Molecular Subtypes Of Bladder Cancer Research Articles

UROSCAN and UROSCANSEQ: a large-scale multicenter effort towards translation of molecular bladder cancer subtypes into clinical practice – from biobank to RNA-sequencing in real time

Background Bladder cancer is molecularly one of the most heterogenous malignancies characterized by equally heterogenous clinical outcomes. Standard morphological assessment with pathology and added immunohistochemical analyses is unable to fully address the heterogeneity, but up to now treatment decisions have been made based on such information only. Bladder cancer molecular subtypes will likely provide means for a more personalized bladder cancer care. Methods To facilitate further development of bladder cancer molecular subtypes and clinical translation, the UROSCAN-biobank was initiated in 2013 to achieve systematic biobanking of preoperative blood and fresh frozen tumor tissue in a population-based setting. In a second phase, we established in 2018 a parallel logistic pipeline for molecular profiling by RNA-sequencing, to develop and validate clinical implementation of molecular subtyping and actionable molecular target identification in real-time. Results Until June 2021, 1825 individuals were included in the UROSCAN-biobank, of which 1650 (90%) had primary bladder cancer, 127 (7%) recurrent tumors, and 48 (3%) unknown tumor status. In 159 patients, multiple tumors were sampled, and metachronous tumors were collected in 83 patients. Between 2016 and 2020 the UROSCAN-biobanking included 1122/2999 (37%) of all primary bladder cancer patients in the Southern Healthcare Region. Until June 2021, the corresponding numbers subjected to RNA-sequencing and molecular subtyping was 605 (UROSCANSEQ), of which 52 (9%) samples were not sequenced due to inadequate RNA-quality (n = 47) or technical failure/lost sample (n = 5). Conclusions The UROSCAN-biobanking and UROSCANSEQ-infrastructure for molecular subtyping by real-time RNA-sequencing represents, to our knowledge, the largest effort of evaluating population-wide molecular classification of bladder cancer.

Prognostic Signature GXYLT2 Is Correlated with Immune Infiltration in Bladder Cancer.

Background GXYLT2 (glucoside xylosyltransferase 2) was known as an important gene that regulates classical Notch signaling and is involved in progression in human tumors. However, the correlation between GXYLT2 expression and bladder cancer remains unclear. Methods GXYLT2 expression was analyzed by ONCOMINE database, GEPIA database, and TIMER database. The Cancer Genome Atlas (TCGA) was utilized to confirm relationships between GXYLT2 and molecular subtypes of BLCA (bladder cancer). We discovered prognostic value of GXYLT2 in BLCA using GEPIA, LinkedOmics database, and Kaplan-Meier Plotter database. Subsequently, correlations between GXYLT2 and tumor immune infiltration were investigated through TIMER and TISIDB website. We then downloaded data of patients with BLCA from TCGA website, to conduct functional annotations and to construct protein-protein interaction network through STRING and Enrich web servers. Results Significant differences were observed between GXYLT2 expression of bladder cancer and normal tissues. GXYLT2 was a poor prognostic biomarker in BLCA with impact on diverse clinical characteristics. We found that GXYLT2 was closely related to tumor immune infiltrated cells and immune genes. Functional annotations indicated that GXYLT2 was linked to immune-related pathways. Conclusions The results suggested that GXYLT2 was associated with a poor prognosis and tumor immune cell infiltration of BLCA. GXYLT2 could be a promising therapeutic target in bladder cancer.

An EMT-based risk score thoroughly predicts the clinical prognosis, tumor immune microenvironment and molecular subtypes of bladder cancer.

