Muscle-invasive Bladder Cancer Tumors Research Articles

Abstract PR001: TRIM29 is required for basal bladder cancer invasive progression

Abstract TRIM29 (Tripartite Motif 29) also known ATDC, is an E3 ubiquitin ligase frequently upregulated in muscle-invasive bladder cancer (MIBC). We have previously shown TRIM29 global overexpression in a genetically engineered mouse model (GEMM) is sufficient to ` bladder cancer development and progression to MIBC. Other studies strongly suggest cytokeratin-5/14 (Krt5+/Krt14+) expressing basal-stem progenitor cells in the urothelium are the cells of origin for MIBC in mice. We therefore hypothesized that Trim29 expression in Krt5+ basal urothelial cells is required for development of MIBC in mice. To test this, we generated a GEMM harboring a Trim29 conditional KO allele and tamoxifen responsive Cre under control of the bovine Krt5promoter. Exposure of Krt5-CreERT2 Trim29flox/flox mice to tamoxifen resulted in activation of Cre and loss of Trim29 expression only in the Krt5+ basal urothelium. To induce bladder tumorigenesis, we utilized the urothelial specific carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water at 0.05%. After 6 months, tumor invasion was staged by a genitourinary pathologist based on depth of invasion: T1 (stromal invasive), T2 (muscle invasive) and T3 (deep muscle/peri-vesicular invasion). In agreement with previous studies, BBN exposure resulted in loss of Krt5-negative intermediate and superficial cells, and expansion of Krt5+/Krt14+ basal cells across the urothelium. In combination with a tamoxifen treatment this led to total Trim29 KO in the urothelium. All male Trim29 WT mice exposed to BBN developed Trim29+ bladder tumors, >50% of which demonstrated extensive progression to muscle-invasion (57% n=23). In contrast, only 16% of mice with Trim29 loss in Krt5+ basal cells developed MIBC (n=18). As an E3 ubiquitin ligase TRIM29 is known to target immune adaptor molecules, notably the stimulator of interferon genes (STING), for proteasomal degradation. We hypothesized TRIM29 targets STING during invasive progression and supports the establishment of an immune-privileged niche at the bladder cancer invasive front. We find TRIM29 expression in human bladder tissues is upregulated in the urothelium under stress and in MIBC tumors. In mice, BBN exposure upregulated Trim29 expression in the bladder urothelium which correlated with low urothelial Sting. In contrast, Krt5+ Trim29 loss resulted in significantly elevated Sting in the bladder urothelium. Multiplex immunofluorescence imaging and immunotyping of Trim29 KO mouse tissues revealed peritumoral enrichment of CD4+ and CD8+ T cells, CD206+ immunosuppressive macrophages, and CD4+FoxP3+ regulatory T cells. Taken together, these results suggest that TRIM29-mediated downregulation of STING in the bladder urothelium results in decreased immune surveillance at the tumor-stromal interface which expedites invasion and tumor burden. Citation Format: Alan Kelleher, Luke Broses, Yin Wang, Marian Henderson, Mark Day, Aaron Udager, Phillip Palmbos. TRIM29 is required for basal bladder cancer invasive progression [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 PR001.

Exploring the bladder tissue microbiome in patients with muscle-invasive bladder cancer using 2bRAD-M sequencing

Abstract Objectives The 2bRAD sequencing for Microbiome (2bRAD-M) represents an innovative and streamlined approach for the reconstruction of microbial profiles at the species level. In our investigation, we conducted 2bRAD-M analysis to characterize the microbiome of bladder tissue in patients with muscle-invasive bladder cancer (MIBC). Methods 15 tumor tissues and 15 paired para-carcinoma tissues were obtained from the bladder excised during surgery. 2bRAD-M sequencing was used to assess the abundance of microorganisms in samples. Results The microbial community structure and biodiversity, as assessed at varying taxonomic ranks, exhibited a high degree of similarity between the tumor and paired non-tumor tissues. At the genus level, we observed a notably elevated abundance of Brachybacterium and Haloparvum, coupled with a diminished abundance of Anoxybacillus, Anoxybacillu_A, Deinococcus, NCEH01, and Pseudoxanthomonas_A in the tumor tissues. Meanwhile, at the species level, the non-tumor tissues exhibited an enrichment of Anoxybacillus_A rupiensis, Anoxybacillus flavithermus_G, Klebsiella quasipneumoniae, NCEH01 sp002304505, and Pseudoxanthomonas_A sp004284195. Linear discriminant analysis effect size (LEfSe) identified 29 discriminative features, characterized by significant variations (p<0.5, LDA≥2.0) in relative abundance between the two groups. Furthermore, an analysis of functional predictions utilizing Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) also uncovered disparities in the microbial functional composition. Conclusions This study identified several microorganisms that exhibit differences between MIBC tumor tissue and adjacent non-tumor tissue using 2bRAD-M sequencing, providing some insights into the potential association between the bladder microbiome and cancer.

