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Penile squamous cell carcinoma (PSCC) is a rare malignancy with limited understanding of the tumor immune microenvironment (TIME). The interplay between PSCC and the immune system across disease progression and HPV infection status remains poorly characterized. This study aims to assess the TIME changes from localized to advanced disease and between HPV-positive versus negative tumors to identify potential immune evasion mechanisms in advanced PSCC. scRNA-seq was performed on ten PSCC tissue samples from penile, lymph node and distant metastatic sites with four matched penile and lymph node samples to understand the cellular heterogeneity within PSCC tumors. Analysis of immune cell populations and transcriptional hallmarks were performed stratified by localized (pT1–3, N0) versus advanced (N1–3, M0 or any N, M1) disease states and HPV infection status. We observed significant differences in immune cell infiltration between localized and advanced PSCC disease states and by HPV status. Advanced disease states demonstrated an exhausted immune phenotype, characterized by terminally exhausted CD8 + T cells, M2-like macrophages and hypoxic signature, while localized disease states demonstrated an active innate immune system characterized by increased DCs. HPV-negative tumors displayed low immune cell infiltration while HPV-positive tumors demonstrated an immune exhausted phenotype. These findings offer valuable insights into the evolving PSCC immune landscape, paving the way for the development of potential therapeutic approaches for advanced PSCC.

There is emerging evidence that the adaptive anti-tumor activity may be orchestrated by secondary lymphoid organ-like aggregates residing in the tumor microenvironment. Known as tertiary lymphoid structures, these lymphoid aggregates serve as key outposts for lymphocyte recruitment, priming and activation. They have been linked to favorable outcomes in many tumor types, and more recently, have been shown to be effective predictors of response to immune checkpoint blockade. We have previously described a 12-chemokine (12-CK) transcriptional score which recapitulates an overwhelming enrichment for immune-related and inflammation-related genes in colorectal carcinoma. Subsequently, the 12-CK score was found to prognosticate favorable survival in multiple tumors types including melanoma, breast cancer, and bladder cancer. In the current study, we summarize the discovery and validation of the 12-CK score in various tumor types, its relationship to TLSs found within the tumor microenvironment, and explore its potential role as both a prognostic and predictive marker in the treatment of various cancers.

Immune infiltration is typically quantified using cellular density, not accounting for cellular clustering. Tumor-associated macrophages (TAM) activate oncogenic signaling through paracrine interactions with tumor cells, which may be better reflected by local cellular clustering than global density metrics. Using multiplex immunohistochemistry and digital pathologic analysis we quantified cellular density and cellular clustering for myeloid cell markers in 129 regions of interest from 55 samples from 35 patients with metastatic ccRCC. CD68+ cells were found to be clustered with tumor cells and dispersed from stromal cells, while CD163+ and CD206+ cells were found to be clustered with stromal cells and dispersed from tumor cells. CD68+ density was not associated with OS, while high tumor/CD68+ cell clustering was associated with significantly worse OS. These novel findings would not have been identified if immune infiltrate was assessed using cellular density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors. Significance : Increased clustering of CD68+ TAMs and tumor cells was associated with worse overall survival for patients with metastatic ccRCC. This effect would not have been identified if immune infiltrate was assessed using cell density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors.

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.