BackgroundEpithelial mesenchymal transition (EMT) is closely related to the occurrence, development, metastasis and antitumor immunity of tumors. However, comprehensive studies correlating EMT and prognosis, tumor microenvironment (TME) and molecular subtypes of bladder cancer (BLCA) are lacking.MethodsTCGA-BLCA was chosen as our training cohort, while Xiangya cohort, GSE13507, GSE48075 were selected as our validation cohorts. Prognostic genes were screened out using univariate Cox analysis and the least absolute shrinkage and selection operator (LASSO) algorithm. Then we developed an EMT risk score based on these prognostic genes and systematically correlated the risk score with prognosis, TME and molecular subtypes of BLCA.ResultsBased on EMT related genes, we developed two different EMT patterns, named EMT cluster 1 and cluster 2, and found that cluster 2 showed a worse prognosis and an inflammatory TME phenotype. For personalized prognosis and TME phenotypes predicting, we developed and validated an EMT-based risk score by 7 candidate genes (ANXA10, CNTN1, FAM180A, FN1, IGFL2, KANK4 and TOX3). Patients with high EMT risk scores had lower overall survival (OS) with high predictive accuracy both in the training cohort and validation cohort. In addition, we comprehensively correlated the EMT risk score with TME and molecular subtype, and found that high EMT risk score suggested higher levels of immune cell infiltration and more inclined to present the basal molecular subtype. It was noteworthy that the same results also appeared in the validation of Xiangya cohort.ConclusionsEMT related genes play an important role in tumor progression and immunity in BLCA. Our EMT risk score could accurately predict prognosis and immunophenotype of a single patient, which could guide more effective precision medical strategies.

High-Content Drug Discovery Targeting Molecular Bladder Cancer Subtypes.

Molecular subtypes of muscle-invasive bladder cancer (MIBC) display differential survival and drug sensitivities in clinical trials. To date, they have not been used as a paradigm for phenotypic drug discovery. This study aimed to discover novel subtype-stratified therapy approaches based on high-content screening (HCS) drug discovery. Transcriptome expression data of CCLE and BLA-40 cell lines were used for molecular subtype assignment in basal, luminal, and mesenchymal-like cell lines. Two independent HCSs, using focused compound libraries, were conducted to identify subtype-specific drug leads. We correlated lead drug sensitivity data with functional genomics, regulon analysis, and in-vitro drug response-based enrichment analysis. The basal MIBC subtype displayed sensitivity to HDAC and CHK inhibitors, while the luminal subtype was sensitive to MDM2 inhibitors. The mesenchymal-like cell lines were exclusively sensitive to the ITGAV inhibitor SB273005. The role of integrins within this mesenchymal-like MIBC subtype was confirmed via its regulon activity and gene essentiality based on CRISPR–Cas9 knock-out data. Patients with high ITGAV expression showed a significant decrease in the median overall survival. Phenotypic high-content drug screens based on bladder cancer cell lines provide rationales for novel stratified therapeutic approaches as a framework for further prospective validation in clinical trials.

Prediction of Response to Cisplatin-Based Neoadjuvant Chemotherapy of Muscle-Invasive Bladder Cancer Patients by Molecular Subtyping including KRT and FGFR Target Gene Assessment.

Patients with muscle-invasive urothelial carcinoma achieving pathological complete response (pCR) upon neoadjuvant chemotherapy (NAC) have improved prognosis. Molecular subtypes of bladder cancer differ markedly regarding sensitivity to cisplatin-based chemotherapy and harbor FGFR treatment targets to various content. The objective of the present study was to evaluate whether preoperative assessment of molecular subtype as well as FGFR target gene expression is predictive for therapeutic outcome—rate of ypT0 status—to justify subsequent prospective validation within the “BladderBRIDGister”. Formalin-fixed paraffin-embedded (FFPE) tissue specimens from transurethral bladder tumor resections (TUR) prior to neoadjuvant chemotherapy and corresponding radical cystectomy samples after chemotherapy of 36 patients were retrospectively collected. RNA from FFPE tissues were extracted by commercial kits, Relative gene expression of subtyping markers (e.g., KRT5, KRT20) and target genes (FGFR1, FGFR3) was analyzed by standardized RT-qPCR systems (STRATIFYER Molecular Pathology GmbH, Cologne). Spearman correlation, Kruskal–Wallis, Mann–Whitney and sensitivity/specificity tests were performed by JMP 9.0.0 (SAS software). The neoadjuvant cohort consisted of 36 patients (median age: 69, male 83% vs. female 17%) with 92% of patients being node-negative during radical cystectomy after 1 to 4 cycles of NAC. When comparing pretreatment with post-treatment samples, the median expression of KRT20 dropped most significantly from DCT 37.38 to 30.65, which compares with a 128-fold decrease. The reduction in gene expression was modest for other luminal marker genes (GATA3 6.8-fold, ERBB2 6.3-fold). In contrast, FGFR1 mRNA expression increased from 33.28 to 35.88 (~6.8-fold increase). Spearman correlation revealed positive association of pretreatment KRT20 mRNA levels with achieving pCR (r = 0.3072: p = 0.0684), whereas pretreatment FGFR1 mRNA was associated with resistance to chemotherapy (r = −0.6418: p < 0.0001). Hierarchical clustering identified luminal tumors of high KRT20 mRNA expression being associated with high pCR rate (10/16; 63%), while the double-negative subgroup with high FGFR1 expression did not respond with pCR (0/9; 0%). Molecular subtyping distinguishes patients with high probability of response from tumors as resistant to neoadjuvant chemotherapy. Targeting FGFR1 in less-differentiated bladder cancer subgroups may sensitize tumors for adopted treatments or subsequent chemotherapy.

