The Prognostic Implications of Immune Classification Using IDO1 Expression in Extrahepatic Bile Duct Carcinoma

The Prognostic Implications of Immune Classification Using IDO1 Expression in Extrahepatic Bile Duct Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Prognostic Implications of Immune Classification Using IDO1 Expression in Extrahepatic Bile Duct Carcinoma Byeong-Joo Noh, Gun Moo Choi, Hyuk Jai Jang, Chung Hyeun Ma, Ho-Suk Oh, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-743421/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Indoleamine 2, 3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme that catalyzes the degradation of tryptophan to kynurenine (Kyn) and induces immune tolerance in tumour cells. The effects of IDO1 on extrahepatic bile duct carcinoma (EHBDC) are poorly understood. This study aimed to investigate the expression and prognostic significance of IDO1 in EHBDC. Methods: An immunohistochemical microarray analysis of IDO1 expression was performed for 77 surgically resected cases of EHBDC, and the results were compared with various clinicopathologic variables including survival data. CD8 + tumour infiltrating lymphocytes (TILs) were also investigated to elucidate their relationship with IDO1 expression and prognosis through a combination analysis with IDO1 expression. Results: 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 IDO1 high /CD8 low subgroup showed the worst prognosis in terms of overall survival (P = 0.025, Cox risk ratio = 2.168) and disease-free survival (P = 0.015, Cox risk ratio = 2.460) in a multivariate analysis. Conclusions: Our study confirmed that high IDO1 expression was correlated 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 prediction tool in the patients with EHBDC. Cancer Biology Oncology Indoleamine 2 3-dioxygenase bile duct cancer tumour infiltrating lymphocytes Figures Figure 1 Figure 2 Figure 3 1. Introduction Extrahepatic bile duct carcinoma (EHBDC) is an epithelial tumour 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 tumour 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 tumour [ 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 tumour cells and the peritumoural immune cells (tumour microenvironment) (TME) have received much attention. However, difficult obstacles remain for deciphering TME because of tumour heterogeneity and immune system complexity, especially in immune suppression mechanisms. Local immune suppression of the tumour microenvironment is crucial for cancer development, metastasis, and even tumour 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 a 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 tumours [ 8 – 10 ]. 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 tumour immune evasion through tumour infiltrating lymphocyte (TIL) inhibition [ 11 ]. To date, contradictory results for prognostic implication of IDO1 expression has been reported in various carcinomas [ 12 – 19 ], but there is no study in EHBDC. In this study, we investigated intratumoural IDO1 expression in epithelial tumour cells in 77 patients with EHBDC who had undergone surgical resection. In addition, we defined the correlation between CD8 + TILs and IDO1 expression and examined prognostic differences between subgroups by performing stratification combining CD8 TIL status and IDO1 expression. 2. Materials And Methods 2.1 Patients and clinicopathologic data This study was approved by the Institutional Review Board of Gangneung Asan Hospital. A total of 77 consecutive patients with bile duct carcinoma who underwent surgical treatment between January 2001 and December 2017 were investigated after obtaining each patient's informed consent for use of their clinical records and pathological specimens at Gangneung Asan Hospital. 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, tumour location, operation date, American Joint Committee on Cancer (AJCC) TNM stage (8th edition), most recent follow-up date, recurrence, and survival status. Pathological data included the tumour size, depth of invasion, tumour location, histologic subtype, tumour differentiation, nodal metastasis, and perineural or lymphovascular invasion. 2.2 Tissue microarray (TMA) Formalin-fixed, paraffin-embedded tissue samples of randomly selected bile duct adenocarcinoma (n = 77) were obtained and arrayed using a tissue-arraying instrument (Quick-Ray, Unitma Co., Ltd., Seoul, Korea). Briefly, areas with invasive adenocarcinomas were identified on the corresponding H&E-stained slides, and sections were indicated as representative tumours. Three cores from each tumour sample were sampled using a 2.0-mm punch. Four-µm-thick slides were cut from the TMA blocks for immunohistochemical staining. 2.3 Immunohistochemical staining and interpretation Immunohistochemical staining was performed using primary mouse monoclonal anti-IDO1 antibody (Abcam, Cambridge, Ma, USA; Ab13248, 1:100) and CD8 (SP16; Thermo Fisher Scientific, Runcorn, UK; 1:100). Staining was conducted on a Bond-Max automatic immunostaining device (Leica Biosystem, Newcastle, UK), according to the manufacturer’s instructions. The tumour epithelial immunoreactivity for IDO1 was semiquantitatively interpreted by means of light microscopic examination and evaluated without prior knowledge of the clinicopathologic data. Cytoplasmic immunostaining was evaluated as the percentage and intensity of positive epithelial cells, as previously described [ 20 ]. 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. CD8 was estimated for TILs in the tumour bed including the tumour epithelium and intratumoural stroma using an automatic image analysis software ( https://oncoimmunoquantifier.com ). The images of each case were captured using a digital camera (Jenoptik ProgRes speedXT) attached to an Olympus light microscope (BX51; Olympus, Tokyo, Japan). To reduce the effect of sampling error, whole areas of 2-mm TMA cores were included in analysis using a low power view (x4) according to recommendations of the software guide. CD8-positive cells were automatically detected, segmented, and counted. The mean numbers (176.5/mm 2 ) were used as cut-off values to determine high expression of CD8 in TILs. 2.4 Statistical analysis Statistical analyses were performed with SPSS software (version 23.0; SPSS Inc., Chicago, IL, USA). Categorical data were assessed using χ2 or Fisher’s exact tests. The mean number of CD8 + TILs was examined using a t-test. Survival curves were estimated using the Kaplan-Meier method, and the log-rank test was used to calculate associations between survival rates and various clinicopathologic factors in univariate analyses. We also investigated the prognostic significance using Cox proportional hazards modelling. P-values less than 0.05 were considered to denote statistical significance. 3. Results 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/mm 2 ± 268.26) than IDO1-high expressing tissue samples (98.38/mm 2 ± 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): IDO1 high /CD8 low (18.4%, 14/76); IDO1 low /CD8 low (32.9%, 25/76); IDO1 high /CD8 high (13.2%, 10/76); IDO1 low /CD8 high (35.5%, 27/76). The patients whose carcinomas were IDO1 high /CD8 low 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 IDO1 low /CD8 low and IDO1 high /CD8 high expression were 19.40 months and 33.70 months, respectively. The median disease-free survival time of patients with IDO1 low /CD8 low and IDO1 high /CD8 high expression were 18.80 months and 26.1 months, respectively. Finally, patients with IDO1 low /CD8 high expression revealed the best survival (medial overall survival, 47.50 months; disease-free survival, 47.50 months). Next, a multivariate statistical analysis of the IDO1 high /CD8 low 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, IDO1 high /CD8 low 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, IDO1 high /CD8 low 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. 