Cuproptosis-Related lncRNAs Modulate the Prognosis of MIBC by Regulating the Expression Pattern of Immunosuppressive Molecules Within the Tumor Microenvironment.

Cuproptosis-related gene and long non-coding RNA (lncRNA) modulation of cancer regulation is well-established. This investigation aimed to elucidate the prognostic implications of cuproptosis-associated lncRNAs in muscle-invasive bladder cancer (MIBC). Employing the Cancer Genome Atlas (TCGA) and IMvigor210 cohorts, bioinformatics and statistical analyses probed the prognostic relevance of cuproptosis-related lncRNAs. Co-expression analysis revealed tight associations between lncRNA expression and cuproptosis-linked genes, with 13 cuproptosis-related lncRNAs found to correlate with MIBC prognosis. Lasso regression identified a six-lncRNA prognostic signature, enabling patient stratification into high- and low-risk categories. Tissue validation substantiated differential expression of FAM13A-AS1, GHRLOS, LINC00456, OPA1-AS1, RAP2C-AS1, and UBE2Q1-AS1 between MIBC tumor and normal tissues. Comparative analyses of tumor microenvironments and immune profiles between risk groups disclosed elevated immunosuppressive molecule expression, including programmed cell death-1 (PD-L1) and T-cell immunoglobulin-3 (TIM-3), in high-risk individuals. These findings suggest that cuproptosis-related lncRNAs may modulate the expression of immunosuppressive molecules, thereby influencing MIBC tumorigenesis and progression. Further exploration is warranted to unveil novel therapeutic targets for MIBC based on the expression patterns of cuproptosis-related lncRNAs and their impact on immune responses in the tumor microenvironment.

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.

STAG2 expression is associated with adverse survival outcomes and regulates cell phenotype in muscle-invasive bladder cancer.

STAG2 (Stromal Antigen 2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n=119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n=346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for MIBC patients. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy number variations, or cell cycle distribution. However, unbiased RNA sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a non-canonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC.

Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping.

Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting <200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response.

Abstract 5285: Antitumor and mechanism of selective serotonin reuptake inhibitor fluoxetine on muscle invasive bladder cancer

Abstract Purpose/Introduction: Bladder cancer can be broadly divided into two type, including non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Among them, MIBC has been recognized as hard to treatment type of cancer for many decades. Therefore, to develop a new treatment strategy for MIBC is urgently needed. Transcription factor nuclear factor-kappaB (NF-κB) plays important role in various cancer progression, including hepatocellular carcinoma (HCC), lung adenocarcinoma (LUAD), and MIBC. Thus, NF-κB may be a potential target to suppress the progression of MIBC. Fluoxetine is FDA approval antidepressant agent, belongs to selective serotonin reuptake inhibitor (SSRI). Interestingly, recent study indicated that Fluoxetine inhibited NF-κB and induced apoptosis in the HCC both in vivo and in vitro. However, whether Fluoxetine may regulate NF-κB and inhibit MIBC tumor progression is remaining unknown. Therefore, the aim of present study is to verify the treatment efficacy and underlying mechanism of fluoxetine on MIBC. Methods: Human MIBC cells TSGH8301 and T24 cells and mice bearing with MIBC MB49 cells was used in this study. Cytotoxicity, apoptosis, and metastasis ability of cells after fluoxetine treatment was validated through MTT assay, cleaved caspase-3, -8, -9 activation using flow cytometry and transwell migration/invasion assay, respectively. In in vivo study murine MB49 cells were subcutaneous inoculated into right flank of C57B6/J mice and treated with fluoxetine for 15 days. Result: Fluoxetine markedly induced cytotoxicity, cleaved caspase-3, -8, -9 expression, and inhibited migration and invasion in both TSGH8301 and T24 cells. Western blotting and reporter gene assay indicated that fluoxetine may suppress the phosphorylation and activity of NF-κB. In in vivo results, fluoxetine inhibited tumor growth and the expression of NF-κB within tumor. Tumor immunohistochemistry (IHC) staining results proved that the apoptosis marker such as Caspase-3, -8, -9 were all increased by fluoxetine. AST and ALT value that represent as liver function showed no significant difference between vehicle group and fluoxetine treatment group. Body weight and H&amp;E results also proved the safety of fluoxetine usage. Conclusion: Fluoxetine induced cytotoxicity via both extrinsic/intrinsic apoptotic pathway and inhibited metastasis ability as well. Fluoxetine may also suppress the phosphorylation and activity NF-κB in vitro and in vivo. In sum, we suggested that fluoxetine may effectively suppress tumor progression via inactivate NF-κB signaling pathway. Citation Format: Zhao-Lin Tan, Yuan Chang, I-Tsang Chiang, Fei-Ting Hsu, Fei-Ting Hsu. Antitumor and mechanism of selective serotonin reuptake inhibitor fluoxetine on muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5285.