CD93 orchestrates the tumor microenvironment and predicts the molecular subtype and therapy response of bladder cancer

BackgroundCD93 is newly reported to normalize vasculature and attenuate pancreatic cancer therapy response, but its role in bladder cancer (BLCA) is unknown. MethodThe immunologic role of CD93 is analyzed across TCGA pan-cancers. The correlation between CD93 and BLCA clinical and tumor microenvironment features, predicted immunotherapy pathways, molecular subtypes, therapeutic signatures and mutation status was evaluated in TCGA-BLCA and other two BLCA cohorts. The impact of CD93 on immunotherapy response was validated by five real-world cohorts, and chemotherapy response was assessed with IC50. CD93-based risk model was constructed with LASSO regression and validated by seven independent cohorts. ResultCD93 is positively correlated with immunomodulators, tumor-infiltrating lymphocytes (TILs) and immune checkpoints across pan-cancers. In BLCA, CD93 leads to higher T cell inflamed score and expression of immune checkpoints. However, CD93 is indicative of more aggressive clinical features, worse survival, more tumor-associated macrophages and regulatory T cells recruitment, less recognition and killing of cancer cells by T cells, lower predicted chemotherapy and immunotherapy response, which is further validated by immunotherapy cohorts (IMvigor210: 16.11% vs 29.53%; GSE176307: 15.56% vs 20.93%). Notably, CD93 correlates with enriched neuroendocrine subtype and epithelial-mesenchymal transition differentiation, while CD93-low group has enriched luminal subtype. Pathways including hypoxia and Wnt-β-catenin are enriched along with CD93 expression, and more frequent FGFR3 mutation is also observed. Lastly, the CD93-based risk model, validated by seven independent cohorts, is powerful in distinguishing the survival probability of BLCA (3-year AUC 0.808). ConclusionCD93 plays a critical role in tumor immune regulation. CD93 expression indicates more aggressive clinicopathological status and molecular subtypes of BLCA and worse therapy response, which implies that combing anti-CD93 therapy with immunotherapy (or chemotherapy) may be potentially beneficial for BLCA in clinical practice.

Heterogeneity Analysis of Bladder Cancer Based on DNA Methylation Molecular Profiling.

Bladder cancer is a highly complex and heterogeneous malignancy. Tumor heterogeneity is a barrier to effective diagnosis and treatment of bladder cancer. Human carcinogenesis is closely related to abnormal gene expression, and DNA methylation is an important regulatory factor of gene expression. Therefore, it is of great significance for bladder cancer research to characterize tumor heterogeneity by integrating genetic and epigenetic characteristics. This study explored specific molecular subtypes based on DNA methylation status and identified subtype-specific characteristics using patient samples from the TCGA database with DNA methylation and gene expression were measured simultaneously. The results were validated using an independent cohort from GEO database. Four DNA methylation molecular subtypes of bladder cancer were obtained with different prognostic states. In addition, subtype-specific DNA methylation markers were identified using an information entropy-based algorithm to represent the unique molecular characteristics of the subtype and verified in the test set. The results of this study can provide an important reference for clinicians to make treatment decisions.