4. Discussion 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 tumours showing contradictory biologic behaviors. IDO1 expression is a predictor of poor clinical outcomes in many kinds of solid tumours, such as ovarian adenocarcinomas, colorectal adenocarcinomas, laryngeal squamous cell carcinomas, and endometrial and esophageal cancers [ 12 – 17 ]. In contrast, patients with high IDO1 expression in some tumours, such as basal cell-like breast carcinoma, hepatocellular carcinoma, and renal cell carcinoma, have increased survival [ 13 , 18 , 19 ]. In the present study of 77 EHBDC surgical specimens, we demonstrated that high IDO1 expression in the tumour epithelial cells was positively correlated with tumour 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 tumour progression has been examined in a variety of human malignancies [ 21 – 25 ]. Most research has revealed a beneficial prognostic effect of high intratumoural/intraepithelial CD8 + T lymphocyte infiltration. In accordance with our results, there are a few reports of a favourable prognostic effect of CD8 + TILs in patients with EHBDC [ 21 , 26 , 27 ]. IDO1 expression in tumour epithelial cells was inversely associated with number of CD8 + TILs in the present study. Similar to our results, Ino K et al. reported that tumoural 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 G et al. also reported that high IDO1 expression was associated with a significant reduction in CD3 + TILs in colon cancer as compared to tumour samples with low IDO1 expression [ 28 ]. 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 [ 29 ]. Recently, functional inactivation of tumour-reactive T cells has been considered to be an essential mechanism of tumour immune evasion [ 30 ]. The upregulation of IDO1 occurs in tumour cells in response to interferon-γ (IFN- γ ) secreted by CD8 + T cells [ 31 ], while the increased expression of IDO1 suppresses the CD8 + T cell response, resulting in tumour immune evasion and tumour growth, suggesting a possible negative feedback loop to regulate T-cell activation [ 5 ]. 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 tumour growth is likely needed. Capitalizing on this background and the negative correlation between IDO1 expression and CD8 + TILs as shown in our study, patients with IDO1 high /CD8 low or IDO1 low /CD8 high subgroups can represent a stronger interaction between IDO1-expressing tumour and CD8 + TILs compared to other subgroups. Therefore, IDO1 blockers are expected to be more effective for the IDO1 high /CD8 low subgroup with worst prognosis by inhibiting a patent link between IDO1-expressing tumour 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 tumour heterogeneity, remains unresolved. In summary, IDO1 was highly expressed in the tumour epithelial cells in approximately one-third of cases of EHBDC. High expression of IDO1 was correlated 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 IDO1 high /CD8 low 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 a IDO1 blocker-targetable candidate in patients with EHBDC. Declarations Ethics approval and consent to participate This study was approved by the Institutional Review Board of Gangneung Asan Hospital. 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. Consent for publicatiaon Not applicable. Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding The corresponding author (DAE-WOON EOM) has received a research grant (NRF-2020R1F1A1067158) of the Basic Science Research Program from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, and a grant (2019-B08) from the Gangneung Asan Hospital Biomedical Research Center Promotion Fund. Author’s contributions BJN, GMC, HJJ, CHM, HSO, MHK, and DWE conceived and designed the research. BJN (the first author) and DWE (the corresponding author) performed the experiments, analyzed the data, and wrote the manuscript. GMC, HJJ, CHM, HSO, and MHK reviewed the manuscript and approved the final version. Acknowledgements Not applicable. References Bridgewater JA, Goodman KA, Kalyan A, Mulcahy MF. Biliary Tract Cancer: Epidemiology, Radiotherapy, and Molecular Profiling. Am Soc Clin Oncol Educ Book. 2016;35:e194-203. http://doi.org/10.1200/edbk_160831. Rizvi S, Gores GJ. Pathogenesis, diagnosis, and management of cholangiocarcinoma. Gastroenterology. 2013;145:1215-29. http://doi.org/10.1053/j.gastro.2013.10.013. Nagino M, Ebata T, Yokoyama Y, Igami T, Sugawara G, Takahashi Y, et al. Evolution of surgical treatment for perihilar cholangiocarcinoma: a single-center 34-year review of 574 consecutive resections. Ann Surg. 2013;258:129-40. http://doi.org/10.1097/SLA.0b013e3182708b57. 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J Exp Med. 1994;180:489-95. http://doi.org/10.1084/jem.180.2.489. Tables Table 1. Clinicopathologic correlation of IDO1 expression Parameters IDO1 expression (%) P Low High Age (years) Less than 70 25(67.6) 12(32.4) 0.933 More than 70 26(66.7) 13(33.3) Sex Male 34(64.2) 19(35.8) 0.441 Female 17(73.9) 6(26.1) Size (cm) Less than 2.3 28(70.0) 12(30.0) 0.571 More than 2.3 23(63.9) 13(36.1) Location distal 28(62.2) 17(37.8) 0.200 proximal 23(74.2) 8(25.8) Differentiation Well 15(71.4) 6(28.6) 0.618 Moderately 31(66.0) 16(34.0) Poorly 3(50.0) 3(50.0) LVI Absent 35(71.4) 14(28.6) 0.422 Present 14(60.9) 9(39.1) PNI Absent 9(60.0) 6(40.0) 0.555 Present 36(67.9) 17(32.1) LN metastasis Absent 35(74.5) 12(25.5) 0.082 Present 16(55.2) 13(44.8) pT stage § pT1 10(83.3) 2(16.7) 0.384 pT2 30(62.5) 18(37.5) pT3 11(68.8) 5(31.3) pN stage § pN0 35(74.5) 12(25.5) 0.007 pN1 15(68.2) 7(31.8) pN2 1(14.3) 6(85.7) pTNM stage § I 9(100) 0(0) 0.001 II 41(68.3) 19(31.9) III 1(14.3) 6(85.7) CD8 + TIL/mm 2 Mean 241.78 98.38 0.008 Recurrence Negative 20(87.0) 3(13.0) 0.018 Positive 31(58.5) 22(41.5) Death Alive 20(83.3) 4(16.7) 0.065 Death 31(59.6) 21(40.4) AJCC Stage grouping, 8 th edition § . Abbreviations: LVI, lymphovascular invasion; PNI, perineural invasion; LN, lymph node; TNM, tumour node metastasis; TIL, tumour-infiltrating lymphocyte. Table 2. Univariate analyses (log-rank test) for overall and disease-free survival Parameters Mean OS (Months) CI (95%) P Mean DFS (Months) CI (95%) P Lower Upper Lower Upper Age (years) Less than 70 36.8 8.41 65.19 0.056 24.7 4.43 44.97 0.082 More than 70 20.9 10.67 31.13 18.8 12.13 25.47 Sex Male 20.9 9.54 32.26 0.769 20.8 11.26 30.34 0.827 Female 26.1 9.11 43.09 21.6 15.91 27.09 Size (cm) Less than 2.3 47.3 13.54 81.16 0.030 47.3 8.15 86.45 0.011 More than 2.3 20.8 17.56 24.04 18.4 8.45 28.35 Location distal 36.3 23.55 49.05 0.007 26.1 9.43 42.77 0.007 proximal 13.4 4.39 22.41 12.9 10.31 15.49 Differentiation Well 20.9 0.00 44.36 0.303 20.8 12.35 29.26 0.184 Moderately 26.2 11.50 40.90 26.1 15.10 37.10 Poorly 8.1 4.14 12.06 8.1 2.10 14.10 LVI Absent 26.2 8.09 44.31 0.392 24.7 7.98 41.42 0.151 Present 26.1 13.70 38.49 15.7 9.76 21.64 PNI Absent 56.2 7.56 104.84 0.026 92.2 38.03 146.37 0.005 Present 20.9 14.84 26.96 16.6 6.22 26.98 LN metastasis Absent 36.0 21.29 50.71 0.100 36.0 19.53 52.47 0.012 Present 20.8 12.76 28.84 11.4 1.55 21.25 pT stage § pT1 41.5 0.00 115.37 0.439 20.8 0.00 95.00 0.573 pT2 24.3 17.87 30.73 22.9 14.20 31.61 pT3 18.4 10.95 25.85 18.4 4.09 32.71 pN stage § pN0 36.0 21.29 50.71 0.092 36.0 19.53 52.47 0.017 pN1 20.9 17.61 24.19 15.7 7.92 23.48 pN2 10.9 3.75 18.09 8.1 0.66 15.54 pTNM stage § I 73.0 7.37 138.63 0.112 73.0 0.00 150.25 0.063 II 26.1 11.68 40.52 22.3 16.17 28.43 III 10.9 3.72 18.09 8.1 0.66 15.42 IDO1 expression Low 26.3 7.94 44.67 0.097 26.3 9.23 43.87 0.026 High 21.2 5.68 36.72 16.3 7.98 24.62 CD8 + TIL Low 19.4 15.42 23.38 0.135 16.3 7.03 25.57 0.023 High 36.0 8.20 63.80 47.3 20.93 73.67 IDO1/ CD8 + TIL, 2 tiers Other than High/Low 33.7 17.50 49.90 0.013 26.3 10.19 42.42 <0.001 High/Low 16.3 0.00 34.45 8.1 2.42 13.78 IDO1/ CD8 + TIL, 4 tiers Low/Low 19.4 14.50 24.30 0.097 18.8 5.75 31.85 0.005 High/Low 16.3 0.00 34.45 8.1 2.42 13.78 High/High 33.7 18.36 49.04 26.1 0.00 56.16 Low/High 47.5 5.98 89.02 47.5 0.00 97.81 AJCC Stage grouping, 8th edition § . Abbreviations: OS, overall survival; DFS, disease free survival; CI, confidence interval; LVI, lymphovascular invasion; PNI, perineural invasion; LN, lymph node; TNM, tumour node metastasis; TIL, tumour-infiltrating lymphocyte. Table 3. Multivariate analyses (Cox proportional hazards model) Overall survival Disease-free survival HR 95% CI P HR 95% CI P Location Distal vs. Proximal 1.349 1.004 - 1.812 0.047 1.449 1.073 - 1.957 0.016 PNI Presence vs. Absence 2.159 0.984 - 4.737 0.055 2.486 1.060 - 5.830 0.036 LN metastasis Presence vs. Absence – – – 1.956 1.026 - 3.732 0.042 IDO1/CD8 + TIL IDO1 high /CD8 low vs. Others 2.168 1.1 - 4.272 0.025 2.460 1.195 - 5.065 0.015 Abbreviations: CI: confidence interval; HR: hazard ratio; PNI, perineural invasion; LN, lymph node; TNM, tumour node metastasis; TIL, tumour-infiltrating lymphocyte. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-743421","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":44095029,"identity":"9daa487a-8776-47f3-a70e-57b89b611591","order_by":0,"name":"Byeong-Joo Noh","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Byeong-Joo","middleName":"","lastName":"Noh","suffix":""},{"id":44095030,"identity":"bb623907-4002-41b6-93f4-77be58009762","order_by":1,"name":"Gun Moo Choi","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gun","middleName":"Moo","lastName":"Choi","suffix":""},{"id":44095031,"identity":"1617ff7f-f4cf-43c3-87b2-8838f4fabc7e","order_by":2,"name":"Hyuk Jai Jang","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyuk","middleName":"Jai","lastName":"Jang","suffix":""},{"id":44095032,"identity":"10920d0f-4f26-44e4-9e5e-7fb489f5c703","order_by":3,"name":"Chung Hyeun Ma","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chung","middleName":"Hyeun","lastName":"Ma","suffix":""},{"id":44095033,"identity":"0e29a53d-b817-41fb-a433-b4555e82f6d3","order_by":4,"name":"Ho-Suk Oh","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ho-Suk","middleName":"","lastName":"Oh","suffix":""},{"id":44095036,"identity":"d4cc60c7-afbc-47ad-b777-a9b43a2fd205","order_by":5,"name":"Moonho Kim","email":"","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Moonho","middleName":"","lastName":"Kim","suffix":""},{"id":44095038,"identity":"8acc5c3e-b230-4e4b-bc51-40e61a7b6b49","order_by":6,"name":"Dae-Woon Eom","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYFCCBAYDxgYbIIMHzDUgVksaiVoYGBsOk6CFnz3HoJh3x3m77exnDzD8qGAwNm8goEWy542BMe+Z28k7e/ISGHvOMJjJHCCgxeBG7gZj3rbbyQYHcgyYGdsYbCQIOcweouVcssH5N0RqMZAAazlgZ3ADYosZQS0SZ95/MJzblpxgcOONwcGeMxLGBLXwt6elGbxts7M3OJ9j+OBHhY3hDEJagIDNCBgjiQ1A1gGgrURoYGBgfvgDGAxEKR0Fo2AUjIKRCQAOGz6jy80kzAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Ulsan College of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Dae-Woon","middleName":"","lastName":"Eom","suffix":""}],"badges":[],"createdAt":"2021-07-22 14:59:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-743421/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-743421/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12266759,"identity":"35202aaf-6155-47d7-b9af-1229d9878292","added_by":"auto","created_at":"2021-08-09 23:05:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3983081,"visible":true,"origin":"","legend":"Immunohistochemical stains for IDO1 and CD8 expression. \nA case with high IDO1 expression in the cytoplasm of tumour epithelial cells (A) with rare CD8-positive TILs (B). Another case with low IDO1 expression (C) with frequent CD8-positive TILs (D) (original magnification ×200).","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-743421/v1/aaa539011305506575361008.png"},{"id":12266766,"identity":"ee5e8683-0427-40fa-b79c-a5953e709652","added_by":"auto","created_at":"2021-08-09 23:08:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":702988,"visible":true,"origin":"","legend":"Kaplan-Meier curves according to IDO1 and CD8+ TILs expression. \nA-B. Patients with high IDO1 expression shows poor clinical outcomes for disease-free survival and overall survival than low IDO1 expression. C-D. Patients with high CD8+ TIL expression tends to have favourable disease free survival and overall survival than those with low CD8+ TIL expression.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-743421/v1/29b855a85dc69b8644d8c02d.png"},{"id":12266757,"identity":"63c926db-4bd1-4bb3-bae5-105cbdf23301","added_by":"auto","created_at":"2021-08-09 23:05:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":829468,"visible":true,"origin":"","legend":"Kaplan-Meier curves according to immune subgroups using combined IDO1 and CD8+ TILs expression. \nA-B. The IDO1high/CD8low subgroup shows the worst prognosis, while the IDO1low/CD8high subgroup demonstrates the best clinical outcome for disease-free survival and overall survival compared to IDO1low/CD8low and IDO1high/CD8high subgroups. C-D. Patients with IDO1high/CD8low expression shows statistically favourable survival outcome for disease-free survival and overall survival than those in the other three groups. ","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-743421/v1/d8ff31fca1708f6fe429d875.png"},{"id":21268008,"identity":"4bbb1580-efa6-4cb9-918b-9eb81867479d","added_by":"auto","created_at":"2022-05-10 07:59:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3734198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-743421/v1/31d23d28-e556-497d-98e6-6fae2265a9c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Prognostic Implications of Immune Classification Using IDO1 Expression in Extrahepatic Bile Duct Carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExtrahepatic bile duct carcinoma (EHBDC) is an epithelial tumour 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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite recent advances in diagnostic and therapeutic techniques, complete surgical resection of the tumour 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 tumour [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, with the advent of immunotherapy, studies for the interactive relationships between tumour cells and the peritumoural immune cells (tumour microenvironment) (TME) have received much attention. However, difficult obstacles remain for deciphering TME because of tumour heterogeneity and immune system complexity, especially in immune suppression mechanisms. Local immune suppression of the tumour microenvironment is crucial for cancer development, metastasis, and even tumour immune escape [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIDO1 has been emerging as a 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 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. 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 tumours [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. IDO1 is known to be involved in maternal-fetal immune tolerance in the mouse placenta [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and has been shown to be linked in a mechanism of response to tumour immune evasion through tumour infiltrating lymphocyte (TIL) inhibition [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To date, contradictory results for prognostic implication of IDO1 expression has been reported in various carcinomas [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], but there is no study in EHBDC.\u003c/p\u003e \u003cp\u003eIn this study, we investigated intratumoural IDO1 expression in epithelial tumour cells in 77 patients with EHBDC who had undergone surgical resection. In addition, we defined the correlation between CD8\u003csup\u003e+\u003c/sup\u003e TILs and IDO1 expression and examined prognostic differences between subgroups by performing stratification combining CD8 TIL status and IDO1 expression.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patients and clinicopathologic data\u003c/h2\u003e \u003cp\u003e This study was approved by the Institutional Review Board of Gangneung Asan Hospital. A total of 77 consecutive patients with bile duct carcinoma who underwent surgical treatment between January 2001 and December 2017 were investigated after obtaining each patient's informed consent for use of their clinical records and pathological specimens at Gangneung Asan Hospital.\u003c/p\u003e \u003cp\u003eThe patients\u0026rsquo; medical records were reviewed for clinical information, and histological parameters were evaluated on hematoxylin \u0026amp; eosin (H\u0026amp;E)-stained slides. Clinical data included the patient\u0026rsquo;s sex and age, tumour location, operation date, American Joint Committee on Cancer (AJCC) TNM stage (8th edition), most recent follow-up date, recurrence, and survival status. Pathological data included the tumour size, depth of invasion, tumour location, histologic subtype, tumour differentiation, nodal metastasis, and perineural or lymphovascular invasion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Tissue microarray (TMA)\u003c/h2\u003e \u003cp\u003eFormalin-fixed, paraffin-embedded tissue samples of randomly selected bile duct adenocarcinoma (n\u0026thinsp;=\u0026thinsp;77) were obtained and arrayed using a tissue-arraying instrument (Quick-Ray, Unitma Co., Ltd., Seoul, Korea). Briefly, areas with invasive adenocarcinomas were identified on the corresponding H\u0026amp;E-stained slides, and sections were indicated as representative tumours. Three cores from each tumour sample were sampled using a 2.0-mm punch. Four-\u0026micro;m-thick slides were cut from the TMA blocks for immunohistochemical staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Immunohistochemical staining and interpretation\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining was performed using primary mouse monoclonal anti-IDO1 antibody (Abcam, Cambridge, Ma, USA; Ab13248, 1:100) and CD8 (SP16; Thermo Fisher Scientific, Runcorn, UK; 1:100). Staining was conducted on a Bond-Max automatic immunostaining device (Leica Biosystem, Newcastle, UK), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe tumour epithelial immunoreactivity for IDO1 was semiquantitatively interpreted by means of light microscopic examination and evaluated without prior knowledge of the clinicopathologic data. Cytoplasmic immunostaining was evaluated as the percentage and intensity of positive epithelial cells, as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. 