenue to novel mechanisms of pathogenesis. OBJECTIVE: To identify and characterize aberrant SVs enriched in ccRCC. DESIGN, SETTING, AND PARTICIPANTS: Using RNA-seq data from the Cancer Cell Line Encyclopedia, we identified neojunctions uniquely expressed in ccRCC. Candidate SVs were then checked for expression across normal tissue in the Genotype-Tissue Expression Project and primary tumor tissue from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and our institutional Total Cancer Care database. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Clinicopathologic, genomic, and survival data were available for all cohorts. Epigenetic data were available for the TCGA and CPTAC cohorts. Proteomic data were available for the CPTAC cohort. The association of aberrant SV expression with these variables was examined using the Kruskal-Wallis test, pairwise t test, Spearman correlation test, and Cox regression analysis. RESULTS AND LIMITATIONS: Our pipeline identified 16 ccRCC-enriched SVs. EGFR, HPCAL1-SV and RNASET2-SV expression was negatively correlated with gene-specific CpG methylation. We derived a survival risk score based primarily on the expression of five SVs (RNASET2, FGD1, PDZD2, COBLL1, and PTPN14), which was consistent and applicable across multiple cohorts on multivariate analysis. The splicing factor RBM4, which modulates splicing of HIF-1α, exhibited significantly lower expression at the protein level in the high-risk group, as defined by our SV-based score. CONCLUSIONS: We describe 16 aberrant SVs enriched in ccRCC, many of which are associated with disease biology and/or clinical outcomes. This study provides a novel strategy for identifying and characterizing disease-specific aberrant SVs. PATIENT SUMMARY: We describe a method to identify disease targets and biomarkers using transcriptomic analysis beyond somatic mutations or gene expression. Kidney tumors express unique splice variants that may provide additional prognostic information following surgery.

A Targetable Myeloid Inflammatory State Governs Disease Recurrence in Clear-Cell Renal Cell Carcinoma.

It is poorly understood how the tumor immune microenvironment influences disease recurrence in localized clear-cell renal cell carcinoma (ccRCC). Here we performed whole-transcriptomic profiling of 236 tumors from patients assigned to the placebo-only arm of a randomized, adjuvant clinical trial for high-risk localized ccRCC. Unbiased pathway analysis identified myeloid-derived IL6 as a key mediator. Furthermore, a novel myeloid gene signature strongly correlated with disease recurrence and overall survival on uni- and multivariate analyses and is linked to TP53 inactivation across multiple data sets. Strikingly, effector T-cell gene signatures, infiltration patterns, and exhaustion markers were not associated with disease recurrence. Targeting immunosuppressive myeloid inflammation with an adenosine A2A receptor antagonist in a novel, immunocompetent, Tp53-inactivated mouse model significantly reduced metastatic development. Our findings suggest that myeloid inflammation promotes disease recurrence in ccRCC and is targetable as well as provide a potential biomarker-based framework for the design of future immuno-oncology trials in ccRCC. SIGNIFICANCE: Improved understanding of factors that influence metastatic development in localized ccRCC is greatly needed to aid accurate prediction of disease recurrence, clinical decision-making, and future adjuvant clinical trial design. Our analysis implicates intratumoral myeloid inflammation as a key driver of metastasis in patients and a novel immunocompetent mouse model. This article is highlighted in the In This Issue feature, p. 2221.

Survival Outcomes Associated With Cytoreductive Nephrectomy in Patients With Metastatic Clear Cell Renal Cell Carcinoma.

