Nano Pom-Poms Prepared Exosomes for Highly Specific Cancer Biomarker Detection

Extracellular vesicles (EVs), particularly nano-sized small EV exosomes, are emerging biomarker sources. However, due to heterogeneous populations secreted from diverse cell types, mapping exosome multi-omic molecular information specifically to their pathogenesis origin for cancer biomarker identification is still extraordinarily challenging. Herein, we introduced a novel 3D-structured nanographene immunomagnetic particles (NanoPoms) with unique flower pom-poms morphology and photo-click chemistry for specific marker-defined capture and release of intact exosome. This specific exosome isolation approach leads to the expanded identification of targetable cancer biomarkers with enhanced specificity and sensitivity, as demonstrated by multi-omic exosome analysis of bladder cancer patient tissue fluids using the next generation sequencing of somatic DNA mutations, miRNAs, and the global proteome. The NanoPoms prepared exosomes also exhibit distinctive in vivo biodistribution patterns, highlighting the highly viable and integral quality. The developed method is simple and straightforward, which is applicable to nearly all types of biological fluids and amenable for enrichment, scale up, and high-throughput exosome isolation.

901 Bacillus Calmette-Guerin can subvert patients antitumor immune response by downregulating HLA-I expression on cancer cells

BackgroundPatients with high-risk non muscle-invasive bladder cancer (NMIBC) frequently relapse after standard BCG immunotherapy and have a dismal outcome after progression to muscle-invasive bladder cancer (MIBC).1 2 The mechanisms of tumor resistance to such immunotherapy remain elusive.MethodsWe performed functional assays of fresh human bladder tumors mixed with BCG, reinforced with in vitro experiments and in situ transcriptomics analyses together with immune profiling by immunohistochemistry (IHC) in a cohort of T1 NMIBC pre- and post BCG therapy.ResultsWe found two distinct patterns of BCG-induced immune subversion. In the first pattern, intracellular infection by live BCG was associated with HLA-I loss and epithelial-to-mesenchymal transition characteristics. Mechanistically, LC3-GFP reporter cell line showed a significant induction of autophagy upon BCG exposure. HLA-I deficient tumors displayed a myeloid immunosuppressive microenvironment together with an upregulation of autophagy-related genes, and dismal outcome. Conversely, HLA-I+ BCG-treated tumors generated a Th1 type of immune response associated with an upregulation of exhaustion markers. Such patients had a very favorable outcome upon radical surgery.ConclusionsWe surmise that HLA-I expression in bladder cancers does not result from immunoediting but rather from HLA-I molecules endocytosis related to autophagy induction in infected cancer cells. Cancer cells HLA-I scoring by immunohistochemistry staining can be easily implemented by pathologists in routine practice to stratify future bladder cancer patient treatment strategies.ReferencesPietzak EJ, Zabor EC, Bagrodia A, et al. Genomic differences between “primary” and “secondary” muscle-invasive bladder cancer as a basis for disparate outcomes to cisplatin-based neoadjuvant chemotherapy. Eur Urol 2019;75(2):231–239.. Patrick J Hensley, Kelly K Bree, Matthew T Campbell, et al. Progression of disease after BCG therapy: refining patient selection for neoadjuvant chemotherapy before radical cystectomy. J Urol 2021 June 29;101097JU0000000000001943.Ethics ApprovalOur study obtained ethics approval from the Foch Hospital Ethics Committee (IRB00012437). All the participants gave informed consent before taking part.ConsentWe surmise that HLA-I expression in bladder cancers does not result from immunoediting but rather from HLA-I molecules endocytosis related to autophagy induction in infected cancer cells. Cancer cells HLA-I scoring by immunohistochemistry staining can be easily implemented by pathologists in routine practice to stratify future bladder cancer patient treatment strategies.

82 Single-cell RNA sequencing and CITE-Seq analysis of bladder cancer patient urine with matched tumor and peripheral blood suggests urine as a window into the tumor immune microenvironment

