Single-Cell Sequencing-Enabled Hexokinase 2 Assay for Noninvasive Bladder Cancer Diagnosis and Screening by Detecting Rare Malignant Cells in Urine
Single-Cell Sequencing-Enabled Hexokinase 2 Assay for Noninvasive Bladder Cancer Diagnosis and Screening by Detecting Rare Malignant Cells in Urine
复制标题
单细胞测序支持的己糖激酶 2 检测通过检测尿液中的罕见恶性肿瘤细胞进行非侵袭性膀胱癌诊断和筛查。
DOI：
10.1021/acs.analchem.0c04282
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发表时间：
2020-12-15
影响因子：
7.4
通讯作者：
Shi, Qihui
中科院分区：
文献类型：
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作者：
Wang, Zhuo;Chen, Jie;Shi, Qihui
膀胱癌（BC）是最常见的肿瘤之一，复发率高，需要无创且灵敏的诊断方法。准确检测尿液中脱落的肿瘤细胞 (ETC) 对于无创性 BC 诊断至关重要，但当 ETC 很少且与反应性、再生性或修复性细胞混淆时，其灵敏度就会受到限制。单细胞测序 (SCS) 通过调查致癌驱动突变或全基因组拷贝数变异，能够准确检测 ETC。为了克服 SCS 的低通量限制，我们报告了一种经过 SCS 验证的细胞标记物己糖激酶 2 (HK2)，用于高通量筛选尿液中的细胞并检测参与糖酵解升高的 ETC。在基于 SCS 的训练集中，对来自 8 名尿路上皮癌 (UC) 患者尿液样本的总共 385 个细胞进行了测序，以建立 HK2 阈值，该阈值实现了 ETC 检测 >90% 的特异性。这种基于尿液的 HK2 检测方法在盲法患者组 (n = 384) 中进行了测试，其中包括 UC 和良性泌尿生殖系统疾病，作为前瞻性评估诊断准确性的验证队列。该检测的敏感性、特异性、阳性预测值和阴性预测值分别为90%、88%、83%和93%，优于尿细胞学检查。为了研究筛选测试的潜力，我们对一组健康个体 (n = 846) 进行了 HK2 检测测试，并进行了 6 个月的随访。该健康组的特异性为 98.4%。发现三名参与者有超过 5 个假定的 ETC，这些 ETC 经测序显示出恶性细胞的反复拷贝数变化特征，这证明了在当前临床方法之前的早期 BC 检测。
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.