Importance: Level I evidence has failed to demonstrate an overall survival (OS) advantage for cytoreductive nephrectomy in patients with metastatic clear cell renal cell carcinoma (ccRCC) in the modern era, which is at odds with observational studies reporting a marked OS benefit associated with these operations. These observational studies were not designed to adjust for unmeasured confounding. Objective: To assess whether cytoreductive nephrectomy is associated with improved OS in patients with metastatic ccRCC. Design, Setting, and Participants: This cohort study identified patients with metastatic ccRCC in the National Cancer Database from January 1, 2006, to December 31, 2016, who received systemic targeted therapy. The analysis was finalized on July 23, 2021. Exposures: Receipt of cytoreductive nephrectomy. Main Outcomes and Measures: The primary outcome was OS from the date of diagnosis to death or censoring at last follow-up. Distance from the patients' zip code of residence to the treating facility was identified as a valid instrument and was used in a 2-stage residual inclusion instrumental variable analysis. Conventional adjustments for selection bias, multivariable Cox proportional hazards regression, and propensity score matching were performed for comparison. Measured covariates adjusted for in all analyses included age, sex, race, Charlson-Deyo score, facility type, year of diagnosis, clinical T stage, and clinical N stage. Results: The final study population included 12 766 patients (median age, 63 years; IQR, 56-70 years; 8744 [68%] male; 11 206 [88%] White). Cytoreductive nephrectomy was performed in 5005 patients (39%). Conventional adjustments for selection bias demonstrated a significant OS benefit associated with cytoreductive nephrectomy (multivariable Cox proportional hazards regression: hazard ratio [HR], 0.49; 95% CI, 0.47-0.51; propensity score matching: HR, 0.48; 95% CI, 0.46-0.50). Instrumental variable estimates did not demonstrate an association between cytoreductive nephrectomy and OS (HR, 0.92; 95% CI, 0.78-1.09). Conclusions and Relevance: Instrumental variable analysis did not demonstrate a survival advantage associated with cytoreductive nephrectomy for patients with metastatic ccRCC. This discrepancy likely reflects the fact that surgical indication for cytoreductive nephrectomy is primarily driven by factors that are not commonly measured or available in observational data sets.

Evaluation of Patient-Reported Outcomes (PROs) Protocol Content and Reporting for Clinical Trials that Lead to the approval of frontline Immune Checkpoint Blockade Combination for Patients with Advanced Renal Cell Carcinoma - The Patients' Voice or a Missed Opportunity.

INTRODUCTION: Immune checkpoint blockade (ICB) is a rapidly emerging field of oncology that has revolutionized the metastatic renal cell carcinoma (mRCC) treatment. Four recent treatment regimens Nivolumab-Ipilimumab, Pembrolizumab-Axitinib, Nivolumab-Cabozantinib, and Pembrolizumab-Lenvatinib-have demonstrated improved clinical endpoints compared to standard of care and are endorsed by NCCN (2021). However, data on patient-reported outcomes (PROs) for patients receiving these regimens are limited. We conducted a comparative assessment of the quality and standardization of PROs endpoints and data reported for these randomized controlled trials (RCTs). PATIENTS AND METHODS: We systematically identified all RCTs evaluating combination ICB for ccRCC. PROs-specific data were abstracted from the final version of 4 RCT protocols, as well as clinical and PROs specific manuscripts published between April 2018 and April 2021. We used 3 previously published guides standardizing PROs research to objectively score the data: (i) 24-point PROEAS; (ii) 12-point SPIRIT-PRO; and (iii) 14-point CONSORT-PRO. RESULTS: The CheckMate 214, KEYNOTE 426, CheckMate 9ER, and CLEAR studies had PROEAS scores of 88% (21/24), 37% (9/24), 83% (20/24), and 16% (4/24), respectively, and SPIRIT-PRO scores of 50% (6/12), 75% (9/12), 66% (8/12), and 41% (5/12) respectively. The CONSORT-PRO scores were 86% (12/14) for CheckMate 214 and 43% (6/14) for CheckMate 9ER, but scores were not available for the CLEAR and KEYNOTE 426 studies because of a lack of sufficient data. The average SPIRIT-PRO score across the 4 RCTs was 58%, indicating a reasonable adoption of PROs research in data management and analysis. The CheckMate 214 trial had the longest follow-up and most comprehensive published PROs data. CONCLUSION: Our analysis identified the limitations of current PROs data in combination ICB approved for mRCC. This analysis will enable clinicians to better interpret the current PROs results and emphasize the importance of better incorporation of PROs endpoints in future mRCC trial design.

