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. 2025 Mar 20;26(6):2794.
doi: 10.3390/ijms26062794.

Harnessing Plasma Biomarkers to Predict Immunotherapy Outcomes in Hepatocellular Carcinoma: The Role of cfDNA, ctDNA, and Cytokines

Elena Vargas-Accarino  1   2 , Mónica Higuera  1 , María Bermúdez-Ramos  1   2   3 , Agnès Soriano-Varela  1   4 , María Torrens  1   4 , Mònica Pons  3   4 , Ana María Aransay  5 , José Ezequiel Martín  5 , Francisco Rodríguez-Frías  3   6 , Xavier Merino  7 , Beatriz Mínguez  1   2   3   4
Affiliations

Harnessing Plasma Biomarkers to Predict Immunotherapy Outcomes in Hepatocellular Carcinoma: The Role of cfDNA, ctDNA, and Cytokines

Elena Vargas-Accarino et al. Int J Mol Sci. .

Abstract

Immunotherapy has improved survival in patients with advanced hepatocellular carcinoma (HCC); yet, objective radiological responses occur in only about 20% of cases, suggesting variable benefits. This study aimed to identify serologic markers predictive of response to immune checkpoint inhibitors (ICIs). A cohort of 38 advanced HCC patients receiving immunotherapy was prospectively analyzed. Levels of cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), and cytokines were measured pre-treatment and three months post-treatment initiation. Genomic profiling of ctDNA was also conducted. Baseline levels of cfDNA and ctDNA effectively discriminated HCC patients based on their radiological response to ICIs. Additionally, individuals with pathologic mutations in the CDKN2A gene exhibited significantly reduced survival. Patients with progressive disease (PD) as their best radiological response had significantly fewer copy number variations (CNVs) than those with a radiological response. Furthermore, levels of IL10, PD1, and TGFβ assessed after three months of treatment showed significant variations correlating with survival status. In conclusion, the analysis of cfDNA, ctDNA, and cytokines may improve treatment selection for HCC patients by predicting their expected response to immunotherapies.

Keywords: biomarkers; cfDNA; ctDNA; hepatocellular carcinoma; immunotherapy; liquid biopsy.

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Conflict of interest statement

B.M. received consultancy fees from Bayer–Shering Pharma, Roche, Astra Zeneca and Eisai–Merck, lectures/speaker fees from Eisai–MSD, Astra Zeneca, Bayer–Shering, and Roche, and a research grant from Laboratorios Viñas S.L. The remaining authors have no conflicts of interest to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Clinical outcomes. (A) Best radiological response assessed by mRECIST. (B) Kaplan–Meier curve of overall survival. CR: complete response, PR: partial response, SD: stable disease, PD: progressive disease, mRECIST: Modified Response Evaluation Criteria in Solid Tumors.
Figure 2
Figure 2
cfDNA levels. (A) Basal cfDNA levels in alive vs. death patients. The statistical method used was the t-test. (B) ROC curve distinguishing patients with more than 3.32 ng/µL. (C) Kaplan–Meier curve of overall survival for HCC patients stratified by baseline cfDNA levels; p-value from the log-rank test. (D) Basal cfDNA levels in patients with CR, PR, SD, and PD. CR: complete response, PR: partial response, SD: stable disease, PD: progressive disease. The statistical method used was one-way ANOVA followed by Tukey’s multiple comparisons test.
Figure 3
Figure 3
ctDNA levels. (A) Basal ctDNA levels in patients with CR, PR, SD, and PD. The statistical method used was the one-way ANOVA followed by Tukey’s multiple comparisons test. (B) ROC curve distinguishing patients with more than 2.09 ng/µL. (C) Kaplan–Meier curve of overall survival for HCC patients stratified by baseline ctDNA level; p-value from the log-rank test. CR: complete response, PR: partial response, SD: stable disease, PD: progressive disease.
Figure 4
Figure 4
(A) Genomic ctDNA profiling of HCC patients treated with ICI. (B).Kaplan–Meier curves of overall survival according to the presence or absence of CDKN2A mutations. (C) Number of copy number variations (CNVs), the statistical method used was the T-test. AFP: alpha-fetoprotein, TMB: tumor mutational burden, NR: no response, OR: objective response, CR: complete response, PR: partial response, SD: stable disease, PD: progressive disease, HCV: hepatitis C virus, HBV: hepatitis B virus, OH: alcohol, MASLD: metabolic-associated steatotic liver disease, CNV: copy number variation.
Figure 5
Figure 5
Cytokine levels. (A) Basal levels of IL10 in patients alive and death. (B,C) Levels of PD1 and TGF-β, respectively, measured after three months (3 m) of receiving ICI treatment according to status (Alive vs. Death). The statistical method used was the t-test.
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