Abstract 3092: Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment

Abstract The outcome of patients with Muscle Invasive Bladder Cancer (MIBC) is highly variable with many patients experiencing progressive disease despite multimodal therapy. A key variable that contributes to poor patient outcomes lies in our inability to predict therapeutic response due to the molecular heterogeneity of MIBC tumors. There is, therefore, a significant unmet clinical need to accurately predict effective drug combinations and therapeutic doses in individual patients with MIBC. Patient-derived tumor organoids (PDOs) have the potential to serve as an ex vivo model of urothelial carcinoma (UC) capable of functionally predicting patient-specific treatment responses. However, current culture procedures typically rely on the expansion of UC organoids from resected tumors or biopsy specimens, which is inherently invasive and hence limits clinical application. We hypothesized that culture of MIBC PDOs can be achieved from urine samples, as is the case in dogs with MIBC, since histopathological features, tumor heterogeneity and subtypes, metastatic activity, and gene expression profiles of canine MIBC mimic those of human MIBC. Herein, we successfully cultured MIBC organoids from both human and canine patients using first-catch urine. Briefly, after centrifuging urine samples to pellet cells, the cells were washed with basal culture media containing FBS and Pen/Strep before being cultured in canine or human culture media containing essential growth factors based on their species of origin. Paraffin-embedded samples were used for molecular characterization by RNA in situ hybridization or immunohistochemistry. Organoids from canine MIBC patients were analyzed for expression of the urothelial differentiation marker (FOXA1) associated with high grade bladder cancer, CD44, a non-kinase transmembrane glycoprotein expressed in UC cancer stem cells, and CK7, an epithelial cell marker that is upregulated in neoplastic UC cells. FOXA1 expression was undetectable in canine MIBC, although CD44 expression was enhanced compared to healthy dog bladder organoids. All canine MIBC organoids had significantly enhanced CK7 expression. Similarly, in human-derived UC organoids, GATA binding protein 3 (GATA3), transcription factor paired box 8 (Pax8), and tumor protein p63 were utilized as markers, with a Pax8-/p63+/GATA3+ profile indicating UC origin of the organoids. Functional response to chemotherapy (cisplatin and gemcitabine) was further assessed using cell viability assay in canine and human UC organoids. Our data suggest that organoids cultured from the urine of patients with MIBC can recapitulate organ structures and maintain marker expression profiles of the original tumor tissues. 3D organoids may provide novel insights to predict therapeutic response to chemotherapy and serve as early diagnostic biomarkers of bladder cancer in both canine and human patients. Citation Format: Dipak Kumar Sahoo, Allison P. Mosichuk, Fabrice Lucien, Igor Frank, John C. Cheville, Karin Allenspach, Jonathan P. Mochel. Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3092.

