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  • Clinical Research Article
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Density of tertiary lymphoid structures predict clinical outcome in hepatoblastoma

Pediatric Research (2025)Cite this article

Abstract

Background

Tertiary lymphoid structures (TLSs) have emerged as critical regulators of antitumor immunity and prognostic indicators in various malignancies. However, the distribution patterns and prognostic significance of TLSs in hepatoblastoma (HB) remain poorly understood. This study aimed to investigate the presence, distribution, and prognostic value of TLSs in HB patients following neoadjuvant chemotherapy and to explore the underlying mechanisms linking TLSs to the tumor immune microenvironment.

Methods

A total of 112 HB patients who underwent neoadjuvant chemotherapy and surgical resection at Shandong Provincial Hospital between 2015 and 2024 were retrospectively enrolled. The presence of TLSs was evaluated using hematoxylin and eosin (H&E) staining, and patients were classified into TLS-positive and TLS-negative groups. Univariate and multivariate Cox regression analyses were performed to identify independent prognostic factors for overall survival (OS). In addition, transcriptome data from the GEO database (GSE133039) were analyzed to construct a TLS gene signature score and explore immune-related mechanisms associated with TLS presence.

Results

TLSs were identified in 45 out of 112 hepatoblastoma patients (40.2%). Kaplan-Meier survival analysis demonstrated that TLS-positive patients had significantly longer overall survival (OS) compared to TLS-negative patients (p = 0.0017). Multivariate Cox regression analysis further confirmed the presence of TLSs as an independent favorable prognostic factor (HR = 0.061, p = 0.027). In contrast, advanced PRETEXT stage (III/IV), vascular invasion, and distant metastasis were identified as independent adverse prognostic factors, indicating that patients diagnosed at later stages tended to have a worse prognosis. Transcriptomic analysis revealed that TLS-positive tumors exhibited higher expression of antigen presentation and immune activation-related genes (e.g., HLA-DQA1, HLA-DQB1, SLAMF7), along with enriched infiltration of B cells, CD8+ T cells, and NK cells, suggesting a more active antitumor immune microenvironment.

Conclusion

The presence of TLSs is significantly associated with favorable prognosis in HB patients and may contribute to enhanced antitumor immunity by recruiting and activating cytotoxic immune cells. TLSs represent a promising prognostic biomarker and potential immunotherapeutic target for HB patients.

Impact

  • Tertiary lymphoid structures (TLSs) serve as a promising prognostic biomarker in hepatoblastoma (HB).

  • Our study demonstrates that TLS-positive patients exhibit significantly prolonged overall survival.

  • TLSs contribute to the tumor immune microenvironment by recruiting cytotoxic immune cells.

  • These findings provide new insights into TLSs as a potential immunotherapeutic target for HB patients.

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Fig. 1: Representative histological images of tertiary lymphoid structures (TLS) in hepatoblastoma tissue.
Fig. 2: Kaplan-Meier survival curves depicting the association between recurrence-free survival (RFS), overall survival (OS) and tertiary lymphoid structure (TLS) status in hepatoblastoma patients.
Fig. 3: Kaplan-Meier survival curves illustrating the relationship between PRETEXT stage and survival outcomes in hepatoblastoma patients.
Fig. 4: Heatmap of normalized expression (Z-score) for 12 TLS-associated chemokine genes—CCL2, CCL18, CCL21, CXCL9, CXCL10, CCL19, CCL5, CCL4, CCL3, CCL8, CXCL11, and CXCL13—in hepatoblastoma samples from GSE133039.
Fig. 5: Volcano plot depicting the results of differential gene expression analysis.
Fig. 6: GO enrichment analysis results of up-regulated genes.
Fig. 7: Differential immune cell infiltration in high- vs. low-TLS score groups.

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Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

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Funding

This study was supported by the Shandong Provincial Natural Science Foundation (No. ZR2024QH349) and the Incubation Foundation of Shandong Provincial Hospital (No. 2023FY051).

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Author notes

  1. These authors contributed equally: Ruiwen Sun, Zhipeng Liu, Yisu Zhang.

Authors and Affiliations

  1. Department of Pediatric Surgery, Jinan Maternal and Child Health Hospital, Shandong University, Jinan, China

    Ruiwen Sun

  2. Department of Hepatobiliary and Pancreatic Surgery, Shandong Provincial Hospital affiliated to Shandong First Medical University, Jinan, China

    Zhipeng Liu, Faji Yang, Qirong Jiang, Yupeng Jiang, Shizhe Zhang, Qixuan Zheng, Zheyu Niu, Jun Lu, Hengjun Gao & Yijie Hao

  3. Department of Hepatobiliary and Pancreatic Surgery, Shandong Provincial Hospital, Shandong University, Jinan, China

    Yisu Zhang

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Contributions

S.R.W. wrote the article, L.Z.P. and Z.Y.S. collected case data, G.H.J. and H.Y.J. were responsible for designing the research plan, Y.F.J., J.Q.R. and J.Y.P. conducted statistical analysis, Z.S.Z. and Z.Q.X. assisted in patient coordination, N.Z.Y. and J.L. provided guidance for the article writing.

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Correspondence to Hengjun Gao or Yijie Hao.

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The authors declare no competing interests.

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Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. This retrospective study was approved by the Biomedical Ethics Committee of Shandong Prov incial Hospital (Approval No. SWYX2025-217). The need for informed consent was waived.

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Sun, R., Liu, Z., Zhang, Y. et al. Density of tertiary lymphoid structures predict clinical outcome in hepatoblastoma. Pediatr Res (2025). https://doi.org/10.1038/s41390-025-04210-x

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