BackgroundFDA-approved immunotherapies for early and advanced stage bladder cancer have response rates of 15–65% in bladder cancer, suggesting that tumor-associated resistance mechanisms undermine their efficacy. Accordingly, there is an unmet need to identify accessible biomarkers that predict response. Urine, which is in direct contact with urothelial tumors, represents an easily accessible patient material that may reflect cellular and/or genetic signatures related to immune resistance. It has been demonstrated that urine from bladder cancer patients contains not only tumor cells, which are routinely assessed by clinical urinalyses, but also immune cells that previous studies suggest may reflect the tumor microenvironment (TME).1 However, the concordance between cells in the urine and those in bladder tumors is unknown., Here, we characterized patient urine in an unbiased fashion by performing the first single-cell RNA sequencing (scRNAseq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on matched bladder cancer patient urine, tumor, and peripheral blood.MethodsMatched tumor tissue, urine, and peripheral blood were collected from bladder cancer patients (n=7) during surgery; either trans-urethral resection of bladder tumor or cystectomy. All three tissues were processed to single-cell suspensions and sequenced using the 10X Genomics platform (scRNAseq: 17 samples, CITE-seq: 3 samples). These sequencing approaches permitted quantification of both transcriptomic and surface protein expression of 54,469 cells total.2 3 Analysis was performed using Seurat, Enrichr, and Monocle packages and platforms.4 5 6Results scRNAseq of urine from bladder cancer patients revealed several immune populations including CD4+ and CD8+ T cells, Treg cells, NK cells, B cells, neutrophils, dendritic cells, monocytes, and macrophages in addition to non-hematopoietic lineages including bladder epithelial cells, neuronal cells, prostate epithelial cells, fibroblasts, myofibroblasts, and endothelial cells. The composition and transcriptional profiles of urine immune cells were more similar to TME immune cells than to peripheral blood immune cells. Urine immune cells expressed gene signatures associated with hypoxia, anergy, pro-inflammation, and glucose deprivation that were more similar to tumor immune cells than those in the peripheral blood.ConclusionsOur work represents the first scRNAseq and CITEseq profiling of cancer patient urine. Our study suggests several viable immune cells shed in bladder cancer patient urine that look more transcriptionally and phenotypically similar to the TME than peripheral blood cells. This important finding has several implications for future research and clinical applications as urine can be sampled non-invasively in scenarios when tumor resection may not be feasible.ReferencesWong YNS, Joshi K, Khetrapal P, et al. Urine-derived lymphocytes as a non-invasive measure of the bladder tumor immune microenvironment. Journal of Experimental Medicine. 2018; 215:2748–59.Zheng GXY, Terry JM, Belgrader P, et al. Massively parallel digital transcriptional profiling of single cells. Nature Communications 2017; 8.Stoeckius M, Hafemeister C, Stephenson W, et al. Simultaneous epitope and transcriptome measurement in single cells. Nature Methods 2017;14, 865–68.Butler A, Hoffman P, Smibert, P, et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nature Biotechnology 2018; 36: 411–20.Xie Z, Bailey A, Kuleshov MV, et al. Gene set knowledge discovery with Enrichr. Current Protocols 2021.Trapnell C, Cacchiarelli D, Grimsby J, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nature Biotechnology 2014; 32: 381–6.Ethics ApprovalThe study was approved by Mount Sinai Institution’s Ethics Board, approval number 10–1180. Participants gave informed consent before taking part in the study.

Magnetic-resonance-guided Radiation Therapy With Simultaneous Integrated Boost at Mid-bladder Volume for Bladder Cancer

Background and Purpose: To report the workflow and dose accumulation for bladder preservation for a bladder cancer patient, using magnetic-resonance-guided radiation therapy (MRgRT) and the simultaneous integrated boost (SIB) technique at mid-bladder volume.Materials and Methods: A muscle-invasive bladder cancer patient was treated with MRgRT. The patient was treated with the SIB technique at mid-bladder volume, with 45 Gy to the whole bladder (CTV WB) and 55 Gy to the tumor bed (CTV boost) in 20 fractions. Daily re-optimization with an adapt-to-position (ATP) strategy was utilized for dose adjustment to encompass the bladder within anisotropic planning target volume (PTV WB and PTV boost). Results: The mean daily treatment time was 55 minutes (range, 35–73). The actual whole-bladder and tumor-bed-boost doses were 45.74 ± 5.91 and 54.1 ± 4.62 Gy, respectively. PTV WB encompassing CTV WB was 95.69% ± 5.36%. PTV boost encompassing CTV boost was 97.52% ± 6.05%. The actual rectal and bowel doses were below the reference plan doses.Conclusions: The use of MRgRT with the SIB and ATP strategy proved feasible for bladder cancer treatment. Mid-bladder volume allowed treatment with the SIB technique under MR monitoring.

Myositis/Myasthenia after Pembrolizumab in a Bladder Cancer Patient with an Autoimmunity-Associated HLA: Immune–Biological Evaluation and Case Report

Pembrolizumab (mAb to PD-1) has been recently approved for the therapy of pretreated urothelial cancer. Despite the efficacy, it is often accompanied by unpredictable and sometime severe immune-related (ir) adverse events (AEs). Here, we report the clinical and immune–biological characterization of a patient with a metastatic bladder cancer who developed myositis signs (M) and a myasthenia-like syndrome (MLS) during treatment with pembrolizumab. The patient presented an autoimmunity-associated HLA haplotype (HLA-A*02/HLA-B*08/HLA-C*07/HLA-DRB1*03) and experienced an increase in activated CD8 T-cells along the treatment. The symptomatology regressed after pembrolizumab discontinuation and a pyridostigmine and steroids-based therapy. This is the first report of concurrent M and MLS appearance in cancer patients receiving pembrolizumab. More efforts are needed to define early the risk and the clinical meaning of irAEs in this setting.

Circulating tumor cell-driven use of neoadjuvant chemotherapy in patients with muscle-invasive bladder cancer.

