Investigating cellular similarities and differences between upper tract urothelial carcinoma and bladder urothelial carcinoma using single-cell sequencing.

Background: Upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BLCA) both originate from uroepithelial tissue, sharing remarkably similar clinical manifestations and therapeutic modalities. However, emerging evidence suggests that identical treatment regimens may lead to less favorable outcomes in UTUC compared to BLCA. Therefore, it is imperative to explore molecular processes of UTUC and identify biological differences between UTUC and BLCA. Methods: In this study, we performed a comprehensive analysis using single-cell RNA sequencing (scRNA-seq) on three UTUC cases and four normal ureteral tissues. These data were combined with publicly available datasets from previous BLCA studies and RNA sequencing (RNA-seq) data for both cancer types. This pooled analysis allowed us to delineate the transcriptional differences among distinct cell subsets within the microenvironment, thus identifying critical factors contributing to UTUC progression and phenotypic differences between UTUC and BLCA. Results: scRNA-seq analysis revealed seemingly similar but transcriptionally distinct cellular identities within the UTUC and BLCA ecosystems. Notably, we observed striking differences in acquired immunological landscapes and varied cellular functional phenotypes between these two cancers. In addition, we uncovered the immunomodulatory functions of vein endothelial cells (ECs) in UTUC, and intercellular network analysis demonstrated that fibroblasts play important roles in the microenvironment. Further intersection analysis showed that MARCKS promote UTUC progression, and immunohistochemistry (IHC) staining revealed that the diverse expression patterns of MARCKS in UTUC, BLCA and normal ureter tissues. Conclusion: This study expands our multidimensional understanding of the similarities and distinctions between UTUC and BLCA. Our findings lay the foundation for further investigations to develop diagnostic and therapeutic targets for UTUC.

The role of immunotherapy in urological cancers.

Immunotherapy is defined as a therapeutic approach that targets or manipulates the immune system. A deeper understanding of the cellular and molecular composition of the tumour environment, as well as the mechanisms controlling the immune system, has made possible the development and clinical investigation of many innovative cancer therapies. Historically, immunotherapy has played an essential role in treating urologic malignancies, while in the modern era, the development of immune checkpoint inhibitors (ICIs) has been critical to urology. Urothelial carcinoma is a common type of cancer in the genitourinary system, and treatment strategies in this area are constantly evolving. Intravesical and systemic immunotherapeutic agents have begun to be used increasingly frequently in treating urothelial carcinoma. These agents increase the anti-tumour response by affecting the body's defence mechanisms. Immunotherapeutic agents used in urothelial carcinoma include various options such as BCG, interferon, anti-PD-1 (pembrolizumab, nivolumab) and anti-PD-L1 (atezolizumab, avelumab, durvalumab). Renal cell carcinoma (RCC) has been known for many years as a tumour with unique sensitivity to immunotherapies. The recent emergence of ICIs that block PD-1/PD-L1 (pembrolizumab, nivolumab, atezolizumab) or CTLA4 (ipilimumab) signalling pathways has reestablished systemic immunotherapy as central to the treatment of advanced RCC. In light of randomized clinical trials conducted with increasing interest in the application of immunotherapies in the adjuvant setting, combination therapies (nivolumab/ipilimumab, nivolumab/cabozantinib, pembrolizumab/ axitinib, pembrolizumab/lenvantinib) have become the standard first-line treatment of metastatic RCC. Prostate cancer is in the immunologically "cold" tumour category; on the contrary, in recent years, immunotherapeutic agents have come to the fore as an essential area in the treatment of this disease. Especially in the treatment of castration-resistant prostate cancer, immunotherapeutic agents constitute an alternative treatment method besides androgen deprivation therapy and chemotherapy. Ipilimumab, nivolumab, pembrolizumab, atezolizumab, and Sipuleucel T (Vaccine-based) are promising alternative treatment options. Considering ongoing randomized clinical trials, immunotherapeutic agents promise to transform the uro-oncology field significantly. In this review, we aimed to summarize the role of immunotherapy in urothelial, renal and prostate cancer in the light of randomized clinical trials.

