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. 2023 May 10:14:1166497.
doi: 10.3389/fimmu.2023.1166497. eCollection 2023.

Tertiary lymphoid structures predict the prognosis and immunotherapy response of cholangiocarcinoma

Taiyu Shang  1   2 , Tianyi Jiang  1 , Tao Lu  3 , Hui Wang  4 , Xiaowen Cui  1 , Yufei Pan  1 , Mengyou Xu  1 , Mengmiao Pei  1   5 , Zhiwen Ding  1 , Xiaofan Feng  1 , Yunkai Lin  1 , Xin Li  4 , Yexiong Tan  1 , Feiling Feng  5 , Hui Dong  3 , Hongyang Wang  1   2   6 , Liwei Dong  1   6
Affiliations

Tertiary lymphoid structures predict the prognosis and immunotherapy response of cholangiocarcinoma

Taiyu Shang et al. Front Immunol. .

Abstract

Introduction: Cholangiocarcinoma (CCA) is a malignant tumor of the biliary epithelium with a poor prognosis. The lack of biomarkers to predict therapeutic response and prognosis is one of the major challenges for CCA treatment. Tertiary lymphoid structures (TLS) provide a local and pivotal microenvironment for tumor immune responses. The prognostic value and clinical relevance of TLS in CCA remain unclear. We aimed to explore the characteristics and clinical significance of TLS in CCA.

Methods: We investigated the prognostic value and clinical relevance of TLS in CCA using a surgery cohort containing 471 CCA patients (cohort 1) and an immunotherapy cohort containing 100 CCA patients (cohort 2). Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to evaluate the maturity of TLS. Multiplex IHC (mIHC) was employed to characterize the composition of TLS.

Results: Different maturity of TLS were observed in CCA tissue sections. Strong staining of the four-gene signature including PAX5, TCL1A, TNFRSF13C, and CD79A were found in TLS regions. A high density of intra-tumoral TLS (T-score high) were significantly correlated with longer overall survival (OS) both in CCA cohort 1 (p = 0.002) and cohort 2 (p = 0.01), whereas a high density of peri-tumoral TLS (P-score high) were associated with shorter OS in these two cohorts (p = 0.003 and p = 0.03, respectively).

Conclusion: The established four-gene signature efficiently identified the TLS in CCA tissues. The abundance and spatial distribution of TLS were significantly correlated with the prognosis and immune checkpoint inhibitors (ICIs) immunotherapy response of CCA patients. The presence of intra-tumoral TLS are positive prognostic factors for CCA, which provide a theoretical basis for the future diagnosis and treatment of CCA.

Keywords: cholangiocarcinoma; immune checkpoint inhibitors; prognosis; tertiary lymphoid structures; tumor microenvironment.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Immune microenvironment (IME) compositions of CCA, surrounding liver, and normal intrahepatic bile duct tissues in GSE26566. (A) IME compositions between CCA and surrounding liver tissues detected by MCP-counter. (B, C) Expressions of TLS-related factors and chemokines in CCA, surrounding liver, and normal intrahepatic bile duct tissues. Indicators for P values: **** P ≤ 0.0001, ** P ≤ 0.01, * P ≤ 0.05.
Figure 2
Figure 2
Features of TLS in CCA. (A) H&E and IHC staining for a typical TLS in CCA tissue. TLS appeared as clusters of B-cell follicles surrounded by T-cell zones. Scale bars, 100 μm. (B) Multiplex immunohistochemistry (mIHC) staining for TLS identification. Markers for follicular dendritic cells (CD23), B cells (CD20), macrophages (CD68), and natural killer cells (CD56) are used. (C) H&E staining for intra-tumoral (T) and peri-tumoral (P) regions. Triangles indicate TLS.
Figure 3
Figure 3
Different maturity and corresponding features of TLS in CCA. (A) Different maturity of TLS present in CCA. GC center can be found in the secondary TLS in H&E staining sections. (B) Primary TLS. CD20+ B cells were diffusely distributed without CD23+ follicular structures formation. (C) Secondary TLS. CD20+ B cells and CD23+ FDCs were formed into a follicular structure, with many T cells aggregated around and within the follicles. Scale bars, 200 μm.
Figure 4
Figure 4
Discovery and validation of four-gene signature for TLS identification. (A) Representative H&E image of TLS in CCA. (B) Correlation between TLS presence and genetic mutation. The p values were assessed by chi-square tests. (C) Differential gene expression of TLS-positive versus TLS-negative CCA cases in the TCGA database. (D, E) Representative images of PAX5, TCL1A, TNFRSF13C, and CD79A staining in CCA sections. Scale bars in D, 300 μm. Scale bars in E, 100 μm.
Figure 5
Figure 5
TLS in different regions predicts a distinct prognosis of CCA. (A, B) Kaplan-Meier analyses of PFS and OS according to P-score in CCA surgery cohort (n = 471). (C, D) Kaplan-Meier analyses of PFS and OS according to T-score in CCA surgery cohort. P values were determined by log-rank tests. OS, overall survival; PFS, progression-free survival.
Figure 6
Figure 6
TLS was associated with the response to immunotherapy. (A, B) Kaplan-Meier analyses of PFS and OS according to P-score in CCA immunotherapy cohort (n = 100). (C, D) Kaplan-Meier analyses of PFS and OS according to T-score in CCA immunotherapy cohort. P values were determined by log-rank tests.
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