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\u0026ndash;24%), 2 (25\u0026ndash;49%), 3 (50\u0026ndash;74%), or 4 (\u0026ge;\u0026thinsp;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\u0026thinsp;\u0026gt;\u0026thinsp;3.\u003c/p\u003e \u003cp\u003eCD8 was estimated for TILs in the tumour bed including the tumour epithelium and intratumoural stroma using an automatic image analysis software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://oncoimmunoquantifier.com\u003c/span\u003e\u003c/span\u003e). The images of each case were captured using a digital camera (Jenoptik ProgRes speedXT) attached to an Olympus light microscope (BX51; Olympus, Tokyo, Japan). To reduce the effect of sampling error, whole areas of 2-mm TMA cores were included in analysis using a low power view (x4) according to recommendations of the software guide. CD8-positive cells were automatically detected, segmented, and counted. The mean numbers (176.5/mm\u003csup\u003e2\u003c/sup\u003e) were used as cut-off values to determine high expression of CD8 in TILs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with SPSS software (version 23.0; SPSS Inc., Chicago, IL, USA). Categorical data were assessed using χ2 or Fisher\u0026rsquo;s exact tests. The mean number of CD8\u003csup\u003e+\u003c/sup\u003e TILs was examined using a t-test. Survival curves were estimated using the Kaplan-Meier method, and the log-rank test was used to calculate associations between survival rates and various clinicopathologic factors in univariate analyses. We also investigated the prognostic significance using Cox proportional hazards modelling. P-values less than 0.05 were considered to denote statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Clinicopathologic characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026ndash;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Clinicopathologic correlation IDO1 and CD8 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIDO1 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHigh IDO1 expression was associated with a higher pN category (P = 0.007), an advanced overall TNM stage (P = 0.001),\u0026nbsp;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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCD8 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).\u003c/p\u003e\n\u003cp\u003eComparing IDO1 expression with CD8expression in TILs, IDO1 expression was inversely correlated with the number of CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eTILs. IDO1-low expressing tumours exhibited a significantly higher proportion of intratumoural CD8\u003csup\u003e+\u003c/sup\u003e cells (mean \u0026plusmn; standard error (SE), 214.78/mm\u003csup\u003e2\u003c/sup\u003e \u0026plusmn; 268.26) than IDO1-high expressing tissue samples (98.38/mm\u003csup\u003e2\u003c/sup\u003e \u0026plusmn; 100.26) (P = 0.008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Univariate and multivariate analyses for overall Survival and disease-free survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh 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\u003csup\u003e+\u003c/sup\u003e TILs tended to have longer disease-free survival (median survival time: 47.30 months vs. 16.30 months,\u0026nbsp;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\u003csup\u003e+\u003c/sup\u003e TILs.\u003c/p\u003e\n\u003cp\u003ePatient survival was further stratified according to four expression combinations of IDO1 and CD8\u0026nbsp;arranged in order of adverse prognostic value\u0026nbsp;as follows (Fig. 3): IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e (18.4%, 14/76); IDO1\u003csup\u003elow\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e (32.9%, 25/76); IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003ehigh\u003c/sup\u003e (13.2%, 10/76); IDO1\u003csup\u003elow\u003c/sup\u003e/CD8\u003csup\u003ehigh\u003c/sup\u003e (35.5%, 27/76). The patients whose carcinomas were IDO1\u003csup\u003ehigh\u0026nbsp;\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e 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 IDO1\u003csup\u003elow\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e and IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003ehigh\u003c/sup\u003e expression were 19.40 months and 33.70 months, respectively. The median disease-free survival time of patients with IDO1\u003csup\u003elow\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e and IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003ehigh\u003c/sup\u003e expression were 18.80 months and 26.1 months, respectively. Finally, patients with IDO1\u003csup\u003elow\u003c/sup\u003e/CD8\u003csup\u003ehigh\u003c/sup\u003e expression revealed the best survival (medial overall survival, 47.50 months; disease-free survival, 47.50 months).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, a multivariate statistical analysis of the IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e 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, IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e 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, IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e 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.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSince 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 tumours showing contradictory biologic behaviors. IDO1 expression is a predictor of poor clinical outcomes in many kinds of solid tumours, such as ovarian adenocarcinomas, colorectal adenocarcinomas, laryngeal squamous cell carcinomas, and endometrial and esophageal cancers [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In contrast, patients with high IDO1 expression in some tumours, such as basal cell-like breast carcinoma, hepatocellular carcinoma, and renal cell carcinoma, have increased survival [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the present study of 77 EHBDC surgical specimens, we demonstrated that high IDO1 expression in the tumour epithelial cells was positively correlated with tumour 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).\u003c/p\u003e \u003cp\u003eThe role of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in tumour progression has been examined in a variety of human malignancies [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Most research has revealed a beneficial prognostic effect of high intratumoural/intraepithelial CD8\u003csup\u003e+\u003c/sup\u003e T lymphocyte infiltration. In accordance with our results, there are a few reports of a favourable prognostic effect of CD8\u003csup\u003e+\u003c/sup\u003e TILs in patients with EHBDC [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. IDO1 expression in tumour epithelial cells was inversely associated with number of CD8\u003csup\u003e+\u003c/sup\u003e TILs in the present study. Similar to our results, \u003cem\u003eIno K\u003c/em\u003e et al. reported that tumoural 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 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eBrandacher G\u003c/em\u003e et al. also reported that high IDO1 expression was associated with a significant reduction in CD3\u003csup\u003e+\u003c/sup\u003e TILs in colon cancer as compared to tumour samples with low IDO1 expression [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIDO1 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Recently, functional inactivation of tumour-reactive T cells has been considered to be an essential mechanism of tumour immune evasion [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The upregulation of IDO1 occurs in tumour cells in response to interferon-γ (IFN-\u003cem\u003eγ\u003c/em\u003e) secreted by CD8\u003csup\u003e+\u003c/sup\u003e T cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while the increased expression of IDO1 suppresses the CD8\u003csup\u003e+\u003c/sup\u003e T cell response, resulting in tumour immune evasion and tumour growth, suggesting a possible negative feedback loop to regulate T-cell activation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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 tumour growth is likely needed. Capitalizing on this background and the negative correlation between IDO1 expression and CD8\u003csup\u003e+\u003c/sup\u003e TILs as shown in our study, patients with IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e or IDO1\u003csup\u003elow\u003c/sup\u003e/CD8\u003csup\u003ehigh\u003c/sup\u003e subgroups can represent a stronger interaction between IDO1-expressing tumour and CD8\u003csup\u003e+\u003c/sup\u003e TILs compared to other subgroups. Therefore, IDO1 blockers are expected to be more effective for the IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e subgroup with worst prognosis by inhibiting a patent link between IDO1-expressing tumour and CD8\u003csup\u003e+\u003c/sup\u003e TILs.