The identification of a N6-methyladenosin-modifed immune pattern to predict immunotherapy response and survival in urothelial carcinoma.

BACKGROUND: Dysregulation of the immune system and N6-methyladenosine (m6A) contribute to immune therapy resistance and cancer progression in urothelial carcinoma (UC). This study aims to identify immune-related molecules, that are m6A-modified, and that are associated with tumor progression, poor prognosis, and immunotherapy response. METHODS: We identified prognostic immune genes (PIGs) using Cox analysis and random survival forest variable hunting algorithm (RSF-VH) on immune genes retrieved from the Immunology Database and Analysis Portal database (ImmPort). The RM2Target database and MeRIP-seq analysis, combined with a hypergeometric test, assessed m6A methylation in these PIGs. We analyzed the correlation between the immune pattern and prognosis, as well as their association with clinical factors in multiple datasets. Moreover, we explored the interplay between immune patterns, tumor immune cell infiltration, and m6A regulators. RESULTS: 28 PIGs were identified, of which the 10 most significant were termed methylated prognostic immune genes (MPIGs). These MPIGs were used to create an immune pattern score. Kaplan-Meier and Cox analyses indicated this pattern as an independent risk factor for UC. We observed significant associations between the immune pattern, tumor progression, and immune cell infiltration. Differential expression analysis showed correlations with m6A regulators expression. This immune pattern proved effective in predicting immunotherapy response in UC in real-world settings. CONCLUSION: The study identified a m6A-modified immune pattern in UC, offering prognostic and therapeutic response predictions. This emphasizes that immune genes may influence tumor immune status and progression through m6A modifications.

Effect of FGFR alteration on prognosis in 1963 urothelial carcinoma patients with immune checkpoint inhibitors: Implying combination of FGFR inhibitor and immunotherapy for FGFR-altered urothelial carcinoma.

Immune checkpoint inhibitors (ICIs) are essential for urothelial carcinoma (UC) treatment. Fibroblast growth factor receptor (FGFR) alterations, as common oncogenic drivers in UC, have been reported to drive T cell depletion of UC immune microenvironment via up-regulating FGFR signaling, which indicated FGFR alterations potentially result in reduced response to ICIs. In addition, the selective pan-FGFR inhibitor showed better clinical benefit in clinical trials, indicating FGFR has emerged as critical therapeutic target via inhibiting FGFR signaling. The present study aims to evaluate prognosis and response to ICIs between FGFR-altered UC patients and FGFR-wildtype UC patients via 1963 UC patients and offers new insights into personalized precision therapy and combination therapy for UC.

Nivolumab + Ipilimumab as Immunotherapeutic Boost in Metastatic Urothelial Carcinoma: A Nonrandomized Clinical Trial.