A classification based on tumor-stroma ratio and tumor budding for patients with muscle-invasive bladder cancer

Background Tumor-stroma ratio (TSR) and tumor budding (TB) play important roles in muscle-invasive bladder cancer (MIBC). We developed a rating system (TSR-TB type) based on the morphological evaluation of TSR and TB for predicting patient outcome and using individualized care. Research design and methods TSR and TB were assessed in publicly accessible MIBC tumor slides from the TCGA database. MIBC patients were classified as low stromal type or high stromal type based on TSR, and high stromal type was further classified as compartmentalized stromal type or mixed stromal type based on TB. Results TSR-TB type was an independent adverse prognostic factor for OS in MIBC (P<0.001). Low stromal type had a greater prognosis than the other subtypes (P<0.001) and were enriched for FGFR3 mutations (P=0.001), which could be susceptible to targeted therapy. The mixed stromal type was distinguished by increased M2 macrophage penetration (P<0.001), anti-tumor immune activity, DNA repair pathway mutations, and poor survival. GSEA showed that certain cancer-related pathways, such as mitotic spindle, PI3K-AKT-MTOR signaling, and the G2M checkpoint, were hyperactivated in high stromal type (all FDR<0.05). Furthermore, mixed stromal type demonstrated enhanced activation of epithelial mesenchymal transformation (EMT), inflammatory response, KRAS signaling, and angiogenesis (all FDR<0.05). Conclusion TSR and TB-based MIBC classification coincides with patient survival and molecular alterations. The identified subtypes may have important implications for individualized MIBC therapy.

Single-Cell Proteomic Analysis Dissects the Complexity of Tumor Microenvironment in Muscle Invasive Bladder Cancer.

Simple SummaryThe tumor microenvironment (TME) is considered to play a key role in the development of many types of tumors. Muscle invasive bladder cancer (MIBC), which is well known for its heterogeneity, has a highly complex TME. Herein, we integrated mass cytometry and imaging mass cytometry to systematically investigate the complexity of the MIBC TME. Our investigation revealed tumor and immune cells with diverse phenotypes. We identified a specific cancer stem-like cell cluster (ALDH+PD-L1+ER-β−), which is associated with poor prognosis and highlighted the importance of the spatial distribution patterns of MIBC TME components. The present study comprehensively elucidated the complexity of the MIBC TME and provides potentially valuable information for future research.Muscle invasive bladder cancer (MIBC) is a malignancy with considerable heterogeneity. The MIBC tumor microenvironment (TME) is highly complex, comprising diverse phenotypes and spatial architectures. The complexity of the MIBC TME must be characterized to provide potential targets for precision therapy. Herein, an integrated combination of mass cytometry and imaging mass cytometry was used to analyze tumor cells, immune cells, and TME spatial characteristics of 44 MIBC patients. We detected tumor and immune cell clusters with abnormal phenotypes. In particular, we identified a previously overlooked cancer stem-like cell cluster (ALDH+PD-L1+ER-β−) that was strongly associated with poor prognosis. We elucidated the different spatial architectures of immune cells (excluded, infiltrated, and deserted) and tumor-associated collagens (curved, stretched, directionally distributed, and chaotic) in the MIBC TME. The present study is the first to provide in-depth insight into the complexity of the MIBC TME at the single-cell level. Our results will improve the general understanding of the heterogeneous characteristics of MIBC, potentially facilitating patient stratification and personalized therapy.

Urine tumor DNA detection of minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy: A cohort study.

BackgroundThe standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis.Methods and findingsWe applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD–positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4–38.9; p = 0.02) compared to utDNA MRD–negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD–positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point.ConclusionsutDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future.

Abstract 547: Urine tumor DNA MRD detection and correlation with pathologic complete response in muscle-invasive bladder cancer treated with curative-intent radical cystectomy

