Open Access

Prognostic implications of immune classification using IDO1 expression in extrahepatic bile duct carcinoma

  • Authors:
    • Byeong-Joo Noh
    • Gun Moo Choi
    • Hyuk Jai Jang
    • Chung Hyeun Ma
    • Ho-Suk Oh
    • Moonho Kim
    • Dae-Woon Eom
  • View Affiliations

  • Published online on: September 5, 2022
  • Article Number: 373
  • Copyright: © Noh et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Indoleamine 2, 3‑dioxygenase 1 (IDO1) is an immunomodulatory enzyme that catalyzes the degradation of tryptophan to kynurenine and induces immune tolerance in tumor cells. The effects of IDO1 on extrahepatic bile duct carcinoma (EHBDC) are poorly understood. Therefore, the present study aimed to investigate the expression and prognostic significance of IDO1 in EHBDC. An immunohistochemical microarray analysis of IDO1 expression was performed for 76 surgically resected cases of EHBDC. CD8+ tumor infiltrating lymphocytes (TILs) were also investigated through a combination analysis with IDO1 expression. IDO1 was highly expressed in 25 of 76 (32.9%) cases. High expression of IDO1 was associated with decreased numbers of CD8+ TILs (P=0.008), a higher pN category (P=0.007), an advanced overall stage (P=0.001) and frequent recurrence (P=0.018). When IDO1 expression was further stratified with CD8+ TIL state, the IDO1high/CD8low subgroup was decreased in terms of overall survival (P=0.025) and disease‑free survival (P=0.015) compared with IDO1high/CD8high, IDO1low/CD8high and IDO1low/CD8low subgroups. High IDO1 expression was associated with a decreased number of CD8+ TILs and associated with a poor prognosis. As IDO1 may be a new target of immunotherapy applications, IDO1/CD8+ TIL subgrouping can be a useful prognostic and predictive tool in patients with EHBDC.


Extrahepatic bile duct carcinoma (EHBDC) is an epithelial tumor that forms in the bile ducts outside the liver, and its incidence ranges from 0.53 to 2.0 per 100,000 in the western world, but can be much higher (0.97 to 85.0) in Asian countries including Thailand, Republic of Korea and China (1). Despite recent advances in diagnostic and therapeutic techniques, complete surgical resection of the tumor remains the best treatment for EHBDC. However, EHBDC patients are often diagnosed at an advanced stage. Even in patients who have undergone therapeutic resection, the prognosis is dismal due to the high recurrence rate of this tumor (2,3). A late diagnosis and frequent recurrence in EHBDC compromise surgical resection, the only potentially curative treatment. Current chemotherapeutic drugs for these EHBDC patients have a limited efficacy because more than 50% have relapsed early (4,5).

Recently, with the advent of immunotherapy, studies for the interactive relationships between tumor cells and the peritumoral immune cells (tumor microenvironment) (TME) have received much attention. However, difficult obstacles remain for deciphering TME because of tumor heterogeneity and immune system complexity, especially in immune suppression mechanisms. Local immune suppression of the tumor microenvironment is crucial for cancer development, metastasis, and even tumor immune escape (6,7). Therefore, elucidation of the immune suppression mechanisms involved in the carcinogenesis, cancer progression, and metastasis can improve the accuracy of predicting its prognosis and provide a target for therapeutic intervention, contributing to the improvement of survival rate.

IDO1 has been emerging as an important biomarker associated with the immunosuppressive mechanism in addition to PD-L1/PD-1 and B7/CTLA-4 interactions. IDO1 is a 403 amino acid and cytosolic heme enzyme encoded by the INDO gene on human chromosome 8p22 that catalyzes the initial and rate-limiting steps in the kynurenine (Kyn) pathway (8). It is expressed ubiquitously in various normal tissues including the small intestine, epididymis, lungs, female genital tract, and placenta as well as various types of solid tumors (810). IDO1 is known to be involved in maternal-fetal immune tolerance in the mouse placenta (9) and has been shown to be linked in a mechanism of response to tumor immune evasion through tumor infiltrating lymphocyte (TIL) inhibition (11). To date, contradictory results for prognostic implication of IDO1 expression has been reported in various carcinomas (1219), but there is no study in EHBDC.

In this study, we investigated intratumoral IDO1 expression in epithelial tumor cells in 76 patients with EHBDC who had undergone surgical resection. In addition, we defined the association between CD8+ TILs and IDO1 expression and examined prognostic differences between subgroups by performing stratification combining CD8 TIL status and IDO1 expression.

