Muscle-invasive Bladder Cancer Samples Research Articles

Abstract PR002: Sarcomatoid Histological Variants of Canine Muscle-Invasive Bladder Cancer: Transcription Factor Activation Highlights Pathways of Epithelial-Mesenchymal Transformation Similar to Humans

Abstract The histologic heterogeneity of human Muscle Invasive Bladder Cancer (MIBC) is one of the main contributing factors to poor treatment outcomes. Recent reports on MIBC histological variants have highlighted increased levels of transcription factors (TF) associated with epithelial-mesenchymal transformation (EMT) and epigenetic regulation in the sarcomatoid histology variant of MIBC. Dogs with spontaneously developing MIBC have been shown to display similar histological and molecular features, in addition to closely mimicking the clinical behavior of human MIBC. Here, we add to previously reported data on histological variants in canine MIBC by evaluating 21 tumor biopsies for variant histology and performing bulk RNAseq analyses on 3 organoid cell lines derived from one sarcomatoid subvariant and two conventional urothelial carcinoma (UC). MIBC biopsies from 21 dogs were reviewed for variant histology by a human urologic pathologist. Organoids derived from the urine of canine MIBC patients were cultured, preserved in RNA later, and sequenced for mRNA expression to identify TF associated with sarcomatoid variants versus conventional UC. All canine MIBC biopsies were classified as invasive, high-grade UC. Histologic variants were identified as glandular differentiation in 7/21, sarcomatoid differentiation in 3/21, squamous differentiation in 3/21, micropapillary carcinoma in 1/21 MIBC samples and small nested variants in 1/21 samples; with the rest of the tumors classified as conventional UC. Raw RNAseq analyses revealed no clear clustering of expression profiles between the three organoid cell lines, indicating distinct RNA expression profiles between the sarcomatoid variants and conventional UC of canine MIBC. To evaluate differences in the expression of TF targets between the organoid cell lines, pathway analyses and VIPER algorithms were used. Organoids derived from sarcomatoid variant UC showed increased expression of Snail familiy transcription repressor 1 (SNAI1) along with decreased expression of cadherin 1 (CDH1), indicating a signature of EMT, similar to that previously identified in human MIBC organoids with sarcomatoid variant histology. Canine MIBC organoids derived from conventional UC histology samples showed a TF profile associated with proliferation, metastasis, and progression, such as increased Zinc finger E-box binding homeobox 1 (ZEB1), E2F transcription factor 3 (E2F3), and centromere protein F (CENPF), as previously described in humans with high grade, invasive UC. In conclusion, the frequency of some histologic variants in canine MIBC, such as sarcomatoid UC, appear to be higher than reported in human patients. TF analyses of canine MIBC-derived organoid cell lines showed evidence of EMT, similar to the sarcomatoid variants of MIBC in humans. Organoids derived from canine MIBC could potentially serve as a valuable model for evaluating the efficacy of novel treatment modalities for the rare histologic variants of UC. Citation Format: Karin Allenspach, Mohamed Elbadawy, Hannah Nocholson, Christopher Zdyrski, John Cheville, Eugene Douglass, Elizabeth W. Howerth, Lilian Oliveira, Jonathan P. Mochel. Sarcomatoid Histological Variants of Canine Muscle-Invasive Bladder Cancer: Transcription Factor Activation Highlights Pathways of Epithelial-Mesenchymal Transformation Similar to Humans [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR002.

Abstract LB_B16: Quantification of immune-oncology drug responses in autologous co-cultures of ex vivo patient tumor tissues with PBMCs

Abstract Introduction Immuno-oncology drug treatments, including immune checkpoint inhibitors, represent a huge promise for cancer patients and have been clinically approved for some cancer indications. However, patient response rates stay relatively low (about 10-20% responders across indications) hampering the success of such drugs in clinical trials. In addition, current in vitro and in vivo pre-clinical models often lack the (immunological) complexity necessary to predict the clinical effectiveness of such drugs or drug combinations. To bridge this gap in translational models, we have recently developed the 3D Ex vivo Patient Tissue (EVPT) platform. The EVPT platform is leveraging short-term 3D ex vivo tumor culture system containing micro-tissues generated from fresh human tumor material and high content imaging analysis. The platform is preserving the innate tumor microenvironment including resident immune cells, which are crucial for addressing immunological drug responses. In this study we aimed to further boost immune compartment of the assay by addition of autologous immune cells (PBMCs) isolated from whole blood of the patient in parallel with ex vivo tumor processing. Methods Patient tumor tissues from resected NSCLC and muscle-invasive bladder cancer (MIBC) tumors and matching peripheral blood were obtained from commercial tissue providers and processed within 24 hours to preserve the TME. PBMCs were isolated from whole blood and pre-stained with cell tracker. Fresh, minimally processed tumor material was embedded in a protein-rich hydrogel with or without matching PBMCs and exposed to mono- and combination immunotherapies at various doses. Next, the phenotypic screening using our proprietary automated HCI analysis platform was performed. Where applicable, IHC biomarker, flow cytometry and cytokine measurements were performed on fixed tissues or culture supernatants. Results 3D ex vivo co-cultures were executed in 22 NSCLC and 5 MIBC patient samples. Immunological treatment responses varied between tested patients. When comparing co-culture to mono-culture conditions, one third of the samples showed baseline killing associated with PBMCs addition. About 20% of tissues from both indications responded to antiPD1 treatment (Pembrolizumab) and on average 50% of tissues responded to immunostimulatory control SEA. Overall best effects were observed for one test article that was used in three doses in combination with antiPD1. Five out five tested NSLCLC samples showed functional responses in these conditions as wells as two out five MIBC samples. In addition, it induced immunostimulatory effects on the PBMCs in the assay – increased numbers and tumor infiltration rates compared to vehicle control. Conclusions 3D Ex vivo Patient Tissue platform combined with autologous PBMCs offers supplementary readouts such as tumor infiltration and immune cell proliferation while providing TME-rich environment for functional immune-oncology drug testing. Citation Format: Niels Meesters, Sander Basten, Ezgi Kaya Aksoy, Mariusz Madej, Kuan Yan, Emma Spanjaard, Leo S. Price, Nataliia Beztsinna. Quantification of immune-oncology drug responses in autologous co-cultures of ex vivo patient tumor tissues with PBMCs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B16.

