Liquid Biopsy-Based Accurate Diagnosis and Genomic Profiling of Hard-to-Biopsy Tumors via Parallel Single-Cell Genomic Sequencing of Exfoliated Tumor Cells

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.