Background The positive prediction of prognosis and immunotherapy within tertiary lymphoid structure (TLS) in cancerous tissue has been well demonstrated, including liver cancer. However, the relationship between TLS and prognosis in the peritumoral region of hepatocellular carcinoma (HCC) has received less attention. Few studies on whether TLS, as a typical representative of acquired immune cell groups, is associated with innate immune cells. The aim of this paper was to identify the prognostic role of peritumor TLS in HCC and to simply explore the relationship with neutrophils infiltration. Methods This study included cancerous and paracancerous tissue from 170 patients after surgical resection of HCC. TLS was examined and identified by pathological H&E examination, and the impact on prognosis was further classified by determination of total TLS area. Immunohistochemical staining of CD15+ neutrophils was also performed on half of the cases. The obtained results were validated by external public database, as TLS has been widely shown to be tagged with 12 chemokines. Results In peritumoral tissue, the TLS− group had better overall survival (OS) and disease‐free survival (DFS) outcomes compared with the TLS+ group. On the contrary, the intratumor TLS+ group showed better DFS outcomes. When further investigating the relationship between TLS area distribution and DFS, progressively worse prognosis was only found in the peritumor region with increasing TLS density (TLS− vs. TLS L vs. TLS H ). In addition, neutrophil infiltration increased in parallel with TLS density in the peritumoral region, which was not observed in the intratumoral region. Conclusions TLS might have a dual prognostic role in different regions of HCC. The abundance of peritumoral TLS is an independent influence of DFS. The inconsistent correlation between neutrophils and corresponding TLS in different regions may indicate different pathways of immune aggregation and may serve as an explanation for the different prognosis of TLS, which needs to be specifically explored.

Background: Tertiary lymphoid structures (TLSs) are formed by the aggregation of tumour-infiltrating lymphocytes (TILs), which is driven by chemokines or cytokines in the tumour microenvironment. Studies have shown that TLSs are associated with good prognosis in patients with various solid tumours and can improve patient responses to immunotherapy. However, the role of TLSs in hepatocellular carcinoma (HCC) remains controversial, and the underlying molecular mechanism is unclear. Methods: According to haematoxylin-eosin (HE) staining results, HCC patients in Xijing Hospital data and TCGA data were divided into TLS+ and TLS- groups, and Kaplan–Meier (KM) analysis was performed to assess overall survival (OS) and recurrence-free survival (RFS). Immunofluorescence (IF) and immunohistochemistry (IHC) were used to identify TILs in the TLS+ group. Lymphocyte-specific protein tyrosine kinase (LCK), a molecule involved in TLS formation, was explored in LinkedOmics. TILs were divided into two groups by drawing receiver operating characteristic (ROC) curves to calculate cut-off values. Spearman correlation analysis was used to calculate the correlation between LCK and TILs, and the molecular pathways by which LCK regulates immunotherapy were clarified through enrichment analysis. The half-maximal inhibitory concentration (IC50) distribution of sorafenib was observed in groups that varied in LCK expression. Results: According to the HE results, 61 cases in the Xijing Hospital cohort and 195 cases in the TCGA cohort had TLSs, while 89 cases and 136 cases did not. The KM results showed that TLSs had no effect on the OS of HCC patients but significantly affected RFS. The IF/IHC results showed that higher TIL numbers in TLSs were correlated with better prognosis in HCC patients. Spearman correlation analysis showed that LCK expression was positively correlated with TIL numbers. Enrichment analysis showed that upregulation of LCK expression mainly regulated the cytokine signalling pathway, the chemokine signalling pathway and T-cell activation. The IC50 scores of sorafenib in HCC patients with high LCK expression were lower, and the sensitivity was higher. Conclusion: TLSs mainly affected the early RFS of HCC patients but had no effect on OS. The high expression of the TLS formation-related gene LCK can increase the sensitivity of HCC patients to ICIs.