Predictive Value of Minimal Residual Disease for Postoperative Recurrence and Adjuvant PD-1 Inhibitor in HCC

Hepatocellular carcinoma (HCC) is a leading global malignancy and a primary cause of mortality in patients with chronic liver disease. While curative resection offers a critical treatment strategy, postoperative recurrence remains a major obstacle. Emerging evidence suggests adjuvant therapy with immune checkpoint inhibitors (ICIs), such as PD-1/PD-L1 inhibitors, may improve disease-free survival; however, identifying the patient subgroup most likely to benefit remains challenging. Theoretically, patients at high risk for minimal residual disease (MRD) post-surgery represent the ideal target for adjuvant treatment. Current clinical practice relies on indirect pathological surrogates of MRD-such as microvascular invasion, poor differentiation, or satellite nodules-which lack sufficient sensitivity and specificity.

Cell-free DNA (cfDNA), with its short half-life, dynamically mirrors tumor evolution, making it a promising tool for monitoring recurrence. The genes GNB4 and Riplet are located within CpG islands strongly associated with hepatocarcinogenesis. We developed specific primers and fluorescent probes targeting their bisulfite-converted sequences to enable selective PCR-based detection of methylated alleles. Monitoring MRD via *GNB4/Riplet* methylation status thus offers a potential method for dynamic assessment of tumor progression and recurrence, optimizing patient risk stratification and guiding therapeutic decisions.

To evaluate this approach, we will conduct a prospective, multi-center cohort study to investigate the predictive value of MRD for recurrence and adjuvant therapy benefit. The study comprises two cohorts: 1) an Active Surveillance Cohort, undergoing a pre-operative MRD assessment followed by regular post-operative surveillance and serial MRD testing; and 2) an Adjuvant Therapy Cohort, receiving pre-operative MRD assessment followed by standard adjuvant PD-1 inhibitor therapy alongside serial MRD monitoring. This study aims to validate a more effective tool for predicting recurrence and to identify patients most likely to benefit from adjuvant immunotherapy.

Inclusion criteria

• Age between 18 and 75 years, inclusive, regardless of gender.
• Newly diagnosed, treatment-naïve patients with HCC.
• Received radical treatments, such as liver resection or microwave ablation.
• Combine at least one of the risk factors for tumor recurrence, such as microvascular/macrovascular invasion, poor differentiation, satellite nodules, multiple tumors, and tumor diameter greater than 5 cm.
• Child-Pugh liver function score ≤ 7.
• Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
• Absence of severe organic diseases affecting the heart, lungs, brain, or other major organs.

Exclusion criteria

• History of other malignancies.
• Recurrent HCC.
• Prior systemic therapy for HCC.
• Unable to complete the follow-up and dynamic MRD monitoring.
• Having an immune deficiency disorder.
• Allergic to PD-1 inhibitors or unable to tolerate related treatments.