Abstract Objective: Standard-of-care for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy. The inability to assess molecular residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. We seek to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. Methods: We applied uCAPP-Seq, a targeted next-generation sequencing method for detecting utDNA, to urine cell-free DNA samples acquired on the day of curative-intent radical cystectomy from 42 patients with non-metastatic bladder cancer, 30 of whom had a confirmed diagnosis of MIBC and 19 of whom received neoadjuvant chemotherapy. utDNA mutational calling was performed noninvasively without tumor tissue sequencing knowledge. The overall utDNA level for each patient was represented by the non-silent mutation with the highest duplex-supported variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy donors. The concordance between urine cell-free DNA and tumor tissue was determined for a subset of patients. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of mutations detected in urine cell-free DNA by applying a linear relationship derived from TCGA whole-exome sequencing of 409 MIBC tumors. Results: utDNA analysis revealed a median utDNA level of 0% in healthy donors and 2.4% in non-metastatic bladder cancer patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 87%. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (n = 26), median utDNA levels were 4.3% vs. 0%, respectively (P = 0.002). The lack of utDNA MRD detection was highly correlated with pathologic complete response by Fisher's exact test (P = 0.0001) with Youden's index-determined sensitivity of 81% and specificity of 81%. Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival compared to utDNA MRD-negative patients (HR = 7.2; P = 0.03) with a median follow-up time of 200 days. Leveraging data from TCGA, the median inferred TMB in utDNA MRD-positive patients was 198 mutations per exome. We suggest that 58% of these patients with inferred TMB ≥ this level might have been candidates for early immune checkpoint blockade. Conclusion: The lack of utDNA MRD detection prior to curative-intent radical cystectomy for muscle-invasive bladder cancer correlated significantly with pathologic complete response. utDNA MRD detection status also correlated significantly with progression-free survival. Furthermore, utDNA results can be used to noninvasively infer TMB, which may facilitate the targeted use of adjuvant immunotherapy. Citation Format: Kevin Chen, Pradeep S. Chauhan, Ramandeep K. Babbra, Wenjia Feng, Peter K. Harris, Katherine Dienstbach, Andrew Atkocius, Lenon Maguire, Faridi Qaium, Armaan Nallicheri, Brian C. Baumann, Melissa A. Reimers, Eric H. Kim, Zachary L. Smith, Vivek K. Arora, Aadel A. Chaudhuri. Urine tumor DNA MRD detection and correlation with pathologic complete response in muscle-invasive bladder cancer treated with curative-intent radical cystectomy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 547.

A comprehensive molecular characterization of the 8q22.2 region reveals the prognostic relevance of OSR2 mRNA in muscle invasive bladder cancer.

Technological advances in molecular profiling have enabled the comprehensive identification of common regions of gene amplification on chromosomes (amplicons) in muscle invasive bladder cancer (MIBC). One such region is 8q22.2, which is largely unexplored in MIBC and could harbor genes with potential for outcome prediction or targeted therapy. To investigate the prognostic role of 8q22.2 and to compare different amplicon definitions, an in-silico analysis of 357 patients from The Cancer Genome Atlas, who underwent radical cystectomy for MIBC, was performed. Amplicons were generated using the GISTIC2.0 algorithm for copy number alterations (DNA_Amplicon) and z-score normalization for mRNA gene overexpression (RNA_Amplicon). Kaplan-Meier survival analysis, univariable, and multivariable Cox proportional hazard ratios were used to relate amplicons, genes, and clinical parameters to overall (OS) and disease-free survival (DFS). Analyses of the biological functions of 8q22.2 genes and genomic events in MIBC were performed to identify potential targets. Genes with prognostic significance from the in silico analysis were validated using RT-qPCR of MIBC tumor samples (n = 46). High 8q22.2 mRNA expression (RNA-AMP) was associated with lymph node metastases. Furthermore, 8q22.2 DNA and RNA amplified patients were more likely to show a luminal subtype (DNA_Amplicon_core: p = 0.029; RNA_Amplicon_core: p = 0.01). Overexpression of the 8q22.2 gene OSR2 predicted shortened DFS in univariable (HR [CI] 1.97 [1.2; 3.22]; p = 0.01) and multivariable in silico analysis (HR [CI] 1.91 [1.15; 3.16]; p = 0.01) and decreased OS (HR [CI] 6.25 [1.37; 28.38]; p = 0.0177) in RT-qPCR data analysis. Alterations in different levels of the 8q22.2 region are associated with manifestation of different clinical characteristics in MIBC. An in-depth comprehensive molecular characterization of genomic regions involved in cancer should include multiple genetic levels, such as DNA copy number alterations and mRNA gene expression, and could lead to a better molecular understanding. In this study, OSR2 is identified as a potential biomarker for survival prognosis.

