Zoom in on antibody aggregates:A potential pitfall in the search of rare EV populations

High-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre-treated labels. We found a varying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs

Genome-wide Association Study of Bladder Cancer Reveals New Biological and Translational Insights

BACKGROUND: Genomic regions identified by genome-wide association studies (GWAS) for bladder cancer risk provide new insights into etiology. OBJECTIVE: To identify new susceptibility variants for bladder cancer in a meta-analysis of new and existing genome-wide genotype data. DESIGN, SETTING, AND PARTICIPANTS: Data from 32 studies that includes 13,790 bladder cancer cases and 343,502 controls of European ancestry were used for meta-analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSES: Log-additive associations of genetic variants were assessed using logistic regression models. A fixed-effects model was used for meta-analysis of the results. Stratified analyses were conducted to evaluate effect modification by sex and smoking status. A polygenic risk score (PRS) was generated on the basis of known and novel susceptibility variants and tested for interaction with smoking. RESULTS AND LIMITATIONS: Multiple novel bladder cancer susceptibility loci (6p.22.3, 7q36.3, 8q21.13, 9p21.3, 10q22.1, 19q13.33) as well as improved signals in three known regions (4p16.3, 5p15.33, 11p15.5) were identified, bringing the number of independent markers at genome-wide significance (p \u3c 5 × 10 CONCLUSIONS: We report novel loci associated with risk of bladder cancer that provide clues to its biological underpinnings. Using 24 independent markers, we constructed a PRS to stratify lifetime risk. The PRS combined with smoking history, and other established risk factors, has the potential to inform future screening efforts for bladder cancer. PATIENT SUMMARY: We identified new genetic markers that provide biological insights into the genetic causes of bladder cancer. These genetic risk factors combined with lifestyle risk factors, such as smoking, may inform future preventive and screening strategies for bladder cancer

Comprehensive genomic characterization of early-stage bladder cancer

Understanding the molecular landscape of nonmuscle-invasive bladder cancer (NMIBC) is essential to improve risk assessment and treatment regimens. We performed a comprehensive genomic analysis of patients with NMIBC using whole-exome sequencing (n = 438), shallow whole-genome sequencing (n = 362) and total RNA sequencing (n = 414). A large genomic variation within NMIBC was observed and correlated with different molecular subtypes. Frequent loss of heterozygosity in FGFR3 and 17p (affecting TP53) was found in tumors with mutations in FGFR3 and TP53, respectively. Whole-genome doubling (WGD) was observed in 15% of the tumors and was associated with worse outcomes. Tumors with WGD were genomically unstable, with alterations in cell-cycle-related genes and an altered immune composition. Finally, integrative clustering of multi-omics data highlighted the important role of genomic instability and immune cell exhaustion in disease aggressiveness. These findings advance our understanding of genomic differences associated with disease aggressiveness in NMIBC and may ultimately improve patient stratification

Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations

High-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre-treated labels. We found a varying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs.</jats:p

Single-nucleus and Spatially Resolved Intratumor Subtype Heterogeneity in Bladder Cancer

Background: Current bulk transcriptomic classification systems for bladder cancer do not consider the level of intratumor subtype heterogeneity. Objective: To investigate the extent and possible clinical impact of intratumor subtype heterogeneity across early and more advanced stages of bladder cancer. Design, setting, and participants: We performed single-nucleus RNA sequencing (RNA-seq) of 48 bladder tumors and additional spatial transcriptomics for four of these tumors. Total bulk RNA-seq and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients. Outcome measurements and statistical analysis: The primary outcome was progression-free survival for non–muscle-invasive bladder cancer. Cox regression analysis, log-rank tests, Wilcoxon rank-sum tests, Spearman correlation, and Pearson correlation were used for statistical analysis. Results and limitations: We found that the tumors exhibited varying levels of intratumor subtype heterogeneity and that the level of subtype heterogeneity can be estimated from both single-nucleus and bulk RNA-seq data, with high concordance between the two. We found that a higher class 2a weight estimated from bulk RNA-seq data is associated with worse outcome for patients with molecular high-risk class 2a tumors. The sparsity of the data generated using the DroNc-seq sequencing protocol is a limitation. Conclusions: Our results indicate that discrete subtype assignments from bulk RNA-seq data may lack biological granularity and that continuous class scores may improve clinical risk stratification of patients with bladder cancer. Patient summary: We found that several molecular subtypes can exist within a single bladder tumor and that continuous subtype scores can be used to identify a subgroup of patients with poor outcomes. Use of these subtype scores may improve risk stratification for patients with bladder cancer, which can help in making decisions on treatment

Field cancerization impacts tumor development, T-cell exhaustion and clinical outcomes in bladder cancer

AbstractBladder field cancerization may be associated with disease outcome in patients with bladder cancer. To investigate this, we analyzed biopsies from bladder urothelium and urine samples by genomics and proteomics analyses. Samples were procured from multiple timepoints from 134 patients with early stage bladder cancer and detailed long term follow-up. We measured the field cancerization in normal-appearing bladder biopsies and found that high levels were associated with high tumor mutational burden, high neoantigen load, and high tumor-associated CD8 T-cell exhaustion. Non-synonymous mutations in known bladder cancer driver genes such asKDM6AandTP53were identified as early disease drivers in normal urothelium. High field cancerization was associated with worse outcome but not with response to BCG. The level of urinary tumor DNA (utDNA) reflected the bladder tumor burden and originated from both tumors and field cancerization. High utDNA levels after BCG were associated with worse clinical outcomes for the patients. Our results indicate that the level of field cancerization may affect clinical outcome, tumor development and immune responses. utDNA measurements have significant prognostic value and reflect the disease status of the bladder.</jats:p