A Pilot Study of Plasma Cell-Free DNA Fragmentomics in Gallbladder Cancer

Gallbladder cancer (GBC) is a rare and highly lethal biliary tract cancer with limited treatment options and lack of diagnostic and prognostic noninvasive biomarkers. Circulating cell-free DNA (cfDNA) offers a noninvasive means to capture fragmentomics alterations that may assist with diagnosis and biological characterization. This pilot study aimed to identify cfDNA-based features that differentiate GBC from individuals with gallstones and healthy controls.

cfDNA was extracted from archived plasma samples from 67 individuals in two case-control studies from China and Chile, followed by low coverage whole genome sequencing to evaluate fragmentomics and related features. cfDNA computational packages were leveraged to generate features and create a classification model. External cfDNA datasets including other hepatopancreatobiliary disease groups were processed and compared to the current study.

In this pilot, individuals with GBC displayed significantly altered cfDNA features compared to healthy controls and individuals with gallstones (P<0.05). Key differences were observed in fragment lengths, end motif patterns, estimated tumor fractions, detectable copy number alterations, and transcription factor binding site accessibility, all of which discriminated GBC from a combined non-cancer group (AUC: 0.852). Many of these cfDNA alterations were consistent with the results from paired and unpaired tissue genomics datasets and other related cancer groups (liver cancer, pancreatic cancer, and non-GBC biliary tract cancer).

This proof-of-concept study demonstrates that archived plasma samples can be successfully used for cfDNA sequencing. These methods capture biologically meaningful alterations in GBC that are consistent with tissue-based genomics data. Collectively, these findings highlight the potential of cfDNA profiling for biological characterization and as a promising noninvasive diagnostic tool for GBC.

This pilot study demonstrates that archived plasma EDTA samples can be used for cfDNA sequencing and reinforces the utility of cfDNA fragmentomics analyses for studying gallbladder disease. By assessing biologically relevant cfDNA features across related hepatopancreatobiliary cancers, we identified common and distinct features that may be used for classification and risk stratification. In high-risk settings for GBC, cfDNA fragmentomics might offer complementary information that could be used to guide clinical decision-making or help optimize waitlists for cholecystectomy.