Importance: Studies with nivolumab, an approved therapy for metastatic urothelial carcinoma (mUC) after platinum-based chemotherapy, demonstrate improved outcomes with added high-dose ipilimumab. Objective: To assess efficacy and safety of a tailored approach using nivolumab + ipilimumab as an immunotherapeutic boost for mUC. Design, Setting, and Participants: In this phase 2 nonrandomized trial, patients with mUC composed 2 cohorts. Cohort 1 received first-line or second-/third-line nivolumab with escalating doses of ipilimumab, and cohort 2 received second-/third-line nivolumab with high-dose ipilimumab. Recruitment spanned 26 sites in Germany and Austria from August 8, 2017, to February 18, 2021. All patients had a 70% or higher Karnofsky Performance Score and measurable disease per Response Evaluation Criteria in Solid Tumours, version 1.1. Interventions: All patients initiated 4 doses of 240-mg nivolumab (1× every 2 wk). Week 8 nonresponders received nivolumab + ipilimumab (1× every 3 wk). Cohort 1 received 2 doses of 3-mg/kg nivolumab + 1-mg/kg ipilimumab followed by 2 doses of 1-mg/kg nivolumab + 3-mg/kg ipilimumab if no response. Due to safety concerns, cohort 1 treatment was halted, and first-line cohort 2 treatment was not pursued. Cohort 2 received 2 to 4 doses of 1-mg/kg nivolumab + 3-mg/kg ipilimumab. Responders continued with nivolumab maintenance but could receive nivolumab + ipilimumab for later progression. Main Outcomes and Measures: The primary end point was objective response rate. Results: The study comprised 169 patients (118 [69.8%] men; median [range] age, 68 [37-84] years): 86 in cohort 1 (42 first-line; 44 second-/third-line) and 83 in cohort 2. The median (IQR) follow-up times were 10.4 (4.2-23.5) months (first-line cohort 1), 7.5 (3.1-23.8) months (second-/third-line cohort 1), and 6.2 (3.2-22.7) months (cohort 2). Response rates to nivolumab induction were 12/42 (29%, first-line cohort 1), 10/44 (23%, second-/third-line cohort 1), and 17/83 (20%, cohort 2). Response rates to a tailored approach were 20/42 (48% [90% CI, 34%-61%], first-line cohort 1), 12/44 (27% [90% CI, 17%-40%], second-/third-line cohort 1), and 27/83 (33% [90% CI, 23%-42%], cohort 2). Three-year overall survival rates for first-line cohort 1, second-/third-line cohort 1, and cohort 2 using the Kaplan-Meier method were 32% (95% CI, 17%-49%), 19% (95% CI, 8%-33%), and 34% (95% CI, 23%-44%), respectively. Conclusions and Relevance: In this nonrandomized trial, although first-line cohort 1 treatment improved objective response rates, considerable progression events urge caution with this as a first-line therapy. Second-/third-line cohort 1 treatment did not improve response rates compared with nivolumab monotherapy. However, added high-dose ipilimumab may improve tumor response and survival in patients with mUC. Trial Registration: ClinicalTrials.gov Identifier: NCT03219775.

High serum sodium predicts immunotherapy response in metastatic renal cell and urothelial carcinoma.

OBJECTIVES: The development of reliable biomarkers for the prediction of immune checkpoint inhibition (ICI) response in patients with metastatic renal cell carcinoma (mRCC) and urothelial carcinoma (mUC) remains an unresolved challenge. Conventional ICI biomarkers typically focus on tumor-related factors such as PD-L1 expression. However, a comprehensive evaluation of the predictive value of serum electrolyte levels, a so far widely unexplored area, is still pending. METHODS: We conducted a post-hoc analysis of baseline sodium, potassium, chloride, magnesium and calcium levels in two independent phase 3 clinical trials: IMvigor211 for mUC comparing atezolizumab to chemotherapy, and IMmotion151 for mRCC comparing atezolizumab+bevacizumab to sunitinib. This analysis aimed to evaluate the prognostic and predictive value of these electrolyte levels in these clinical settings. A total of 1787 patients (IMvigor211 n = 901; IMmotion151 n = 886) were analyzed. RESULTS: We found a linear correlation of baseline serum sodium and chloride with prognosis across both trials, which was not found for potassium, magnesium and calcium. In multivariate analysis, the prognostic capacity of sodium was limited to patients receiving ICI as compared to the control group. Interestingly, in both studies, the chance of achieving an objective response was highest in the patient subgroup with high baseline serum sodium levels of > 140 mmol/L (IMmotion151: Complete response in 17.9% versus 2.0% in patients with mRCC with baseline sodium < 135 mmol/L). Serum sodium outperformed tumor PD-L1 expression as a predictor for immunotherapy efficacy. CONCLUSIONS: Patients exhibiting elevated serum sodium levels derive the greatest benefit from immunotherapy, suggesting that baseline serum concentration could serve as a valuable and cost-effective predictive biomarker for immunotherapy across entities.