Uroplakin II as a single marker for luminal versus basal molecular subtypes in muscle invasive urothelial carcinoma.

Bladder cancer is a heterogeneous disease classified into two broad molecular subtype categories, basal and luminal, with critical treatment and prognostic implications. Recent studies have shown the utility of immunohistochemistry in predicting bladder cancer molecular subtypes, with a two-marker approach using GATA3 and CK5/6 showing over 80% reliability. In the current study, we calculated the accuracy of uroplakin II (UPII), a marker of urothelial differentiation, with different scores (0: <1%, 1+: 1-10%, 2+: 10-50%, 3+: >50%) to predict RNA-based luminal versus basal subtypes in a cohort of muscle-invasive bladder cancer-received neoadjuvant chemotherapy followed by radical cystectomy. The 1% cutoff of the UPII stain predicts the luminal subtype with the sensitivity and specificity of 95% and 56%, respectively. With a UPII cutoff of 10%, the sensitivity and specificity were 93% and 81%, respectively, and with a UPII cutoff of 50%, the sensitivity and specificity were 91% and 96%, respectively. The prediction performance of UPII was better than either GATA3 or CK5/6. There was no significant difference in prognoses between UPII 0-2+ and UPII 3+ patients in this cohort. The current study shows that evaluating the staining proportion score of UPII can accurately predict basal and luminal subtypes of muscle-invasive bladder cancer.

GATA3 Predicts the Tumor Microenvironment Phenotypes and Molecular Subtypes for Bladder Carcinoma.

AimsGATA3 is a key player in antitumor immunology, and continuous studies show that it might be a key biomarker for bladder cancer (BLCA). Thus, we lucubrate the immunological role of GATA3 in BLCA.Main MethodsWe initially used pan-cancer analysis to analyze the expression pattern and immunological function of GATA3 with data gathered from the TCGA (The Cancer Genome Atlas). Then, in the BLCA tumor microenvironment (TME), we comprehensively associated GATA3 with immunomodulators, cancer immune cycles, tumor-infiltrating immune cells (TIICs), immune checkpoints, and T-cell inflamed scores(TIS). The role of GATA3 in predicting BLCA molecular subtypes and responsiveness to various treatment regimens was also investigated. We confirmed our findings in an external cohort and the Xiangya-Pingkuang cohort to guarantee the correctness of our study.Key FindingsGATA3 was preferentially expressed in the TME of numerous malignancies, including BLCA. High GATA3 expression was adversely connected with immunological aspects such as immunomodulators, cancer immune cycles, TIICs, immune checkpoints, and TIS in the BLCA TME. In addition, high GATA3 was more likely to be a luminal subtype, which meant it was less susceptible to cancer immunotherapy and neoadjuvant chemotherapy but more sensitive to targeted treatments.SignificanceGATA3 may aid in the precision treatment for BLCA because it can accurately predict the clinical outcomes and the TME characteristics of BLCA.

Identification of glycolysis-associated long non-coding RNA regulatory subtypes and construction of prognostic signatures by transcriptomics for bladder cancer.

