Prognostic Significance of Immune Cell Infiltration in Muscle-invasive Bladder Cancer Treated with Definitive Chemoradiation: A Secondary Analysis of RTOG 0524 and RTOG 0712.

Chemoradiation therapy (CRT) is a treatment for muscle-invasive bladder cancer (MIBC). Using a novel transcriptomic profiling panel, we validated prognostic immune biomarkers to CRT using 70 pretreatment tumor samples from prospective trials of MIBC (NRG/RTOG 0524 and 0712). Disease-free survival (DFS) and overall survival (OS) were estimated via the Kaplan-Meier method and stratified by genes correlated with immune cell activation. Cox proportional-hazards models were used to assess group differences. Clustering of gene expression profiles revealed that the cluster with high immune cell content was associated with longer DFS (hazard ratio [HR] 0.53, 95% confidence interval [CI] 0.26-1.10; p = 0.071) and OS (HR 0.48, 95% CI 0.24-0.97; p = 0.040) than the cluster with low immune cell content. Higher expression of T-cell infiltration genes (CD8A and ICOS) was associated with longer DFS (HR 0.40, 95% CI 0.21-0.75; p = 0.005) and OS (HR 0.49, 95% CI 0.25-0.94; p = 0.033). Higher IDO1 expression (IFNγ signature) was also associated with longer DFS (HR 0.44, 95% CI 0.24-0.88; p = 0.021) and OS (HR 0.49, 95% CI 0.24-0.99; p = 0.048). These findings should be validated in prospective CRT trials that include biomarkers, particularly for trials incorporating immunotherapy for MIBC. PATIENT SUMMARY: We analyzed patient samples from two clinical trials (NRG/RTOG 0524 and 0712) of chemoradiation for muscle-invasive bladder cancer using a novel method to assess immune cells in the tumor microenvironment. Higher expression of genes associated with immune activation and high overall immune-cell content were associated with better disease-free survival and overall survival for patients treated with chemoradiation.

Loss of LPAR6 and CAB39L dysregulates the basal-to-luminal urothelial differentiation program, contributing to bladder carcinogenesis.

We describe a strategy that combines histologic and molecular mapping that permits interrogation of the chronology of changes associated with cancer development on a whole-organ scale. Using this approach, we present the sequence of alterations around RB1 in the development of bladder cancer. We show that RB1 is not involved in initial expansion of the preneoplastic clone. Instead, we found a set of contiguous genes that we term "forerunner" genes whose silencing is associated with the development of plaque-like field effects initiating carcinogenesis. Specifically, we identified five candidate forerunner genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) mapping near RB1. Two of these genes, LPAR6 and CAB39L, are preferentially downregulated in the luminal and basal subtypes of bladder cancer, respectively. Their loss of function dysregulates urothelial differentiation, sensitizing the urothelium to N-butyl-N-(4-hydroxybutyl)nitrosamine-induced cancers, which recapitulate the luminal and basal subtypes of human bladder cancer.

Inferring Bladder Cancer Evolution from Mucosal field Effects by Whole-Organ Spatial Mutational, Proteomic, and Metabolomic Mapping.

Multi-platform mutational, proteomic, and metabolomic spatial mapping was used on the whole-organ scale to identify the molecular evolution of bladder cancer from mucosal field effects. We identified complex proteomic and metabolomic dysregulations in microscopically normal areas of bladder mucosa adjacent to dysplasia and carcinoma in situ. The mutational landscape developed in a background of complex defects of protein homeostasis which included dysregulated nucleocytoplasmic transport, splicesome, ribosome biogenesis, and peroxisome. These changes were combined with altered urothelial differentiation which involved lipid metabolism and protein degradations controlled by PPAR. The complex alterations of proteome were accompanied by dysregulation of gluco-lipid energy-related metabolism. The analysis of mutational landscape identified three types of mutations based on their geographic distribution and variant allele frequencies. The most common were low frequency α mutations restricted to individual mucosal samples. The two other groups of mutations were associated with clonal expansion. The first of this group referred to as ß mutations occurred at low frequencies across the mucosa. The second of this group called γ mutations increased in frequency with disease progression. Modeling of the mutations revealed that carcinogenesis may span nearly 30 years and can be divided into dormant and progressive phases. The α mutations developed gradually in the dormant phase. The progressive phase lasted approximately five years and was signified by the advent of ß mutations, but it was driven by γ mutations which developed during the last 2-3 years of disease progression to invasive cancer. Our study indicates that the understanding of complex alterations involving mucosal microenvironment initiating bladder carcinogenesis can be inferred from the multi-platform whole-organ mapping.