Prognostic role of circulating cytokines and inflammation indexes for avelumab maintenance in metastatic urothelial carcinoma.

Background: Avelumab maintenance after first-line platinum-based chemotherapy represents a cornerstone for the treatment of metastatic urothelial carcinoma (mUC). However, identifying prognostic biomarkers is paramount for optimizing patients' benefits while minimizing toxicity. Cytokines represent circulating mediators of the complex interaction between cancer, the immune system, and inflammation. Inflammation, a hallmark of cancer, can be expressed by circulating factors. In different tumor subtypes, peripheral blood biomarkers, such as circulating cytokines, and systemic inflammatory indexes, have been addressed as potential prognostic factors for immune checkpoint inhibitors. However, their role in mUC still needs to be determined. Methods: Between February 2021 and April 2023, we prospectively collected plasma cytokines and inflammation indexes in 28 patients with mUC before starting avelumab as first-line maintenance. The primary endpoint was the relationship between baseline cytokines and inflammatory indexes with the clinical benefit (CB), defined as the number of Responders. Secondary endpoints included the correlation of baseline cytokines and inflammatory indexes with progression-free survival (PFS), overall survival (OS), and the number and grade of immune-related adverse events. Results: High pre-treatment levels of interferon (IFN)-γ and interleukin (IL)-2, and low levels of IL-6, IL-8, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and systemic-inflammation index (SII) were associated with clinical benefit and longer survival. In the multivariate analysis, low IL-8, NLR, and SII levels maintained a positive prognostic value for OS. Conclusion: Our data suggest that, in mUC patients receiving avelumab, pre-treatment levels of plasma cytokines and inflammatory indexes may serve as potential prognostic biomarkers for response and efficacy. In particular, patients with signs of pre-therapeutic inflammation showed a significantly lower response and survival to avelumab. On the contrary, low systemic inflammation and high levels of cytokines characterized responders and longer survivors.

Prognostic Impact and Spatial Interplay of Immune Cells in Urothelial Cancer.

BACKGROUND AND OBJECTIVE: Quantity and the spatial relationship of specific immune cell types can provide prognostic information in bladder cancer. The objective of the study was to characterize the spatial interplay and prognostic role of different immune cell subpopulations in bladder cancer. METHODS: A total of 2463 urothelial bladder carcinomas were immunostained with 21 antibodies using BLEACH&STAIN multiplex fluorescence immunohistochemistry in a tissue microarray format and analyzed using a framework of neuronal networks for an image analysis. Spatial immune parameters were compared with histopathological parameters and overall survival data. KEY FINDINGS AND LIMITATIONS: The identification of > 300 different immune cell subpopulations and the characterization of their spatial relationship resulted in numerous spatial interaction patterns. Thirty-nine immune parameters showed prognostic significance in univariate analyses, of which 16 were independent from pT, pN, and histological grade in muscle-invasive bladder cancer. Among all these parameters, the strongest association with prolonged overall survival was identified for intraepithelial CD8+ cytotoxic T cells (time-dependent area under receiver operating characteristic curve [AUC]: 0.70), while stromal CD8+ T cells were less relevant (AUC: 0.65). A favorable prognosis of inflamed cancers with high levels of "exhaustion markers" suggests that TIM3, PD-L1, PD-1, and CTLA-4 on immune cells do not hinder antitumoral immune response in tumors rich of tumor infiltrating immune cells. CONCLUSIONS AND CLINICAL IMPLICATIONS: The density of intraepithelial CD8+ T cells was the strongest prognostic feature in muscle-invasive bladder cancer. Given that tumor cell killing by CD8+ cytotoxic T lymphocytes through direct cell-to-cell-contacts represents the "terminal end route" of antitumor immunity, the quantity of "tumor cell adjacent CD8+ T cells" may constitute a surrogate for the efficiency of cancer recognition by the immune system that can be measured straightaway in routine pathology as the CD8 labeling index. PATIENT SUMMARY: Quantification of intraepithelial CD8+ T cells, the strongest prognosticfeature identified in muscle-invasive bladder cancer, can easily be assessed by brightfield immunohistochemistry and is therefore "ready to use" for routine pathology.