4523 Background: International guidelines for the treatment of non-metastatic muscle-invasive bladder cancer (MIBC) recommend neoadjuvant chemotherapy (NAC), which, however, is underutilized in practice. We hypothesized that the absence of circulating tumour cells (CTCs), an established prognostic marker in MIBC, may identify patients with such a favourable prognosis that NAC may be withheld. Methods: The CirGuidance study included adults with clinical stage T2-T4aN0-N1M0 muscle-invasive urothelial carcinoma of the bladder who were fit to undergo radical cystectomy. CTCs were enumerated using the CellSearch system. CTC-negative patients (no CTCs detectable) underwent radical surgery without NAC; CTC-positive patients (≥1 detectable CTCs) were advised to receive NAC followed by radical surgery, but NAC could be withheld at the discretion of the treating physician. The primary endpoint was the two-year overall survival (OS) in the CTC-negative group, analysed in the intention-to-treat population. The prespecified criterion for trial success was a two-year OS of minimally 75% (95% confidence interval (CI) ±5%) in the CTC-negative group. Results: Of 315 patients screened for eligibility, 273 were enrolled in the study. The median age was 69 years; the median follow-up was 36 months. The two-year OS in the CTC-negative group was 69.5% (n = 203; 95% CI 62.6%-75.5%); in the CTC-positive group it was 58.2% (n = 70; 95% CI 45.5%-68.9%). CTC-positive patients had a higher rate of cancer-related mortality (hazard ratio (HR) 1.61, 95% CI 1.05-2.45, p = 0.03) and disease relapse (HR 1.87, 95% CI 1.28-2.73, p = 0.001) than CTC-negative patients. Explorative analyses suggested that CTC-positive patients who had received NAC (n = 22) survived longer than CTC-positive patients who had not (n = 48), with a two-year OS of 74.8% (95%CI 49.5%-88.8%) versus 52.0% (95% CI 37.2%-65.0%), respectively. Conclusions: The two-year OS in the CTC-negative group did not meet the prespecified criterion for trial success. However, given the trial population’s advanced age and high rate of non-cancer related mortality, the benefit of NAC is likely to be limited in CTC-negative MIBC patients. CTC enumeration at the moment of diagnosis could aid in the decision to prescribe neoadjuvant chemotherapy for a muscle-invasive bladder cancer patient as a criterion in addition to clinical characteristics. Clinical trial information: NL3954.

The Role of Molecular Imaging in a Muscle-Invasive Bladder Cancer Patient: A Narrative Review in the Era of Multimodality Treatment

Diagnostic imaging in bladder cancer plays an important role since it is needed from pretreatment staging to follow-up, but a morphological evaluation performed with both CT and MRI showed low sensitivities and specificities in detecting pathologic lymph nodes, due to the occurrence of false positive results. Implementation of functional information provided by PET/CT could be a determinant in the management of patients with muscle-invasive bladder cancer. A focus on the role of 18F-FDG PET/CT and alternative tracers in patients with muscle-invasive bladder cancer is provided in this analysis in order to outline its potential applications in staging settings and response evaluation after neoadjuvant chemotherapy.

Dualmarker: a flexible toolset for exploratory analysis of combinatorial dual biomarkers for clinical efficacy

Background An increasing number of clinical trials require biomarker-driven patient stratification, especially for revolutionary immune checkpoint blockade therapy. Due to the complicated interaction between a tumor and its microenvironment, single biomarkers, such as PDL1 protein level, tumor mutational burden (TMB), single gene mutation and expression, are far from satisfactory for response prediction or patient stratification. Recently, combinatorial biomarkers were reported to be more precise and powerful for predicting therapy response and identifying potential target populations with superior survival. However, there is a lack of dedicated tools for such combinatorial biomarker analysis. Results Here, we present dualmarker, an R package designed to facilitate the data exploration for dual biomarker combinations. Given two biomarkers, dualmarker comprehensively visualizes their association with drug response and patient survival through 14 types of plots, such as boxplots, scatterplots, ROCs, and Kaplan–Meier plots. Using logistic regression and Cox regression models, dualmarker evaluated the superiority of dual markers over single markers by comparing the data fitness of dual-marker versus single-marker models, which was utilized for de novo searching for new biomarker pairs. We demonstrated this straightforward workflow and comprehensive capability by using public biomarker data from one bladder cancer patient cohort (IMvigor210 study); we confirmed the previously reported biomarker pair TMB/TGF-beta signature and CXCL13 expression/ARID1A mutation for response and survival analyses, respectively. In addition, dualmarker de novo identified new biomarker partners, for example, in overall survival modelling, the model with combination of HMGB1 expression and ARID1A mutation had statistically better goodness-of-fit than the model with either HMGB1 or ARID1A as single marker. Conclusions The dualmarker package is an open-source tool for the visualization and identification of combinatorial dual biomarkers. It streamlines the dual marker analysis flow into user-friendly functions and can be used for data exploration and hypothesis generation. Its code is freely available at GitHub at https://github.com/maxiaopeng/dualmarker under MIT license.