QUANTITATIVE IMMUNOFLUORESCENCE EVALUATION OF PD-L1 EXPRESSION IN NON-MUSCLE-INVASIVE AND MUSCLE-INVASIVE UROTHELIAL BLADDER CANCER

The expression of PD-L1 protein was quanti ed in 56 samples of urothelial bladder cancer (BC). PD-L1 expression was detected in all BC samples, while in non-muscle-invasive bladder cancer (NMIBC) the median value is 1,5 times higher than in muscle-invasive bladder cancer (MIBC) (p = 0,001). High PD-L1 expression level (≥30%) was observed in 20% of MIBC samples and among NMIBC tumors - in 68% of cases, that is 3,4 times more often (p = 0,0004). The last fact justi es the prospect of starting therapy with immune checkpoint inhibitors already at the onset of the disease - at NMIBC.

Generalizable FGFR prediction across tumor types using self supervised learning.

e15057 Background: Deep Learning models have demonstrated the ability to detect tumors, classify disease state, or infer genetic biomarkers from digital whole slide images (WSI) of Hematoxylin and Eosin (H&E)-stained tissue. Such models can be deployed to improve clinical practice or efficiently recruit for clinical trials. However, the data to train these models is often very limited and image capture protocols vary between labs. We propose to use Self Supervised Learning (SSL) on large unlabeled histopathology data sets to improve generalizability and predictive performance of deep learning models on unseen data. Methods: We pre-trained a Convolutional Neural Network (CNN) using SSL on 25,120 unlabeled, digital WSIs from various sources (multiple scanners, hospital systems, labs, diseases, tissue sites). We then fine-tuned the pre-trained CNN to infer the presence or absence of select FGFR alterations in Muscle Invasive Bladder Cancer (MIBC) from the digital WSI. We applied this model (FGFRSSL) to WSIs of biopsies from other data sources and tumor types and compared with a model (FGFRTRAD) that was trained exclusively on MIBC samples without self-supervised learning. Results: The FGFRSSL model achieved an Area Under ROC Curve (AUC = 0.80) on MIBC WSIs and maintained performance on images from an independent lab (AUC=0.82). We further show that the model generalizes to Non-Muscle Invasive Bladder Cancer (NMIBC) samples with (AUC = 0.76) and Pan-Tumor WSIs (AUC: 0.83). The FGFRTRAD model trained without SSL-based pre-training achieved an Area Under ROC Curve (AUC: 0.76) on MIBC WSIs was less generalizable, showing lower performance for independent data (AUC = 0.72), NMIBC samples (AUC = 0.72), and Pan-Tumor samples (AUC = 0.64). Conclusions: We leveraged SSL pretraining to improve the reliability and generalizability of AI-based models across multiple data sources. We also demonstrate our model’s ability to infer FGFR status across multiple solid tumor types after training on only MIBC samples, suggesting the cell morphology conferred by FGFR alteration in cancer may be shared across diverse tumor types. These models represent a means for efficiently screening patients for actionable clinical biomarkers in a robust manner to guide clinical decisions and inform drug development efforts. [Table: see text]

CDH12 and the development of the neuronal subtype in MIBC.