Materials and methods

Patients and clinicopathological data

This study was approved by the Institutional Review Board of Gangneung Asan Hospital. A total of 76 consecutive patients with bile duct carcinoma between January 2001 and December 2017 were collected at Gangneung Asan Hospital. In detail, inclusion criteria were as follows: i) surgical resection specimen with enough tumor cells, ii) obtaining each patient's informed consent for use of their clinical records and pathological specimen, and iii) without previous history of a cancer other than bile duct carcinoma and chemo-or radiotherapy. Exclusion criteria were as follows: i) histological diagnosis of a tumor type other than bile duct carcinoma, ii) inappropriate amount of tumor sample, iii) insufficient preservation of paraffin blocks for tissue microarray (TMA) construction, iv) Follow-up loss, and v) failure to obtain informed consent.

The patients' medical records were reviewed for clinical information, and histological parameters were evaluated on hematoxylin & eosin (H&E)-stained slides. Clinical data included the patient's sex and age, tumor location, operation date, American Joint Committee on Cancer (AJCC) TNM stage (20), most recent follow-up date, recurrence, and survival status. Pathological data included the tumor size, depth of invasion, tumor location, histologic subtype, tumor differentiation, nodal metastasis, and perineural or lymphovascular invasion.

Tissue microarray (TMA)

Tumor samples collected from clinical cases were fixed with 10% formalin at room temperature for 24 h. Formalin-fixed, paraffin-embedded tissue samples of randomly selected bile duct adenocarcinoma (n=76) were obtained and arrayed using a tissue-arraying instrument (Quick-Ray, Unitma Co., Ltd.). Briefly, areas with invasive adenocarcinomas were identified on the corresponding H&E-stained slides, and sections were indicated as representative tumors. Three cores were sampled from the center and border of invasive areas in each representative tumor block using a 2.0-mm punch. Four-µm-thick slides were cut from the TMA blocks for immunohistochemical and H&E staining. H&E stains for TMA blocks were performed by automated DAKO CoverStainer (Dako Korea Co., Ltd.; temperature, 4°C; duration, 46 min) to correlate with the immunohistochemical TMA slides.

Immunohistochemical staining and interpretation

Five-µm thick sections were cut from the formalin-fixed, paraffin-embedded tumor samples and stained with Leica auto-stainer Bond Max using the Bond Polymer Refine Detection System (Leica Biosystems Newcastle Ltd.) according to the manufacturer's protocols. Briefly, the sections were deparaffinized by Bond Dewax Solution (Leica Biosystems Newcastle Ltd.), followed by heat-induced antigen retrieval using Bond Epitope retrieval solution (Leica Biosystems Newcastle Ltd.) for 20 min at 100°C. The endogenous peroxidase was quenched by incubation with hydrogen peroxide for 15 min. Sections were incubated for 15 min at ambient temperature with primary mouse monoclonal anti-IDO1 antibody (Abcam; Ab13248, 1:100) and CD8 (SP16; Thermo Fisher Scientific; 1:100). Bound primary antibodies were visualized using a biotin-free polymeric horseradish peroxidase-linker antibody conjugate system (Bond Polymer Refine Detection; ready-to-use dilution; cat. no. DS9800; Leica Biosystems, Inc.) in a Bond-Max automatic slide stainer (Leica Biosystems Melbourne Pty. Ltd.). The nuclei of these sections were counterstained with hematoxylin (Biocare, cat: NM-HEM-M) for 4 min at 25°C in the Bond Polymer detection kit (Leica Biosystems).

The tumor epithelial immunoreactivity for IDO1 was semi quantitatively interpreted by means of light microscopic examination and evaluated without prior knowledge of the clinicopathological data. Cytoplasmic immunostaining was evaluated as the percentage and intensity of positive epithelial cells, as previously described (21). Staining intensity was graded as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positively stained cells was graded as 1 (1–24%), 2 (25–49%), 3 (50–74%), or 4 (≥75%). The final immunohistochemical score was calculated by multiplying these 2 grades to yield a score ranging from 0 to 12. We defined high IDO1 expression as a total score >3. The cut-point value is determined using the AUC (area under the ROC (receiver operating characteristic) curve) and Youden's index for patient's survival (22).

CD8 was estimated for TILs in the tumor bed including the tumor epithelium and intratumoral stroma using an automatic image analysis software ( The images of each case were captured using a digital camera (Jenoptik ProgRes speedXT) attached to an Olympus light microscope (BX51; Olympus). To reduce the effect of sampling error, whole areas of 2-mm TMA cores were included in analysis using a low power view (×4) according to recommendations of the software guide. CD8-positive cells were automatically detected, segmented, and counted. The cut-off numbers (176.5/mm2) were used to determine high expression of CD8 in TILs. This cut-off point is referred to as the optimal value that is calculated using AUC and Youden's index for patient's survival (23).

