Optimization of bile preparation for liquid biopsy in cholangiocarcinoma focusing on circulating tumor DNA and protein stability.
Author(s): Han JY, Ahn KS, Kim MJ, Kim TS, Kim YH, Cho KB, Kang KJ
Publication: Sci Rep, 2025, Vol. 15, Page 12090
PubMed ID: 40204863 PubMed Review Paper? No
Purpose of Paper
This paper examined how the duration of room temperature storage (1-7 h) affects protein concentration, copies of mutant and wildtype KRAS cell-free DNA (cfDNA), and E- and N-Cadherin activity in case-matched bile specimens from six patients with extrahepatic cholangiocarcinoma.
Conclusion of Paper
Storage of bile specimens for ≤ 7 h at room temperature (prior to centrifugation) did not affect protein concentrations or copies of mutant and wildtype KRAS cfDNA. Mutant copies of KRAS cfDNA were above the threshold (1.5 copies/μL) in bile from two of the six patients examined at all timepoints. E- and N-Cadherin activity was unaffected by storage duration in the single bile specimen evaluated.
Studies
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Study Purpose
This study examined how the duration of room temperature storage (1-7 h) affects protein concentration, copies of mutant and wildtype KRAS cell-free DNA (cfDNA), and E- and N-Cadherin activity in case-matched bile specimens. Bile was collected from six patients with extrahepatic cholangiocarcinoma more than 3 days after percutaneous transhepatic biliary drainage (no further details provided) and divided into aliquots. Aliquots were stored at room temperature for 1, 3, 5, and 7 h before centrifugation at 16,000 g for 10 min at 4°C. Cell pellets (washed in phosphate buffered saline and air dried) and supernatant were stored at -80°C. cfDNA was extracted from supernatant using the NucleoSpin cfDNA XS Kit and yield and fragment size were evaluated using a 2100 Bioanalyzer. Mutant and wildtype DNA were quantified using the droplet digital PCR (ddPCR) KRAS Screening Multiplex Kit. Protein was quantified using the Bradford assay. E- and N-Cadherin activities were analyzed by ELISA in bile from one patient.
Summary of Findings:
Storage of bile specimens for ≤ 7 h at room temperature (prior to centrifugation) did not affect protein concentrations or copies of mutant and wildtype KRAS cfDNA. Mutant copies of KRAS cfDNA were above the threshold (1.5 copies/μL) in bile from two of the six patients examined at all timepoints. E- and N-Cadherin activity was unaffected by storage duration in the single bile specimen evaluated.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Colorimetric assay DNA Digital PCR DNA Automated electrophoresis/Bioanalyzer Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Digital PCR Specific Targeted nucleic acid Mutant KRAS
Wildtype KRAS
Storage Time at room temperature 1 h
3 h
5 h
7 h