Urinary bladder cancer (BCa) is the 10th most frequent cancer in the world, most commonly found among the elderly population, and becomes highly lethal once cells have spread from the primary tumor to surrounding tissues and distant organs. Cystectomy, alone or with other treatments, is used to treat most BCa patients, as it offers the best chance of cure. However, even with curative intent, 29% of patients experience relapse of the cancer, 50% of which occur within the first year of surgery. This study aims to use the liquid biopsy to noninvasively detect disease and discover prognostic markers for disease progression. Using the third generation high-definition single cell assay (HDSCA3.0), 50 bladder cancer patient samples and 50 normal donor (ND) samples were analyzed for circulating rare events in the peripheral blood (PB), including circulating tumor cells (CTCs) and large extracellular vesicles (LEVs). Here, we show that (i) CTCs and LEVs are detected in the PB of BCa patients prior to cystectomy, (ii) there is a high heterogeneity of CTCs, and (iii) liquid biopsy analytes correlate with clinical data elements. We observed a significant difference in the incidence of rare cells and LEVs between BCa and ND samples (median of 74.61 cells/mL and 30.91 LEVs/mL vs. 34.46 cells/mL and 3.34 LEVs/mL, respectively). Furthermore, using classification models for the liquid biopsy data, we achieved a sensitivity of 78% and specificity of 92% for the identification of BCa patient samples. Taken together, these data support the clinical utility of the liquid biopsy in detecting BCa, as well as the potential for predicting cancer recurrence and survival post-cystectomy to better inform treatment decisions in BCa care.

Urothelial carcinomas (UCs) are a broad and heterogeneous group of malignancies, with the prevalence of upper tract urothelial carcinoma (UTUC) being rare, accounting for only 5–10% of total malignancies. There is a need for additional toolsets to assist the current clinical paradigm of care for patients with UTUC. As a non-invasive tool for the discovery of cancer-related biomarkers, the liquid biopsy has the potential to represent the complex process of tumorigenesis and metastasis. Herein, we show the efficacy of the liquid biopsy as a source of biomarkers for detecting UTUC. Using the third-generation high-definition single-cell assay (HDSCA3.0) workflow, we investigate liquid biopsy samples collected from patients with UTUC and normal donors (NDs) to provide critical information regarding the molecular and morphological characteristics of circulating rare events. We document several important findings from the liquid biopsy analysis of patients diagnosed with UTUC prior to surgery: (1) Large extracellular vesicles (LEVs) and circulating tumor cells (CTCs) are detectable in the peripheral blood. (2) The rare-event profile is highly heterogeneous. (3) Clinical data elements correlate with liquid biopsy analytes. Overall, this study provides evidence for the efficacy of the liquid biopsy in understanding the biology of UTUC with the future intent of informing clinical decision making, ultimately improving patient outcomes.

The liquid biopsy presents potential as a non-invasive cancer screening tool, and its role in diagnosing and monitoring the progression of primary upper tract urothelial carcinoma (UTUC) to reveal the tumor’s kinetics remains to be fully understood. This study includes 22 patients diagnosed with UTUC, from which we collected 1 peripheral blood (PB) sample prior to surgery (pre-op; n = 22) and 1 sample at follow-up (post-op; n = 11). Each patient’s sample was analyzed via our enrichment-free third generation comprehensive High-Definition Single Cell Assay (HDSCA3.0) workflow to detect rare events in the PB. Rare events include circulating tumor cells (CTCs) and large extracellular vesicles (LEVs) and are identified via a 4-channel immunofluorescence assay that captures the following morphometric expressions: pan cytokeratin (CK), vimentin (V), CD45/CD31 (CD), and cellular nucleation (which is absent in LEVs) through DAPI (D). Through a matched sample analysis approach via a Wilcoxon signed-rank test, pre-op and post-op liquid biopsy profiles were compared to each other as well as to samples from 50 normal donors (NDs) who had no known pathology. The HDSCA3.0 analysis discovered a highly heterogeneous spread of rare events in PB samples collected from patients with UTUC in both pre-op and post-op time periods and in ND samples. For pre-op vs ND samples, significant differences were found in total rare events, total rare cells, events expressing D|V, D|CK|V|CD, D|V|CD, total LEVs, total CK+ cells, CK LEVs, and events expressing D and D|CK. For post-op vs ND samples, significant differences were in total rare events, total rare cells, events expressing D|V, D|CK|V|CD, total CK+ cells, and events expressing D|CK|V. For pre-op vs post-op samples, significant differences manifested in total LEVs, CK LEVs, and CK|CD LEVs. At follow-up, 3 patients had an increase in rare events, and 8 patients had a decrease in rare events, 4 of the 8 presenting with ND rare event levels. This study has the potential to inform how primary UTUC evolves in patients over time as well as elucidate if rare event expressions may correlate with specific disease states. By analyzing circulating rare events through channel-type classifications, different frequencies at various time points compared to ND samples can assist in clinical decision making for treating UTUC. Citation Format: Salmaan Sayeed, George Courcoubetis, Alireza Ghoreifi, Amy Huang, Jeremy Mason, Inderbir S. Gill, Peter Kuhn, Hooman Djaladat, Stephanie N. Shishido. Circulating rare events inform disease progression in patients with upper tract urothelial carcinoma undergoing surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2203.