Combining irreversible electroporation and immunotherapy for hepatocellular carcinoma: Reflections and directions for advancement

Abstract

We read with great interest the recent article by Xing et al, which describes the synergy between irreversible electroporation and anti-programmed death-1 therapy in a murine hepatocellular carcinoma model. The study offers valuable mechanistic insights into local ablation, enhancing the efficacy of immune checkpoint blockade. However, critical methodological limitations and an overstatement of mechanistic conclusions warrant cautious interpretation. We recommend clarifying experimental details, optimizing murine models, applying more robust statistical analyses, and tempering conclusions to reflect the correlative nature of the findings. Further work should investigate immune mechanisms, durability of response, and safety in clinically relevant models to maximize translational potential. These refinements will strengthen the study’s impact in advancing ablation-immunotherapy strategies in hepatocellular carcinoma.

Key Words: Irreversible electroporation; Anti-programmed death-1 therapy; Hepatocellular carcinoma; Tumor immunology; Immune checkpoint blockade; Murine cancer model; Translational oncology

Core Tip: A recent study by Xing et al explored how combining a special tumor treatment called irreversible electroporation with a type of immunotherapy (anti-programmed death-1) can help the immune system fight liver cancer in mice. While the results are promising, the study requires clearer explanations of how and where samples were collected, more effective use of statistical methods, and a cautious interpretation of the findings. Future studies should use models more similar to human liver cancer and test long-term effects and safety to understand the treatment’s potential for patients.

TO THE EDITOR

We read with great interest the recent article by Xing et al[1], which examined the immunologic synergy between irreversible electroporation (IRE) and anti-programmed death (PD)-1 therapy in a murine hepatocellular carcinoma (HCC) model. This is a timely and important investigation highlighting the potential of local ablation to augment immune checkpoint blockade. While the study provides valuable insights, several methodological, analytical, and interpretive limitations warrant further consideration.

Experimental transparency

The manuscript described the collection of tumor tissue and peripheral blood, but did not clearly specify which samples were used for which assays and at which time points. For example, flow cytometry was reported to use peripheral blood, yet Figure 1 cites “tumor tissue”. Such inconsistencies compromise reproducibility. Explicit reporting is needed (e.g., peripheral blood for immune cell phenotyping, tumor tissue for transcriptional assays). In addition, the use of subcutaneous H22 tumors does not replicate the cirrhotic microenvironment of human HCC. Orthotopic or patient-derived xenograft models, particularly in fibrotic or humanized immune backgrounds, would provide greater translational validity. Orthotopic pancreatic ductal adenocarcinoma models in that IRE reverses stroma-induced immunosuppression and, with anti-PD-1, induces durable tumor clearance and memory T-cell responses, highlighting the importance of immune microenvironment modulation[2].

Biological and experimental confounders

The H22 HCC cell line is highly immunogenic, potentially exaggerating treatment effects compared with human HCC. Variables such as tumor heterogeneity, operator variability, and anesthesia-related immune effects were not addressed. Similarly, the rationale for IRE parameters (1200 V/cm, 70 pulses) and anti-PD-1 dosing (200 μg/mouse) was insufficiently justified. Providing pharmacokinetic or pilot optimization data would strengthen reproducibility and future clinical translation. Combining IRE with PD-1/PD-ligand 1 (PD-L1) blockade enhances immune activation, increases the number of tumor-infiltrating CD8+ T cells, and improves survival in pancreatic cancer, providing a rationale for testing mechanisms such as dendritic cell activation[3]. Providing pharmacokinetic or pilot optimization data would strengthen reproducibility and future clinical translation.

Statistical rigor and analytical considerations

The study relied on t-tests and one-way ANOVA, which may not account for longitudinal data correlations. More appropriate methods, such as repeated-measures ANOVA or mixed-effects modeling, are recommended. Given multiple comparisons, adjustments such as Bonferroni correction or false discovery rate control should be applied. Sensitivity analyses with non-parametric methods could further improve robustness[4].

Interpretation of findings and discussion balance

The discussion occasionally overstates clinical relevance. For instance, the assertion that “IRE may overcome immunosuppressive tumor microenvironment” should be reframed as a hypothesis, given the absence of direct mechanistic assays. Similarly, statements about apoptosis and tolerance require clarification in the context of immunogenic cell death. A balanced interpretation that acknowledges hypotheses vs established findings would improve scientific rigor. Furthermore, mechanisms such as dendritic cell activation or extracellular matrix remodeling were mentioned but not experimentally supported. Explicit acknowledgment of these as hypotheses rather than conclusions would strengthen scientific integrity[5].

Future directions

In the context of future research, a few recommendations to advance preclinical and translational development: (1) Incorporate assays for dendritic cell activation (CD80/CD86 expression), Treg and myeloid suppressor profiling, or interferon-γ blockade studies to confirm the mechanisms by which IRE modulates immunity directly[6]; (2) Assess a mechanistic validation by performing detailed dendritic cell activation assays and exploring the dynamics of memory T-cell functions. Additionally, incorporate an array of biomarkers, such as PD-L1 expression, cytokine profiles, and tumor mutational burden, to refine and enhance patient selection, creating a more tailored approach to treatment; (3) Embrace the transformation toward more precise models, such as orthotopic models that replicate the natural organ environment, cirrhotic models that reflect advanced liver disease, patient-derived xenografts that incorporate the patient’s biology into the lab, and humanized immune system models that mimic the complexities of human immunity. These innovations are paving the way for a deeper understanding of disease mechanisms and the development of more effective therapies; and (4) Evaluate hepatic toxicity profiles of IRE combined with immunotherapy, including serum transaminases, histopathology, and autoimmunity markers. Integration of predictive biomarkers (PD-L1 expression, circulating cytokines, or tumor mutational burden) will be critical to selecting patients most likely to benefit in clinical translation.

Conclusion

In summary, Xing et al[1] present an innovative preclinical study highlighting the potential synergy between IRE and anti-PD-1 therapy in HCC. The findings are promising; however, methodological inconsistencies, limited statistical rigor, and overinterpretation of the mechanistic scope warrant further refinement. Prioritizing statistical improvements, adopting clinically relevant models, and validating mechanisms through biomarker integration will enhance the translational value. Despite these limitations, the work provides an important foundation for future investigations into ablation-immunotherapy strategies and underscores the need for rigorous translational pipelines to deliver effective immunomodulatory approaches for HCC patients. We commend the authors for their contribution and encourage additional refinement to strengthen both reproducibility and clinical applicability.