Glycolysis-targeted cancer therapy based on long non-coding RNAs (lncRNAs), owing to its high specificity and less toxicity, is at the preclinical stages. Our study aimed to examine the roles of the core glycolysis-associated lncRNAs in bladder cancer (BC). Glycolysis scores of BC were computed by single-sample gene set enrichment analysis (ssGSEA). Glycolysis-associated lncRNAs were screened by Pearson's correlation analysis. Unsupervised consensus clustering using ConsensusClusterPlus assessed the glycolysis-associated lncRNAs for the identification of molecular subtypes of BC. The Kaplan-Meier survival analysis, genomic mutations, and tumor microenvironment (TME) analysis were used to compare the characteristics of different subtypes. Key glycolysis-associated lncRNAs were screened by first-order partial correlation and univariate Cox proportional-hazards model analyses; finally, the lncRNA signature was constructed. Four glycolysis-associated lncRNA-regulated subtypes having differential overall survival (OS), clinical features, genomic mutation profiles, and TME profiles along with nuclear immunotherapeutic responses were identified. Nine lncRNAs localized in the nucleus were identified and transcription factors (TFs) significantly negatively associated with these were found to be enriched in multiple oncogenic signaling pathways. Among them, three lncRNAs (AC093673.5, AC034220.3, and RP11-250B2.3) exerted the most profound effects on glycolysis and constituted the lncRNA signature, which could substantially distinguish the risk levels among different BC patients. Four glycolysis-associated lncRNA-regulated subtypes were identified in this study, reflective of the biological characteristics and heterogeneity of BC. Three key glycolysis-associated lncRNA constituting a signature could predict the risk levels in BC, provide a reference for stratification, and be used as prognostic markers for BC diagnosis and treatment.

Molecular classification of patients with NMIBC predicts the efficacy of intravesical chemotherapy with pirarubicin, pharmorubicin and gemcitabine-immunohistochemistry-based classification.

To investigate the relationships between non-muscle invasive bladder cancer molecular subtypes and predict the efficacy of intravesical chemotherapy with pirarubicin, pharmorubicin and gemcitabine. A total of 160 patients with T1 stage non-muscle invasive bladder cancer were enrolled in this study. Fifty-three patients underwent anthracycline (Pirarubicin and Pharmorubicin) therapy and 107 patients accepted gemcitabine therapy. Uroplakin II and CK20 were categorized as immunohistochemistry (IHC) markers for luminal subtype, whereas CK5/6 and CD44 were categorized as immunohistochemistry markers for basal subtype. The cluster results with immunohistochemical score indicated that non-muscle invasive bladder cancer can be subgrouped into three major classes. Class 2 showed the luminal-like characteristics, whereas class 3 showed the basal-like characteristics. Class 1 showed no high expression of luminal or basal-associated immunohistochemistry markers. The molecular subtype is an independent risk factor for recurrence-free survival (P=0.030) and progression-free survival (P=0.006) in patients with T1 stage non-muscle invasive bladder cancer. In class 1 and class 2 (luminal-like) subtypes, gemcitabine and anthracycline show no difference in recurrence-free survival and progression-free survival. Gemcitabine was associated with reduced recurrence compared with anthracycline (P=0.039) in class 3 (basal-like) subtypes and show no difference in decreasing progression. The molecular classification based on immunohistochemical results is an independent risk factor for the prognosis of non-muscle invasive bladder cancer with T1 stage. Different therapeutic methods should be selected according to different molecular subtypes.

TROP2 Expression Across Molecular Subtypes of Urothelial Carcinoma and Enfortumab Vedotin-resistant Cells

Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with >1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. Patient summaryIn this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.

Association of leucocyte levels in urine with tissue PD-L1 status and immune infiltration into basal bladder cancer subtype in the prospective real-world clinicopathological register trial Bladder BRIDGister.