e16579 Background: We discovered a novel cell epithelial cell population we termed “C3” in muscle-invasive bladder cancer (MIBC) that is negatively prognostic for surgery and chemotherapy. This cell population shares similarities with the neuronal subtype MIBC (NEBC) which has limited treatment options and is driven by unclear molecular mechanisms. The objective of the study was to determine if NEBC is driven by some of the genes that are overexpressed in C3 cells. A top overexpressed and C3 cell specific gene that is also enriched in normal neuronal tissue is Cadherin 12 (N-Cadherin 2, CDH12). Hence, we initiated a study of this gene. Methods: We overexpressed CDH12 in two murine MIBC cell lines (NA13 and MB49) and then inoculated them into mice. Tumor growth kinetics were studied, and single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing were then used to evaluate the tumors. Tissue samples from patients with MIBC were also analyzed using snRNA-seq and Co-detection by Indexing (CODEX) multiplexed immunofluorescence, and our bioinformatics analysis combined transcriptomic profiles from > 2000 MIBC samples from published datasets, to characterize the transcriptional programs induced by enforcing CDH12 expression. Results:Enforced expression of CDH12 led to enhanced tumor growth and the appearance of the neuroendocrine tumor phenotype. NEBC from human samples showed the highest expression of CDH12, and snRNA-seq results showed that NEBC also has the highest proportion of CDH12 epithelial cells and demonstrated co-expression of CDH12 and neuronal markers (NCAM1, CHGA, and SYP). RNA velocity analysis in murine and human tumors implicated CDH12 as a driver for the development of the neuronal subtype. Conclusions: To our knowledge, this study provides the first identification of a genetic driver leading to NEBC and implicates a novel gene, CDH12 in this process.

Abstract 2509: Similar variant histology and immune cell infiltration in canine and human muscle-invasive bladder cancer

Abstract Introduction: Muscle-Invasive Bladder Cancer (MIBC) is associated with a poor overall survival rate. One important factor that contributes to poor treatment outcomes is the histologic heterogeneity of MIBC tumors, which limits the translational value of some rodent models. As opposed to mice, dogs with spontaneously occurring bladder cancer have been shown to present with similar clinical behavior as human MIBC. In addition, canines have an intact immune system, which could provide for an ideal pre-clinical study population to investigate the efficacy of immunotherapy. In this study, we evaluated canine MIBC tumor biopsies for their similarities to human MIBC with regards to histology and immune cell infiltration. Materials & Methods: Nine canine MIBC tumor samples were provided by the ISU pathology service and evaluated by a human uropathologist. Canine MIBC tumors were phenotypically characterized using either immunofluorescence (IF) or RNA in situ hybridization (RNA-ISH), including CD44, KRT7 and FOXA1, as well as specific markers characterizing the immune cell infiltrate, such as CD163 (tumor-associated macrophages), CD8a (cytotoxic T-lymphocytes), PD-L1 (immune-checkpoint protein) and TGFβ2 (cancer-associated fibroblasts). Results: Histologically, all canine MIBCs were classified as invasive, high-grade urothelial carcinomas (UC) with identical morphology to human MIBC. Interestingly, previously described histological variants of human MIBC tumors were also observed within the canine MIBC samples, such as glandular differentiation in 3/9 canine MIBC samples; epithelial-mesenchymal transition with sarcomatoid differentiation 1/9, squamous differentiation in 2/9 and micropapillary carcinoma in 1/9, all of which have been associated with poor clinical outcomes in human patients. IF was strongly positive for KRT7, FOXA1 and CD44, and immune cell markers CD163 and TGFβ2. However, CD8a was only weakly expressed (5/9) or negative, and PD-L1 was only positive in 4/9 canine MIBC biopsies. Conclusions: Canine MIBC tumors showed significant heterogeneity with highly similar histological variant morphologies to those reported in humans with MIBC. Furthermore, phenotypic characterization of immune cell infiltrates and checkpoint proteins suggest that the tumors were typically immunologically “cold”. These data further establish the utility of the spontaneous canine model of MIBC for pre-clinical investigation of novel therapeutic drug candidates, including immunotherapies. Citation Format: Karin Allenspach, John Cheville, Hannah Wickham, Christopher Zdyrski, Vojtech Gabriel, Basant Ahmed, Mei-Jyun Ciou, Fabrice Lucien, Igor Frank, Olufemi Fasina, Pablo Pineyro, Johnathan P. Mochel. Similar variant histology and immune cell infiltration in canine and human muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2509.