Molecular profile of bladder cancer progression to clinically aggressive subtypes.

Bladder cancer is a histologically and clinically heterogenous disease. Most bladder cancers are urothelial carcinomas, which frequently develop distinct histological subtypes. Several urothelial carcinoma histological subtypes, such as micropapillary, plasmacytoid, small-cell carcinoma and sarcomatoid, show highly aggressive behaviour and pose unique challenges in diagnosis and treatment. Comprehensive genomic characterizations of the urothelial carcinoma subtypes have revealed that they probably arise from a precursor subset of conventional urothelial carcinomas that belong to different molecular subtypes - micropapillary and plasmacytoid subtypes develop along the luminal pathway, whereas small-cell and sarcomatoid subtypes evolve along the basal pathway. The subtypes exhibit distinct genomic alterations, but in most cases their biological properties seem to be primarily determined by specific gene expression profiles, including epithelial-mesenchymal transition, urothelial-to-neural lineage plasticity, and immune infiltration with distinct upregulation of immune regulatory genes. These breakthrough studies have transformed our view of bladder cancer histological subtype biology, generated new hypotheses for therapy and chemoresistance, and facilitated the discovery of new therapeutic targets.

Oncologic Outcomes in Patients with Residual Upper Tract Urothelial Carcinoma Following Neoadjuvant Chemotherapy.

BACKGROUND AND OBJECTIVE: Growing evidence supports the use of neoadjuvant chemotherapy (NAC) for upper tract urothelial carcinoma (UTUC). However, the implications of residual UTUC at radical nephroureterectomy (RNU) after NAC are not well characterized. Our objective was to compare oncologic outcomes for pathologic risk-matched patients who underwent RNU for UTUC who either received NAC or were chemotherapy-naïve. METHODS: We retrospectively identified 1993 patients (including 112 NAC recipients) who underwent RNU for nonmetastatic, high-grade UTUC between 1985 and 2022 in a large, international, multicenter cohort. We divided the cohort into low-risk and high-risk groups defined according to pathologic findings of muscle invasion and lymph node involvement at RNU. Recurrence-free survival (RFS), overall survival (OS), and cancer-specific survival (CSS) estimates were calculated using the Kaplan-Meier method. Multivariable analyses were performed to determine clinical and demographic factors associated with these outcomes. KEY FINDINGS AND LIMITATIONS: Among patients with low-risk pathology at RNU, RFS, OS, and CSS were similar between the NAC and chemotherapy-naïve groups. Among patients with high-risk pathology at RNU, the NAC group had poorer RFS (hazard ratio [HR] 3.07, 95% confidence interval [CI] 2.10-4.48), OS (HR 2.06, 95% CI 1.33-3.20), and CSS (subdistribution HR 2.54, 95% CI 1.37-4.69) in comparison to the pathologic risk-matched, chemotherapy-naïve group. Limitations include the lack of centralized pathologic review. CONCLUSIONS AND CLINICAL IMPLICATIONS: Patients with residual invasive disease at RNU after NAC represent a uniquely high-risk population with respect to oncologic outcomes. There is a critical need to determine an optimal adjuvant approach for these patients. PATIENT SUMMARY: We studied a large, international group of patients with cancer of the upper urinary tract who underwent surgery either with or without receiving chemotherapy beforehand. We identified a high-risk subgroup of patients with residual aggressive cancer after chemotherapy and surgery who should be prioritized for clinical trials and drug development.

Tumour tissue-derived small extracellular vesicles reflect molecular subtypes of bladder cancer.

