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. Single-cell 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.
Analytical chemistry. 2020 Dec 03 [Epub]
Zhuo Wang, Jie Chen, Liu Yang, Mingzhe Cao, Yanlan Yu, Rulin Zhang, Heng Quan, Qi Jiang, Yingqi Hua, Wei Wei, Peihua Lu, Jun Wu, Qihui Shi
Minhang Branch, Zhongshan Hospital and Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Fudan University, Shanghai 201100, China., Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China., Shanghai Bone Tumor Institute and Department of Orthopedics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China., The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen 518107, China., Department of Urology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China., Department of Laboratory Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China., Department of Urology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China., Institute for Systems Biology, Seattle 98109, Washington, United States., Department of Medical Oncology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China., Department of Clinical Laboratory, Shanghai General Hospital Jiading Branch, Shanghai Jiao Tong University School of Medicine, Shanghai 201803, China.