LBA03-09 CDH12 DRIVES THE DEVELOPMENT OF THE NEURONAL SUBTYPE IN MIBC

You have accessJournal of UrologyCME1 Apr 2023LBA03-09 CDH12 DRIVES THE DEVELOPMENT OF THE NEURONAL SUBTYPE IN MIBC Xingyu Chen, Mukta Basu, Simon Knott, and Dan Theodorescu Xingyu ChenXingyu Chen More articles by this author , Mukta BasuMukta Basu More articles by this author , Simon KnottSimon Knott More articles by this author , and Dan TheodorescuDan Theodorescu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003426.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We discovered a novel cell epithelial cell population we termed “C3” in muscle-invasive bladder cancer (MIBC) that is negatively prognostic for surgery and chemotherapy. This cell population shares similarities with the neuronal subtype MIBC (NEBC) which has limited treatment options and is driven by unclear molecular mechanisms. The objective of the study was to determine if NEBC is driven by some of the genes that are overexpressed in C3 cells. A top overexpressed and C3 cell specific gene that is also enriched in normal neuronal tissue is Cadherin 12 (N-Cadherin 2, CDH12). Hence, we initiated a study of this gene. METHODS: We overexpressed CDH12 in two murine MIBC cell lines (NA13 and MB49) and then inoculated them into mice. Tumor growth kinetics were studied, and single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing were then used to evaluate the tumors. Tissue samples from patients with MIBC were also analyzed using snRNA-seq and Co-detection by Indexing (CODEX) multiplexed immunofluorescence, and our bioinformatics analysis combined transcriptomic profiles from >2000 MIBC samples from published datasets, to characterize the transcriptional programs induced by enforcing CDH12 expression. RESULTS: Enforced expression of CDH12 led to enhanced tumor growth and the appearance of a neuroendocrine tumor phenotype. NEBC from human samples showed the highest expression of CDH12, and snRNA-seq results showed that NEBC also has the highest proportion of CDH12 epithelial cells and demonstrated co-expression of CDH12 and neuronal markers (NCAM1, CHGA, and SYP). RNA velocity analysis in murine and human tumors implicated CDH12 as a driver for the development of the neuronal subtype. CONCLUSIONS: To our knowledge, this study provides the first identification of a genetic driver leading to NEBC and implicates a novel gene, CDH12 in this process. Source of Funding: CA274375 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1195 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xingyu Chen More articles by this author Mukta Basu More articles by this author Simon Knott More articles by this author Dan Theodorescu More articles by this author Expand All Advertisement PDF downloadLoading ...

An innovative risk index based on neutrophils and macrophages can effectively predict prognosis and immunotherapy response in patients with muscle-invasive bladder cancer.

This study aimed to establish an innovative prognostic risk index based on tumor-infiltrating immune cells (TIICs) for patients with muscle-invasive bladder cancer (MIBC). We used the CIBERSORT algorithm to calculate the abundance of immune cells in MIBC samples from the The Cancer Genome Atlas (TCGA) database. Univariate Cox regression analysis, least absolute shrinkage and selection operator (LASSO) regression analysis, and stepwise regression analysis were used to identify the included immune cells. Then, we constructed the risk index based on these cells through multivariate Cox regression analysis. By the risk index formula, we obtained the risk score of each patient and divided the patients into high-risk and low-risk groups. Kaplan-Meier (K-M) curve was used for survival analysis between groups. The receiver operating characteristic (ROC) curve was used to measure the prediction accuracy of the index. We validated the performance of the index in the IMvigor210 immunotherapy cohort. In addition, we established a nomogram combining the index and clinical characteristics. Finally, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential genes between groups. The risk index we constructed consists of three variables, including M0 macrophages, M2 macrophages, and neutrophils. K-M curve showed that the risk score had good risk stratification performance. Moreover, the risk score correlates with the patient's response to chemotherapy and immunotherapy. The nomogram has good accuracy and clinical benefit. GO and KEGG enrichment analysis revealed the tumor-promoting molecular mechanism of the index. The risk index based on TIICs can facilitate individualized treatment of MIBC patients by predicting prognosis and immunotherapy response.

Predictive value of molecular subtypes and APOBEC3G for adjuvant chemotherapy in urothelial bladder cancer.

Although targeted approaches have become available in second- and third-line settings, platinum-based chemotherapy remains the standard first-line treatment for advanced muscle-invasive bladder cancer (MIBC). Therefore, the prediction of platinum resistance is of utmost clinical importance. In this study, we established a routine compatible method for the molecular classification of MIBC samples according to various classification systems and applied this method to evaluate the impact of subtypes on survival after adjuvant chemotherapy. This retrospective study included 191 patients with advanced MIBC (pT≥3 or pN+) who underwent radical cystectomy, with or without adjuvant chemotherapy. A 48-gene panel and classifier rule set were established to determine molecular subtypes according to TCGA, MDA, LundTax, and Consensus classifications. Additionally, 12 single platinum-predictive candidate genes were assessed. The results were correlated with patients' clinicopathological and follow-up data and were validated using independent data sets. Our final evaluation of 159 patients demonstrated better survival in the luminal groups for those who received chemotherapy compared with those who did not. In contrast, no such differences were observed in basal subtypes. The use of chemotherapy was associated with better survival in patients with high APOBEC3G expression (p < 0.002). This association was confirmed using an independent data set of patients who received neoadjuvant platinum therapy. The proposed method robustly replicates the most commonly used transcriptome-based subtype classifications from paraffin-embedded tissue samples. The luminal, but not basal, molecular subtypes had the greatest benefit from adjuvant platinum therapy. We identified and validated APOBEC3G as a novel predictive marker for platinum-treated patients.

CK5/6 and GATA3 Defined Phenotypes of Muscle-Invasive Bladder Cancer: Impact in Adjuvant Chemotherapy and Molecular Subtyping of Negative Cases.