\u003c/p\u003e \u003cp\u003eThis is the first study to demonstrate the detailed clinicopathological and prognostic impact of IDO1 expression associated with decreased numbers of CD8\u003csup\u003e+\u003c/sup\u003e 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.\u003c/p\u003e \u003cp\u003eThe 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 tumour heterogeneity, remains unresolved.\u003c/p\u003e \u003cp\u003eIn summary, IDO1 was highly expressed in the tumour epithelial cells in approximately one-third of cases of EHBDC. High expression of IDO1 was correlated with decreased numbers of CD8\u003csup\u003e+\u003c/sup\u003e TILs, an increased pN category, an advanced overall stage, and frequent recurrence. When further stratified by combining IDO1 expression with CD8\u003csup\u003e+\u003c/sup\u003e TIL status, the IDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e subgroup had the worst prognosis for overall survival and disease-free survival. High IDO1 expression with decreased numbers of CD8\u003csup\u003e+\u003c/sup\u003e TILs is an independent prognostic indicator, and expected to be a IDO1 blocker-targetable candidate in patients with EHBDC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Gangneung Asan Hospital. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publicatiaon\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author (DAE-WOON EOM) has received a research grant\u0026nbsp;(NRF-2020R1F1A1067158)\u0026nbsp;of\u0026nbsp;the Basic Science Research Program from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, and a grant (2019-B08) from the Gangneung Asan Hospital Biomedical Research Center Promotion Fund.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBJN, GMC, HJJ, CHM, HSO, MHK, and DWE conceived and designed the research. BJN (the first author) and DWE (the corresponding author) performed the experiments, analyzed the data, and wrote the manuscript. GMC, HJJ, CHM, HSO, and MHK reviewed the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBridgewater JA, Goodman KA, Kalyan A, Mulcahy MF. Biliary Tract Cancer: Epidemiology, Radiotherapy, and Molecular Profiling. Am Soc Clin Oncol Educ Book. 2016;35:e194-203. http://doi.org/10.1200/edbk_160831.\u003c/li\u003e\n\u003cli\u003eRizvi S, Gores GJ. Pathogenesis, diagnosis, and management of cholangiocarcinoma. Gastroenterology. 2013;145:1215-29. http://doi.org/10.1053/j.gastro.2013.10.013.\u003c/li\u003e\n\u003cli\u003eNagino M, Ebata T, Yokoyama Y, Igami T, Sugawara G, Takahashi Y, et al. 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Nat Immunol. 2002;3:991-8. http://doi.org/10.1038/ni1102-991.\u003c/li\u003e\n\u003cli\u003eMaggi E, Giudizi MG, Biagiotti R, Annunziato F, Manetti R, Piccinni MP, et al. Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J Exp Med. 1994;180:489-95. http://doi.org/10.1084/jem.180.2.489.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Clinicopathologic correlation of IDO1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.28767123287671%\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"26.810176125244617%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 39.5692%;\" width=\"38.74755381604697%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDO1 expression (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.154598825831702%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"52.525252525252526%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"47.474747474747474%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLess than 70\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e25(67.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e12(32.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.933\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 70\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e26(66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e13(33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e34(64.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e19(35.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.441\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e17(73.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e6(26.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSize (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLess than 2.3 \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e28(70.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e12(30.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.571\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 2.3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e23(63.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e13(36.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003edistal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e28(62.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e17(37.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.200\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eproximal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e23(74.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e8(25.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDifferentiation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e15(71.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e6(28.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.618\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eModerately\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e31(66.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e16(34.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePoorly\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e3(50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e3(50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLVI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e35(71.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e14(28.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.422\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePresent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e14(60.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e9(39.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e9(60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e6(40.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.555\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePresent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e36(67.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e17(32.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLN metastasis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e35(74.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e12(25.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.082\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePresent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e16(55.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e13(44.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT stage\u003c/strong\u003e\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e10(83.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e2(16.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.384\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e30(62.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e18(37.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e11(68.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e5(31.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003epN stage\u003c/strong\u003e\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003epN0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e35(74.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e12(25.