mRNA-based molecular subtypes have implications for bladder cancer prognosis and clinical benefit from certain therapies. Whether small extracellular vesicles (sEVs) can reflect bladder cancer molecular subtypes is unknown. We performed whole transcriptome RNA sequencing for formalin fixed paraffin embedded (FFPE) tumour tissues and sEVs separated from matched tissue explants, urine and plasma in patients with bladder cancer. sEVs were separated using size-exclusion chromatography, and characterized by transmission electron microscopy, nano flow cytometry and western blots, respectively. High yield of sEVs were obtained using approximately 1 g of tissue, incubated with media for 30 min. FFPE tumour tissue and tumour tissue-derived sEVs demonstrated good concordance in molecular subtype classification. All urinary sEVs were classified as luminal subtype, while all plasma sEVs were classified as Ba/Sq subtype, regardless of the molecular subtypes indicated by their matched FFPE tumour tissue. The comparison within urine sEVs, which may exclude the sample type specific background, could pick up the different biology between NMIBC and MIBC, as well as the signature genes related to molecular subtypes. Four candidate sEV-related bladder cancer-specific mRNA biomarkers, FAM71E2, OR4K5, FAM138F and KRTAP26-1, were identified by analysing matched urine sEVs, tumour tissue derived sEVs, and adjacent normal tissue derived sEVs. Compared to sEVs separated from biofluids, tissue-derived sEVs may reflect more tissue- or disease-specific biological features. Urine sEVs are promising biomarkers to be used for liquid biopsy-based molecular subtype classification, but the current algorithm needs to be modified/adjusted. Future work is needed to validate the four new bladder cancer-specific biomarkers in large cohorts.

Association of Molecular Subtypes with Pathologic Response, PFS, and OS in a Phase II Study of COXEN with Neoadjuvant Chemotherapy for Muscle-invasive Bladder Cancer.

PURPOSE: The Coexpression Extrapolation (COXEN) gene expression model with chemotherapy-specific scores [for methotrexate, vinblastine, adriamycin, cisplatin (ddMVAC) and gemcitabine/cisplatin (GC)] was developed to identify responders to neoadjuvant chemotherapy (NAC). We investigated RNA-based molecular subtypes as additional predictive biomarkers for NAC response, progression-free survival (PFS), and overall survival (OS) in patients treated in S1314. EXPERIMENTAL DESIGN: A total of 237 patients were randomized between four cycles of ddMVAC (51%) and GC (49%). On the basis of Affymetrix transcriptomic data, we determined subtypes using three classifiers: TCGA (k = 5), Consensus (k = 6), and MD Anderson (MDA; k = 3) and assessed subtype association with path response to NAC and determined associations with COXEN. We also tested whether each classifier contributed additional predictive power when added to a model based on predefined stratification (strat) factors (PS 0 vs. 1; T2 vs. T3, T4a). RESULTS: A total of 155 patients had gene expression results, received at least three of four cycles of NAC, and had pT-N response based on radical cystectomy. TCGA three-group classifier basal-squamous (BS)/neuronal, luminal (Lum), Lum infiltrated, and GC COXEN score yielded the largest AUCs for pT0 (0.59, P = 0.28; 0.60, P = 0.18, respectively). For downstaging (

Definitions, End Points, and Clinical Trial Designs for Bladder Cancer: Recommendations From the Society for Immunotherapy of Cancer and the International Bladder Cancer Group.

PURPOSE: There is a significant unmet need for new and efficacious therapies in urothelial cancer (UC). To provide recommendations on appropriate clinical trial designs across disease settings in UC, the Society for Immunotherapy of Cancer (SITC) and the International Bladder Cancer Group (IBCG) convened a multidisciplinary, international consensus panel. METHODS: Through open communication and scientific debate in small- and whole-group settings, surveying, and responses to clinical questionnaires, the consensus panel developed recommendations on optimal definitions of the disease state, end points, trial design, evaluations, sample size calculations, and pathology considerations for definitive studies in low- and intermediate-risk nonmuscle-invasive bladder cancer (NMIBC), high-risk NMIBC, muscle-invasive bladder cancer in the neoadjuvant and adjuvant settings, and metastatic UC. The expert panel also solicited input on the recommendations through presentations and public discussion during an open session at the 2021 Bladder Cancer Advocacy Network (BCAN) Think Tank (held virtually). RESULTS: The consensus panel developed a set of stage-specific bladder cancer clinical trial design recommendations, which are summarized in the table that accompanies this text. CONCLUSION: These recommendations developed by the SITC-IBCG Bladder Cancer Clinical Trial Design consensus panel will encourage uniformity among studies and facilitate drug development in this disease.

Combined Mek inhibition and Pparg activation Eradicates Muscle Invasive Bladder cancer in a Mouse Model of BBN-induced Carcinogenesis.