Statistical analysis

Statistical analyses were performed with SPSS software (version 23.0; SPSS Inc., Chicago, IL, USA). The average values were utilized as cut-off points for age and tumor size. Categorical data were assessed using Pearson's χ2 or Fisher's exact tests. The mean number of CD8+ TILs was examined using an unpaired Student's t-test. Overall survival (OS) was measured by the amount of time the patient is alive after the primary treatment. Disease-free survival (DFS) was referred to as the length of time that the patient survives without any signs or symptoms of that cancer after primary treatment for a cancer ends. In univariate analyses for survival, survival curves were illustrated and the relationship between survival rates and various clinicopathological factors were compared using the Kaplan-Meier method with the log-rank test. In multivariate analyses for survival, we investigated the prognostic significance using Cox proportional hazards modelling. P-values less than 0.05 were considered to denote statistical significance.


Clinicopathological characteristics

The demographics and clinicopathological correlation of IDO1 expression are summarized in Tables I and II, respectively. The series consisted of 54 men and 23 women with an average 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. Surgical resection and regional lymph node dissection were dependent on the location of the primary tumor. 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. Forty-seven patients (61.0%) showed clear resection margins. In 18 patients (23.4%), resection margins were involved by carcinoma. In addition, low-and high-grade dysplasia at resection margins were identified in 5 (6.5%) and 7 (9.1%) patients, respectively. Thirty-one patients received postoperative chemotherapy. The range of follow-up was from 0.3 months to 96.0 months (median: 20.9 months). No follow-up loss is identified.

Table I.

Patient demographics.

Table I.

Patient demographics.

ParametersGroupingN (%)
Age, years<7037 (48.7)
>7039 (51.3)
SexMale53 (69.7)
Female23 (30.3)
Size, cm<2.340 (52.6)
>2.336 (47.4)
LocationDistal45 (59.2)
Proximal31 (40.8)
DifferentiationaWell21 (27.6)
Moderately47 (61.8)
Poorly6 (7.9)
LVIaAbsent49 (64.5)
Present23 (30.3)
PNIaAbsent15 (19.7)
Present53 (69.7)
LN metastasisAbsent47 (61.8)
Present29 (38.2)
pT stagebpT112 (15.8)
pT248 (63.2)
pT316 (21.1)
pN stagebpN047 (61.8)
pN122 (28.9)
pN27 (9.2)
pTNM stagebI9 (11.8)
II60 (78.9)
III7 (9.2)
RecurrenceNegative23 (30.3)
Positive53 (69.7)
DeathAlive24 (31.6)
Dead52 (68.4)

a Clinicopathological data that can be not obtained were omitted;

b AJCC Stage grouping, 8th edition (20). LVI, lymphovascular invasion; PNI, perineural invasion; LN, lymph node; TNM, tumor node metastasis; TIL, tumor-infiltrating lymphocyte.

Table II.

Clinicopathological correlation of IDO1 expression.

Table II.

Clinicopathological correlation of IDO1 expression.

IDO1 expression (%)

Age, years<7025 (67.6)12 (32.4)0.933
>7026 (66.7)13 (33.3)
SexMale34 (64.2)19 (35.8)0.441
Female17 (73.9)6 (26.1)
Size, cm<2.328 (70.0)12 (30.0)0.571
>2.323 (63.9)13 (36.1)
LocationDistal28 (62.2)17 (37.8)0.200
Proximal23 (74.2)8 (25.8)
DifferentiationWell15 (71.4)6 (28.6)0.618
Moderately31 (66.0)16 (34.0)
Poorly3 (50.0)3 (50.0)
LVIAbsent35 (71.4)14 (28.6)0.422
Present14 (60.9)9 (39.1)
PNIAbsent9 (60.0)6 (40.0)0.555
Present36 (67.9)17 (32.1)
LN metastasisAbsent35 (74.5)12 (25.5)0.082
Present16 (55.2)13 (44.8)
pT stageapT110 (83.3)2 (16.7)0.384
pT230 (62.5)18 (37.5)
pT311 (68.8)5 (31.3)
pN stageapN035 (74.5)12 (25.5)0.007
pN115 (68.2)7 (31.8)
pN21 (14.3)6 (85.7)
pTNM stageaI9 (100)0 (0)0.001
II41 (68.3)19 (31.9)
III1 (14.3)6 (85.7)
CD8+ TIL/mm2Mean214.7898.380.008
RecurrenceNegative20 (87.0)3 (13.0)0.018
Positive31 (58.5)22 (41.5)
DeathAlive20 (83.3)4 (16.7)0.065
Death31 (59.6)21 (40.4)

a AJCC Stage grouping, 8th edition (20). LVI, lymphovascular invasion; PNI, perineural invasion; LN, lymph node; TNM, tumor node metastasis; TIL, tumor-infiltrating lymphocyte.