Quantification of T- and B-cell Immune Receptor Distribution Diversity Characterizes Immune Cell Infiltration and Lymphocyte Heterogeneity in Clear Cell Renal Cell Carcinoma.

Immune-modulating systemic therapies are often used to treat advanced cancer such as metastatic clear cell renal cell carcinoma (ccRCC). Used alone, sequence-based biomarkers neither accurately capture patient dynamics nor the tumor immune microenvironment. To better understand the tumor ecology of this immune microenvironment, we quantified tumor infiltration across three distinct ccRCC patient tumor cohorts using complementarity determining region-3 (CDR3) sequence recovery counts in tumor-infiltrating lymphocytes and a generalized diversity index (GDI) for CDR3 sequence distributions. GDI can be understood as a curve over a continuum of diversity scales that allows sensitive characterization of distributions to capture sample richness, evenness, and subsampling uncertainty, along with other important metrics that characterize tumor heterogeneity. For example, richness quantified the total unique sequence count, while evenness quantified similarities across sequence frequencies. Significant differences in receptor sequence diversity across gender and race revealed that patients with larger and more clinically aggressive tumors had increased richness of recovered tumoral CDR3 sequences, specifically in those from T-cell receptor alpha and B-cell immunoglobulin lambda light chain. The GDI inflection point (IP) allowed for a novel and robust measure of distribution evenness. High IP values were associated with improved overall survival, suggesting that normal-like sequence distributions lead to better outcomes. These results propose a new quantitative tool that can be used to better characterize patient-specific differences related to immune cell infiltration, and to identify unique characteristics of tumor-infiltrating lymphocyte heterogeneity in ccRCC and other malignancies. SIGNIFICANCE: Assessment of tumor-infiltrating T-cell and B-cell diversity in renal cell carcinoma advances the understanding of tumor-immune system interactions, linking tumor immune ecology with tumor burden, aggressiveness, and patient survival. See related commentary by Krishna and Hakimi, p. 764.

Comparative effectiveness analysis of first-line immunotherapy versus chemotherapy in metastatic urothelial carcinoma of the bladder.

BACKGROUND: Clinical trials have not shown a significant overall survival (OS) difference between chemotherapy and immunotherapy as first-line agents in metastatic urothelial carcinoma (UC). However, the generalizability of these findings in a real-world setting has not yet been evaluated in comparative effectiveness studies. OBJECTIVE: To assess the effectiveness of first-line immunotherapy compared with chemotherapy regimens on OS in patients with metastatic UC of the bladder. DESIGN, SETTING, AND PARTICIPANTS: This retrospective propensity-matched study identified metastatic bladder UC patients in the National Cancer Database from 2014 to 2017 who received either first-line immunotherapy-monotherapy or multi-agent chemotherapy, and who were not treated on a clinical trial protocol. OUTCOME MEASURES AND ANALYSIS: The primary outcome was OS from the date of diagnosis to date of death or censoring at last follow-up. Patients were stratified into first-line immunotherapy and chemotherapy treatment groups. After 1:1 nearest-neighbor caliper-matching of propensity scores, the survival analysis was conducted using Cox regression modeling and Kaplan-Meier estimates. RESULTS AND LIMITATIONS: A total of 2,796 patients were included in the final study population, and 960 in the matched cohort (480 per treatment group). Utilization of immunotherapy increased over the time period studied as chemotherapy decreased (Immunotherapy: 3%-37%; Chemotherapy: 97%-63%; P < 0.001). In the overall cohort, patients who received first-line immunotherapy were older and more comorbid than those who received first-line chemotherapy (Age: 73 v. 67, respectively, P < 0.001; Charlson-Deyo score ≥2: 17% v. 11.5%, respectively, P < 0.001). In the matched cohort, patients who were treated with first-line immunotherapy had similar OS to those who were treated with first-line chemotherapy (HR: 0.91, 95CI 0.72-1.15). Due to the retrospective nature of the study, interpretation is limited by potential selection bias from unmeasured confounding. CONCLUSIONS AND RELEVANCE: Metastatic bladder UC patients who received first-line immunotherapy had similar OS to those who received first-line chemotherapy.