Introduction and ObjectiveIdentifying patients that benefit from cisplatin-based adjuvant chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC). The purpose of this study is to correlate “luminal” and “basal” type protein expression with histological subtypes, to investigate the prognostic impact on survival after adjuvant chemotherapy and to define molecular consensus subtypes of “double negative” patients (i.e., without expression of CK5/6 or GATA3).Materials and MethodsWe performed immunohistochemical (IHC) analysis of CK5/6 and GATA3 for surrogate molecular subtyping in 181 MIBC samples. The mRNA expression profiles for molecular consensus classification were determined in CK5/6 and GATA3 (double) negative cases using a transcriptome panel with 19.398 mRNA targets (HTG Molecular Diagnostics). Data of 110 patients undergoing radical cystectomy were available for survival analysis.ResultsThe expression of CK5/6 correlated with squamous histological subtype (96%) and expression of GATA3 was associated with micropapillary histology (100%). In the multivariate Cox-regression model, patients receiving adjuvant chemotherapy had a significant survival benefit (hazard ratio [HR]: 0.19 95% confidence interval [CI]: 0.1–0.4, p < 0.001) and double-negative cases had decreased OS (HR: 4.07; 95% CI: 1.5–10.9, p = 0.005). Double negative cases were classified as NE-like (30%), stroma-rich (30%), and Ba/Sq (40%) consensus molecular subtypes and displaying different histological subtypes.ConclusionImmunohistochemical-based classification was associated with histological subtypes of urothelial MIBC. IHC markers like CK5/6 and GATA3 that are used in pathological routine could help to identify patients with basal and luminal tumor characteristics. However, a two-sided classification system might not sufficiently reflect the heterogeneity of bladder cancer to make treatment decisions. Especially the group of IHC-double negative cases, as further analyzed by mRNA expression profiling, are a heterogeneous group with different implications for therapy.

Single-Cell Sequencing Reveals that DBI is the Key Gene and Potential Therapeutic Target in Quiescent Bladder Cancer Stem Cells.

Background: Drug resistance and recurrence often develop during the treatment of muscle-invasive bladder cancer (MIBC). The existence of cancer stem cells (CSCs) in MIBC makes the formulation of effective treatment strategies extremely challenging. We aimed to use single-cell RNA sequencing approaches to identify CSCs and evaluate their molecular characteristics and to discover possible therapeutic measures. Methods: GEO data sets GSE130001 and GSE146137 were used to construct an expression matrix. After cells were identified by type, malignant epithelial cells inferred by InferCNV were extracted for stemness evaluation. The subset of cells with the highest stemness was subjected to weighted gene coexpression network analysis (WGCNA) and pseudotime analysis to identify key genes. In addition, we predicted drug sensitivity relationships for key genes in CTD and predicted the correlation between drugs and survival through siGDC. Results: We found that there were some CSCs in MIBC samples. The CSC population was heterogeneous during tumor development and was divided into quiescent and proliferating CSCs. We identified DBI as the key gene in quiescent CSCs. Analysis of a TCGA data set showed that higher DBI expression indicated higher histological grade. In addition, we predicted that acetaminophen can reduce DBI expression, thereby reducing the stemness of CSCs. Thus, we identified a potential new use of acetaminophen. Conclusion: We systematically explored CSCs in tumors and determined that DBI may be a key gene and potential therapeutic target in quiescent CSCs. In addition, we confirmed that acetaminophen may be a candidate drug targeting CSCs, improving our understanding of CSC-targeting therapeutic strategies.

Classification of Muscle Invasive Bladder Cancer to Predict Prognosis of Patients Treated with Immunotherapy.

Background Recently, immunotherapies have been approved for advanced muscle invasive bladder cancer (MIBC) treatment, but only a small fraction of MIBC patients could achieve a durable drug response. Our study is aimed at identifying tumor microenvironment (TME) subtypes that have different immunotherapy response rates. Methods The mRNA expression profiles of MIBC samples from seven discovery datasets (GSE13507, GSE31684, GSE32548, GSE32894, GSE48075, GSE48276, and GSE69795) were analyzed to identify TME subtypes. The identified TME subtypes were then validated by an independent dataset (TCGA-MIBC). The subtype-related biomarkers were discovered using computational analyses and then utilized to establish a random forest predictive model. The associations of TME subtypes with immunotherapy therapeutic responses were investigated in a group of patients who had been treated with immunotherapy. A prognostic index model was constructed using the subtype-related biomarkers. Two nomograms were built by the subtype-related biomarkers or the clinical parameters. Results Two TME subtypes, including ECM-enriched class (EC) and immune-enriched class (IC), were found. EC was associated with greater extracellular matrix (ECM) pathways, and IC was correlated with immune pathways, respectively. Overall survival was significantly greater for tumors classified as IC, whereas the EC subtype had a worse prognosis. A total of nine genes (AKAP12, APOL3, CXCL13, CXCL9, GBP4, LRIG1, PEG3, PODN, and PTPRD) were selected by computational analyses to construct the random forest model. The area under the curve (AUC) values for this model were 0.827 and 0.767 in the testing and external validation datasets, respectively. Therapeutic response rates were greater in IC patients than in EC patients (28 percent vs. 18 percent). Patients with a high prognostic index had a poorer prognosis than those with a low prognostic index. The nomogram constructed from nine genes and stage achieved a C-index of 0.71. Conclusion The present investigation defined two distinct TME subtypes and developed models to assess immunotherapeutic treatment outcomes.

