Pancreatic circulating tumor cell detection by targeted single-cell next-generation sequencing.

Area of Science:

• Genomics
• Oncology
• Biotechnology

Background:

• Single-cell next-generation sequencing (scNGS) is crucial for genomic profiling but whole-genome amplification (WGA) introduces errors, particularly for single nucleotide variant (SNV) detection.
• Existing scNGS methods are less accurate for SNV analysis due to WGA limitations.
• Targeted scNGS for SNV detection without WGA has not been previously described.

Purpose of the Study:

• To develop and validate a novel targeted scNGS method for detecting circulating tumor cells (CTCs) based on DNA SNVs.
• To assess the method's accuracy in identifying SNVs in key cancer genes (KRAS/TP53/SMAD4) within CTCs.

Main Methods:

• A targeted scNGS approach was developed to detect SNVs in KRAS, TP53, and SMAD4 genes without whole-genome amplification.
• The method was tested on the AsPC-1 pancreatic cancer cell line and spiked-in cells in healthy blood.
• Validation was performed on blood samples from patients with metastatic pancreatic ductal adenocarcinoma (PDAC) and controls.

Main Results:

• The targeted scNGS method successfully detected single CTCs with specific SNVs in pancreatic cancer cell lines and patient samples.
• Blood samples from metastatic PDAC patients showed CTCs with KRAS/TP53/SMAD4 SNVs, confirmed by immunofluorescence.
• Concordant results between targeted scNGS and CTC enumeration were observed in early-stage disease, benign cysts, and healthy controls.

Conclusions:

• The novel targeted scNGS technique, performed without pre-amplification, shows significant promise for identifying and characterizing CTCs.
• This method offers improved accuracy for SNV analysis in CTCs, advancing liquid biopsy applications in oncology.