Correlating Immune Cell Infiltration Patterns with Recurrent Somatic Mutations in Advanced Clear Cell Renal Cell Carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) tumors have low frequencies of genetic alterations compared with other malignancies, but very high levels of immune cell infiltration and favorable response rates to immunotherapy. Currently, the interplay between specific ccRCC somatic mutations and immune infiltration pattern is unclear. OBJECTIVE: To analyze the associations between common ccRCC somatic mutations and immune cell infiltration patterns within the tumor immune microenvironment (TIME). DESIGN, SETTING, AND PARTICIPANTS: The study included tumor samples (24 primary and 24 metastatic) from 48 patients with stage IV ccRCC. Targeted sequencing was performed for well-characterized recurrent somatic mutations in ccRCC, with the analysis focusing on the six most common ones: VHL, BAP1, PBRM1, SETD2, TP53, and KDM5C. For each sample, multiplex immunofluorescence (IF) was performed in lymphoid and myeloid panels, for seven regions of interest in three zones (tumor core, stroma, and tumor-stroma interface). IF-derived cellular densities were compared across patients, stratified by their somatic mutation status, using a linear mixed-model analysis. External validation was pursued using RNA-seq enrichment scoring from three large external data sources. RESULTS AND LIMITATIONS: Tumors with SETD2 mutations demonstrated significantly decreased levels of FOXP3+ T cells in the tumor core, stroma, and tumor-stroma interface. PBRM1 mutations were associated with decreased FOXP3+ T cells in the tumor core. Primary KDM5C mutations were associated with significantly increased CD206+ macrophage tumor infiltration in the tumor core. A computational method estimating immune cell types in the TIME using bulk RNA-seq data, xCell scoring, failed to validate associations from the IF analysis in large external data sets. A major limitation of the study is the relatively small patient population studied. CONCLUSIONS: This study provides evidence that common somatic mutations in ccRCC, such as SETD2, PBRM1, and KDM5C, are associated with distinct immune infiltration patterns within the TIME. PATIENT SUMMARY: In this study, we analyzed tumor samples from patients with metastatic kidney cancer to determine whether common genetic mutations that arise from the cancer cells are associated with the density of immune cells found within those tumors. We found several distinct immune cell patterns that were associated with specific genetic mutations. These findings provide insight into the interaction between cancer genetics and the immune system in kidney cancer.

spatialTIME and iTIME: R package and Shiny application for visualization and analysis of immunofluorescence data.

SUMMARY: Multiplex immunofluorescence (mIF) staining combined with quantitative digital image analysis is a novel and increasingly used technique that allows for the characterization of the tumor immune microenvironment (TIME). Generally, mIF data is used to examine the abundance of immune cells in the TIME; however, this does not capture spatial patterns of immune cells throughout the TIME, a metric increasingly recognized as important for prognosis. To address this gap, we developed an R package spatialTIME that enables spatial analysis of mIF data, as well as the iTIME web application that provides a robust but simplified user interface for describing both abundance and spatial architecture of the TIME. The spatialTIME package calculates univariate and bivariate spatial statistics (e.g. Ripley's K, Besag's L, Macron's M and G or nearest neighbor distance) and creates publication quality plots for spatial organization of the cells in each tissue sample. The iTIME web application allows users to statistically compare the abundance measures with patient clinical features along with visualization of the TIME for one tissue sample at a time. AVAILABILITY AND IMPLEMENTATION: spatialTIME is implemented in R and can be downloaded from GitHub (https://github.com/FridleyLab/spatialTIME) or CRAN. An extensive vignette for using spatialTIME can also be found at https://cran.r-project.org/web/packages/spatialTIME/index.html. iTIME is implemented within a R Shiny application and can be accessed online (http://itime.moffitt.org/), with code available on GitHub (https://github.com/FridleyLab/iTIME). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Risk factors and survival outcomes for upstaging after inguinal lymph node dissection for cN1 penile squamous cell carcinoma.