560 Background: The objective of the present study was to evaluate whether preoperative standard urine parameters such as leucocyte count in urine samples dominate in particular molecular subtypes of bladder cancer and reflect the immune infiltration status in the tissue in matched urine and TUR biopsy samples from patients being suspicious of bladder cancer and undergoing first TURB within the prospective Real World Experience registry trial "BRIDGister". Methods: For this pilot study paraffin fixed pretreatment tissue samples from the first TURB of 48 pts participating in the BRIDGister trial and matched urine samples were prospectively collected and analyzed. RNA from FFPE tissues were extracted by commercial kits- Relative gene expression of subtyping markers (KRT5, KRT20) by standardized RT-qPCR systems for PD-1, PD-L1, CTLA4 (STRATIFYER Molecular Pathology GmbH, Cologne). In addition urine samples were analyzed for leucocyte and erythrocyte count. Spearman correlation, Kruskal-Wallis, MannWhitney and Sensitivity/Specificity tests were done by JMP 9.0.0 (SAS software). Results: The pilot cohort of the BRIDGister trial consisted of 48 patients (median age: 77, male 65% vs. female 35%) of diverse clinical stages (Benign lesions/no tumor 38%, pTa 23%, pT1 20%, pT2 19%) and WHO 1973 grade (G1 11%, G2 43%, G3 23%). Presence of leucocytes but not erythrocytes was negatively associated with KRT20 (r=-0.4249, p=0.0216) but positive with KRT5 (r=0.3704, p=0.0479). Moreover high levels of leucocytes in urine were positively associated with tumor expression of PD-L1 (r=0.5081, p=0.0041), PD-1 (r=0.5342, p=0.00024) and CTLA4 (r=0.4244, p=0.0194). In contrast there was no significant association of tumor PDL-1, PD-1 and CTLA4 expression with erythrocyte count in urine. Conclusions: Presence of leucotytes in urine is strongly associated with basal subtype and immune cell infiltration into early bladder cancer. As the presence of erythrcytes did not reveal these significant associations, the presence of leucocytes is not due to simple bleeding or tissue vulnerability. Moreover, the strong tumor subtype specificity further demonstrate the basal tumor specificity of urine leucocytes and therefore may be helpful for detecting and monitoring basal bladder cancer in a non invasive fashion. Given the prognostic value of tissue determination of PD-L1, PD-1 and CTLA4 quantitation on mRNA level, these results warrant further investigation to conclude on its impact on outcome prediction (BCG responsiveness, recurrence, progression, etc.), which will be prospectively analyzed in the framework of the ongoing multicenter BRIDGister Real World Experience trial.

Activation of PPARγ in bladder cancer via introduction of the long arm of human chromosome 9.

Bladder cancer is divided into two molecular subtypes, luminal and basal, which form papillary and nodular tumors, respectively, and are identifiable by gene expression profiling. Although loss of heterozygosity (LOH) of the long arm of human chromosome 9 (9q) has been observed in the early development of both types of bladder cancer, the functional significance of LOH remains to be clarified. The present study introduced human chromosome 9q into basal bladder cancer cell line, SCaBER, using microcell-mediated chromosome transfer to investigate the effect of LOH of 9q on molecular bladder cancer subtypes. These cells demonstrated decreased proliferation and migration capacity compared with parental and control cells. Conversely, transfer of human chromosome 4 did not change the cell phenotype. Expression level of peroxisome proliferator-activated receptor (PPAR)γ, a marker of luminal type, increased 3.0-4.4 fold in SCaBER cells altered with 9q compared with parental SCaBER cells. Furthermore, the expression levels of tumor suppressor PTEN, which regulates PPARγ, also increased in 9q-altered cells. These results suggested that human chromosome 9q may carry regulatory genes for PPARγ that are involved in the progression of neoplastic transformation of bladder cancer.

PMEPA1 Is a Prognostic Biomarker That Correlates With Cell Malignancy and the Tumor Microenvironment in Bladder Cancer.

Prostate transmembrane protein androgen induced 1 (PMEPA1) has been reported to promote cancer progression, but the potential role of PMEPA1 in bladder cancer (BLCA) remains elusive. We assess the role of PMEPA1 in BLCA, via a publicly available database and in vitro study. PMEPA1 was identified from 107 differentially expressed genes (DEGs) to have prognostic value. GO, KEGG, and GSEA analysis indicated that PMEPA1 was involved in cancer progression and the tumor microenvironment (TME). Then bioinformatical analysis in TCGA, GEO, TIMER, and TISIDB show a positive correlation with the inflammation and infiltration levels of three tumor-infiltrating immune cells (TAMs, CAFs, and MDSCs) and immune/stromal scores in TME. Moreover, in vitro study revealed that PMEPA1 promotes bladder cancer cell malignancy. Immunohistochemistry and survival analysis shed light on PMEPA1 potential to be a novel biomarker in predicting tumor progression and prognosis. At last, we also analyzed the role of PMEPA1 in predicting the molecular subtype and the response to several treatment options in BLCA. We found that PMEPA1 may be a novel potential biomarker to predict the progression, prognosis, and molecular subtype of BLCA.

