Although immune checkpoint inhibitors have shown benefit for advanced urothelial carcinoma (aUC) patients, prognostication of treatment efficacy and response duration remains a clinical challenge. We evaluated the expression of immune markers in the tumor microenvironment and assessed their associations with response to and survival after pembrolizumab treatment in 26 aUC patients. High levels of CD8⁺ tumor-infiltrating lymphocytes (TILs) were associated with favorable objective responses (23.0% vs. 15.3%, p = 0.0425), progression-free survival (median, 8.8 vs 2.1 months; hazard ratio (HR), 0.24; 95% confidence interval (CI), 0.07–0.66, p = 0.0060), and overall survival (median, >24.0 vs. 5.3 months; HR, 0.17; 95% CI, 0.04–0.56, p = 0.0034) compared with low levels. High interferon-gamma (IFNγ) expression levels were associated with longer post-progression survival (median, 4.9 vs. 1.0 months; HR, 0.18; 95% CI, 0.04–0.59, p = 0.0027) compared with low expression. Multivariate analysis incorporating clinical prognosticators demonstrated that the coincidence of low CD8⁺ T cells/IFNγ was an independent factor for unfavorable overall survival after pembrolizumab treatment (HR, 4.07; 95% CI, 1.36–12.73; p = 0.0125). The combination of low CD8⁺ TILs and IFNγ expression was an independent prognostic factor with predictive ability equivalent to previously reported clinical prognosticators.

Accurate staging of bladder cancer (BCa) is important for identifying optimal treatment. Currently, clinical tumor staging for BCa relies on computed tomography (CT) scans, but these can lead to under- or overstaging of patients. Recent research suggests that using magnetic resonance imaging (MRI) along with RNA sequencing (RNASeq) gene expression analysis can provide more precise staging. In this study, 31 matched CT scans, MRI images, and formalin-fixed, paraffin-embedded (FFPE) tissues were collected. First, two radiologists reviewed the images for staging BCa. Next, radiomics features were extracted from both CT and MR images, and computational radiogenomics analyses were performed. Subsequently, RNASeq was performed using FFPE tissues of TURBT prior to cystectomy. A radiogenomic analysis was conducted to identify advanced T-stage signatures. Regarding imaging alone, MRI was found to be more accurate in staging >T2 compared to CT scans. Within a retrospective cohort, MRI radiogenomic signatures were more effective in staging patients than CT, with genomic features playing a significant role. Using canonical correlation analysis, we additionally identified radiomic features underlying genomic signatures of advanced tumor stage. When applying these signatures across a small prospective cohort, MRI radiomic data were able to stratify stage; however, the addition of the same genomic features did not improve the sensitivity and specificity of the model. These preliminary results are promising, but additional research with larger sample sizes is needed to draw definitive conclusions and explore further correlations and statistical interactions between genes and imaging features through machine learning techniques as we move radiogenomics to the clinic.

Background/Objectives: Urinalysis accuracy requires reliable sample stability that is dependent on the chosen collection and storage conditions. The multiplex Oncuria bladder cancer immunoassay currently needs urine samples stored at 4 °C until analysis, which requires more effort, equipment, and workflow than storing samples at room temperature. Thus, successful sample storage at room temperature (20 °C) may reduce laboratory handling time and expenses. This study evaluated whether different voided urine sample collection and storage parameters affected subsequent biomarker analysis with Oncuria. The Oncuria simultaneously quantifies 10 protein analytes in urine to generate a bladder cancer diagnostic signature. Methods: Samples were stored at varied temperatures (20 °C, 4 °C, −20 °C) for up to 1 month. The effects of adding two commercial urine sample stabilizers and antibiotics (trimethoprim) were also assessed. Subsequently, multiple potential biospecimen stabilizers were tested in urine samples and evaluated with Oncuria in hopes of allowing the urine sample to remain at room temperature for extended periods of time. Results: First, it was demonstrated that voided urine samples stored at room temperate without such stabilizers had different levels of the 10 analytes associated with the Oncuria test compared to voided urine samples stored at 4 °C. Next, we evaluated the effects of commercially available biospecimen stabilizers. Despite the addition of these stabilizers, the levels of the 10 analytes were altered when the samples were stored at room temperature for prolonged periods of time. Therefore, we could not identify a suitable biospecimen stabilizer that would not require sample refrigeration. Conclusions: To minimize sample degradation/alteration after collection, voided urine samples should be refrigerated until analyzed with Oncuria as the refrigeration is advantageous for the storage and the transport of these urine samples.