Bladder cancers (BCs) can be divided into 2 major subgroups displaying distinct clinical behaviors and mutational profiles: basal/squamous (BASQ) tumors that tend to be muscle invasive, and luminal/papillary (LP) tumors that are exophytic and tend to be non-invasive. Pparg is a likely driver of LP BC and has been suggested to act as a tumor suppressor in BASQ tumors, where it is likely suppressed by MEK-dependent phosphorylation. Here we tested the effects of rosiglitazone, a Pparg agonist, in a mouse model of BBN-induced muscle invasive BC. Rosiglitazone activated Pparg signaling in suprabasal epithelial layers of tumors but not in basal-most layers containing highly proliferative invasive cells, reducing proliferation but not affecting tumor survival. Addition of trametinib, a MEK inhibitor, induced Pparg signaling throughout all tumor layers, and eradicated 91% of tumors within 7-days of treatment. The 2-drug combination also activated a luminal differentiation program, reversing squamous metaplasia in the urothelium of tumor-bearing mice. Paired ATAC-RNA-seq analysis revealed that tumor apoptosis was most likely linked to down-regulation of Bcl-2 and other pro-survival genes, while the shift from BASQ to luminal differentiation was associated with activation of the retinoic acid pathway and upregulation of Kdm6a, a lysine demethylase that facilitates retinoid-signaling. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients. That muscle invasive tumors are populated by basal and suprabasal cell types with different responsiveness to PPARG agonists will be an important consideration when designing new treatments.

Phase 2 Trial of Atezolizumab in Bacillus Calmette-Guérin-unresponsive High-risk Non-muscle-invasive Bladder Cancer: SWOG S1605.

BACKGROUND: Although radical cystectomy (RC) is the standard of care for patients with bacillus Calmette-Guérin (BCG)-unresponsive high-risk non-muscle-invasive bladder cancer (NMIBC), many patients are ineligible for surgery or elect bladder preservation. OBJECTIVE: To evaluate the efficacy and safety of atezolizumab in BCG-unresponsive high-risk NMIBC. DESIGN, SETTING, AND PARTICIPANTS: This was a single-arm phase 2 trial in patients with BCG-unresponsive high-risk NMIBC who were ineligible for or declined RC. INTERVENTION: Intravenous atezolizumab every 3 wk for 1 yr. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was the pathological complete response (CR) rate for patients with carcinoma in situ (CIS) determined via mandatory biopsy at 6 mo. Event-free survival (EFS) at 18 mo for patients with non-CIS tumors and treatment-related adverse events (TRAEs) were key secondary endpoints. RESULTS AND LIMITATIONS: Of 172 patients enrolled in the trial, 166 received at least one dose of atezolizumab (safety analysis) and 129 were eligible (efficacy analysis). Of the 74 patients with CIS, 20 (27%) experienced a CR at 6 mo. The median duration of response was 17 mo, and 56% (95% confidence interval [CI] 34-77%) of the responses were durable to at least 12 mo. The 18-mo actuarial EFS rate among 55 patients with Ta/T1 disease was 49% (90% CI 38-60%). Twelve of 129 eligible patients experienced progression to muscle-invasive or metastatic disease. Grade 3-5 TRAEs occurred in 26 patients (16%), including three treatment-related deaths. The study was limited by the small sample size and a high rate of patient ineligibility. CONCLUSIONS: The efficacy of atezolizumab observed among patients with BCG-unresponsive NMIBC is similar to results from similar trials with other agents, but did not meet the prespecified efficacy threshold. Modest efficacy needs to be balanced with a significant rate of TRAEs and the risk of disease progression when considering systemic immunotherapy in early-stage bladder cancer. PATIENT SUMMARY: We tested intravenous immunotherapy (atezolizumab) in patients with high-risk non-muscle-invasive bladder cancer that recurred after BCG (bacillus Calmette-Guérin) treatment. Although we found similar outcomes to previous trials, the benefit of this therapy is modest and needs to be carefully balanced with the significant risk of side effects. This trial is registered on ClinicalTrials.gov as NCT02844816.

Long-term Outcomes from a Phase 2 Study of Neoadjuvant Chemotherapy for Muscle-invasive Bladder Cancer (SWOG S1314; NCT02177695).