Expression of FOXA1 Is Associated with the Tumor-Infiltrating M2 Macrophage, Cytotoxic T Lymphocyte, and Effect of Chemotherapy in Bladder Cancer

Purpose: Cisplatin-containing combination chemotherapy has been the standard of care since the late 1980s, but the response rate is <50%. Studies have shown that the efficiency of chemotherapy differs among molecular subtypes of bladder cancer. In this study, we aimed to correlate FOXA1, a marker for differentiation of the basal and luminal subtypes, with tumor immune cell infiltration and the effect of chemotherapy in bladder cancer. Materials and Methods: Eighty-three patients with bladder cancer treated with chemotherapy were reviewed. Clinicopathological variables for each case were recorded. FOXA1, M2 tumor-associated macrophage (TAM), dendritic cell (DC), and cytotoxic T lymphocyte (CTL) were examined by immunohistochemistry. The relationship between FOXA1, immune cell infiltration, and clinical response to chemotherapy was assessed. Results: The overall objective response rate was 34%. The objective response rate for tumors with lower FOXA1 expression was 58% and for tumors with higher FOXA1 expression was 12%. Tumors with infiltrated M2 TAM proportion <3% had a higher objective response rate compared with infiltrated M2 TAM proportion >3% tumors (46% vs. 21%, p = 0.02). Tumors with infiltrated CTL proportion >5% had a higher objective response rate compared with infiltrated CTL proportion <5% tumors (50% vs. 17%, p = 0.002). DCs showed no significant differences. We found that the objective response rate for tumors with lower FOXA1 expression, proportion <3% M2 TAM infiltration, and proportion >5% CTL infiltration is 82%. Lower FOXA1 expression was associated with low M2 TAM infiltration and high CTL infiltration. Conclusions: Thus, we showed that in patients with bladder cancer who received chemotherapy, the higher clinical response rate is associated with low FOXA1 expression, low M2 TAM infiltration, and high CTL infiltration.

Abstract 1194: MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer

Abstract There are diverse resources available for data relevant to the drug screening of bladder cancer cell lines in 2D culture, such as the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) databases; however, 3D culture is more representative of in vivo response in patients. Moreover, drug screens in 3D reveal differential drug response and expression of genes and proteins that indicate the potential of more effective selection of drugs for specific patients: the basic tenet of personalized oncology. Therefore, we screened bladder cancer cell lines in 3D culture using a library of curated small-molecule inhibitors to identify compounds and classes of compounds with efficacy in bladder cancer. Genomic and transcriptomic data for these bladder cancer cell lines were used to correlate genomic molecular parameters with drug response with the aim of discerning biomarkers of response or to suggest plausible drug combinations. This allowed us to identify groups of tumors that are vulnerable to specific drugs or classes of drugs. We found that MEK inhibitors were a promising target among basal bladder cancers, an RNA-based molecular subtype of bladder cancer, especially aggressive and exhibiting poor prognosis. Furthermore, trametinib was found to be a “very active” MEK inhibitor in basal cancers, classified using drug sensitivity 3 (DSS3) scoring. PI3K/AKT/MTOR inhibitors target a parallel signaling pathway to MEK inhibition; therefore, we tested PI3K/AKT/MTOR combinations with trametinib in basal bladder cancer to query for synergistic properties. Additionally, we tested PI3K/AKT/MTOR combinations with trametinib using ex vivo organoids of bladder cancer patient samples collected directly at operation. Taken together, MEK inhibition is a promising therapeutic strategy in the basal subtype of bladder cancer, particularly in combination with parallel pathway inhibition. Moving forward, we plan to test PI3K/AKT/MTOR combinations with trametinib in trametinib-resistant basal bladder cancer cell lines to investigate how this affects signaling and re-sensitization to trametinib. Citation Format: Athena M. Apfel, Nathan M. Merrill, Nathalie M. Vandecan, Liwei Bao, Xu Cheng, Lluis A. Lopez-Barcons, Kathleen C. Day, Phillip L. Palmbos, Mark L. Day, Aaron M. Udager, Matthew B. Soellner, Sofia D. Merajver. MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer [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 1194.