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003epN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e15(68.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e7(31.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003epN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e1(14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e6(85.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003epTNM stage\u003c/strong\u003e\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e9(100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e0(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e41(68.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e19(31.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e1(14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e6(85.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD8\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e \u003cstrong\u003eTIL/mm\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e241.78\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e98.38\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.008\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecurrence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e20(87.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e3(13.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.018\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e31(58.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e22(41.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.3808%;\" width=\"23.333333333333332%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDeath\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"22.15686274509804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"20.392156862745097%\"\u003e\n \u003cp\u003e20(83.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"18.431372549019606%\"\u003e\n \u003cp\u003e4(16.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 7.3409%;\" width=\"11.176470588235293%\"\u003e\n \u003cp\u003e0.065\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 29.1845%;\" width=\"33.83233532934132%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDeath\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.5426%;\" width=\"31.137724550898202%\"\u003e\n \u003cp\u003e31(59.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0266%;\" width=\"28.143712574850298%\"\u003e\n \u003cp\u003e21(40.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAJCC Stage grouping, 8\u003csup\u003eth\u003c/sup\u003e edition\u003csup\u003e\u0026sect;\u003c/sup\u003e. Abbreviations: LVI, lymphovascular invasion; PNI, perineural invasion;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLN, lymph node; TNM, tumour node metastasis; TIL, tumour-infiltrating lymphocyte.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Univariate analyses (log-rank test) for overall and disease-free survival\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" width=\"31.34328358208955%\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"11.276948590381426%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean OS\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Months)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"16.417910447761194%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI (95%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.799336650082918%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"10.613598673300165%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean DFS\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Months)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"15.422885572139304%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCI (95%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.12603648424544%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.225130890052355%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLower\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.607329842931936%\"\u003e\n \u003cp\u003e\u003cstrong\u003eUpper\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.083769633507853%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLower\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.083769633507853%\"\u003e\n \u003cp\u003e\u003cstrong\u003eUpper\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLess than 70\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e36.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e8.41\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e65.19\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.056\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e24.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e4.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e44.97\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e0.082\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 70\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e20.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e10.67\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e31.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e18.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e12.13\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e25.47\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e20.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e9.54\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e32.26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.769\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e20.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e11.26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e30.34\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e0.827\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e26.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e9.11\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e43.09\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e21.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e15.91\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e27.09\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSize (cm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLess than 2.3 \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e47.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e13.54\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e81.16\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.030\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e47.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e8.15\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e86.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e0.011\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMore than 2.3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e20.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e17.56\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e24.04\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e18.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e8.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e28.35\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003edistal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e36.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e23.55\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e49.05\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e26.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e9.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e42.77\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eproximal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e13.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e4.39\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e22.41\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e12.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e10.31\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e15.49\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDifferentiation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e20.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e44.36\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.303\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e20.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e12.35\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e29.26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e0.184\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eModerately\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e26.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e11.50\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e40.90\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e26.