BACKGROUND: The COXEN gene expression model was evaluated for prediction of response to neoadjuvant chemotherapy for muscle-invasive bladder cancer (MIBC). OBJECTIVE: To conduct a secondary analysis of the association of each COXEN score with event-free survival (EFS) and overall survival (OS) and by treatment arm. DESIGN, SETTING, AND PARTICIPANTS: This was a randomized phase 2 trial of neoadjuvant gemcitabine-cisplatin (GC) or dose-dense methotrexate-vinblastine-adriamycin-cisplatin (ddMVAC) in MIBC. INTERVENTION: Patients were randomized to ddMVAC (every 14 d) or GC (every 21 d), both for four cycles. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: EFS events were defined as progression or death before scheduled surgery, a decision to not undergo surgery, recurrence, or death due to any cause after surgery. Cox regression was used to evaluate the COXEN score or treatment arm association with EFS and OS. RESULTS AND LIMITATIONS: A total of 167 evaluable patients were included in the COXEN analysis. The COXEN scores were not significantly prognostic for OS or EFS in the respective arms, but the GC COXEN score had a hazard ratio (HR) of 0.45 (95% confidence interval [CI] 0.20-0.99; p = 0.047) when the arms were pooled. In the intent-to-treat analysis (n = 227), there was no significant difference between ddMVAC and GC for OS (HR 0.87, 95% CI 0.54-1.40; p = 0.57) or EFS (HR 0.86, 95% CI 0.59-1.26; p = 0.45). Among the 192 patients who underwent surgery, pathologic response (pT0 vs downstaging vs no response) was strongly correlated with superior postsurgical survival (5-yr OS 90%, 89% and 52%, respectively). CONCLUSIONS: The COXEN GC score has prognostic value for patients receiving cisplatin-based neoadjuvant treatment. The randomized, prospective design provides estimates of OS and EFS for GC and ddMVAC in this population. Pathologic response (

Identification of Lineage-specific Transcriptional Factor-defined Molecular Subtypes in Small Cell Bladder Cancer.

Small cell/neuroendocrine bladder cancers (SCBCs) are rare and highly aggressive tumors that are associated with poor clinical outcomes. We discovered that lineage-specific transcription factors (ASCL1, NEUROD1, and POU2F3) defined three SCBC molecular subtypes that resemble well-characterized subtypes in small cell lung cancer. The subtypes expressed various levels of neuroendocrine (NE) markers and distinct downstream transcriptional targets. Specifically, the ASCL1 and NEUROD1 subtypes had high NE marker expression and were enriched with different downstream regulators of the NE phenotype (FOXA2 and HES6, respectively). ASCL1 was also associated with the expression of delta-like ligands that control oncogenic Notch signaling. POU2F3, a master regulator of the NE low subtype, targeted TRPM5, SOX9, and CHAT. We also observed an inverse association between NE marker expression and immune signatures associated with sensitivity to immune checkpoint blockade, and the ASCL1 subtype had distinct targets for clinically available antibody-drug conjugates. These findings provide new insight into molecular heterogeneity in SCBCs with implications for the development of new treatment regimens. PATIENT SUMMARY: We investigated the levels of different proteins in a specific type of bladder cancer (small cell/neuroendocrine; SCBC). We could identify three distinct subtypes of SCBC with similarity to small cell/neuroendocrine cancers in other tissues. The results may help in identifying new treatment approaches for this type of bladder cancer.

Cell-free DNA Methylation as a Predictive Biomarker of Response to Neoadjuvant Chemotherapy for Patients with Muscle-invasive Bladder Cancer in SWOG S1314.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care in muscle-invasive bladder cancer (MIBC). However, treatment is intense, and the overall benefit is small, necessitating effective biomarkers to identify patients who will benefit most. OBJECTIVE: To characterize cell-free DNA (cfDNA) methylation in patients receiving NAC in SWOG S1314, a prospective cooperative group trial, and to correlate the methylation signatures with pathologic response at radical cystectomy. DESIGN, SETTING, AND PARTICIPANTS: SWOG S1314 is a prospective cooperative group trial for patients with MIBC (cT2-T4aN0M0, ≥5 mm of viable tumor), with a primary objective of evaluating the coexpression extrapolation (COXEN) gene expression signature as a predictor of NAC response, defined as achieving pT0N0 or ≤pT1N0 at radical cystectomy. For the current exploratory analysis, blood samples were collected prospectively from 72 patients in S1314 before and during NAC, and plasma cfDNA methylation was measured using the Infinium MethylationEPIC BeadChip array. INTERVENTION: No additional interventions besides plasma collection. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Differential methylation between pathologic responders (≤pT1N0) and nonresponders was analyzed, and a classifier predictive of treatment response was generated using the Random Forest machine learning algorithm. RESULTS AND LIMITATIONS: Using prechemotherapy plasma cfDNA, we developed a methylation-based response score (mR-score) predictive of pathologic response. Plasma samples collected after the first cycle of NAC yielded mR-scores with similar predictive ability. Furthermore, we used cfDNA methylation data to calculate the circulating bladder DNA fraction, which had a modest but independent predictive ability for treatment response. In a model combining mR-score and circulating bladder DNA fraction, we correctly predicted pathologic response in 79% of patients based on their plasma collected at baseline and after one cycle of chemotherapy. Limitations of this study included a limited sample size and relatively low circulating bladder DNA levels. CONCLUSIONS: Our study provides the proof of concept that cfDNA methylation can be used to generate classifiers of NAC response in bladder cancer patients. PATIENT SUMMARY: In this exploratory analysis of S1314, we demonstrated that cell-free DNA methylation can be profiled to generate biomarker signatures associated with neoadjuvant chemotherapy response. With validation in additional cohorts, this minimally invasive approach may be used to predict chemotherapy response in locally advanced bladder cancer and perhaps also in metastatic disease.

