Evaluation of metabolite biomarker candidates in detecting HCC in patients with liver cirrhosis

Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, ranks as the third leading cause of mortality globally. Patients diagnosed with HCC exhibit a dismal prognosis, mostly due to the emergence of symptoms in the advanced stages of the disease. Moreover, conventional biomarkers demonstrate insufficient efficacy in the early detection of HCC, hence highlighting the need for the identification of novel and more effective biomarkers. This study aims to evaluate a selected panel of serum biomarker candidates for the detection of HCC in patients with liver cirrhosis (CIRR). This is accomplished by targeted quantitation of the candidates using a triple quadrupole mass spectrometer. Serum samples from 50 HCC cases (27 Stage I HCC), 50 patients with CIRR, and 25 healthy controls were analyzed using ultra-high-performance liquid chromatography-TSQ Altis Plus triple quadrupole mass spectrometry (UHPLC-MS/MS) by multiple reaction monitoring (MRM). Absolute quantification of 13 endogenous metabolites selected from previous studies was performed using the surrogate matrix approach by creating calibration curves for each metabolite. Statistical analyses included univariate testing with false discovery rate (FDR) correction, multivariable logistic regression adjusted for clinical covariates, and receiver operating characteristic (ROC) curves. Six metabolites primarily involving amino acid and bile acid metabolism were significantly altered in HCC vs. CIRR, with four of these also significant in Stage I HCC vs. CIRR. While AFP alone achieved AUCs of 0.773 ± 0.106 in HCC vs. CIRR and 0.804 ± 0.093 in Stage I HCC vs. CIRR. The combination of AFP with a six-metabolite panel improved discrimination (AUCs 0.870 ± 0.083 and 0.877 ± 0.059, respectively). Among the six metabolites, ornithine and proline remained associated with HCC after adjusting for confounding factors such as age, sex, BMI, MELD score, and HCV status. Targeted metabolomics reveals reproducible metabolic alterations in HCC, including early-stage disease; however, substantial overlap with cirrhosis limits their independent diagnostic utility. Integration with AFP provides modest improvement, supporting a complementary multi-marker approach for HCC detection.