[Tumor cell-based glycolytic metabolism and single-cell sequencing of urinary exfoliated cells for the diagnosis and molecular profiling of urothelial carcinoma].

Objective: To explore the diagnostic values of HK2 testing and single-cell sequencing in the urothelial carcinoma (UC). Methods: The qualified urine specimens of 265 suspected UC patients or postoperative patients from the Cancer Hospital of Chinese Academy of Medical Sciences, Beijing, China were collected. Both exfoliative cytology and HK2 testing were performed on clinically suspected UC or postoperative patients. The performance of diagnostic cytology and HK2, including consistency, sensitivity, specificity, positive predictive value and negative predictive value, was evaluated based on histopathological, clinical and imaging diagnosis. Isolated HK2 metabolically abnormal cells were subject to single-cell sequencing to verify the reliability of HK2 detection performance and to explore the molecular characteristics of UC. Results: The concordance rate of HK2 testing and cytology for detecting UC was 90.3% (102/113, Kappa=0.604). Compared with cytology, the sensitivity of HK2 was significantly higher (85.2% versus 75.6%, P=0.024). The detection sensitivity of combined HK2 testing and cytology was increased to 91.1%. HK2 testing was significantly more sensitive than cytology for diagnosing UC in the upper urinary tract (81.8% versus 65.5%, P=0.022). It was also more sensitive than cytology for diagnosing early-stage UC (82.6% versus 69.5%, P=0.375) and low-grade UC (69.6% versus 47.8%, P=0.125). Single-cell sequencing of the ten patients, whose samples were positive for HK2, demonstrated highly concordant copy number variations (CNVs) in tumor cells from the same UC patient, with heterogeneity in CNV profiles among different patients. Deletion of chromosome 8p was found in 3 of the 4 urine samples of renal pelvis UC. The 2 patients with benign lesions had no CNVs in all sequenced cells. Conclusions: The test for abnormal urinary glycolytic HK2 metabolism can assist urine cytology to improve the sensitivity of UC diagnosis, and it provides a novel and reliable approach for early detection of upper urinary tract UC and lower grade UC. Meanwhile, this study has preliminarily revealed the feasibility of single-cell sequencing in urinary samples, which is expected to improve the diagnostic specificity of HK2 testing.目的： 探讨己糖激酶-2（hexokinase2，HK2）检测和单细胞测序在尿路上皮癌（urothelial carcinoma，UC）中的诊断价值。 方法： 收集2021年6月至2022年4月就诊于中国医学科学院肿瘤医院疑似UC或UC术后复查患者的合格尿液标本265例，对其同时行脱落细胞学和HK2检测。基于组织病理、临床及影像学诊断，通过交叉列联表及分层分析评价2种检测方法的一致性、特异度、灵敏度、阳性预测值和阴性预测值。分离HK2代谢异常细胞行单细胞测序，从分子层面验证HK2检测性能的可靠性。 结果： HK2及细胞学检测诊断UC的阳性符合率为90.3%（102/113），两者一致性较高（Kappa=0.604）。HK2检测较细胞学可明显提高UC诊断灵敏度（85.2%比75.6%，P=0.024），两者联合后灵敏度可提高至91.1%。HK2对上尿路上皮癌诊断灵敏度明显高于细胞学（81.8%比65.5%，P=0.022）；HK2检测较细胞学在早期病变（82.6%比69.5%，P=0.375）及低级别UC（69.6%比47.8%，P=0.125）中同样具有更高的灵敏度。对10例HK2检测阳性样本行单细胞测序，结果显示同一UC患者的肿瘤细胞表现出高度一致的拷贝数变异，不同患者间拷贝数变异谱存在异质性。4例肾盂癌尿液样本中发现3例均存在8p染色体缺失；2例良性病变患者所有测序细胞均无拷贝数变异，提示细胞为良性。 结论： 尿液HK2检测可辅助尿液细胞学提高UC诊断的灵敏度，为上尿路上皮癌、低级别UC及早期病变提供一种全新可靠的手段。同时本研究初步揭示了在尿液样本中行单细胞测序的可行性，有望从基因层面提高HK2检测的特异度。.