Enfortumab Vedotin-related Cutaneous Toxicity and Radiographic Response in Patients with Urothelial Cancer: A Single-center Experience and Review of the Literature.

Enfortumab vedotin (EV) is an antibody-drug conjugate approved for the treatment of refractory advanced urothelial cancer. Cutaneous toxicity is well described but has not been correlated with response. In this retrospective single-center study, data from patients treated with more than one dose of EV between December 2017 and June 2022 were analyzed. Of 56 patients with a median age of 69 yr, 41 (73.2%) were male and 27 (48.2%) had any-grade skin toxicity. For all 51 patients evaluable by physician-assessed Response Evaluation Criteria in Solid Tumors (RECIST) criteria, the response rate was 41.2%. For those with cutaneous toxicity, the response rate was 57.7%; for those without cutaneous toxicity, it was 24.0% (p = 0.0145). All three patients with complete response experienced cutaneous toxicity, and two of these responses remain durable 5 and 24 mo off EV. The median starting weight and body mass index (BMI) were, respectively, 80.86 kg and 26.53 kg/m2 among patients with cutaneous toxicity, and 69.37 kg and 23.29 kg/m2 in patients without (p = 0.0129 and 0.0014, respectively). In this small dataset, EV-related cutaneous toxicity was more common in patients with higher weight and BMI at baseline, and was associated with disease response. Confirmation in prospective trials may confirm this association and lead to an important clinical biomarker of response. Patient summary: We evaluated patients with urothelial cancer who were treated at our institution with enfortumab vedotin (EV). We found that patients who experienced the common side effect of any type of skin toxicity, such as rash or itching, were more likely to have improvement in their cancer from EV treatment than those who did not experience skin toxicity. Patients with higher weight and body mass index when starting EV tended to have more skin toxicity. We conclude that presence of skin toxicity might help doctors make decisions about how to manage the care of patients with EV in the future.

Comparison of clinicopathological characteristics, gene expression profiles, mutational analysis, and clinical outcomes of pure and mixed small-cell carcinoma of the bladder.

AIMS: Small cell bladder carcinoma (SCBC) is a rare, divergent form of urothelial carcinoma (UC). We aimed to determine whether pure (n = 16) and mixed (SCBC and UC; n = 30) tumours differed in pathology, gene expression characteristics, genetic alterations, and clinical outcomes. METHODS AND RESULTS: Forty (87%) patients received first-line chemotherapy. Twenty-nine patients had no metastatic disease at diagnosis and underwent radical cystectomy. There were no differences in age, sex, race distribution, tumour size, stage at presentation, therapy response with pathological downstaging to ≤ypT1N0, or overall or progression-free survival (PFS) between pure and mixed tumours. There was a longer PFS among downstaged chemotherapy-responding tumours ≤ypT2N0M0 than among unresponsive tumours ≥ypT2 ≥ yN1M1 (P = 0.001). Patients who achieved pathological downstaging with neoadjuvant chemotherapy (n = 10) were stage cT2N0M0 at the time of diagnosis and were alive at the last follow-up (median 37 months), while 46% of patients who failed to achieve pathological downstaging were alive at the last follow-up (median 38 months; P = 0.008). RNA sequencing showed that the UC of mixed SCBC had similar neural expression signatures to pure SCBC. DNA sequencing revealed alterations in TERT (83%), P53 (56%), ARID1A (28%), RB1 (22%), and BRCA2 (11%). Immunohistochemistry for RB1 showed loss of expression in 18/19 (95%) patients, suggesting frequent pathway downregulation despite a low prevalence of RB1 mutation. CONCLUSION: Patients with pure and mixed SCBC have similar outcomes and these outcomes are determined by the pathological stage at RC and are best among patients who have pathological downstaging after NAC.

