Tumor-agnostic ctDNA methylation profiling as an enabler of preoperative risk stratification and pathological response assessment in hepatocellular carcinoma.

Abstract

e16291 Background: Clinical management of hepatocellular carcinoma (HCC) is becoming increasingly complex, encompassing curative resection for early-stage disease and multimodal treatment strategies across disease stages. However, preoperative risk stratification and timely evaluation of treatment response remain challenging due to the lack of reliable blood-based tools. Tumor-agnostic circulating tumor DNA (ctDNA) methylation profiling may help address these unmet clinical needs by enabling real-time, noninvasive assessment of tumor biology and therapeutic efficacy. Methods: We prospectively enrolled 174 HCC patients undergoing curative hepatectomy and collected plasma samples within 24 hours prior to surgery (Ts). In parallel, an independent neoadjuvant therapy cohort of 27 HCC patients treated with immune checkpoint inhibitor-based systemic therapy was included, in which plasma samples were obtained at baseline before treatment initiation (Tb) and prior to surgery (Ts). All plasma samples were assayed using the GutSeer. A tumor-agnostic machine learning model was developed based on 342 HCC patients and 1,415 healthy subjects to generate a methylation-based cancer score (MCS) for prognostic stratification and pathological response assessment. Results: The hepatectomy cohort was followed for a median of 26.3 months with 47 recurrences. Preoperative Ts MCS was significantly associated with advanced tumor stage, larger tumor size, and vascular invasion, indicating its ability to reflect tumor burden and underlying biological aggressiveness. Based on Ts MCS, patients were stratified into low-risk (n = 45) and high-risk (n = 129) groups, yielding a hazard ratio (HR) of 9.36 (95%CI: 2.27–38.59; p < 0.001) and a negative predictive value of 95.6%. Multivariate analysis confirmed Ts MCS as an independent predictor of recurrence (HR = 5.09, 95%CI: 1.18-21.92; p < 0.05). In the neoadjuvant therapy cohort, MCS decreased significantly from Tb to Ts (p < 0.001). The change of MCS (Tb-Ts) was strongly negatively correlated with the proportion of residual viable tumor (R = -0.70, p < 0.001), indicating MCS changes closely mirror pathological response. Consistently, MCS change accurately discriminated major pathological response (MPR, ≤50% viable tumor cells) from non-MPR (AUC = 0.859), outperforming changes of alpha-fetoprotein (AUC = 0.731) and des-gamma-carboxy prothrombin (AUC = 0.686). Conclusions: This study demonstrates that tumor-agnostic ctDNA methylation profiling enables effective preoperative risk stratification in resectable HCC. Furthermore, dynamic changes in MCS provide a precise, noninvasive biomarker for assessing pathological response to systemic therapy. These findings highlight the broad clinical utility of methylation-based liquid biopsy approaches across surgical and systemic treatment settings in HCC. Clinical trial information: NCT06178809 .