00005 Urine tumor DNA detects minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy

ABSTRACT IMPACT: Urine tumor DNA non-invasively detects minimal residual disease and infers tumor mutational burden in locally advanced bladder cancer prior to radical cystectomy, which may potentially enable the selection of patients for bladder-sparing treatment or facilitate personalized adjuvant immunotherapy. OBJECTIVES/GOALS: Standard-of-care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy. The inability to assess minimal residual disease (MRD) non-invasively limits our ability to offer bladder-sparing treatment. We sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. METHODS/STUDY POPULATION: We applied uCAPP-Seq, a targeted sequencing method for detecting utDNA, to urine cell-free DNA samples acquired on the day of radical cystectomy from 42 patients with bladder cancer. utDNA variant-calling was performed non-invasively without prior tumor mutational knowledge. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. Tumor mutational burden (TMB) was inferred from the number of non-silent mutations detected in urine cell-free DNA by applying a linear relationship derived from TCGA whole exome sequencing of 409 MIBC tumors. RESULTS/ANTICIPATED RESULTS: utDNA levels were significantly higher in patients with residual disease detected in their surgical pathology compared to those who achieved a pathologic complete response (P = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD significantly predicted the absence of pathologic complete response with a sensitivity of 81% and specificity of 81%. Positive utDNA MRD also portended significantly worse progression-free survival (HR = 7.4; P = 0.03) compared to negative utDNA MRD. Furthermore, we applied a linear relationship (Pearson r = 0.84; P &lt; 0.0001) to identify patients with high inferred TMB who may have been candidates for early immune checkpoint blockade. DISCUSSION/SIGNIFICANCE OF FINDINGS: utDNA MRD analysis prior to surgery correlated significantly with pathologic response and progression-free survival, which may help select patients for bladder-sparing treatment. utDNA can also non-invasively infer TMB, which could facilitate personalized adjuvant therapy for patients in the future.

Poor clinical outcomes and immunoevasive contexture in interleukin-9 abundant muscle-invasive bladder cancer.

Chemotherapy and immunotherapy yield survival benefits for muscle-invasive bladder cancer (MIBC) patients, in which tumor microenvironment has been found to exert crucial roles through tipping the balance between antitumor immunity and immune evasion. Our study aims to explore the clinical significance and therapeutic role of intratumoral interleukin-9-producing cells (IL-9+ cells) in MIBC. Two hundred fifty-nine MIBC patients from two independent clinic centers were utilized for retrospective analysis in the study. Sixty-five fresh MIBC tumor tissues were used to evaluate the infiltration and function of immune cells via flow cytometry and ex vivo intervention experiments. Three hundred ninety-one MIBC patients of The Cancer Genome Atlas were applied for bioinformatics analysis. It was found that patients with high IL-9+ cells infiltration had worse overall survival and relapse-free survival. pT2 patients with low IL-9+ cells infiltration could benefit more from adjuvant chemotherapy (ACT). IL-9+ cells infiltration was correlated with decreased expression of granzyme B from CD8+ T cells and natural killer (NK) cells and perforin from CD8+ T cells, while blockade of IL-9 reactivated the antitumor capacity of both cells leading to tumor regression. Furthermore, IL-9+ cells infiltration could be a biomarker for predicting anti-PD-1 efficacy. In conclusion, IL-9+ cells infiltration could be applied as an independent prognosticator for clinical outcome and ACT/anti-PD-1 effectiveness. IL-9+ cells infiltration diminished the cytotoxicity of CD8+ T cells and NK cells resulting in tumor immune evasion, and thus targeting IL-9 could be a potential therapeutic strategy for MIBC.

Abstract A11: Generation and characterization of immune-competent syngeneic model of muscle-invasive bladder cancer (MIBC)

Abstract Immunologically, MIBC tumors can be divided into inflamed and noninflamed phenotype. Inflamed tumors show high immune cell infiltration. In contrast, noninflamed tumors have little to no immune infiltration. Clinical evidence shows that response to therapy, particularly immune therapy, is associated with the immune phenotype of the tumor. In our preliminary analysis of the TCGA tumor expression data, we observed that patients with high CD8 T-cell infiltration expression signature show an improved overall survival irrespective of treatment. To study the molecular and immunologic drivers of CD8+ T-cell tumor infiltration in MIBC, we have generated immune-competent syngeneic models of bladder cancer. The mouse models were generated by inducing invasive disease by treating C57BL/6 mice with carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The induced tumors were grafted into sex-matched immune-competent syngeneic hosts establishing allograft tumor lines. The models represent different clinical subtypes both molecularly and histologically. Currently we have 8 different models with squamous, sarcamatoid, or a more differentiated phenotype. RNA-seq analysis of the tumor lines was used to classify the models into basal-like and luminal-like tumors using the TCGA classifier system. Using the RNA-seq data, we applied a deconvolution algorithm to identify models that had inflamed or noninflamed immune phenotype. Flow cytometry data indicate that the inflamed tumors have a higher infiltration of the myeloid population. We treated 4/8 models with cisplatin, the current standard of care. The models show variable response to cisplatin, indicating the variability between the different models. In conclusion, we have successfully developed immune-competent syngeneic mouse models of MIBC that can be used to study the molecular and immune-driven processes of the disease. Citation Format: Shruti D. Shah, Bryan Gillard, Ellen Karasik, Michelle Wrobel, Michael T. Moser, Barbara A. Foster. Generation and characterization of immune-competent syngeneic model of muscle-invasive bladder cancer (MIBC) [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A11.