TP53 disruptive mutation predicts platinum-based chemotherapy and PD-1/PD-L1 blockade response in urothelial carcinoma.

TP53 mutation is one of the most common genetic alterations in urothelial carcinoma (UrCa), and heterogeneity of TP53 mutants leads to heterogeneous clinical outcomes. This study aimed to investigate the clinical relevance of specific TP53 mutations in UrCa. In this study, a total of eight cohorts were enrolled, along with matched clinical annotation. TP53 mutations were classified as disruptive and nondisruptive according to the degree of disturbance of p53 protein function and structure. We evaluated the clinical significance of TP53 mutations in our local datasets and publicly available datasets. The co-occurring events of TP53 mutations in UrCa, along with their therapeutic indications, functional effects, and the tumor immune microenvironment, were also investigated. TP53 mutations were identified in 49.7% of the UrCa patients. Within this group, 25.1% of patients carried TP53Disruptive mutations, a genetic alteration correlated with a significantly poorer overall survival (OS) when compared to individuals with TP53Nondisruptive mutations and those with wild-type TP53. Significantly, patients with TP53Disruptive mutations exhibit an increased probability of responding favorably to PD-1/PD-L1 blockade and chemoimmunotherapy. Meanwhile, there was no noteworthy distinction in OS among patients with varying TP53 mutation status who underwent chemotherapy. Samples with TP53Disruptive mutations showed an enriched APOBEC- and POLE-related mutational signature, as well as an elevated tumor mutation burden. The sensitivity to immunotherapy in tumors carrying TP53Disruptive mutation may be attributed to the inflamed tumor microenvironment characterized by increased CD8+T cell infiltration and interferon-gamma signaling activation. In conclusion, UrCa patients with TP53Disruptive mutations have shown reduced survival rates, yet they may respond well to PD-1/PD-L1 blockade therapy and chemoimmunotherapy. By distinguishing specific TP53 mutations, we can improve risk stratification and offer personalized genomics-guided therapy to UrCa patients. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Aspectos prácticos sobre la determinación de PD-L1 en el tratamiento de carcinoma urotelial. Consenso del grupo de uropatología de la SEAP / Practical aspects of PD-L1 assessment in the treatment of urothelial carcinoma: Consensus of the uropathology group of the SEAP

La reciente llegada de nuevos fármacos de inmunoterapia para el tratamiento del carcinoma urotelial hace necesario establecer criterios para armonizar la determinación de PD-L1 mediante inmunohistoquímica como factor pronóstico y para la selección de pacientes a tratar. En este escenario, un grupo de uropatólogos de la Sociedad Española de Anatomía Patológica, junto con un oncólogo médico como colaborador externo subespecializado en urooncología, ha elaborado este documento de recomendaciones basadas en la evidencia disponible. En la determinación de PD-L1 son especialmente relevantes la selección de la muestra analizada, su procesamiento, la plataforma de inmunohistoquímica y anticuerpo empleados, así como el algoritmo que se aplique para la lectura. Todos estos aspectos deben indicarse en el informe de resultados, que debería poder ser fácilmente interpretable en un contexto de rápida evolución de terapias inmunológicas.(AU)

The recent addition of novel immunotherapy drugs for the treatment of urothelial carcinoma makes it necessary the establishment of criteria to harmonize the immunohistochemical assessment of PD-L1, both as a prognostic factor and for the selection of patients to be treated. In this scenario, a group of uropathologists from the Spanish Society of Pathological Anatomy, together with a medical oncologist as an external collaborator subspecialized in uro-oncology, have prepared this document of recommendations based on the available evidence. During PD-L1 assessment it is especially relevant the selection of the sample, its processing, the immunohistochemical platform and antibody used, and the algorithm applied in the interpretation of results. All these aspects must be indicated in the results report, which should be easily interpretable in a context of rapid evolution of immunological therapies.(AU)

Mass cytometry reveals immune atlas of urothelial carcinoma.