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 tumor epithelium and stromal mononuclear immune cells. The epithelial IDO1-expressing tumors 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%) tumor specimens. In 51 of 76 (67.1%) cases, IDO1 expression is low. IDO1-high expressing tumors (Fig. 1A) exhibited a significantly low proportion of intratumoral CD8+ cells (mean ± standard error (SE), 98.38/mm2±100.26; Fig. 1B), whereas IDO1-low expressing tissue samples (Fig. 1C) demonstrated high CD8+ cells (214.78/mm2±268.26; Fig. 1D) (P=0.008). Comparing IDO1 expression with CD8 expression in TILs, IDO1 expression was inversely associated with the number of CD8+ TILs.

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). There was non-significant relationship between IDO1 overexpression and disease-specific death (P=0.065) (Table II).

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).

Univariate and multivariate analyses for overall Survival and disease-free survival

High IDO1 expression was significantly associated with shorter disease-free survival (median time: 16.30 months vs. 26.30 months, P=0.026; Fig. 2A) and had a non-significant trend to decreased overall survival (median time: 21.20 months vs. 26.30 months, P=0.097; Fig. 2B). Patients with high CD8+ TILs showed longer disease-free survival (median survival time: 47.30 months vs. 16.30 months, P=0.023; Fig. 2C) than those with low CD8+ TILs. No significant relationship between high CD8+ TILs and overall survival (median survival time: 36.00 months vs. 19.4 months, P=0.135; Fig. 2D) was illustrated.

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 disease-free survival times (Fig. 3A and C) and median overall (Fig. 3B and D) (8.10 and 16.30 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) (Table III).

Table III.

Univariate analyses (log-rank test) for overall and disease-free survival.

Table III.

Univariate analyses (log-rank test) for overall and disease-free survival.

A, Overall survival

CI (95%)

Parameters Mean survival (months)LowerUpperP-value
Age, years<7036.88.4165.190.056
Size, cm<2.347.313.5481.160.03
LN metastasisAbsent3621.2950.710.1
pT stageapT141.50115.370.439
pN stageapN03621.2950.710.092
pTNM stageaI737.37138.630.112
Postoperative chemotherapyNo26.110.5441.670.705
IDO1 expressionLow26.37.9444.670.097
CD8+ TILLow19.415.4223.380.135
IDO1/CD8+ TIL, 2 tiersOther than High/Low33.717.549.90.013
IDO1/CD8+ TIL, 4 tiersLow/Low19.414.524.30.097

B, Disease-free survival

CI (95%)

Parameters Mean survival (months)LowerUpperP-value

Age, years<7024.74.4344.970.082
Size, cm<2.347.38.1586.450.011
LN metastasisAbsent3619.5352.470.012
pT stageapT120.80950.573
pN stageapN03619.5352.470.017
pTNM stageaI730150.250.063
Postoperative chemotherapyNo21.513.629.40.728
IDO1 expressionLow26.39.2343.870.026
CD8+ TILLow16.37.0325.570.023
IDO1/CD8+ TIL, 2 tiersOther than High/Low26.310.1942.42<0.001
IDO1/CD8+ TIL, 4 tiersLow/Low18.85.7531.850.005

a AJCC Stage grouping, 8th edition (20). CI, confidence interval; LVI, lymphovascular invasion; PNI, perineural invasion; LN, lymph node; TNM, tumor node metastasis; TIL, tumor-infiltrating lymphocyte.

In univariate analyses (Table III), larger tumor size (P=0.030), proximal location (P=0.007), perineural invasion (P=0.026), and the IDO1high/CD8low subgroup (P=0.013) revealed significantly shorter overall survival. Similarly, worse disease-free survival was significantly related to proximal location (P=0.007), perineural invasion (P=0.005), lymph node metastasis (P=0.012), higher pN stage (P=0.017), high IDO1 expression (P=0.026), low CD8+ TILs (P=0.023), and the IDO1high/CD8low expression subgroup (P<0.001). Multivariate statistical analyses of the IDO1high/CD8low expression subgroup were performed with the significant prognostic valuables examined by univariate analyses. As shown in Table IV, IDO1high/CD8low expression (P=0.025, Cox hazard ratio=2.168) in addition to tumor proximal 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 tumor proximal location, lymph node metastasis, and perineural invasion was an independent prognosticator for disease-free survival.

Table IV.

Multivariate analyses (Cox proportional hazards model).

Table IV.

Multivariate analyses (Cox proportional hazards model).