MP55-02 CCR8 BLOCKADE PRIMES ANTI-TUMOR IMMUNITY THROUGH INTRATUMORAL REGULATORY T CELLS DESTABILIZATION IN MUSCLE-INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVE: Regulatory T cells (Tregs) play a major role in the development of an immunosuppressive tumor microenvironment. CCR8 has recently been identified as an important chemokine receptor on intratumoral Tregs and is known to be critical for CCR8+Treg-mediated immunosuppression. This study aimed to investigate the clinical significance and inherent molecular mechanisms of intratumoral CCR8+ Tregs in Muscle-invasive bladder cancer (MIBC). METHODS: 259 MIBC patients from two independent clinic centers were included in the study. 83 fresh MIBC tumor tissues were used to evaluate the proportion and function of immune cells via flow cytometry and ex vivo intervention experiments. 396 MIBC patients of TCGA were applied for bioinformatics analysis. RESULTS: We found that the CCR8 expression by intratumoral Tregs maintained the stability and potentiated their suppressive function by upregulating the expression of transcript factors Foxo1 and c-Maf. High level of CCR8+ Tregs is associated with immune tolerance and predicts poor survival and inferior therapeutic responsiveness to chemotherapy. Moreover, we revealed that CCR8 blockade using neutralizing antibody destabilized intratumoral Tregs into a fragile phenotype, reactivated the antitumor immunity and augmented the therapeutic benefits of the anti-PD-1 antibody in MIBC. CONCLUSIONS: Our study presents the mechanism of CCR8 in maintaining the stability of intratumoral Tregs in MIBC and indicates that therapeutic targeting of CCR8 could provide a more specific modulation of Tregs and augment immunotherapy in patients with MIBC.Source of Funding: This study was funded by grants from National Natural Science Foundation of China (81570607, 81671628, 31770851, 81702496, 81702497, 81702805, 81772696, 81871306, 81872082, 81902556, 81902563, 81902898, 81974393), Three-year action plan for promoting clinical skills and clinical innovation in municipal hospitals of Shanghai Shenkang (16CR2003A), National Natural Science Foundation for Young Scholars of China (81902566), Shanghai Jiaotong University Medical-Engineering Cross Research Fund (YG2019QNA53), National Key R&D Program of China (2017YFC0114303), Shanghai Municipal Natural Science Foundation (16ZR1406500, 17ZR1405100, 19ZR1431800), Guide Project of Science and Technology Commission of Shanghai Municipality (17411963100), Shanghai Sailing Program (18YF1404500, 19YF1407900, 19YF1427200), Shanghai Municipal Commission of Health and Family Planning Program (20174Y0042, 201840168, 20184Y0151), Fudan University Shanghai Cancer Center for Outstanding Youth Scholars Foundation (YJYQ201802) and Shanghai Cancer Research Charity Center. All these study sponsors have no roles in the study design, in the collection, analysis and interpretation of data.

Distribution of Molecular Subtypes in Muscle-invasive Bladder Cancer Is Driven by Sex-specific Differences

Muscle-invasive bladder cancer (MIBC) is a sex-biased cancer with a higher incidence in men but worse outcomes in women. The root cause behind these observations remains unclear. To investigate whether sex-specific tumor biology could explain the differences in clinical behavior of MIBC, we analyzed the transcriptome profiles from transurethral resected bladder tumors of 1000 patients. Female tumors expressed higher levels of basal- and immune-associated genes, while male tumors expressed higher levels of luminal markers. Using molecular subtyping, we found that the rates of the basal/squamous subtype were higher in females than in males. Males were enriched with tumors of the luminal papillary (LumP) and neuroendocrine-like subtypes. Male MIBC tumors had higher androgen response activity across all luminal subtypes and male patients with LumP tumors were younger. Taken together, these data confirm differences in molecular subtypes based on sex. The role of the androgen response pathway in explaining subtype differences between men and women should be studied further. Patient summaryWe explored the sex-specific biology of bladder cancer in 1000 patients and found that women had more aggressive cancer with higher immune activity. Men tended toward less aggressive tumors that showed male hormone signaling, suggesting that male hormones may influence the type of bladder cancer that a patient develops.