BackgroundEmerging evidence from studies in sarcoma and melanoma immune checkpoint blockade(ICB) trials demonstrated enhanced efficacy in tumors harbouring tertiary lymphoid structures(TLS)1–3 - lymph node like aggregates in the tumor microenvironment postulated to recruit lymphocyte infiltration and coordinate tumor antigen presentation and lymphocyte priming. Previously, our group described a 12-chemokine metagene signature that reflects strong intratumoral chemotactic signalling and accurately predicts the presence of TLS in colorectal carcinoma.4 Grounded in these works, we attempted to correlate high 12CK signature with TLS formation in the context of muscle invasive bladder cancer(MIBC), and then sought to define its prognostic implications and its ability to predict response to ICB.MethodsA total of 130 MIBC samples were arrayed on Affymetrix microarrays and 12CK scores were assessed. Scores were normalized using 12CK>1 to denote 12CK-High(n=24). We then investigated the the presence of TLS with the associated immune cellular infiltration evaluated by immunohistochemistry and gene signature deconvolution method (xCell).5 12CK scores were also correlated with survival in our institutional cohort and validated using data from TCGA. Finally, 12CK scores were extracted from the IMVIGOR210 study to examine its ability to predict response to ICB.6 ResultsType III TLS, consisting of germinal center-like structures and discrete T-cell zones were found in 7/22 12CK-High vs. 0/21 12CK-Low tumors (p=0.009) (figure 1a). Additionally, a more robust immuno-environment was seen in 12CK-High tumors, consisting of increased infiltration of CD4+ T lymphocytes (p=0.1), CD8+ T lymphocytes (p=0.02), activated dendritic cells (p=0.047), and B lymphocytes (p=0.006) on immunohistochemistry (figure 1b). Furthermore, on xCell deconvolution, M1 macrophage, NK cells, CD8+ Tem, CD4+ Tem, and memory B cells were enriched in 12CK-High tumors, suggesting both a heightened innate and adaptive immune response (figure 1c, d). Kaplan-Meier survival analyses of our internal cohort revealed improved PFS (HR 0.25, p=0.003 ), CSS (HR 0.25, p=0.003), and OS (HR 0.55, p=0.03) amongst 12CK-High patients (figure 2a-c). From the TCGA, similar improvements were found in PFS (HR 0.55, p=0.007), CSS (HR 0.40, p=0.002), and OS (HR 0.59, p=0.01) in 12CK-High patients (figure 2d-f). From the IMVIGOR 210 study, complete responders exhibited significantly higher 12-CK scores than all other groups (figure 2g). Strikingly, the 12CK-High signature conferred a median overall survival benefit of almost 1 year in the atezolizumab-treated patients (figure 2h).Abstract 68 Figure 1High 12-chemokine score (12-CK) is associated with the presence of tertiary lymphoid structures (TLS) in the tumor microenvironment (TME) and a more robust peritumoral inflammatory response. (A) H&E and immunohistochemistry (IHC) stains were performed against CD4, CD8, CD20, and LAMP3 to define populations of CD4+ T lymphocytes, CD8+ T lymphocytes, CD20+ B lymphocytes, and activated dendritic cells in the TME, respectively. Type III tertiary lymphoid structures, consisting of prominent B-cell follicles with germinal center-like structures and discrete T-cell zones, were found within tumors as well as at the tumor invasive front. (B) Higher densities of CD4+ T lymphocytes (p=0.1), CD8+ T lymphocytes (p=0.02), B lymphocytes (p=0.008), and activated dendritic cells (p=0.047) were found in the 12CK-High TME. (C) Findings using the deconvolution tool xCell demonstrated heightened signatures related to immune cells found within the (C) adaptive and (D) innate immune responses. 12-CK – 12-Chemokine score; TLS – Tertiary lymphoid structure; GC – germinal center; * p < 0.05; ** p < 0.01; *** p < 0.001; *** p < 0.0001Abstract 68 Figure 2The prognostic and predictive implications of 12-CK score. Kaplan-Meier survival analyses revealed improved overall survival (OS, HR 0.55, p=0.03) (A), disease-specific survival (DSS, HR 0.25, p=0.003) (B), and progression-free survival (PFS, HR 0.25, p=0.003) (C) in 12CK-High muscle invasive bladder cancer patients treated with radical cystectomy at Moffitt Cancer Center. The favorable prognosis in OS (HR 0.59, p=0.010) (D), DSS (HR 0.40, p=0.002) (E), and PFS (HR 0.55, p=0.007) (F) were confirmed in patients from TCGA. (G) From the IMVIGOR-210 study testing the efficacy of atezolizumab in chemotherapy refractory locally advanced or metastatic bladder cancer patients, complete responders were found to have significantly higher 12CK scores than the other cohorts. (H) Stratified by the 12-CK score, 12CK-High patients were found to have a 11.2mo overall survival benefit after treatment using atezolizumabConclusionsIn muslce invasive bladder cancer, 12CK-High scores corresponded with formation of TLS in the TME; favourable prognosis in surgically treated MIBC patients; and CR in atezolizumab-treated patients.ReferencesCabrita R, Lauss M, Sanna A, et al. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature 2020;577(7791):561–565.Helmink BA, Reddy SM, Gao J, et al. B cells and tertiary lymphoid structures promote immunotherapy response. Nature 2020;577(7791):549–555.Petitprez F, de Reyniès A, Keung EZ, et al. B cells are associated with survival and immunotherapy response in sarcoma. Nature 2020;577(7791):556–560.Aran D, Hu Z, Butte AJ. xCell: digitally portraying the tissue cellular heterogeneity landscape. Genome biology 2017;18(1):220.Rosenberg JE, Hoffman-Censits J, Powles T, et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet (London, England). 2016;387(10031):1909–1920.