A Phase 1 Trial of Durvalumab in Combination with Bacillus Calmette-Guerin (BCG) or External Beam Radiation Therapy in Patients with BCG-unresponsive Non-muscle-Invasive Bladder Cancer: The Hoosier Cancer Research Network GU16-243 ADAPT-BLADDER Study.

BACKGROUND: Novel treatments and trial designs remain a high priority for bacillus Calmette-Guerin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC) patients. OBJECTIVE: To evaluate the safety and preliminary efficacy of anti-PD-L1 directed therapy with durvalumab (D), durvalumab plus BCG (D + BCG), and durvalumab plus external beam radiation therapy (D + EBRT). DESIGN, SETTING, AND PARTICIPANTS: A multicenter phase 1 trial was conducted at community and academic sites. INTERVENTION: Patients received 1120 mg of D intravenously every 3 wk for eight cycles. D + BCG patients also received full-dose intravesical BCG weekly for 6 wk with BCG maintenance recommended. D + EBRT patients received concurrent EBRT (6 Gy × 3 in cycle 1 only). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Post-treatment cystoscopy and urine cytology were performed at 3 and 6 -mo, with bladder biopsies required at the 6-mo evaluation. The recommended phase 2 dose (RP2D) for each regimen was the primary endpoint. Secondary endpoints included toxicity profiles and complete response (CR) rates. RESULTS AND LIMITATIONS: Twenty-eight patients were treated in the D (n = 3), D + BCG (n = 13), and D + EBRT (n = 12) cohorts. Full-dose D, full-dose BCG, and 6 Gy fractions × 3 were determined as the RP2Ds. One patient (4%) experienced a grade 3 dose limiting toxicity event of autoimmune hepatitis. The 3-mo CR occurred in 64% of all patients and in 33%, 85%, and 50% within the D, D + BCG, and D + EBRT cohorts, respectively. Twelve-month CRs were achieved in 46% of all patients and in 73% of D + BCG and 33% of D + EBRT patients. CONCLUSIONS: D combined with intravesical BCG or EBRT proved feasible and safe in BCG-unresponsive NMIBC patients. Encouraging preliminary efficacy justifies further study of combination therapy approaches. PATIENT SUMMARY: Durvalumab combination therapy can be safely administered to non-muscle-invasive bladder cancer patients with the goal of increasing durable response rates.

Improved bladder cancer antitumor efficacy with a recombinant BCG that releases a STING agonist.

Despite the introduction of several new agents for the treatment of bladder cancer (BC), intravesical BCG remains a first line agent for the management of non-muscle invasive bladder cancer. In this study we evaluated the antitumor efficacy in animal models of BC of a recombinant BCG known as BCG-disA-OE that releases the small molecule STING agonist c-di-AMP. We found that compared to wild-type BCG (BCG-WT), in both the orthotopic, carcinogen-induced rat MNU model and the heterotopic syngeneic mouse MB-49 model BCG-disA-OE afforded improved antitumor efficacy. A mouse safety evaluation further revealed that BCG-disA-OE proliferated to lesser degree than BCG-WT in BALB/c mice and displayed reduced lethality in SCID mice. To probe the mechanisms that may underlie these effects, we found that BCG-disA-OE was more potent than BCG-WT in eliciting IFN-ß release by exposed macrophages, in reprogramming myeloid cell subsets towards an M1-like proinflammatory phenotypes, inducing epigenetic activation marks in proinflammatory cytokine promoters, and in shifting monocyte metabolomic profiles towards glycolysis. Many of the parameters elevated in cells exposed to BCG-disA-OE are associated with BCG-mediated trained innate immunity suggesting that STING agonist overexpression may enhance trained immunity. These results indicate that modifying BCG to release high levels of proinflammatory PAMP molecules such as the STING agonist c-di-AMP can enhance antitumor efficacy in bladder cancer.

Lentiviral interferon: A novel method for gene therapy in bladder cancer.

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.