Integrative analysis of immune molecular subtypes and microenvironment characteristics of bladder cancer.

The emergence of immunotherapy has provided an option of treatment methods for bladder cancer (BC). However, the beneficiaries of immunotherapy are still limited to small‐scale patients, and immunotherapy‐related adverse events often occur. It is a major challenge for clinical work to study the immune subtypes of BC and the molecular mechanism of immune escape, and identify the immune responders accurately. Here, we explore the immune molecular subtypes of bladder cancer and potential escape mechanisms. First, we screened the expression profiles of 303 differentially expressed immune‐related genes in BC patients from the Cancer Genome Atlas (TCGA) database, and successfully identified 4 molecular subtypes of BC. By comparing the clinical characteristics, immune cells infiltration, the expression of checkpoint genes, human leukocyte antigen (HLA) genes, and gene mutation status of different subtypes, we identified different clinical and immunological characteristics of 4 subtypes. Among 4 subtypes, Cluster 2 met the general characteristics of immunotherapy responders and responded well to immunotherapy, while Cluster 4 had the highest expression of immune characteristics, and is similar to the immune environment of normal bladder tissue. Then, the weighted gene co‐expression network analysis (WGCNA) of immune‐related genes revealed that brown module was positively correlated with subtypes. Pathway enrichment analysis explored the major pathways associated with subtypes, which are also associated with immune escape mechanisms. Moreover, the decision tree model, which was constructed by the principle of random forest screening factors, was also validated in internal validation set and external validation set from the Gene Expression Omnibus (GEO) cohort (GSE133624), and could achieve accurate subtypes prediction for BC patients with high‐throughput sequencing. Taken together, we explored the immune molecular subtypes and their mechanisms of BC, and these results may provide guidance for the development of new BC immunotherapy strategies.

Heterogeneity in NECTIN4 Expression Across Molecular Subtypes of Urothelial Cancer Mediates Sensitivity to Enfortumab Vedotin.

Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance. Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays. NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance. Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950.

Heterogeneity in Nectin-4 expression across molecular subtypes of urothelial cancer mediates sensitivity to enfortumab vedotin.

463 Background: Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting Nectin-4 (encoded by the PVRL4/NECTIN4 gene ) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In the present study, we sought to 1) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and 2) determine if Nectin-4 expression mediates EV sensitivity or resistance. Methods: NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1912 total patients) were used to compare relative NECTIN4 expression across molecular subtypes. The outcome of the gene expression analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in multiple bladder cancer cell lines. NECTIN4 was stably over-expressed or knocked down in basal (TCCSUP and UMUC-3) and luminal (HT-1376, HT-1197 and UMUC-9) bladder cancer cell lines, respectively, and EV dose-response assays were performed, as measured by cell proliferation and clonogenic assays. Results: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes (p &lt; 0.001). NECTIN4 expression is positively correlated with the luminal markers GATA3, FOXA1, and PPARG across cohorts (Spearman’s rank correlation r = 0.57, p &lt; 0.0001 for GATA3, r = 0.37, p &lt; 0.0001 for FOXA1, and r = 0.56, p &lt; 0.0001 for PPARG). NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 led to EV resistance, and EV-resistant cell lines expressed decreased levels of Nectin-4. Conclusions: Results of this pre-clinical study suggest that sensitivity to EV is mediated by expression of NECTIN4, which is significantly enriched in luminal subtypes of bladder cancer. Downregulation of NECTIN4 leads to resistance to EV. These findings have implications for biomarker development, patient selection and the inclusion of molecular subtyping in ongoing and future EV clinical trials. Further investigation into Nectin-4 loss as a mechanism of resistance in patients treated on EV is warranted.