OBJECTIVES: To identify incidence and risk factors for upstaging from cN1 to pN2/N3 at inguinal lymphadenectomy (ILND) for penile cancer (pSCC). Our secondary objective is to assess survival outcomes and associations for cN1 patients undergoing ILND. SUBJECTS/PATIENTS AND METHODS: Patients with pT≥1cN1cM0 pSCC who underwent bilateral ILND and had complete data were identified in a multi-institutional international cohort from 8 referral centers in 7 countries diagnosed from 1980 to 2017. Upstaging was defined as pN2/N3 at ILND. Multivariable logistic regression analysis was used to determine associations with upstaging, and Cox multivariable logistic regression analysis to determine associations with overall survival (OS). RESULTS: Of 144 patients were included in the final study population. 84 patients (58%) were upstaged from cN1 to pN2/N3, and 25 (17%) were down staged to pN0. Upstaging was associated with pT3/T4 (OR 4.1, 95%CI 1.5-11.7, P < 0.01) and pTX (OR 7.1, 95CI 1.6-51.1, P = 0.02). Age, smoking status, HPV status, and LVI were not associated with upstaging. Age (HR 1.03/y, 95%CI 1.01-1.06, P < 0.01) and upstaging (HR 2.8, 95%CI 1.3-5.9, P < 0.01) were associated with worse OS. Upstaged patients had a 5-year OS of 49%, compared with 86% for patients who were not upstaged. CONCLUSION: The majority of cN1 pSCC patients harbor a higher-risk disease state than their clinical staging suggests, especially those with higher pT stages. More intensive pre-operative workup may be warranted for these patients to identify upstaging prior to ILND and potentially qualify them for neoadjuvant chemotherapy or clinical trials.

Geospatial Cellular Distribution of Cancer-Associated Fibroblasts Significantly Impacts Clinical Outcomes in Metastatic Clear Cell Renal Cell Carcinoma.

Cancer-associated fibroblasts (CAF) are highly prevalent cells in the tumor microenvironment in clear cell renal cell carcinoma (ccRCC). CAFs exhibit a pro-tumor effect in vitro and have been implicated in tumor cell proliferation, metastasis, and treatment resistance. Our objective is to analyze the geospatial distribution of CAFs with proliferating and apoptotic tumor cells in the ccRCC tumor microenvironment and determine associations with survival and systemic treatment. Pre-treatment primary tumor samples were collected from 96 patients with metastatic ccRCC. Three adjacent slices were obtained from 2 tumor-core regions of interest (ROI) per patient, and immunohistochemistry (IHC) staining was performed for αSMA, Ki-67, and caspase-3 to detect CAFs, proliferating cells, and apoptotic cells, respectively. H-scores and cellular density were generated for each marker. ROIs were aligned, and spatial point patterns were generated, which were then used to perform spatial analyses using a normalized Ripley's K function at a radius of 25 µm (nK(25)). The survival analyses used an optimal cut-point method, maximizing the log-rank statistic, to stratify the IHC-derived metrics into high and low groups. Multivariable Cox regression analyses were performed accounting for age and International Metastatic RCC Database Consortium (IMDC) risk category. Survival outcomes included overall survival (OS) from the date of diagnosis, OS from the date of immunotherapy initiation (OS-IT), and OS from the date of targeted therapy initiation (OS-TT). Therapy resistance was defined as progression-free survival (PFS) <6 months, and therapy response was defined as PFS >9 months. CAFs exhibited higher cellular clustering with Ki-67+ cells than with caspase-3+ cells (nK(25): Ki-67 1.19; caspase-3 1.05; p = 0.04). The median nearest neighbor (NN) distance from CAFs to Ki-67+ cells was shorter compared to caspase-3+ cells (15 µm vs. 37 µm, respectively; p < 0.001). Multivariable Cox regression analyses demonstrated that both high Ki-67+ density and H-score were associated with worse OS, OS-IT, and OS-TT. Regarding αSMA+CAFs, only a high H-score was associated with worse OS, OS-IT, and OS-TT. For caspase-3+, high H-score and density were associated with worse OS and OS-TT. Patients whose tumors were resistant to targeted therapy (TT) had higher Ki-67 density and H-scores than those who had TT responses. Overall, this ex vivo geospatial analysis of CAF distribution suggests that close proximity clustering of tumor cells and CAFs potentiates tumor cell proliferation, resulting in worse OS and resistance to TT in metastatic ccRCC.