There is a critical unmet need for safe and efficacious neoadjuvant treatment for cisplatin-ineligible patients with muscle-invasive bladder cancer. Here we launched a phase 1b study using the combination of intravesical cretostimogene grenadenorepvec (oncolytic serotype 5 adenovirus encoding granulocyte–macrophage colony-stimulating factor) with systemic nivolumab in cisplatin-ineligible patients with cT2-4aN0-1M0 muscle-invasive bladder cancer. The primary objective was to measure safety, and the secondary objective was to assess the anti-tumor efficacy as measured by pathologic complete response along with 1-year recurrence-free survival. No dose-limiting toxicity was encountered in 21 patients enrolled and treated. Combination treatment achieved a pathologic complete response rate of 42.1% and a 1-year recurrence-free survival rate of 70.4%. Pathologic response was associated with baseline free E2F activity and tumor mutational burden but not PD-L1 status. Although T cell infiltration was broadly induced after intravesical oncolytic immunotherapy, the formation, enlargement and maturation of tertiary lymphoid structures was specifically associated with complete response, supporting the importance of coordinated humoral and cellular immune responses. Together, these results highlight the potential of this combination regimen to enhance therapeutic efficacy in cisplatin-ineligible patients with muscle-invasive bladder cancer, warranting additional study as a neoadjuvant therapeutic option. ClinicalTrials.gov identifier: NCT04610671 . In a phase 1b trial, treatment of patients with muscle-invasive bladder cancer with intravesical oncolytic virus cretostimogene grenadenorepvec in combination with nivolumab was safe and led to encouraging preliminary clinical response rates.

Purpose To evaluate if the presence of penile intraepithelial neoplasia (PeIN) at the final surgical margin increases the risk of local recurrence in men with penile squamous cell carcinoma (PSCC) undergoing penile sparing surgery (PSS), we performed an analysis of our contemporary PSS database. Methods After IRB approval, data was retrieved from our organizational PSS database. Surgical margin status was verified by a genitourinary pathologist and patients were divided into groups with negative margins and patients with PeIN at the final surgical margins. The primary endpoint of the study was local recurrence free survival. Results A total of 62 patients, including 34 with PeIN positive margins (55%) and 28 with negative surgical margins (45%) were identified. The cohort had a median follow-up of 27 (IQR 11–57) months. In our cohort, 29 patients (46.7%) had pTis disease. The active management of surgical margin versus observation was not associated with a decreased risk of local, regional, or metastatic recurrence (HR 1.13, 95% CI 0.23–5.4, p  = 0.87). Local recurrences were seen in 21 (33.9%) patients but the presence of PeIN at the surgical margin was not associated with an increase in local recurrence rates (HR 0.33, 95% CI 0.13–0.82, p  = 0.01). Conclusions In this series of contemporary management of PSCC we found that presence of PeIN at the surgical margin was not associated with higher rates of local recurrence. Patients with PeIN at the surgical margins can be safely observed without need for immediate intervention unless a clinical recurrence of penile cancer occurs.

