Erythrocyte phosphatidylserine externalization shows greater discriminatory performance than red cell distribution width for early recurrence or progression in hepatocellular carcinoma

Red cell distribution width (RDW) is associated with adverse outcomes in hepatocellular carcinoma (HCC), but whether this association reflects an HCC-associated erythrocyte injury phenotype remains unclear. We investigated whether phosphatidylserine (PS) externalization identifies an HCC-associated erythrocyte injury phenotype and is associated with tumor burden and early recurrence or progression.

This prospective two-center clinical study included 97 patients with newly diagnosed HCC, 30 healthy donors, 13 patients with chronic hepatitis B, and 17 patients with HBV-related cirrhosis as disease controls. Erythrocyte injury phenotypes were characterized by flow cytometry, metabolomics, and proteomics. Findings were examined in orthotopic HCC mouse models and erythrocyte-HCC co-culture systems.

PS externalization was elevated in patients with HCC and correlated with tumor diameter, multiplicity, metastasis, and Barcelona Clinic Liver Cancer stage. HCC also showed higher PS levels than chronic hepatitis B and HBV-related cirrhosis controls. Higher PS externalization remained independently associated with early recurrence or progression within 6 months after adjustment for maximum tumor diameter, tumor number, and lymph-node and/or distant metastasis (OR = 1.382; 95% CI 1.112-1.717; P = 0.004). PS showed greater discriminatory performance than RDW-CV (AUC 0.804 versus 0.642; paired DeLong P = 0.003); bootstrap internal validation of the primary multivariable model yielded an optimism-corrected AUC of 0.780. Spatial analyses revealed progressive erythrocyte injury from peripheral blood to adjacent non-tumor tissue to primary tumor. In orthotopic HCC-bearing mice, PS externalization preceded RDW elevation. Direct co-culture with HCC cells induced glutathione depletion and oxidative membrane injury, which were attenuated by N-acetylcysteine and reproduced by glutathione synthesis inhibition. Multi-omics analyses revealed alterations in pentose phosphate pathway (PPP)-NADPH/glutathione metabolism and membrane-skeletal proteins.

PS externalization identifies an HCC-associated erythrocyte injury phenotype and is a candidate marker for 6-month recurrence or progression. Experimental findings support a functional contribution of glutathione depletion to membrane injury, accompanied by PPP-NADPH-related metabolic and membrane-skeletal alterations.