Educational value of the transplant experience in urology residency.

INTRODUCTION To evaluate the educational value of transplant rotation in urology residency. In the United States, exposure to kidney transplantation during urology residency has declined significantly over the past few decades. At our institution, transplantation has been a core component of urology residency since its inception in 1959. MATERIALS AND METHODS: A 15-question anonymous survey was developed. The first 8 questions queried demographics and the last 7 were a set of questions with a Likert Scale response. The survey was electronic- mailed to past and current urology residents who had completed the transplant rotation, dating back to 1972. RESULTS: A total of 61 out of 98 (62%) individuals responded. The majority (59%) were general urologists, and one (2%) had completed a transplant fellowship. In their practices, 17% performed kidney transplants and 28% performed donor nephrectomies. Overall, 100% responded that the skills learned on the transplant rotation were beneficial for urology training, 100% had learned valuable vascular surgical techniques, and 93% felt that urology residents should have clinical transplant experience during their training. There was no statistical difference between the younger and older graduates in Likert scale responses. CONCLUSION: The majority of graduates did not perform transplants in their practice, yet, all of responders agreed that the skills learned on the transplant rotation were beneficial and 93% expressed that urology residents should have clinical transplant experience during residency. Kidney transplantation should be an integral part of urology residency training.

Perioperative stroke and myocardial infarction in urologic surgery.

INTRODUCTION Perioperative stroke and myocardial infarction are uncommon but devastating thromboembolic complications. There is no comprehensive study detailing these complications for urologic procedures. The primary aim of this study is to determine which urologic procedures and patients carry the highest risk of perioperative stroke and myocardial infarction. MATERIALS AND METHODS: The National Surgical Quality Improvement Program data set was reviewed from 2008-2017. Procedures coded under the urology specialty were included and patients who had a perioperative stroke or myocardial infarction were identified. CPTs were stratified into clinically relevant procedure groups. Two multivariable logistic regression analyses were performed to determine preoperative and procedural risk factors for developing perioperative stroke or myocardial infarction. A multivariable logistic regression analysis was performed to determine the association between these complications and 30-day mortality. RESULTS: A total of 281,744 cases were included, identifying 392 strokes (0.14%) and 1,016 myocardial infarctions (0.36%). Age ≥ 70, hypertension, and disseminated cancer were the strongest preoperative risk factors for perioperative stroke or myocardial infarction. Cystectomy was the highest risk urologic procedure (stroke: OR 3.3, 95%CI 2.3-4.8; MI: OR 7.2, 95%CI 5.6-9.1). Thirty-day mortality was dramatically worse for patients who had a perioperative stroke or myocardial infarction. CONCLUSIONS: Perioperative stroke and myocardial infarction were confirmed to be uncommon but devastating complications of urologic surgery, with incidence of 0.14% and 0.36%, respectively. Cystectomy was the highest risk urologic procedure. Perioperative stroke and myocardial infarction were strongly associated with age ≥ 70, hypertension, and disseminated cancer.