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e15.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e37.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePoorly\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e4.14\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e12.06\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e2.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e14.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLVI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbsent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e26.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e8.09\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e44.31\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.392\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e24.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e7.98\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e41.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e0.151\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePresent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e26.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e13.70\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e38.49\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e15.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e9.76\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e21.64\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n 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width=\"7.641196013289036%\"\u003e\n \u003cp\u003e19.53\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e52.47\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.012\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePresent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e20.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e12.76\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e28.84\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e11.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e1.55\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e21.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT stage\u003c/strong\u003e\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003epT1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e41.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e115.37\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" width=\"6.810631229235881%\"\u003e\n 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width=\"11.442786069651742%\"\u003e\n \u003cp\u003e4.09\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e32.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003epN stage\u003c/strong\u003e\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003epN0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e36.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e21.29\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e50.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" width=\"6.810631229235881%\"\u003e\n 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\u003cp\u003e40.52\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e22.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e16.17\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e28.43\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e10.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e3.72\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e18.09\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e0.66\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e15.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDO1 expression\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e26.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e7.94\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e44.67\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.097\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e26.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e9.23\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e43.87\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.026\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e21.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e5.68\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e36.72\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e16.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e7.98\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e24.62\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD8\u003csup\u003e+\u003c/sup\u003e TIL \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e19.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e15.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e23.38\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.135\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e16.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e7.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e25.57\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.023\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e36.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e8.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e63.80\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e47.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e20.93\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e73.67\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDO1/\u003c/strong\u003e\u003cstrong\u003eCD8\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;TIL,\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2 tiers \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eOther than High/Low\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e33.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e17.50\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e49.90\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.013\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e26.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e10.19\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e42.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh/Low\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e16.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e34.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e2.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e13.78\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" width=\"18.272425249169434%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIDO1/\u003c/strong\u003e\u003cstrong\u003eCD8\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;TIL,\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4 tiers \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.122923588039868%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow/Low\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.295681063122924%\"\u003e\n \u003cp\u003e19.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.637873754152825%\"\u003e\n \u003cp\u003e14.50\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.807308970099668%\"\u003e\n \u003cp\u003e24.30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" width=\"6.810631229235881%\"\u003e\n \u003cp\u003e0.097\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.631229235880399%\"\u003e\n \u003cp\u003e18.8\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e5.75\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.641196013289036%\"\u003e\n \u003cp\u003e31.85\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" width=\"8.13953488372093%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.005\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh/Low\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e16.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e34.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e2.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e13.78\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh/High\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e33.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e18.36\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e49.04\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e26.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e56.16\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.65174129353234%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow/High\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.915422885572138%\"\u003e\n \u003cp\u003e47.