3.1 Clinicopathologic characteristics
The clinicopathologic features of the patients are summarized in Table 1. The series consisted of 54 men and 23 women with a median age of 70 years (range, 40–87 years). The carcinomas were located in the proximal bile duct (26 cases), including 6 cases in the perihilar area, and in the distal (intrapancreatic head) bile duct (45 cases). Histologically, 73 cases were adenocarcinoma, not otherwise specified (NOS), and four cases were classified as adenocarcinoma arising in intraductal papillary neoplasm of the bile duct. Two cases were associated with a congenital biliary abnormality. Curative resection and regional lymph node dissection were dependent on the location of the primary tumour. Pancreaticoduodenectomy or pylorus-preserving pancreaticoduodenectomy was performed in 45 patients, bile duct resection in 26 patients, and combined hepatectomy with bile duct resection in six patients.
3.2 Clinicopathologic correlation IDO1 and CD8 expression
IDO1 expression was evaluated in 76 cases except for one case with no assessable staining due to loss of the cores in the TMA block. IDO1 was expressed in the cytoplasm and/or nuclei of the tumour epithelium and stromal mononuclear immune cells. The epithelial IDO1-expressing tumours were grouped into two categories of high and low expression according to the proportion and intensity score (Fig. 1). IDO1 was highly expressed in 25 of 76 (32.9%) tumour specimens, whereas 54 of 76 (67.1%) cases showed low IDO1 expression levels.
High IDO1 expression was associated with a higher pN category (P = 0.007), an advanced overall TNM stage (P = 0.001), and more frequent lymph node metastasis with marginal statistical significance (P = 0.082). Patients with high IDO1 expression experienced more frequent recurrence (P = 0.018) and disease-specific death (P = 0.065) (Table 1).
CD8 expression in TILs was evaluated in all 76 cases. Although the difference was not statistically significant, high CD8 expression in TILs tended to be associated with less frequent recurrence (61.5%) and a lower disease-specific death rate (64.1%) than low CD8 expression (78.4% and 73.0%, respectively).
Comparing IDO1 expression with CD8expression in TILs, IDO1 expression was inversely correlated with the number of CD8+ TILs. IDO1-low expressing tumours exhibited a significantly higher proportion of intratumoural CD8+ cells (mean ± standard error (SE), 214.78/mm2 ± 268.26) than IDO1-high expressing tissue samples (98.38/mm2 ± 100.26) (P = 0.008).
3.3. Univariate and multivariate analyses for overall Survival and disease-free survival
High IDO1 expression was associated with shorter disease-free survival (median time: 16.30 months vs. 26.30 months, P = 0.026; Fig. 2A) and overall survival (median time: 21.20 months vs. 26.30 months, P = 0.097; Fig. 2B). Patients with high CD8+ TILs tended to have longer disease-free survival (median survival time: 47.30 months vs. 16.30 months, P = 0.023; Fig. 2C) and overall survival (median survival time: 36.00 months vs. 19.4 months, P = 0.135; Fig. 2D) than those with low CD8+ TILs.
Patient survival was further stratified according to four expression combinations of IDO1 and CD8 arranged in order of adverse prognostic value as follows (Fig. 3): IDO1high/CD8low (18.4%, 14/76); IDO1low/CD8low (32.9%, 25/76); IDO1high/CD8high (13.2%, 10/76); IDO1low/CD8high (35.5%, 27/76). The patients whose carcinomas were IDO1high /CD8low showed the worst median overall and disease-free survival times (16.30 and 8.10 months, respectively). The median overall survival time of patients with IDO1low/CD8low and IDO1high/CD8high expression were 19.40 months and 33.70 months, respectively. The median disease-free survival time of patients with IDO1low/CD8low and IDO1high/CD8high expression were 18.80 months and 26.1 months, respectively. Finally, patients with IDO1low/CD8high expression revealed the best survival (medial overall survival, 47.50 months; disease-free survival, 47.50 months).
Next, a multivariate statistical analysis of the IDO1high/CD8low expression group was performed, and other significant prognostic valuables were examined by a univariate analysis (Table 2). Tumour size, tumour location, lymph node metastasis, perineural invasion, and age were included as cofactors. As shown in Table 3, IDO1high/CD8low expression (P = 0.025, Cox hazard ratio = 2.168) in addition to tumour distal location and perineural invasion was an independent prognostic factor for overall survival. Furthermore, IDO1high/CD8low expression (P=0.015, Cox hazard ratio = 2.460) with tumour distal location, lymph node metastasis, and perineural invasion was an independent prognosticator for disease-free survival.