Poor clinical outcomes and immunoevasive contexture in SIRPα+ tumor-associated macrophages enriched muscle-invasive bladder cancer patients.

In tumor immune microenvironment, the functions of tumor-associated macrophages (TAMs), including phagocytosis and immunomodulatory, have attracted increasing attention recently. With the discovery of CD47-signal regulatory protein-α (SIRPα) as "don't eat me" signaling pathway, the role of novel subpopulation of TAMs expressing SIRPα has not been fully elucidated in a wide spectrum of solid tumors including bladder cancer. In this study, we investigated the prognostic and predictive implication of SIRPα+ TAMs regarding clinical outcomes and adjuvant chemotherapeutic benefit in muscle-invasive bladder cancer (MIBC), and preliminarily characterized the phenotypic features of SIRPα+ TAMs and its relationship with immune contexture. A total of 141 histochemical MIBC samples from Zhongshan Hospital (ZS), 45 fresh tissue samples, and 391 MIBC patients from TCGA database were enrolled in this study. SIRPα+ TAMs was evaluated by immunohistochemical staining of CD68 and SIRPα, and flow cytometry fluorescence staining. Our results illustrated that SIRPα+ TAMs were enriched in MIBC specimens. Patients with high SIRPα+ TAMs infiltration suffered significant poor overall survival and recurrence-free survival (P = 0.0030 and P = 0.0282). SIRPα+ TAMs infiltration was an independent prognosticator in multivariate Cox model. Moreover, adjuvant chemotherapy (ACT) application showed significantly survival benefit in patients with low SIRPα+ TAMs infiltration (P = 0.0135). SIRPα+ TAMs with suppressive phenotype exhibited a positive correlation with immune tolerance and dysfunctional CD8+ T cells in MIBC. SIRPα+ TAMs infiltration indicated poor prognosis and ACT resistance in MIBC. Immunosuppressive SIRPα+ TAMs is closely related to immune evasion with exhausted T cells states, suggesting the prospect of SIRPα+ TAMs as a potential therapeutic target in MIBC.

LCK and CD3E Orchestrate the Tumor Microenvironment and Promote Immunotherapy Response and Survival of Muscle-Invasive Bladder Cancer Patients.

Background: Studies have demonstrated the significance of multiple biomarkers for bladder cancer. Here, we attempt to present biomarkers potentially predictive of the prognosis and immunotherapy response of muscle-invasive bladder cancer (MIBC). Method: Immune and stromal scores were calculated for MIBC patients from The Cancer Genome Atlas (TCGA). Core differential expression genes (DEGs) with prognostic value were identified and validated using an independent dataset GSE31684. The clinical implications of prognostic genes and the inter-gene correlation were presented. The distribution of tumor-infiltrating immune cells (TICs), the correlation with tumor mutation burden (TMB), and the expression of eight immune checkpoint–relevant genes and CD39 were accordingly compared. Two bladder cancer cohorts (GSE176307 and IMvigor210) receiving immunotherapy were recruited to validate the prognostic value of LCK and CD3E for immunotherapy. Results: 361 MIBC samples from TCGA revealed a worse overall survival for higher stromal infiltration (p = 0.009) but a better overall survival for higher immune infiltration (p = 0.042). CD3E and LCK were independently validated by TCGA and GSE31684 to be prognostic for MIBC. CD3E was the most correlative gene of LCK, with a coefficient of r = 0.86 (p < 0.001). CD8+ T cells and macrophage M1 are more abundant in favor of a higher expression of CD3E and LCK in MIBC and across pan-cancers. Immune checkpoints like CTLA4, CD274 (PD-1), and PDCD1 (PD-L1) were highly expressed in high-CD3E and high-LCK groups for MIBC and also for pan-cancers, except for thymoma. LCK and CD3E had a moderate positive correlation with CD39 expression. Importantly, high-LCK and high-CD3E groups had a higher percentage of responders than the low-expression groups both in GSE176307 (LCK: 22.73vs. 13.64%, CD3E: 22.00 vs. 13.16%) and IMvigor210 cohorts (LCK: 28.19 vs. 17.45%, CD3E: 25.50 vs. 20.13%). Conclusion: CD3E and LCK were potential biomarkers of MIBC. CD3E and LCK were positively correlated with several regular immunotherapy biomarkers, which is supported by real-world outcomes from two immunotherapy cohorts.

A quantitative polymerase chain reaction based method for molecular subtype classification of urinary bladder cancer-Stromal gene expressions show higher prognostic values than intrinsic tumor genes.