Urine-based assays currently have a limited role in the management of patients with upper tract urothelial carcinoma (UTUC), due to insufficient accuracy. This study evaluated a multiplex urine assay designed to accurately and non-invasively diagnose UTUC. Oncuria-Detect is a multiplex immunoassay capable of detecting urothelia cancer using a single voided urine specimen. In the current multi-institutional study, we tested the assay's utility in identifying UTUC in 60 subjects presenting for evaluation of upper tract mass compared with samples from 60 age-, sex-, and race-matched controls. Cancer status was confirmed via histopathological assessment of tissue specimens obtained during ureteroscopy/biopsy or at the time of definitive surgical intervention (nephroureterectomy or ureterectomy). For detecting UTUC, Oncuria-Detect provided an AUC of 0.77 (95% CI: 0.70-0.84) with an overall sensitivity of 93%, specificity of 55%, negative predictive value (NPV) 89%, and positive predictive value (PPV) 67%. Assay sensitivity values for low-grade, high-grade, non-invasive, and invasive UTUC were 94%, 93%, 93% and 92%, respectively. Voided urine cytology showed an overall sensitivity of 54%, specificity of 100%, NPV 31.0%, and PPV 100%. Selective (upper tract) cytology had a sensitivity of 80%. Oncuria-Detect outperformed both non-invasive voided urinary cytology and invasive selective cytology in overall assay performance. Oncuria-Detect accurately identified and categorized UTUC and discriminated patients with cancer from non-tumor bearing individuals. The Oncuria-Detect test provides a rapid, accurate, and non-invasive method for detecting UTUC.

Bladder cancer (BC) remains a common malignancy, with muscle-invasive bladder cancer (MIBC) comprising 20 % of cases and a poor 5-year survival rate of ∼50 %. While neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard treatment for locally advanced disease, NAC is limited by toxicity and non-response in many patients. Predictive biomarkers are urgently needed to guide treatment decisions. We developed and analytically validated a multiplex qPCR array to measure mRNA expression of 10 NAC response-associated biomarkers in MIBC. RNA from 8 formalin-fixed paraffin-embedded (FFPE) and 4 fresh-frozen (FF) MIBC specimens was analyzed to assess the effects of tissue type, necrosis, and pre-analytical variables on assay performance. The assay showed high concordance between FF and FFPE samples, with stable performance using FFPE curls stored at ≤4 °C for up to two weeks. It remained robust across RNA input levels (5-100 ng) and required a minimum quality threshold (DV200 >15 %). Necrosis had minimal impact on gene expression, and reproducibility was confirmed across technicians and time points. In conclusion, this qPCR array-based assay offers a reliable platform for evaluating key biomarkers in FFPE tissues and holds promise as a potential clinical tool to predict NAC response in MIBC patients.

Background: Bladder cancer (BC) is a prevalent malignancy with muscle-invasive bladder cancer (MIBC) accounting for 20% of cases and a poor prognosis with a 5-year survival of ~50%. Although neoadjuvant chemotherapy (NAC) followed by radical cystectomy is recognized as the optimal approach for treating locally advanced bladder cancer, it is important to acknowledge the limitations of NAC, which include toxicity and a lack of response in certain individuals. There is an unmet need for predictive assays to stratify patients who are likely to benefit from NAC. We identified 10 mRNA bladder cancer associated biomarkers that can predict response to NAC. This study aimed to develop and analytically validate a multiplex qPCR array-based assay to evaluate the mRNA expression of these 10 biomarkers in MIBC. Methods: We analyzed RNA extracted from 8 formalin-fixed paraffin-embedded (FFPE) and 4 fresh-frozen (FF) specimens of MIBC. The effect of procedures and tissue quality on RNA yield and quality were assessed under various pre-analytical and analytical conditions. Gene expression was evaluated using a custom qPCR array targeting 10 biomarkers and 3 reference genes. The robustness, reproducibility, and effect of necrosis on RNA yield and quality were also examined. Results: The assay demonstrated high concordance between FFPE and FF specimens with no significant difference in gene expression patterns. FFPE curls stored at or below 4°C maintained assay accuracy for up to 2 weeks. The assay was robust to variations in RNA input (5–100 ng) with a quality cut-off (DV200 of >15%) ensuring reliable analysis. Reproducibility studies revealed consistent biomarker profiles across independent technicians and dates, confirming the assay’s reliability in clinical settings. Necrotic tissue proportions up to a certain level had minimal impact on gene expression results. Conclusions: The optimized qPCR array-based assay provides a reliable platform for evaluating 10 mRNA bladder cancer associated biomarkers in FFPE tissues, offering potential as a diagnostic tool to predict clinical response to NAC. This assay represents a step forward in precision medicine for managing advanced BC.