液体活检通过分析脱落的肿瘤细胞 (ETC) 或肿瘤来源的无细胞 DNA (cfDNA)，为无法进行活检的肿瘤进行诊断和基因组分析提供了一种方便、更安全的方法。然而，其主要挑战在于与基于组织的方法相比其准确性有限。我们报告了一种并行单 ETC 基因组测序 (Past-Seq) 方法，用于对胆管癌 (CCA) 和上尿路尿路上皮癌 (UTUC) 等难以活检的肿瘤进行准确诊断和基因组分析。对于 CCA，研究了可疑胆道狭窄患者 (n = 36) 的前瞻性队列。对胆汁 ETC 进行并行单细胞全基因组测序和全外显子组测序，分别用于 CCA 诊断和解析突变谱，并对胆汁 cfDNA 进行测序以进行比较。多个 ETC 中一致的单细胞拷贝数改变 (CNA) 谱为恶性诊断提供了令人信服的证据。 Past-Seq 产生了基于胆汁的准确 CCA 诊断（96% 敏感性、100% 特异性和阳性预测值），超越了病理评估（56% 敏感性）和胆汁 cfDNA CNA 分析（13% 敏感性），并在恢复组织突变。为了进一步探索 Past-Seq 的适用性，我们对 10 名可疑 UTUC 患者进行了尿液标本调查，Past-Seq 在诊断 UTUC 方面表现出 90% 的敏感性，证明了其在各种液体活检和癌症类型中的广泛适用性。

Liquid biopsy provides a convenient and safer procedure for the diagnosis and genomic profiling of tumors that are inaccessible to biopsy by analyzing exfoliated tumor cells (ETCs) or tumor-derived cell-free DNA (cfDNA). However, its primary challenge lies in its limited accuracy in comparison to tissue-based approaches. We report a parallel single-ETC genomic sequencing (Past-Seq) method for the accurate diagnosis and genomic profiling of hard-to-biopsy tumors such as cholangiocarcinoma (CCA) and upper tract urothelial carcinoma (UTUC). For CCA, a prospective cohort of patients with suspicious biliary strictures (n = 36) was studied. Parallel single-cell whole genome sequencing and whole exome sequencing were performed on bile ETCs for CCA diagnosis and resolving mutational profiles, respectively, along with bile cfDNA sequenced for comparison. Concordant single-cell copy number alteration (CNA) profiles in multiple ETCs provided compelling evidence for generating a malignant diagnosis. Past-Seq yielded bile-based accurate CCA diagnosis (96% sensitivity, 100% specificity, and positive predictive value), surpassing pathological evaluation (56% sensitivity) and bile cfDNA CNA analysis (13% sensitivity), and generated the best performance in the retrieval tissue mutations. To further explore the applicability of Past-Seq, 10 suspicious UTUC patients were investigated with urine specimens, and Past-Seq exhibited 90% sensitivity in diagnosing UTUC, demonstrating its broad applicability across various liquid biopsies and cancer types.