Molecular characterization of advanced muscle invasive bladder cancer (MIBC): Comparison of low-input targeted RNA sequencing (RNAseq) strategies.

564 Background: 5-year survival for advanced MIBC is poor (~5%), but newer targeted agents (e.g., PD-1/PD-L1 or FGFR) have improved survival over traditional chemotherapy. To better select patients suited for targeted therapies, molecular characterization is more important than ever. The following study compares RNAseq using two low-RNA-input methods (SureSelect hybridization-capture and AmpliSeq PCR amplification, using 200 ng and 10 ng extracted RNA, respectively), as part of a retrospective study of advanced MIBC patients treated with anti-PD-1/PD-L1 therapy (Mayhew et al, 2019 ASCO). RNAseq results, including correlation between methods and molecular subtype agreement are presented herein. Methods: RNA extracts from 24 representative FFPE MIBC tumor samples were analyzed using SureSelect XT RNA (Agilent) and AmpliSeq transcriptome (Illumina) by NovaSeq6000 paired end sequencing. Samples were split between 4 molecular subtypes using the GeneCentric 60-gene MIBC nearest centroid subtyper (n = 6/subtype; luminal, luminal infiltrated, basal, basal infiltrated) (Mayhew et al, 2019 ASCO). Pearson correlation analysis was performed for all genes, as well as the 60 suptyper genes. Expression subtype calls were compared using RNAseq data obtained from both sequencing methods. Results: Of 58,000 genes queried (GrCH38.v22), 27,671 (55%) were found using both methods and 13,992 (28%) were not found using either method. 15,575 unique genes were found using SureSelect and 1,149 unique genes were found using AmpliSeq. The two methods were well correlated: median of 0.87 (0.73-0.89) overall and 0.80 (0.54-0.86) for the 60 subtyper genes. 22 of the 24 samples (92%) had congruent subtype calls using the 60-gene MIBC subtyper; one luminal-infiltrated called basal and one basal called basal-infiltrated. Conclusions: This study demonstrated comparable RNAseq analysis using two low-input FFPE-compatible methods. Using AmpliSeq, MIBC tumors were able to be molecularly characterized, including subtype analysis, with only 1/20th of the extracted RNA needed for SureSelect. Further validation of AmpliSeq methodology in MIBC and other tumor types is warranted.

Association of next generation sequencing characteristics of MIBC specimens with clinical outcomes: The USC experience.

397 Background: Muscle invasive bladder cancer (MIBC) is an aggressive disease. Several molecular subtypes have been identified. Commercial next generation sequencing (NGS) panels now provide significant genetic information on tumors. We conducted an exploratory analysis on MIBC tumor samples at our institution with commercial NGS data to evaluate if these assays provide novel prognostic information. Methods: NGS data (FoundationOne) from all locally advanced and metastatic bladder specimens were collected and combined with information on demographics, tumor characteristics (stage, grade, histology). Overall Survival (OS), post-cystectomy disease free survival (DFS), and Progression Free Survival (PFS) data were also compiled. Mutations in 353 genes were examined for alterations in TP53, Rb, FGF3-RAS-RAF and PI3K-mTOR-AKT pathways. Results: 48 samples were analyzed. Most samples were pure TCC (68.8%) followed by Squamous (16.8%) , Adenocarcinoma (6.3%), Small Cell (6.3%) and Plasmacytoid variants (2.1%). Most patients (91.5%) were locally advanced at diagnosis. Pathologic alterations were found in TP53 (54.2%) , Rb (60.4%), FGF3-RAS-RAF(89.6%) and PI3K-mTOR-AKT(70.8%) pathways. Squamous variants were more likely to have TP53 pathway alterations compared with others (100% vs 45% , p &lt; 0.001) and predicted for poor overall survival. TP53 exons did not predict outcomes. In multivariable adjusted cox-regression models Rb alterations appeared to predict for improved post cystectomy DFS (p = 0.01). TP53 mutations predicted for worse PFS (p=0.01) while PI3K-mTOR-AKT pathway alterations were associated with poorer OS (p=0.02) after adjusting for all other predictors. Conclusions: In this limited single institution sample analyzed by commercial available NGS panels, some prognostic information was available. Analysis of a larger population could result in the validation of other predictive or prognostic markers.[Table: see text]