Intra-Tumoral Secondary Follicle-like Tertiary Lymphoid Structures Are Associated with a Superior Prognosis of Overall Survival of Perihilar Cholangiocarcinoma

SIMPLE SUMMARY: Tertiary lymphoid structures (TLSs) are reported to play an antitumor role in a variety of tumors and are a predictive marker of immunotherapy efficacy and survival outcomes but have not been reported in perihilar cholangiocarcinoma (pCCA). In the present study, hematoxylin and eosin, immunohistochemical staining and multiplex immunofluorescence were performed on tissue sections from 93 pCCA patients to analyze the maturity and number of intra-tumoral TLSs and their relevance to clinical characteristics, overall survival (OS), and recurrence-free survival. The results suggest that intra-tumoral mature TLSs, or secondary TLSs (S-TLSs), significantly improve OS in pCCA patients, suggesting that S-TLSs contribute to effective antitumor immunity and hold promise for diagnostic and therapeutic development in pCCA. ABSTRACT: Ectopic lymphoid structures termed tertiary lymphoid structures (TLSs) have an immunomodulatory function and positively affect prognosis in certain cancers. However, their clinical relevance and prognostic utility in perihilar cholangiocarcinoma (pCCA) are unknown. Therefore, determining the involvement and prognostic utility of TLSs in pCCA is the aim of this study. Ninety-three patients with surgically resected pCCA were included retrospectively. Hematoxylin and eosin and immunohistochemical staining identified and classified the TLSs, and multiplex immunofluorescence determined the TLS composition in the pCCA sample. The correlations between clinical features and TLSs were analyzed using either Fisher’s exact test or the Chi-squared test. Recurrence-free survival (RFS) and overall survival (OS) correlations with TLSs were analyzed using Cox regression and Kaplan–Meier analyses. We identified TLSs in 86% of patients with pCCA, including lymphoid aggregates (6.45%), primary (13.98%) and secondary follicles (65.59%). Patients with intra-tumoral secondary follicle-like TLSs (S-TLSs) had better OS (p = 0.003) and RFS (p = 0.0313). The multivariate analysis identified the presence of S-TLSs as a good independent prognostic indicator for OS but not for RFS. Interestingly, the presence of S-TLS only indicated better 5-year OS in 54 patients without lymph node metastasis (LNM(−), p = 0.0232) but not in the 39 patients with lymph node metastasis (LNM(+), p = 0.1244). Intra-tumoral S-TLSs predicted longer OS in patients with surgically resected pCCA, suggesting intra-tumoral S-TLSs’ contribution to effective antitumor immunity and that S-TLSs hold promise for diagnostic and therapeutic development in pCCA.