Single-Cell Sequencing-Enabled Hexokinase 2 Assay for Noninvasive Bladder Cancer Diagnosis and Screening by Detecting Rare Malignant Cells in Urine

Abstract

Bladder cancer (BC) is among the most common tumors with a high recurrence rate, necessitating noninvasive and sensitive diagnostic methods. Accurate detection of exfoliated tumor cells (ETCs) in urine is crucial for noninvasive BC diagnosis but suffers from limited sensitivity when ETCs are rare and confounded by reactive, regenerative, or reparative cells. Singlecell sequencing (SCS) enables accurate detection of ETCs by surveying oncogenic driver mutations or genome-wide copy number alternations. To overcome the low-throughput limitation of SCS, we report a SCS-validated cellular marker, hexokinase 2 (HK2), for high-throughput screening cells in urine and detecting ETCs engaging elevated glycolysis. In the SCS-based training set, a total of 385 cells from urine samples of eight urothelial carcinoma (UC) patients were sequenced to establish a HK2 threshold that achieved >90% specificity for ETC detection. This urine-based HK2 assay was tested with a blinded patient group (n = 384) including UC and benign genitourinary disorders as a validation cohort for prospectively evaluating diagnostic accuracy. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 90, 88, 83, and 93%, respectively, which were superior to urinary cytology. For investigating the potential to be a screening test, the HK2 assay was tested with a group of healthy individuals (n = 846) and a 6-month follow-up. The specificity was 98.4% in this health group. Three participants were found to have >5 putative ETCs that were sequenced to exhibit recurrent copy number alternations characteristic of malignant cells, demonstrating early BC detection before current clinical methods.

An Overview on Single-Cell Technology for Hepatocellular Carcinoma Diagnosis

“…For instance, SCT has been designated as a robust technique to diagnose (through a noninvasive method) bladder cancer by screening urine for rare malignant cells. This technique surveys malignant cells' copy number alternations or tumor (oncogenic) driver gene mutations of characteristic cancer [18]. SCT is indispensable for the identification of biomarkers of gastric cancer, lung cancer, colorectal cancer, breast cancer, and various other tumors, thus contributing to early diagnosis and prognostic monitoring of cancer [16].…”

Immunotherapy in the Treatment of Urothelial Bladder Cancer: Insights From Single-Cell Analysis

“…Moreover, patient-derived organoid (PDO) provides an ex vivo platform for studying tumor evolution and drug response (72,73). Of special note, the urine from UBC patients, compared to peripheral blood, is a faithful and rich source of tumor-derived materials including DNA, protein, and exfoliated cells (74)(75)(76)(77). Thus, single-cell analysis of urinary lymphocytes can be potentially employed as a noninvasive strategy to monitor tumor immune microenvironment at the cellular level.…”

Current Development and Application of Anaerobic Glycolytic Enzymes in Urothelial Cancer

“…In BC, targeting HK2 suppresses BC progression [ 18 ]. Moreover, single-cell sequencing has been used to assess the activity of hexokinase 2 in urine for the non-invasive diagnosis and screening of BC [ 19 ].…”