Tumour-infiltrating inflammatory and immune cells in patients with extrahepatic cholangiocarcinoma

Abstract

Background: Inflammation and immune characteristics of the tumour microenvironment have therapeutic significance. The aim of this study was to investigate the clinical impact on disease progression in human extrahepatic cholangiocarcinoma (ECC).

Methods: A total of 114 consecutive ECC patients with curative resection between 2000 and 2014 were enrolled. Tumour infiltrating CD66b⁺ neutrophils (TANs; tumour associated neutrophils), CD163⁺ M2 macrophages (TAMs; tumour associated macrophages), CD8⁺ T cells, and FOXP3⁺ regulatory T cells (Tregs) were assayed by immunohistochemistry, and their relationships with patient clinicopathological characteristics and prognosis were evaluated.

Results: Tumour associated neutrophils were inversely correlated with CD8⁺ T cells (P=0.0001) and positively correlated with Tregs (P=0.001). High TANs (P=0.01), low CD8⁺ T cells (P=0.02), and high Tregs (P=0.04) were significantly associated with poor overall survival (OS). A high-risk signature, derived from integration of intratumoural inflammatory and immune cells, was significantly associated with poor recurrence-free survival (P=0.01) and OS (P=0.0008). A high-risk signature was correlated with postoperative distant metastases. Furthermore, a high-risk signature was related to the resistance to gemcitabine-based chemotherapy used after recurrence.

Conclusions: Our data showed that tumour infiltrating inflammatory and immune cells may play a pivotal role in ECC progression and a high-risk signature predicted poor prognosis in ECC patients.

Figure 1

Immunohistochemistry on tumour-filtrating CD66b⁺, CD163⁺, CD8⁺ and FOXP3⁺ cells. (A) Expression of CD66b⁺ cells in normal ducts and ductal cancer in patients with extrahepatic cholangiocarcinoma. (B) Representative pictures of immunohistochemical staining (CD66b, CD8, CD163 and FOXP3) with serial sections in extrahepatic cholangiocarcinoma are shown. (C) Representative cases with intratumoural exclusive expression patterns of CD66b⁺ and CD8⁺ cells.

Figure 2

Distribution and relationships of tumour-infiltrating immune cells. (A) Distributions of tumour-infiltrating CD66b⁺, CD163⁺, CD8⁺ and FOXP3⁺ cells are shown in box plots. (B–D) The relationships of (B) CD66b⁺ and CD8⁺, (C) CD66b⁺ and FOXP3⁺, (D) CD66b⁺ and CD163⁺ expression are shown. One circle corresponds to one patient. TAM = tumour associated macrophage; TAN = tumour associated neutrophil; Treg = regulatory T cells.

Figure 3

Overall survival and tumour-infiltrating immune cell status. Overall survival and (A) CD66b⁺ cell, (B) CD163⁺ cell, (C) CD8⁺ cell and (D) FOXP3⁺ cell status are shown. CI = confidence interval; HR = hazard ratio.

Figure 4

Risk signature model of tumour-infiltrating inflammatory and immune cells and postoperative outcome. (A) Risk signature is derived from the integration of tumour-infiltrating inflammatory and immune cell expression and shown as a heat map. (B) Overall survival based on the risk signature model of tumour-infiltrating inflammatory and immune cells is shown. (C) Comparison of recurrence sites between high- and low-risk signature. (D) Change in CA19-9 level between the initiation of gemcitabine-based chemotherapy and 3 months after the start in patients with recurrence. (E) The effect of chemotherapy on recurrent disease was assessed by RECIST criteria. Progressive disease (PD) was shown as ‘not effective’, whereas stable disease (SD) and partial response (PR) are shown as ‘effective’. CI = confidence interval; HR = hazard ratio; TAM = tumour associated macrophage; TAN = tumour associated neutrophil; Treg = regulatory T cells. *P = 0.005; **P = 0.02.