Immunotherapy has emerged as a robust clinical strategy for cancer treatment. PD1/PD-L1 inhibitors have been used as second-line therapy for urothelial carcinoma due to the high tumor mutational burden. Despite the efficacy of the treatment is significant, the response rate is still poor. The tumor immune microenvironment plays a key role in the regulation of immunotherapeutic efficacy. However, a comprehensive understanding of the intricate microenvironment in clinical samples remains unclear. To obtain detailed systematic tumor immune profile, we performed an in-depth immunoassay on 12 human urothelial carcinoma tissue samples and 14 paratumor tissue samples using mass cytometry. Among the large number of cells assayed, we identified 71 T-cell phenotypes, 30 tumor-associated macrophage phenotypes. T cell marker expression profiles showed that almost all T cells in the tumor tissue were in a state of exhaustion. CD38 expression on tumor-associated macrophages (TAMs) was significantly higher than PDL1, and CD38+ TAMs were closely associated with immunosuppression. CD38 may be a more suitable target for immunotherapy in urothelial carcinoma compared to PD1/PDL1. This single-cell analysis of clinical samples expands our insights into the immune microenvironment of urothelial carcinoma and reveals potential biomarkers and targets for immunotherapy development.

Cancer immunohistogram representing cancer-immunity cycle by immunohistochemistry predicts the efficacy of immune checkpoint inhibitors in urological cancer patients.

We developed an immunohistogram representing an individual cancer-immunity cycle based on immunohistochemical analyses. We evaluated its ability to predict the efficacy of immune checkpoint inhibitors (ICI) in 11 patients with urothelial carcinoma and 7 patients with renal cell carcinoma who underwent surgery and received ICIs for disease recurrence. Immunohistochemical analyses for CD8, TIA-1, HLA class I, HLA-DR, and PD-L1 were performed and scored 0-3. T-cell infiltration pattern was classified into desert, excluded, partially inflamed, and inflamed. Tumors with an inflamed or partially inflamed pattern and positive scores (score ≥ 1) for all five immune markers were classified as "immune-hot" and others as "immune-cold." Association between the immunohistogram and ICI treatment efficacy was evaluated with objective response rate, disease control rate (DCR), progression-free survival (PFS), and cancer-specific survival (CSS). Eight (44%) and 10 (56%) patients had immune-hot and immune-cold tumors, respectively. Immune-hot tumors showed a higher DCR (100% vs. 40%, p < 0.01), longer PFS (median unreached for hot, 1.3 months for cold, p < 0.01), and longer CSS (median unreached for hot, 3.3 months for cold, p < 0.01) than immune-cold tumors. The immunohistogram could be clinically useful as an accessible biomarker for precision cancer immunotherapy in urological cancer.

Identification of an IDO1-based immune classifier for survival prediction of upper tract urothelial carcinoma.

The limited response rate of immunotherapy in upper tract urothelial carcinoma (UTUC) might be attributed to additional immunosuppressive mechanisms in vivo. As a promising immune checkpoint target, the expression and prognostic role of indoleamine 2,3-dioxygenase 1 (IDO1) in UTUC remains unknown. In this study, the expression and prognostic value of IDO1 was analyzed in 251 patients from 3 independent cohorts. The least absolute shrinkage and selection operator (LASSO) Cox regression model was used to construct an IDO1-based immune classifier and external validation was performed to further validate the classifier. RNA sequencing and immunofluorescence were used to explore the immune contexture of different risk groups stratified by classifier. We found that high IDO1 expression on tumor cells (TC) indicated a poorer overall survival and disease-free survival in all cohorts. Patients with high expression of IDO1 TC possessed increased infiltration of CD4+ , CD8+ and Foxp3+ T cells. An immune classifier based on intratumoral CD8+ lymphocytes, IDO1 TC, and stromal PD-L1 expression status was developed, with its area under the curves (AUCs) values for overall survival at 5 y being 0.79 (95% confidence interval [CI] 0.65-0.93) in the discovery cohort, 0.75 (95% CI 0.58-0.92) and 0.78 (95% CI 0.65-0.92) in the internal and external validation cohorts, respectively. The high-risk group stratified by the immune classifier was associated with immunosuppressive contexture, accompanied by enhanced CD8+ T cells exhaustion patterns. Our IDO1-based immune classifier can provide a superior accuracy for survival prediction and lead to individual stratification of UTUC immune subtypes.