Transcriptome-based molecular subtypes of muscle-invasive bladder cancer (MIBC) have been shown to be both prognostic and predictive, but are not used in routine clinical practice. We aimed to develop a feasible, reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based method for molecular subtyping. First, we defined a 68-gene set covering tumor intrinsic (luminal, basal, squamous, neuronal, epithelial-to-mesenchymal, in situ carcinoma) and stromal (immune, extracellular matrix, p53-like) signatures. Then, classifier methods with this 68-gene panel were developed in silico and validated on public data sets with available subtype class information (MD Anderson [MDA], The Cancer Genome Atlas [TCGA], Lund, Consensus). Finally, expression of the selected 68 genes was determined in 104 frozen tissue samples of our MIBC cohort by RT-qPCR using the TaqMan Array Card platform and samples were classified by our newly developed classifiers. The prognostic value of each subtype classification system and molecular signature scores were assessed. We found that the reduced marker set combined with the developed classifiers were able to reproduce the TCGA II, MDA, Lund and Consensus subtype classification systems with an overlap of 79%, 76%, 69% and 64%, respectively. Importantly, we could successfully classify 96% (100/104) of our MIBC samples by using RT-qPCR. Neuronal and luminal subtypes and low stromal gene expressions were associated with poor survival. In conclusion, we developed a robust and feasible method for the molecular subtyping according to the TCGA II, MDA, Lund and Consensus classifications. Our results suggest that stromal signatures have a superior prognostic value compared to tumor intrinsic signatures and therefore underline the importance of tumor-stroma interaction during the progression of MIBC.

MP66-14 DEVELOPMENT &amp; VALIDATION OF A METHOD FOR DETECTION OF CIRCULATING TUMOUR DNA IN MUSCLE-INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP66)1 Sep 2021MP66-14 DEVELOPMENT & VALIDATION OF A METHOD FOR DETECTION OF CIRCULATING TUMOUR DNA IN MUSCLE-INVASIVE BLADDER CANCER Ruchira Nandurkar, Pavel Sluka, Hady Wardan, Ian Davis, and Shomik Sengupta Ruchira NandurkarRuchira Nandurkar More articles by this author , Pavel SlukaPavel Sluka More articles by this author , Hady WardanHady Wardan More articles by this author , Ian DavisIan Davis More articles by this author , and Shomik SenguptaShomik Sengupta More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002106.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Survival from muscle-invasive Bladder Cancer (MIBC) remains variable despite optimal therapy. Current clinical and pathological variables are limited in their prognostic and predictive utility. Identifying the presence and amount of circulating tumour DNA (ctDNA) in the bloodstream is of great interest as an additional tool. Currently, this relies on next-generation sequencing of tumour samples, which is expensive and can take a long time for results, thus precluding responsive clinical decision-making. This study aimed to develop a quick, sensitive, inexpensive and reliable method for detection of ctDNA in MIBC using droplet digital PCR (ddPCR). METHODS: We selected a combination of 10 mutations found in MIBC that were not only common, but when sequentially added to a gene panel, each would increase the panel’s capacity to detect a wider cohort of MIBC patients, thereby maximising the detection rate whilst maintaining assay feasibility. Mutations were selected based upon data published by The Cancer Genome Atlas (TCGA). Our 10-gene panel was tested against archival MIBC specimens. DNA extracted from archival paraffin-embedded tissue blocks was run against the gene panel using ddPCR to assess for presence and fractional abundance of the mutation in each sample. RESULTS: To date, 66 MIBC samples have been processed, of which 65 possessed at least one of the mutations within our gene panel, conferring a 98.5% detection rate, with a median of five panel mutations detected per sample (range 0-9). Our use of ddPCR may be accountable for our higher detection rate compared to the 40% predicted from the TCGA, which used Next Generation Sequencing technology. Further analyses are being undertaken to compare samples before and after neo-adjuvant chemotherapy. CONCLUSIONS: This technique utilising a 10 gene panel appears to be sensitive enough to detect mutations in the majority of patients with MIBC. Prospective validation is underway to determine the potential clinical utility of this technique. Source of Funding: Robert Bulley Research and Innovation Grant, Eastern Health Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1134-e1135 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ruchira Nandurkar More articles by this author Pavel Sluka More articles by this author Hady Wardan More articles by this author Ian Davis More articles by this author Shomik Sengupta More articles by this author Expand All Advertisement Loading ...

Robust Prognostic Subtyping of Muscle-Invasive Bladder Cancer Revealed by Deep Learning-Based Multi-Omics Data Integration.

Muscle-invasive bladder cancer (MIBC) is the most common urinary system carcinoma associated with poor outcomes. It is necessary to develop a robust classification system for prognostic prediction of MIBC. Recently, increasing omics data at different levels of MIBC were produced, but few integration methods were used to classify MIBC that reflects the patient’s prognosis. In this study, we constructed an autoencoder based deep learning framework to integrate multi-omics data of MIBC and clustered samples into two different subgroups with significant overall survival difference (P = 8.11 × 10-5). As an independent prognostic factor relative to clinical information, these two subtypes have some significant genomic differences. Remarkably, the subtype of poor prognosis had significant higher frequency of chromosome 3p deletion. Immune decomposition analysis results showed that these two MIBC subtypes had different immune components including macrophages M1, resting NK cells, regulatory T cells, plasma cells, and naïve B cells. Hallmark gene set enrichment analysis was performed to investigate the functional character difference between these two MIBC subtypes, which revealed that activated IL-6/JAK/STAT3 signaling, interferon-alpha response, reactive oxygen species pathway, and unfolded protein response were significantly enriched in upregulated genes of high-risk subtype. We constructed MIBC subtyping models based on multi-omics data and single omics data, respectively, and internal and external validation datasets showed the robustness of the prediction model as well as its ability of prognosis (P < 0.05 in all datasets). Finally, through bioinformatics analysis and immunohistochemistry experiments, we found that KRT7 can be used as a biomarker reflecting MIBC risk.

