A circulating cfDNA-derived 5hmC signature that non-invasively predicts preoperative MVI status, with potential clinical utility in the management of resectable HCC patients is developed and validated.
Abstract
Background
&
Aim
Microvascular invasion (MVI) is a critical prognostic risk factor in hepatocellular carcinoma (HCC). This study evaluated the performance of 5-hydroxymethylcytosine (5hmC) modifications in circulating cell-free DNA (cfDNA) in preoperative assessment of MVI.
Methods
A total of 907 patients with HCC were enrolled from two centers, including 671 in the training cohort, 152 in the internal validation cohort, and 84 in the external validation cohort. Preoperative clinical data, laboratory parameters, and cfDNA-derived 5hmC profiles were collected. Feature selection was performed using XGBoost, and modeling was conducted using a multilayer perceptron (MLP) neural network. Survival analyses were performed to evaluate the prognostic significance of the MVI prediction model. RNA sequencing analysis was performed to explore the potential mechanism underlying the proposed model.
Results
The 181-5hmC-modification signature demonstrated strong discriminatory performance, achieving an area under curve (AUC) of 0.852 in the training cohort, 0.862 in the internal validation cohort, and 0.864 in the external validation cohort, respectively. Univariate and multivariate analyses identified the α-fetoprotein (AFP) level (odds ratio [OR] 1.576, P = 0.039), Barcelona Clinical Liver Cancer (BCLC) stage (OR 3.051, P < 0.001), and the 5hmC signature (OR 46.891, P < 0.001) as independent predictors of MVI. The 5hmC signature demonstrated significantly higher predictive accuracy than AFP levels or BCLC stage alone. Survival analysis showed that the 5hmC signature significantly stratified both recurrence-free and overall survival in resectable HCC patients. Additionally, interpretability analysis based on RNA sequencing revealed that lower MVI prediction scores were associated with immune-related pathways and immune infiltration levels.
Conclusions
We developed and validated a circulating cfDNA-derived 5hmC signature that non-invasively predicts preoperative MVI status, with potential clinical utility in the management of resectable HCC.
IMPACT AND IMPLICATIONS
This study presents the first integration of cfDNA-derived 5hmC profiling with machine learning for preoperative MVI prediction in resectable HCC. The proposed 5hmC signature demonstrates potential for predicting MVI status and prognosis before surgery. Integration of RNA sequencing analysis provides biological support for the model's predictions, strengthening its clinical relevance. As a blood-based assay, this approach offers practical advantages for potential routine clinical implementation.
Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence and mortality rates worldwide. Given the poor prognosis of patients with HCC, it is crucial to explore the molecular mechanisms underlying HCC development and to evaluate prognostic markers. Differential expression analysis followed by univariate Cox, LASSO, and multivariate Cox regression identified four genes (EPO, SOCS2, IL18RAP, and KPNA2), and a Cox-based risk score was evaluated in the TCGA-LIHC cohort and externally in GSE14520 using Kaplan–Meier and time-dependent ROC analyses. Bulk, single-cell, and protein resources provided convergent expression context. Survival machine-learning analysis using observed overall-survival time and censoring status identified Cox–Ridge as the best-performing model in TCGA-LIHC, with more modest performance in GSE14520, and immune profiling revealed risk-group-associated differences in estimated immune and stromal components, immune-cell composition, and immune-checkpoint expression. The oncoPredict/GDSC2 screen highlighted five potential drug candidates for experimental prioritization. Because the drug screen is based on computationally predicted sensitivities, these findings should be regarded as hypothesis-generating and require validation in prospective cohorts and experimental systems before clinical translation.
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OBJECTIVE
To investigate FUBP1 expression, its prognostic value, impact on the tumor microenvironment (TME), and drug sensitivity in colorectal cancer (CRC), and to explore its potential underlying mechanisms.
METHODS
Using data from The Cancer Genome Atlas (TCGA), we analyzed FUBP1 expression in CRC, evaluated its prognostic value via survival analysis and nomogram construction, performed pathway enrichment analysis, and assessed immune cell infiltration and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) scores. We further conducted immunohistochemistry (IHC) validation on an independent institutional cohort and analyzed the Gene Expression Omnibus (GEO) single-cell RNA sequencing dataset GSE132465 (n = 63,689 cells) to examine the association between FUBP1 expression and the tumor immune microenvironment at single-cell resolution.
RESULTS
FUBP1 was highly expressed in CRC (P < 0.001), particularly in younger patients. High FUBP1 expression was associated with improved overall survival in univariate analysis (HR = 0.68, P = 0.028), yet this association was not maintained as an independent prognostic factor after adjustment for other clinical covariates (HR = 0.722, P = 0.098). Immunohistochemistry results from the independent cohort confirmed upregulated FUBP1 protein in 90% of CRC specimens. Single-cell analysis revealed that FUBP1-high cell clusters exhibited markedly reduced immune cell infiltration (35.97%vs 62.96%, P < 0.001), indicating an immunosuppressive "cold" tumor microenvironment. Tumors with high FUBP1 expression also displayed elevated PD-L1, PD-1, and CTLA4 expression, lower half-maximal inhibitory concentration (IC50) values for oxaliplatin, irinotecan, and 5-fluorouracil, and higher Immune Phenotype Score (IPS) for anti-PD-1 monotherapy or combined anti-CTLA-4 immunotherapy. FUBP1 expression was correlated with increased expression of MYC, TP53, and their downstream target genes (CCND1, CDK4, BAX, CDKN1A).
CONCLUSION
FUBP1 is highly expressed in CRC and associated with an immunosuppressive "cold" tumor microenvironment characterized by decreased immune cell infiltration, while it correlates with favorable chemotherapeutic sensitivity. FUBP1 may serve as a potential predictive biomarker for responses to chemotherapy and immunotherapy, rather than an independent prognostic indicator for survival. Its linkage to MYC and TP53 signaling pathways warrants further mechanistic investigation.
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Functional enrichment analysis revealed that IFFO2 is significantly associated with immune regulation, cell cycle, and complement activation pathways, and its strong association with adverse prognosis and its potential to modulate the immune microenvironment underscore its dual potential as a diagnostic biomarker and a promising therapeutic target.
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This research demonstrated that the mRNA and protein levels of SAMM50 in HCC tissues were elevated compared to those in normal liver and adjacent tissues, suggesting its potential as a diagnostic marker for HCC, though further validation in independent cohorts is needed.