Plasmacytoid urothelial carcinoma (UC) are luminal tumors with similar CD8+ Tcell density and PD-L1 protein expression on immune cells as compared to conventional UC.

BACKGROUND: Plasmacytoid urothelial carcinoma (UC) is a rare pathological variant of UC with low chemotherapeutic sensitivity and dismal outcomes. The molecular and immune profiles of such tumors remain poorly investigated. METHODS: Herein, we investigated the phenotypical features of a cohort of plasmacytoid UC (n=32) by comparison to a control group of conventional high-grade UC with matched clinicopathological characteristics (n=30). Histopathological analysis included the following antibodies: p63, GATA3, CK5/6, CK20 and HER2. In addition, the density of intra-tumor CD8+ lymphocytes, and PD-L1 expression in tumor (TC) and immune cells (IC) were evaluated. RESULTS: Plasmacytoid UC expressed GATA3 (97% vs 86% P=0.18), CK20 (59% vs 36% P=0.08) markers and showed a significantly higher rate of HER2 overexpression (2+ and 3+ score: 25% vs 0%, P<0.01) compared to controls. A significantly lower expression of CK5/6 (22% vs 56%, P<0.05) and p63 (41% vs 80%, P<0.05) was observed in plasmacytoid UC compared to controls. The density of tumor-infiltrating CD8+ cells was similar between plasmacytoid and conventional UC (P=0.9). PD-L1 expression on IC was similar compared to conventional UC (P=0.3). CONCLUSIONS: Together, our study demonstrated that plasmacytoid UC belong to the luminal subtype and display a rather inflamed microenvironment similar to conventional UC. These data support the inclusion of plasmacytoid variant of UC in clinical trials evaluating immune checkpoint inhibitors monotherapy or combination immunotherapeutic strategies.

Development of a multiplex immuno-oncology biomarker and digital pathology workflow for assessment of urothelial carcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICI) therapies have demonstrated significant benefit in the treatment of many tumors including high grade urothelial cancer (HGUC) of the bladder. However, variability in patients' clinical responses highlights the need for biomarkers to aid patient stratification. ICI relies on an intact host immune response. In this context, we hypothesize that key players in the antitumor immune response such as markers of activated cytotoxic T lymphocytes (CD8, granzyme-B) and immune suppression (FOXP3) may help to identify patients who will derive the greatest therapeutic benefit from ICI. A major obstacle for deployment of such a strategy is the limited quantities of tumor-derived biopsy material. Therefore, in this technical study, we develop a multiplex biomarker with digital workflow. We explored the (1) concordance of conventional single stain results using digital image analysis, and (2) agreement between digital scoring versus manual analysis. METHODS: (1) For concordance study of single and multiplex stains, triplicate core tissue microarrays of 207 muscle invasive, HGUC of bladder had sequential 4-micron sections cut and stained with CD8, FOXP3 and granzyme-B. An inhouse developed tri-chromogen multiplex immunohistochemistry (m-IHC) assay consisting of CD8 (green), granzyme B (brown), and FOXP3 (red) was used to stain the next sequential tissue section. (2) Agreement between manual and digital analysis was performed on 19 whole slide sections of HGUC cystectomy specimens. All slides were scanned using Aperio ScanScope AT Digital Scanner at 40X. Quantitative digital image analysis was performed using QuPath version 0.2.3 open-source software. Scores from triplicate cores were averaged for each HGUC specimen for each marker. Intraclass correlation coefficients were used to compare percent positive cells between the single- and multi-plex assays. Lin's concordance correlation coefficients were used for manual versus digital analysis. RESULTS AND CONCLUSIONS: m-IHC offers significant advantages in characterizing the host immune microenvironment particularly in limited biopsy tissue material. Utilizing a digital image workflow resulted in significant concordance between m-IHC and individual single stains (p < 0.001 for all assessments). Moderate to good agreements were achieved between manual and digital scoring. Our technical work demonstrated potential uses of multiplex marker in assessing the host immune status and could be used in conjunction with PD-L1 as a predictor of response to ICI therapy.

