Prognostic Value of N1/N2 Neutrophils Heterogeneity and Tertiary Lymphoid Structure in Hepatocellular Carcinoma Patients

Abstract

Background: The tumor immune microenvironment, including neutrophils and tertiary lymphoid structures (TLSs), is pivotal for HCC prognosis assessment. Tumor-associated neutrophils exhibit plasticity, adopting either an antitumorigenic N1 (MPO+ CD206-) or a pro-tumorigenic N2 (MPO+ CD206+) phenotype. We explored the prognostic value of neutrophil plasticity and TLS maturity in HCC in both tumor and peritumoral tissues and addressed their interaction.

Methods: A retrospective cohort of 79 HCC patients who underwent radical resection from 2015 to 2018 was analyzed, with complete clinical characteristics and survival data of more than 5 years. Multiplex immunohistochemistry identified N1/N2 neutrophils and TLS maturity. Survival differences and correlations with clinical features were assessed.

Results: HCC patients were divided into high- and low-level groups on the basis of the N1 and N2 classifications of neutrophils, revealing a positive correlation with prognosis in tumor tissues and a negative one in peritumoral tissues. TLS maturity stages were associated with prognosis, with a higher proportion of secondary TLS (SFL-TLS) in peritumoral tissues correlating positively with survival. Further analysis of the correlation between neutrophils and TLSs revealed that most neutrophils infiltrated outside of the TLS in the peritumoral tissues of patients with HCC, and the proportions of SFL-TLSs and N1 cells in the peritumoral tissue were negatively correlated and positively correlated with survival. Both univariate and multivariate analyses revealed that the N1/N2 ratio in peritumoral tissues was an independent prognostic predictor of HCC.

Conclusions: The N1/N2 ratio of neutrophils and the proportion of SFL-TLS are considered important prognostic indicators that may reflect the immune microenvironment of HCC patients.

Keywords: hepatocellular carcinoma; neutrophil; prognosis; secondary follicle‐like TLS; tertiary lymphoid structure.

FIGURE 1

Kaplan–Meier and log‐rank tests revealed the predictive value of neutrophils for HCC prognosis. (A–D) Kaplan–Meier OS plots by neutrophil counts in HCC peritumor. (E–H) Kaplan–Meier OS plots for HCC tumor neutrophils.

FIGURE 2

Identification of N1 and N2 neutrophils within HCC peritumor and tumor tissues. Representative images of IF staining for N1 and N2 (white arrows) for the tumors. (B, C) Unpaired analysis of N1, N2 neutrophils in HCC peritumor and tumor tissues. (D, E) Paired analysis of N1 and N2 neutrophils in HCC peritumor and tumor tissues. (F–H) Unpaired analysis of N1, N2, N1/N2 in HCC peritumor (n = 79) and tumor tissues (n = 79) by unpaired analysis. N‐group: Peritumoral tissues; T‐group: Tumor tissues. ****p < 0.0001.

FIGURE 3

TLS maturation status in HCC peritumor and tumor tissues. IHC assessed lymphocyte aggregates in HCC tissues, identifying TLS stages via CD markers (n = 79). N‐group: Peritumoral tissues; T‐group: Tumor tissues. E‐TLSs: Early TLS lacked FDCs and GCs; PFL‐TLSs: Primary follicle‐like TLS had FDCs; SFL‐TLSs: Secondary follicle‐like TLS showed FDCs and GCs. Unpaired t‐tests compared TLS stages in peritumor vs. tumor tissues. ns, not significant; ****p < 0.0001.

FIGURE 4

Kaplan–Meier and log‐rank tests revealed the predictive value of TLS maturation. (A–C) Kaplan–Meier plots for OS according to the proportions of different TLS maturation stages in the peritumoral region. (D–F) Kaplan–Meier plots for OS according to the proportions of different TLS maturation stages. E‐TLS%, proportion of E‐TLSs; PFL‐TLS%, proportion of PFL‐TLSs; SFL‐TLS%, proportion of SFL‐TLSs.

FIGURE 5

Correlation analysis of neutrophils and TLSs in peritumoral and tumor tissues. (A–C) TLS density comparison in HCC tissues from different N1/N2 ratios. (D–F) TLS count differs among neutrophil groups in HCC. (G–I) E‐TLS proportions vary by N1, N2, N1/N2 in HCC. (J–L) PFL‐TLS ratios by N1, N2, and N1/N2 in HCC tissues. (M–O) SFL‐TLS distribution by N1, N2, and N1/N2 in HCC. ns, not significant; *p < 0.05.

FIGURE 6

The distribution of neutrophils intra‐ and extra‐TLSs in peritumor and tumor tissues. (A) N1, N2 neutrophils in/out TLS in HCC tissues. The white circle: TLS; the white square showed neutrophils inside/outside TLS. (B) Neutrophil count comparison in peritumor TLS groups. (C) Neutrophil count in tumor intra/extra‐TLS groups. (D) N1/N2 ratio in intra/extra‐TLS groups in tissues. N‐group: Peritumoral tissues; T‐group: Tumor tissues. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Correlations between TLS maturation and the number of neutrophils in peritumor and tumor tissues. (A) mIHC analyzed TLS and neutrophil relations in HCC tissues. Left panel: Red circle = tertiary lymphatic area; white square = enlarged view. Right panel: Red circle = TLS area; white square = neutrophil infiltration. (B–D) Neutrophil counts by E‐TLS%, PFL‐TLS%, SFL% in peritumor. (E–G) Neutrophil counts by E‐TLS%, PFL‐TLS%, SFL% in tumor. (H–J) N1/N2 ratios in E‐TLS%, PFL‐TLS%, SFL% groups. N‐group: Peritumoral tissues; T‐group: Tumor tissues. ns, not significant; **p < 0.01, ***p < 0.001.