Overall survivalDisease-free survival

ParameterComparisonHR95% CIP-valueHR95% CIP-value
LocationProximal vs. Distal1.3491.004-1.8120.0471.4491.073-1.9570.016
PNIPresence vs. Absence2.1590.984-4.7370.0552.4861.060-5.8300.036
LN metastasisPresence vs. Absence---1.9561.026-3.7320.042
IDO1/CD8+ TIL IDO1high/CD8low vs. Others2.1681.1-4.2720.0252.4601.195-5.0650.015

[i] CI, confidence interval; HR, hazard ratio; PNI, perineural invasion; LN, lymph node; TNM, tumor node metastasis; TIL, tumor-infiltrating lymphocyte.


Since the immunosuppressive effects of IDO1 were discovered, IDO1 has been reported to be highly expressed and associated with clinical outcomes in a variety of solid tumors showing contradictory biologic behaviors. IDO1 expression is a predictor of poor clinical outcomes in many kinds of solid tumors, such as ovarian adenocarcinomas, colorectal adenocarcinomas, laryngeal squamous cell carcinomas, and endometrial and esophageal cancers (1217). In contrast, patients with high IDO1 expression in some tumors, such as basal cell-like breast carcinoma, hepatocellular carcinoma, and renal cell carcinoma, have increased survival (13,18,19). In the present study of 76 EHBDC surgical specimens, we demonstrated that high IDO1 expression in the tumor epithelial cells was positively correlated with tumor recurrence and poor patient survival. As is well known, some stromal mononuclear immune cells are also positive for IDO1; however, this expression was not correlated with patient survival (data not shown).

The role of CD8+ T lymphocytes in tumor progression has been examined in a variety of human malignancies (2428). Most research has revealed a beneficial prognostic effect of high intratumoral/intraepithelial CD8+ T lymphocyte infiltration. In accordance with our results, there are a few reports of a favorable prognostic effect of CD8+ TILs in patients with EHBDC (24,29,30). IDO1 expression in tumor epithelial cells was inversely associated with number of CD8+ TILs in the present study. Similar to our results, Ino et al reported that tumoral IDO1 expression was correlated with a reduced number of TILs and natural killer (NK) cells in endometrial cancer, possibly contributing to disease progression and poor clinical outcomes (14). Brandacher et al also reported that high IDO1 expression was associated with a significant reduction in CD3+ TILs in colon cancer as compared to tumor samples with low IDO1 expression (31).

IDO1 is an immunomodulatory enzyme that catalyzes the degradation of tryptophan (Trp) to Kyn. The depletion of Trp and accumulation of Kyn have been reported to induce effector T-cell apoptosis/dysfunction and generate immunosuppressive regulatory T cells (32). Recently, functional inactivation of tumor-reactive T cells has been considered to be an essential mechanism of tumor immune evasion (33). The upregulation of IDO1 occurs in tumor cells in response to interferon-γ (IFN-γ) secreted by CD8+ T cells (34) while the increased expression of IDO1 suppresses the CD8+ T cell response, resulting in tumor immune evasion and tumor growth, suggesting a possible negative feedback loop to regulate T-cell activation (5). Since both PD-L1 and IDO1 are increased in tumor cells by IFN-γ induced by CD8+ T cells (35), IDO1-and PD-L1-expressing tumors are expected to have similar clinical significance (36). However, IDO1 expression may have different clinicopathological implications from PD-L1 expression because there is the isolated mechanism to promote IDO1 secretion by activation pathway of RAS and PAMP (pathogen-associated molecular pattern) that is not shared with PD-L1 (37). In this regard, further research to elucidate the relationship between IDO1 and PD-L1 is needed in the future. These suggest that a more complex mechanism than previously evaluated acts between IDO1 expression and the immune microenvironment. Considering that single-agent treatments with IDO1 enzyme inhibitors have a negligible effect on decreasing the established cancer burden, a combination of select therapies with IDO1 blockade for a synergistic benefit against tumor growth is likely needed. Capitalizing on this background and the negative association between IDO1 expression and CD8+ TILs as shown in our study, patients with IDO1high/CD8low or IDO1low/CD8high subgroups can represent a stronger interaction between IDO1-expressing tumor and CD8+ TILs compared to other subgroups. Therefore, IDO1 blockers are expected to be more effective for the IDO1high/CD8low subgroup with worst prognosis by inhibiting a patent link between IDO1-expressing tumor and CD8+ TILs.

This is the first study to demonstrate the detailed clinicopathological and prognostic impact of IDO1 expression associated with decreased numbers of CD8+ TILs. In the future, our results can be utilized as a novel candidate biomarker in EHBDC for the development of immunotherapeutic drugs by augmenting our understanding of complex immune mechanisms.

The limitations of this study include the retrospective nature of the study design and the relatively small number of cases. In addition, the fundamental problem in TMA studies, such as tumor heterogeneity, remains unresolved.