CD8+ T effector and immune checkpoint signatures predict prognosis and responsiveness to immunotherapy in bladder cancer.

Immune-checkpoint blockade (ICB) has been routinely implemented to treat bladder cancer; however, most patients have little or no clinical benefit. In this study, 348 pretreated metastatic urothelial cancer samples from the IMvigor210 cohort were used to identify important genes significantly associated with CD8+ T effector and immune checkpoint signatures. The immune checkpoint inhibitor score (IMS) scoring system was constructed to predict the immunotherapy responsiveness. Transcriptome analysis confirmed that the high IMS score group had significant immune activation with better prognosis and higher immunotherapy responsiveness, which was a powerful biomarker for predicting the prognosis and responsiveness of ICB. Tumor immune dysfunction and exclusion (TIDE) scores were calculated using 2031 external bladder cancer samples for further validation. We selected the important Hub genes as potential therapeutic targets, and validated the genes using genomic, transcriptomic, immunomic, and other multi-omics methods. In addition, we construct a risk prediction model which could stratify patients with bladder cancer and predict patient prognosis and ICB treatment responsiveness. In conclusion, this study identified effective biomarkers for the prediction of immune checkpoint inhibitor treatment responsiveness in bladder cancer patients and provided new immunotherapeutic targets.

Fibroblast growth factor receptor 3 alterations and response to immune checkpoint inhibition in metastatic urothelial cancer: a real world experience.

BACKGROUND: FGFR3-altered urothelial cancer (UC) correlates with a non-T cell-inflamed phenotype and has therefore been postulated to be less responsive to immune checkpoint blockade (ICB). Preclinical work suggests FGFR3 signalling may suppress pathways such as interferon signalling that alter immune microenvironment composition. However, correlative studies examining clinical trials have been conflicting as to whether FGFR altered tumours have equivalent response and survival to ICB in patients with metastatic UC. These findings have yet to be validated in real world data, therefore we evaluated clinical outcomes of patients with FGFR3-altered metastatic UC treated with ICB and investigate the underlying immunogenomic mechanisms of response and resistance. METHODS: 103 patients with metastatic UC treated with ICB at a single academic medical center from 2014 to 2018 were identified. Clinical annotation for demographics and cancer outcomes, as well as somatic DNA and RNA sequencing, were performed. Objective response rate to ICB, progression-free survival, and overall survival was compared between patients with FGFR3-alterations and those without. RNA expression, including molecular subtyping and T cell receptor clonality, was also compared between FGFR3-altered and non-altered patients. RESULTS: Our findings from this dataset confirm that FGFR3-altered (n = 17) and wild type (n = 86) bladder cancers are equally responsive to ICB (12 vs 19%, p = 0.73). Moreover, we demonstrate that despite being less inflamed, FGFR3-altered tumours have equivalent T cell receptor (TCR) diversity and that the balance of a CD8 T cell gene expression signature to immune suppressive features is an important determinant of ICB response. CONCLUSIONS: Our work in a real world dataset validates prior observations from clinical trials but also extends this prior work to demonstrate that FGFR3-altered and wild type tumours have equivalent TCR diversity and that the balance of effector T cell to immune suppression signals are an important determinant of ICB response.