Validation of Differential Serum Levels of Ten Candidate Metabolites in Gallstone Patients With and Without Gallbladder Cancer

Gallbladder cancer (GBC) is a highly lethal malignant tumour characterized by a low global incidence but a high prevalence in specific populations, particularly in South America. The absence of specific symptoms and validated early detection tools often leads to late diagnosis and poor prognosis. Although altered metabolomic profiles have been described in GBC, and several metabolites have been proposed as potential risk biomarkers, these findings remain largely exploratory and lack consistent validation in independent cohorts, particularly populations with a high incidence of GBC.

Metabolomics provides a comprehensive approach to characterising disease-associated metabolic alterations. Serum metabolites may reflect cancer-related changes in lipid metabolism, bile acid metabolism, amino acid metabolism, and other biological pathways involved in inflammation, carcinogenesis, and tumour progression.

In our previous research, we used the Biocrates platform to obtain serum metabolomics data for 197 Chilean patients, including 86 patients with gallbladder cancer and 111 patients with gallstone disease. The concentrations in serum of more than 1,000 metabolites were processed applying predefined quality-control steps, missing-value filtering, imputation, log2 transformation, and quantile normalisation.

After robust Huber linear regression analysis adjusted for age and sex, the ten metabolites listed below showed the smallest p-values for association with disease status (GBC versus gallstone disease) and a fold-change greater than 1.5. These metabolites have been selected for subsequent validation in the present study:

1. GCA
2. TCDCA
3. GCDCA
4. PG_16:0_20:3
5. PE_44:11
6. PG_16:0_20:4
7. Cer_d18:1/24:1
8. Cer_d18:1/16:0
9. PC_32:0
10. Cer_d18:1/18:0