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.935323383084578%\"\u003e\n \u003cp\u003e5.98\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.691542288557214%\"\u003e\n \u003cp\u003e89.02\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.92039800995025%\"\u003e\n \u003cp\u003e47.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e0.00\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.442786069651742%\"\u003e\n \u003cp\u003e97.81\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAJCC Stage grouping, 8th edition\u003csup\u003e\u0026sect;\u003c/sup\u003e. Abbreviations: OS, overall survival; DFS, disease free survival; CI, confidence interval; LVI, lymphovascular invasion; PNI, perineural invasion; LN, lymph node; TNM, tumour node metastasis; TIL, tumour-infiltrating lymphocyte.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Multivariate analyses (Cox proportional hazards model)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 17.846%;\" width=\"44.46308724832215%\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 15.5774%;\" width=\"27.013422818791945%\"\u003e\n \u003cp\u003e\u003cstrong\u003eOverall survival\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 0.3305%;\" width=\"28.523489932885905%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisease-free survival\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 5.1421%;\" width=\"14.156626506024097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.6375%;\" width=\"22.89156626506024%\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"11.44578313253012%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"15.662650602409638%\"\u003e\n \u003cp\u003e\u003cstrong\u003eHR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"22.89156626506024%\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 3.1004%;\" width=\"11.44578313253012%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.2424%;\" width=\"21.608040201005025%\"\u003e\n \u003cp\u003eLocation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.6035%;\" width=\"22.780569514237857%\"\u003e\n \u003cp\u003eDistal vs. Proximal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.1421%;\" width=\"7.872696817420436%\"\u003e\n \u003cp\u003e1.349\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.6375%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e1.004 - 1.812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.047\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 4.5371%;\" width=\"9.547738693467336%\"\u003e\n \u003cp\u003e1.449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e1.073 - 1.957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 3.1004%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.016\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.2424%;\" width=\"21.608040201005025%\"\u003e\n \u003cp\u003ePNI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.6035%;\" width=\"22.780569514237857%\"\u003e\n \u003cp\u003ePresence vs. Absence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.1421%;\" width=\"7.872696817420436%\"\u003e\n \u003cp\u003e2.159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.6375%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e0.984 - 4.737\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 4.5371%;\" width=\"9.547738693467336%\"\u003e\n \u003cp\u003e2.486\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e1.060 - 5.830\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 3.1004%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.036\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.2424%;\" width=\"21.608040201005025%\"\u003e\n \u003cp\u003eLN metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.6035%;\" width=\"22.780569514237857%\"\u003e\n \u003cp\u003ePresence vs. Absence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.1421%;\" width=\"7.872696817420436%\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.6375%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 4.5371%;\" width=\"9.547738693467336%\"\u003e\n \u003cp\u003e1.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e1.026 - 3.732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 3.1004%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.042\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.2424%;\" width=\"21.608040201005025%\"\u003e\n \u003cp\u003eIDO1/CD8\u003csup\u003e+\u003c/sup\u003e TIL\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.6035%;\" width=\"22.780569514237857%\"\u003e\n \u003cp\u003eIDO1\u003csup\u003ehigh\u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e vs. Others\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.1421%;\" width=\"7.872696817420436%\"\u003e\n \u003cp\u003e2.168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.6375%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e1.1 - 4.272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.025\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 4.5371%;\" width=\"9.547738693467336%\"\u003e\n \u003cp\u003e2.460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 2.7979%;\" width=\"12.73031825795645%\"\u003e\n \u003cp\u003e1.195 - 5.065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 3.1004%;\" width=\"6.365159128978225%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.015\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 43.8587%;\" width=\"100%\"\u003e\n \u003cp\u003eAbbreviations: CI: confidence interval; HR: hazard ratio; PNI, perineural invasion; LN, lymph node; TNM, tumour node metastasis; TIL, tumour-infiltrating lymphocyte.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Indoleamine 2, 3-dioxygenase; bile duct cancer; tumour infiltrating lymphocytes","lastPublishedDoi":"10.21203/rs.3.rs-743421/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-743421/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBackground:\u003c/em\u003e\u003c/strong\u003e Indoleamine 2, 3-dioxygenase 1 (IDO1) is an immunomodulatory enzyme that catalyzes the degradation of tryptophan to kynurenine (Kyn) and induces immune tolerance in tumour cells. The effects of IDO1 on extrahepatic bile duct carcinoma (EHBDC) are poorly understood. This study aimed to investigate the expression and prognostic significance of IDO1 in EHBDC. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMethods:\u003c/em\u003e\u003c/strong\u003e An immunohistochemical microarray analysis of IDO1 expression was performed for 77 surgically resected cases of EHBDC, and the results were compared with various clinicopathologic variables including survival data. CD8\u003csup\u003e+\u003c/sup\u003e tumour infiltrating lymphocytes (TILs) were also investigated to elucidate their relationship with IDO1 expression and prognosis through a combination analysis with IDO1 expression. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResults:\u003c/em\u003e\u003c/strong\u003e IDO1 was highly expressed in 25 of 76 (32.9%) cases. High expression of IDO1 was associated with decreased numbers of CD8\u003csup\u003e+\u003c/sup\u003e 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\u003csup\u003e+\u003c/sup\u003e TIL state, the IDO1\u003csup\u003ehigh \u003c/sup\u003e/CD8\u003csup\u003elow\u003c/sup\u003e subgroup showed the worst prognosis in terms of overall survival (P = 0.025, Cox risk ratio = 2.168) and disease-free survival (P = 0.015, Cox risk ratio = 2.460) in a multivariate analysis. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConclusions:\u003c/em\u003e\u003c/strong\u003e Our study confirmed that high IDO1 expression was correlated with a decreased number of CD8\u003csup\u003e+\u003c/sup\u003e TILs and associated with a poor prognosis. As IDO1 may be a new target of immunotherapy applications, IDO1/CD8\u003csup\u003e+\u003c/sup\u003e TIL subgrouping can be a useful prognostic prediction tool in the patients with EHBDC.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"The Prognostic Implications of Immune Classification Using IDO1 Expression in Extrahepatic Bile Duct Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-09 23:05:30","doi":"10.21203/rs.3.rs-743421/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7d43fb6-85fc-4726-85db-6889536345ef","owner":[],"postedDate":"August 9th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":6307315,"name":"Cancer Biology"},{"id":6307316,"name":"Oncology"}],"tags":[],"updatedAt":"2022-05-10T07:59:23+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-09 23:05:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-743421","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-743421","identity":"rs-743421","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}