Spatially resolved metabolomic characterization of muscle invasive bladder cancer by mass spectrometry imaging.

Muscle invasive bladder cancer (MIBC) is an advanced stage of bladder cancer which poses a severe threat to life. Cancer development is usually accompanied by remarkable alterations in cell metabolism, and hence deep insights into MIBC at the metabolomic level can facilitate the understanding of the biochemical mechanisms involved in the cancer development and progression. In this proof-of-concept study, the optimal cutting temperature (OCT)-embedded MIBC samples were first washed with pure water to remove the polymer compounds which could cause severe signal suppression during mass spectrometry. Further, the tissue sections were analyzed by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging (IR-MALDESI MSI), providing an overview on the spatially resolved metabolomic profiles. The MSI dataenabled the discrimination between not only the cancerous and normal tissues, but also the subregions within a tissue section associated with different disease states. Using t-Distributed Stochastic Neighbor Embedding (t-SNE), the hyperdimensional MSI data was mapped into a two-dimensional space to visualize the spectral similarity, providing evidence that metabolomic alterations might have occurred outside the histopathological tumor border. Least absolute shrinkage and selection operator (LASSO) was further employed to classify sample pathology in a pixel-wise manner, yielding excellent prediction sensitivity and specificity up to 96% based on the statistically characteristic spectral features. The results demonstrate great promise of IR-MALDESI MSI to identify molecular changes derived from cancer and unveil tumor heterogeneity, which can potentially promote the discovery of clinically relevant biomarkers and allow for applications in precision medicine.

Exploration of Prognostic Biomarkers of Muscle-Invasive Bladder Cancer (MIBC) by Bioinformatics.

We aimed to discover prognostic factors of muscle-invasive bladder cancer (MIBC) and investigate their relationship with immune therapies. Online data of MIBC were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO) database. Weighted gene co-expression network analysis (WGCNA) and univariate Cox analysis were applied to classify genes into different groups. Venn diagram was used to find the intersection of genes, and prognostic efficacy was proved by Kaplan-Meier analysis. Heatmap was utilized for differential analysis. Riskscore (RS) was calculated according to multivariate Cox analysis and evaluated by receiver operating characteristic curve (ROC). MIBC samples from TCGA and GEO were analyzed by WGCNA and univariate Cox analysis and intersected at 4 genes, CLK4, DEDD2, ENO1, and SYTL1. Higher SYTL1 and DEDD2 expressions were significantly correlated with high tumor grades. Riskscore based on genes showed great prognostic efficiency in predicting overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in TCGA dataset (P < .001). The area under the ROC curve (AUC) of RS reached 0.671 in predicting 1-year survival and 0.653 in 3-year survival. KEGG pathways enrichment filtered 5 enriched pathways. xCell analysis showed increased T cell CD4+ Th2 cell, macrophage, macrophage M1, and macrophage M2 infiltration in high RS samples (P < .001). In immune checkpoints analysis, PD-L1 expression was significantly higher in patients with high RS. We have, therefore, constructed RS as a convincing prognostic index for MIBC patients and found potential targeted pathways.

Integrative multi-omics analysis of muscle-invasive bladder cancer identifies prognostic biomarkers for frontline chemotherapy and immunotherapy

Only a subgroup of patients with muscle-invasive bladder cancer (MIBC) are responders toward cisplatin-based chemotherapy and PD-L1 blockade immunotherapy. There is a clinical need to identify MIBC molecular subtypes and biomarkers for patient stratification toward the therapies. Here, we performed an integrative clustering analysis of 388 MIBC samples with multi-omics data and identified basal and luminal/differentiated integrative subtypes and derived a 42 gene panel for classification of MIBC. Using nine additional gene expression data (n = 844), we demonstrated the prognostic value of the 42 basal-luminal genes. The basal subtype was associated with worse overall survival in patients receiving no neoadjuvant chemotherapy (NAC), but better overall survival in patients receiving NAC in two clinical trials. Each of the subtypes could be further divided into chr9 p21.3 normal or loss subgroup. The patients with low expression of MTAP/CDKN2A/2B (indicative of chr9 p21.3 loss) had a significantly lower response rate to anti-PD-L1 immunotherapy and worse survival than the patients with high expression of MTAP/CDKN2A/2B. This integrative analysis reveals intrinsic MIBC subtypes and biomarkers with prognostic value for the frontline therapies.