In conclusion, IDO1 was highly expressed in the tumor epithelial cells in approximately one-third of cases of EHBDC. High expression of IDO1 was associated with decreased numbers of CD8+ TILs, an increased pN category, an advanced overall stage, and frequent recurrence. When further stratified by combining IDO1 expression with CD8+ TIL status, the IDO1high/CD8low subgroup had the worst prognosis for overall survival and disease-free survival. High IDO1 expression with decreased numbers of CD8+ TILs is an independent prognostic indicator, and expected to be an IDO1 blocker-targetable candidate in patients with EHBDC.


Not applicable.


Professor Dae-Woon Eom received a research grant (grant no. NRF-2020R1F1A1067158) of the Basic Science Research Program from the National Research Foundation of Korea funded by the Ministry of Science and ICT, and a grant (grant no. 2020-IB008) from the Gangneung Asan Hospital Biomedical Research Center Promotion Fund.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

BJN, GMC, HJJ, CHM, HSO, MK and DWE conceived and designed the research. BJN and DWE performed the experiments, analysed the data and wrote the manuscript. GMC, HJJ, CHM, HSO and MK reviewed the manuscript and approved the final version. All authors have read and approved the final manuscript. BJN and DWE confirm the authenticity of all the raw data.

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of Gangneung Asan Hospital (approval no. GNAH 2020-06-018). All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent to be included in the study, or the equivalent, was obtained from all patients.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



Bridgewater JA, Goodman KA, Kalyan A and Mulcahy MF: Biliary tract cancer: Epidemiology, radiotherapy, and molecular profiling. Am Soc Clin Oncol Educ Book. 35:e194–e203. 2016. View Article : Google Scholar : PubMed/NCBI


Rizvi S and Gores GJ: Pathogenesis, diagnosis, and management of cholangiocarcinoma. Gastroenterology. 145:1215–1229. 2013. View Article : Google Scholar : PubMed/NCBI


Nagino M, Ebata T, Yokoyama Y, Igami T, Sugawara G, Takahashi Y and Nimura Y: Evolution of surgical treatment for perihilar cholangiocarcinoma: A single-center 34-year review of 574 consecutive resections. Ann Surg. 258:129–140. 2013. View Article : Google Scholar : PubMed/NCBI


Malenica I, Donadon M and Lleo A: Molecular and immunological characterization of biliary tract cancers: A paradigm shift towards a personalized medicine. Cancers (Basel). 12:21902020. View Article : Google Scholar : PubMed/NCBI


Lou Q, Liu R, Yang X, Li W, Huang L, Wei L, Tan H, Xiang N, Chan K, Chen J and Liu H: miR-448 targets IDO1 and regulates CD8(+) T cell response in human colon cancer. J Immunother Cancer. 7:2102019. View Article : Google Scholar : PubMed/NCBI


Harden JL and Egilmez NK: Indoleamine 2,3-dioxygenase and dendritic cell tolerogenicity. Immunol Invest. 41:738–764. 2012. View Article : Google Scholar : PubMed/NCBI


Moon YW, Hajjar J, Hwu P and Naing A: Targeting the indoleamine 2,3-dioxygenase pathway in cancer. J Immunother Cancer. 3:512015. View Article : Google Scholar : PubMed/NCBI


Brochez L, Chevolet I and Kruse V: The rationale of indoleamine 2,3-dioxygenase inhibition for cancer therapy. Eur J Cancer. 76:167–182. 2017. View Article : Google Scholar : PubMed/NCBI


Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C and Mellor AL: Prevention of allogeneic fetal rejection by tryptophan catabolism. Science. 281:1191–1193. 1998. View Article : Google Scholar : PubMed/NCBI


Yu CP, Fu SF, Chen X, Ye J, Ye Y, Kong LD and Zhu Z: The clinicopathological and prognostic significance of IDO1 expression in human solid tumors: Evidence from a systematic review and meta-analysis. Cell Physiol Biochem. 49:134–143. 2018. View Article : Google Scholar : PubMed/NCBI


Frumento G, Rotondo R, Tonetti M, Damonte G, Benatti U and Ferrara GB: Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J Exp Med. 196:459–468. 2002. View Article : Google Scholar : PubMed/NCBI


Soliman H, Rawal B, Fulp J, Lee JH, Lopez A, Bui MM, Khalil F, Antonia S, Yfantis HG, Lee DH, et al: Analysis of indoleamine 2–3 dioxygenase (IDO1) expression in breast cancer tissue by immunohistochemistry. Cancer Immunol Immunother. 62:829–837. 2013. View Article : Google Scholar : PubMed/NCBI