Purpose of reviewThis review sought to define the emerging roles of urinary tumor DNA (utDNA) for diagnosis, monitoring, and treatment of bladder cancer. Building from early landmark studies the focus is on recent studies, highlighting how utDNA could aid personalized care.Recent findingsRecent research underscores the potential for utDNA to be the premiere biomarker in bladder cancer due to the constant interface between urine and tumor. Many studies find utDNA to be more informative than other biomarkers in bladder cancer, especially in early stages of disease. Points of emphasis include superior sensitivity over traditional urine cytology, broad genomic and epigenetic insights, and the potential for non-invasive, real-time analysis of tumor biology.SummaryutDNA shows promise for improving all phases of bladder cancer care, paving the way for personalized treatment strategies. Building from current research, future comprehensive clinical trials will validate utDNA's clinical utility, potentially revolutionizing bladder cancer management.

Clinical trials have shown an overall survival (OS) benefit associated with first-line immunotherapy (IT) and combination targeted therapy (TT) and IT regimens compared with TT among patients with metastatic clear cell renal cell carcinoma (RCC). Generalizability of these findings in a real-world cohort outside of a clinical trial setting is unclear. To assess the association of first-line TT, IT, and combination TT and IT regimens with OS in a real-world cohort of patients with metastatic clear cell RCC. This retrospective propensity-matched cohort study identified 5872 patients with metastatic clear cell RCC in the National Cancer Database from January 1, 2015, to December 31, 2017, who received first-line TT, IT, or combination TT and IT and were not treated on a clinical trial protocol. Patients were stratified by first-line systemic treatment. Statistical analysis was conducted from October 1 to December 1, 2020. The primary outcome was OS from the date of diagnosis to death or censoring at last follow-up. After 1:1:1 nearest-neighbor caliper matching of propensity scores, survival analyses were conducted using Cox proportional hazards regression and Kaplan-Meier estimates. The final study population included 5872 patients (TT group: n = 4755 [81%]; 3332 men [70%]; median age, 64 years [interquartile range, 57-71 years]; IT group: n = 638 [11%]; 475 men [74%]; median age, 61 years [interquartile range, 54-69 years]; and combination TT and IT group: n = 479 [8%]; 321 men [67%]; median age, 62 years [interquartile range, 55-69 years]), and the matched cohort included 1437 patients (479 per treatment group). Patients in the IT and combination TT and IT groups were younger than those in the TT group, had fewer comorbid conditions (Charlson-Deyo score of 0, 480 of 638 [75%] in the TT group, 356 of 479 [74%] in the IT group, and 3273 of 4755 [69%] in the combination TT and IT group), and were more often treated at academic centers (315 of 638 [49%], 216 of 479 [45%], and 1935 of 4755 [41%], respectively). Both first-line IT and combination TT and IT were associated with improved OS compared with first-line TT for patients with metastatic clear cell RCC (IT group: hazard ratio [HR], 0.60 [95% CI, 0.48-0.75]; P < .001; combination TT and IT group: HR, 0.74 [95% CI, 0.60-0.91]; P = .005). No survival difference was seen between the IT and combination TT and IT groups (combination TT and IT: HR, 1.24 [95% CI, 0.98-1.56]; P = .08). This study suggests that both first-line IT and combination TT and IT were associated with improved OS compared with first-line TT for patients with metastatic clear cell RCC. These findings are similar to those identified in recently reported clinical trials, lending confidence to the broader applicability of these findings outside of a clinical trial setting.

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. To assess whether cytoreductive nephrectomy is associated with improved OS in patients with metastatic ccRCC. 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. Receipt of cytoreductive nephrectomy. 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. 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). 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.