Diagnostic value of alpha-fetoprotein and prothrombin induced by vitamin K absence-II in serum, bile, and feces in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the most common pathological type of liver cancer and was the third leading cause of cancer-related deaths worldwide in 2020.

Aim: To evaluate the diagnostic potential of key tumor markers in serum, bile, and fecal samples for detecting HCC.

Methods: Blood, bile, and fecal samples were collected from patients (n = 265) with HCC and cholecystitis from Guangxi Medical University's First Affiliated Hospital. Immunohistochemistry was performed on 69 HCC samples, and 16S ribosomal RNA sequencing was conducted on 166 fecal samples. Tumor marker cut-off values in bile and feces were determined using the Youden index, while serum biomarkers followed hospital standards. Diagnostic performance was evaluated using receiver operating characteristic analysis.

Results: The areas under the curve (AUCs) for distinguishing HCC were 0.898, 0.904, and 0.859 for serum alpha-fetoprotein (AFP), prothrombin induced by vitamin K absence-II (PIVKA-II), and bile AFP, respectively. Serum AFP had the highest diagnostic value (80%) for early-stage HCC. Combination analysis found that bile AFP and serum PIVKA-II achieved the highest AUC of 0.965 (P < 0.001), suggesting that bile AFP may serve as a valuable complementary biomarker, particularly in cases where serum AFP is not significantly elevated. Additionally, bile AFP was positively correlated with Actinomyces, which plays a significant role in promoting tumorigenesis; and was negatively correlated with Faecalibacterium, which was associated with robust anticancer immune responses (P < 0.05). These findings suggest the potential role of gut microbiota in modulating AFP levels and HCC progression.

Conclusion: Bile AFP improved the sensitivity of HCC detection, with the combination of bile AFP and PIVKA-II demonstrating the highest AUC for HCC diagnosis. AFP is associated with poorer clinical outcomes.

Keywords: Alpha-fetoprotein; Bile biomarkers; Diagnosis; Gut-liver axis; Hepatocellular carcinoma; Prothrombin induced by vitamin K absence-II; Receiver operating characteristic analysis; Serum biomarkers.

Figure 1

Differences in tumor marker levels among sample types. A-F: Differences in tumor markers in blood, bile, and feces of patients with hepatocellular carcinoma (HCC) and non-HCC; G and H: Comparative analysis of alpha-fetoprotein (AFP) and prothrombin induced by vitamin K absence-II (PIVKA-II) levels in blood and bile among patients with HCC.

Figure 2

Diagnostic efficacy, correlation coefficients of tumor markers, immunohistochemical analysis, and recurrence-free survival analysis. A: Receiver operating characteristic (ROC) curve for hepatocellular carcinoma (HCC) diagnosis using alpha-fetoprotein (AFP) or prothrombin induced by vitamin K absence-II (PIVKA-II) in serum, bile, and feces; B: ROC curve for HCC diagnosis using the combination of AFP and PIVKA-II in serum and bile; C: ROC curve for the HCC diagnosis using carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), CA153, and CA19-9 in serum; D: Correlation coefficient between serum AFP, PIVKA-II, and bile AFP; E: Correlation analysis of AFP expression in tumor tissues and serum AFP levels; F: Correlation analysis between AFP expression in tumor tissues and bile AFP levels; G: Correlation analysis between AFP expression in paracancerous tissue and serum AFP levels; H: Correlation analysis between AFP expression in paracancerous tissue and bile AFP levels; I: Recurrence-free survival analysis using AFP immunohistochemistry in tumor tissue for serum in patients with AFP-positive HCC; J: Recurrence-free survival analysis using AFP immunohistochemistry in paracancerous tissue for serum in patients with AFP-positive HCC. AUC: Area under the curve; CI: Confidence interval.

Figure 3

Immunohistochemical analysis of alpha-fetoprotein in hepatocellular carcinoma and the structural relationship between alpha-fetoprotein and the spatial distribution of bile ducts. A: Alpha-fetoprotein (AFP) immunohistochemistry in tumor tissue; B: AFP immunohistochemistry in paraneoplastic tissue; C: Negative AFP immunohistochemistry result in tumor tissue, with scattered enhancement in some areas; D: AFP immunohistochemistry results; E: Cytokeratin 7 immunohistochemistry results. A-C from left to right: 100 ×, 200 ×, 400 × magnifications; D and E from left to right: 100 ×, 100 ×, 200 × magnifications.

Figure 4

Correlation analysis of gut microbiota and tumor markers. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. AFP: Alpha-fetoprotein; PIVKA-II: Prothrombin induced by vitamin K absence-II.