Riesenberg R, Weiler C, Spring O, Eder M, Buchner A, Popp T, Castro M, Kammerer R, Takikawa O, Hatz RA, et al: Expression of indoleamine 2,3-dioxygenase in tumor endothelial cells correlates with long-term survival of patients with renal cell carcinoma. Clin Cancer Res. 13:6993–7002. 2007. View Article : Google Scholar : PubMed/NCBI


Ino K, Yoshida N, Kajiyama H, Shibata K, Yamamoto E, Kidokoro K, Takahashi N, Terauchi M, Nawa A, Nomura S, et al: Indoleamine 2,3-dioxygenase is a novel prognostic indicator for endometrial cancer. Br J Cancer. 95:1555–1561. 2006. View Article : Google Scholar : PubMed/NCBI


Gao YF, Peng RQ, Li J, Ding Y, Zhang X, Wu XJ, Pan ZZ, Wan DS, Zeng YX and Zhang XS: The paradoxical patterns of expression of indoleamine 2,3-dioxygenase in colon cancer. J Transl Med. 7:712009. View Article : Google Scholar : PubMed/NCBI


Inaba T, Ino K, Kajiyama H, Shibata K, Yamamoto E, Kondo S, Umezu T, Nawa A, Takikawa O and Kikkawa F: Indoleamine 2,3-dioxygenase expression predicts impaired survival of invasive cervical cancer patients treated with radical hysterectomy. Gynecol Oncol. 117:423–428. 2010. View Article : Google Scholar : PubMed/NCBI


Ye J, Liu H, Hu Y, Li P, Zhang G and Li Y: Tumoral indoleamine 2,3-dioxygenase expression predicts poor outcome in laryngeal squamous cell carcinoma. Virchows Arch. 462:73–81. 2013. View Article : Google Scholar : PubMed/NCBI


Jacquemier J, Bertucci F, Finetti P, Esterni B, Charafe-Jauffret E, Thibult ML, Houvenaeghel G, Van den Eynde B, Birnbaum D, Olive D and Xerri L: High expression of indoleamine 2,3-dioxygenase in the tumour is associated with medullary features and favourable outcome in basal-like breast carcinoma. Int J Cancer. 130:96–104. 2012. View Article : Google Scholar : PubMed/NCBI


Ishio T, Goto S, Tahara K, Tone S, Kawano K and Kitano S: Immunoactivative role of indoleamine 2,3-dioxygenase in human hepatocellular carcinoma. J Gastroenterol Hepatol. 19:319–326. 2004. View Article : Google Scholar : PubMed/NCBI


Amin MB, Edge SB, Greene FL, Byrd DR, Brookland RK, Washington MK, Gershenwald JE, Compton CC, Hess KR, Sullivan DC, et al: AJCC Cancer Staging Manual. 8th edition. Springer; New York, USA: pp. 317–325. 2017


Ferdinande L, Decaestecker C, Verset L, Mathieu A, Moles Lopez X, Negulescu AM, Van Maerken T, Salmon I, Cuvelier CA and Demetter P: Clinicopathological significance of indoleamine 2,3-dioxygenase 1 expression in colorectal cancer. Br J Cancer. 106:141–147. 2012. View Article : Google Scholar : PubMed/NCBI


Ma W, Duan H, Zhang R, Wang X, Xu H, Zhou Q and Zhang L: High expression of indoleamine 2, 3-dioxygenase in adenosquamous lung carcinoma correlates with favorable patient outcome. J Cancer. 10:267–276. 2019. View Article : Google Scholar : PubMed/NCBI


Lugli A, Karamitopoulou E, Panayiotides I, Karakitsos P, Rallis G, Peros G, Iezzi G, Spagnoli G, Bihl M, Terracciano L and Zlobec I: CD8+ lymphocytes/tumour-budding index: An independent prognostic factor representing a ‘pro-/anti-tumour’ approach to tumour host interaction in colorectal cancer. Br J Cancer. 101:1382–1392. 2009. View Article : Google Scholar : PubMed/NCBI


Oshikiri T, Miyamoto M, Shichinohe T, Suzuoki M, Hiraoka K, Nakakubo Y, Shinohara T, Itoh T, Kondo S and Katoh H: Prognostic value of intratumoral CD8+ T lymphocyte in extrahepatic bile duct carcinoma as essential immune response. J Surg Oncol. 84:224–228. 2003. View Article : Google Scholar : PubMed/NCBI


Fukunaga A, Miyamoto M, Cho Y, Murakami S, Kawarada Y, Oshikiri T, Kato K, Kurokawa T, Suzuoki M, Nakakubo Y, et al: CD8+ tumor-infiltrating lymphocytes together with CD4+ tumor-infiltrating lymphocytes and dendritic cells improve the prognosis of patients with pancreatic adenocarcinoma. Pancreas. 28:e26–e31. 2004. View Article : Google Scholar : PubMed/NCBI


Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C, Tosolini M, Camus M, Berger A, Wind P, et al: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 313:1960–1964. 2006. View Article : Google Scholar : PubMed/NCBI


Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Grainge MJ, Lee AH, Ellis IO and Green AR: Tumor-infiltrating CD8+ lymphocytes predict clinical outcome in breast cancer. J Clin Oncol. 29:1949–1955. 2011. View Article : Google Scholar : PubMed/NCBI


Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T, Bruneval P, Trajanoski Z, Fridman WH, Pagès F and Galon J: Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol. 29:610–618. 2011. View Article : Google Scholar : PubMed/NCBI


Kitano Y, Okabe H, Yamashita YI, Nakagawa S, Saito Y, Umezaki N, Tsukamoto M, Yamao T, Yamamura K, Arima K, et al: Tumour-infiltrating inflammatory and immune cells in patients with extrahepatic cholangiocarcinoma. Br J Cancer. 118:171–180. 2018. View Article : Google Scholar : PubMed/NCBI


Goeppert B, Frauenschuh L, Zucknick M, Stenzinger A, Andrulis M, Klauschen F, Joehrens K, Warth A, Renner M, Mehrabi A, et al: Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer. Br J Cancer. 109:2665–2674. 2013. View Article : Google Scholar : PubMed/NCBI


Brandacher G, Perathoner A, Ladurner R, Schneeberger S, Obrist P, Winkler C, Werner ER, Werner-Felmayer G, Weiss HG, Göbel G, et al: Prognostic value of indoleamine 2,3-dioxygenase expression in colorectal cancer: Effect on tumor-infiltrating T cells. Clin Cancer Res. 12:1144–1151. 2006. View Article : Google Scholar : PubMed/NCBI


Munn DH and Mellor AL: IDO in the tumor microenvironment: Inflammation, counter-regulation, and tolerance. Trends Immunol. 37:193–207. 2016. View Article : Google Scholar : PubMed/NCBI


Dunn GP, Bruce AT, Ikeda H, Old LJ and Schreiber RD: Cancer immunoediting: From immunosurveillance to tumor escape. Nat Immunol. 3:991–998. 2002. View Article : Google Scholar : PubMed/NCBI


Maggi E, Giudizi MG, Biagiotti R, Annunziato F, Manetti R, Piccinni MP, Parronchi P, Sampognaro S, Giannarini L, Zuccati G and Romagnani S: Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J Exp Med. 180:489–495. 1994. View Article : Google Scholar : PubMed/NCBI


Ludovini V, Bianconi F, Siggillino A, Vannucci J, Baglivo S, Berti V, Tofanetti FR, Reda MS, Bellezza G, Mandarano M, et al: High PD-L1/IDO-2 and PD-L2/IDO-1 Co-expression levels are associated with worse overall survival in resected non-small cell lung cancer patients. Genes (Basel). 12:2732021. View Article : Google Scholar : PubMed/NCBI


Zhang ML, Kem M, Mooradian MJ, Eliane JP, Huynh TG, Iafrate AJ, Gainor JF and Mino-Kenudson M: Differential expression of PD-L1 and IDO1 in association with the immune microenvironment in resected lung adenocarcinomas. Mod Pathol. 32:511–523. 2019. View Article : Google Scholar : PubMed/NCBI


Prendergast GC, Malachowski WP, DuHadaway JB and Muller AJ: Discovery of IDO1 inhibitors: From bench to bedside. Cancer Res. 77:6795–6811. 2017. View Article : Google Scholar : PubMed/NCBI

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Noh B, Choi GM, Jang HJ, Ma CH, Oh H, Kim M and Eom D: Prognostic implications of immune classification using IDO1 expression in extrahepatic bile duct carcinoma. Oncol Lett 24: 373, 2022
Noh, B., Choi, G.M., Jang, H.J., Ma, C.H., Oh, H., Kim, M., & Eom, D. (2022). Prognostic implications of immune classification using IDO1 expression in extrahepatic bile duct carcinoma. Oncology Letters, 24, 373.
Noh, B., Choi, G. M., Jang, H. J., Ma, C. H., Oh, H., Kim, M., Eom, D."Prognostic implications of immune classification using IDO1 expression in extrahepatic bile duct carcinoma". Oncology Letters 24.4 (2022): 373.
Noh, B., Choi, G. M., Jang, H. J., Ma, C. H., Oh, H., Kim, M., Eom, D."Prognostic implications of immune classification using IDO1 expression in extrahepatic bile duct carcinoma". Oncology Letters 24, no. 4 (2022): 373.