Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy after hepatocellular carcinoma. The overall incidence of cholangiocarcinoma has increased progressively worldwide.1,2 Prognoses of ICC are poor. In fact, most patients have advanced-stage disease at presentation, and diagnosing ICC at an early stage remains challenging. Although the Union for International Cancer Control (UICC) staging system predicts the prognosis for ICC, some additional or alternate prognostic factors are desirable, because the capability of predicting the survival of individual patients is limited.3
Tumor budding, which has been designated previously as sprouting, is a morphologic characteristic of carcinomas. The definition of tumor budding is as a single cell or clusters of up to 4 cells at the invasive margin in colorectal cancer.4 Tumor budding is an independent adverse prognostic factor and associated with a higher UICC stage, high tumor grade, lymphovascular invasion, and lymph node metastasis in colorectal cancer. The International Tumor Budding Consensus Conference (ITBCC) was held in Bern in April 2016, and the scoring system was proposed for colorectal carcinoma.5 Tumor budding has been reported further as an independent adverse prognostic factor in oral squamous cell carcinoma,6 esophageal squamous cell carcinoma,7 ampullary carcinoma,8 intestinal-type gastric adenocarcinoma,9 bladder cancer,10 breast carcinoma,11 and pancreatic ductal adenocarcinoma.12
We have frequently encountered findings of tumor budding during routine examinations of surgically resected cholangiocarcinoma specimens. Therefore, for the present study, we evaluated the significance of tumor budding, especially in terms of its predictive power for the postsurgical prognosis for patients with ICC. Cholangiocarcinoma, which arises from different anatomic locations, is classified as intrahepatic, perihilar cholangiocarcinoma, and extrahepatic cholangiocarcinoma (ECC), according to the staging of the American Joint Committee on Cancer (AJCC) and the UICC system.13 We also analyze the frequencies and meanings of tumor budding in perihilar cholangiocarcinoma and ECC for comparison with ICC.
MATERIALS AND METHODS
From the archives of the University of Tokyo Hospital pathology, we reviewed 107 consecutive treated cases of ICC (1995 to 2014), 54 cases of perihilar cholangiocarcinoma (1998 to 2007), and 40 cases of ECC (1998 to 2007). The histologic diagnosis of each lesion was based on the World Health Organization classification.2 Tumor staging was based on UICC TNM Classification of Malignant Tumours, 8th edition.13 Tumor location was classified by the main tumor region (the largest volume and the deepest invasion area). The ICC cases were the mass-forming type or the mass-forming with periductal infiltrative type and were located at the peripheral site of the second-order bile duct. Almost all ICC cases were examined in our previous study.14 The perihilar cholangiocarcinoma and ECC cases showed flat, nodular, or papillary growth pattern. Carcinomas that were clearly associated with intraductal papillary neoplasm of the bile duct (IPN-B) and papillary predominate growth–type ICC were excluded from the study because differentiation between the predominant intraductal papillary growth–type ICC and IPN-B is confusing. Moreover, the therapeutic strategies used for both tumors are expected to be different from that used for conventional cholangiocarcinoma. Positive surgical margin denotes positive for either carcinoma in situ or invasive carcinoma at the surgical stump. The metabolic syndrome was examined using the criteria for the Japanese population.15
The clinicopathologic statuses of ICC cases had the following characteristics: age: 67 years (median) (33 to 88 y); sex: male (51) and female (56); hepatitis B virus–positive (9/107); hepatitis C virus–positive (24/107); tumor size: 45 mm (median) (8 to 122); UICC stage: I to II (73), III to IV (34); poorly differentiated histology (9/107); hilar invasion positive (41/107); major vascular invasion positive (16/107); lymph node metastasis positive (25/107); and surgical margin positive (22/100). The clinicopathologic status of perihilar cholangiocarcinoma cases is as follows: age: 68.5 years (median) (34 to 78 y); sex: male (39) and female (15); tumor size: 37.5 mm (median) (14 to 95 mm); UICC stage: I to II (26), III to IV (28); poorly differentiated histology (13/54); lymphatic invasion positive (37/54); vascular invasion positive (47/54); lymph node metastasis positive (24/54); and surgical margin positive (19/54). The clinicopathologic status of ECC cases was as follows: age: 68.5 years (median) (54 to 81 y), sex: male (27) and female (13); tumor size: 30 mm (median) (12 to 90 mm); UICC stage: I to II (30), III to IV (10); poorly differentiated histology (9/38); lymphatic invasion positive (27/40); vascular invasion positive (36/40); lymph node metastasis positive (22/40); and surgical margin positive (5/40).
All aspects of this study were approved by The University of Tokyo Ethics Committee. Pathology reports and tissue slides were reviewed along with medical charts when necessary. Recurrent cases and cases with neoadjuvant treatment were excluded.
The tumor of the surgically resected specimen was fixed in 10% formalin at room temperature and was sectioned at intervals of 0.5 to 1.0 cm, with all tumor-containing sections processed routinely and embedded in paraffin. Serial sections of each tumor were cut and were stained with hematoxylin and eosin.
Tumor budding was sought in all tissue slides of each carcinoma. Tumor budding was identified on the basis of ITBCC20165 as a single tumor cell or a cell cluster of up to 4 tumor cells on hematoxylin and eosin staining (Figs. 1A, B), in 1 hotspot, in a field measuring 0.785 mm2 (objective magnification, ×20). It was graded as low (0 to 4 buddings), intermediate (5 to 9 buddings), or high (≥10 buddings). We defined intermediate or high grade as tumor-budding–positive cases. We assessed tumor budding in whole tumor areas in cholangiocarcinoma rather than at the invasive front because the determination of the front was frequently difficult. All specimens were reviewed independently by 2 pathologists (M.T. and N.Y.). The concordance rate was 94.5%. As regards disagreement cases, these pathologists discussed the findings and reached a consensus.
Subtyping of ICC as type 1 and type 214 was based on an assessment of the following 3 factors: mucin productivity, S100P immunoreactivity score, and combined scores of N-cadherin and neural cell adhesion molecule immunoreactivity. Typically, type 1 cases showed mucin production and diffuse immunoreactivity to S100P. Type 2 cases showed little mucin production and immunoreactivity to N-cadherin or neural cell adhesion molecule.
Categorical variables were compared using chi-square test or the Fisher exact test with software (JMP Pro 13.0; SAS Institute Inc., Cary, NC). Results for which P-value <0.05 were inferred as significant. Recurrence-free survival (RFS) and overall survival (OS) curves were constructed using the Kaplan-Meier method and were compared using the log-rank test with software (JMP Pro 13.0). Univariate and multivariate Cox regression analyses were applied to ascertain the prognostic factors using EZR software (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html; Saitama Medical Center, Jichi Medical University). The clinicopathologic factors that were included in the statistical analyses were age, sex, liver cirrhosis, biliary disease, alcohol use, smoking habit, cirrhosis, tumor size, UICC stage, histology, lymphovascular invasion, lymph node metastasis, and surgical margin. In ICC, hepatitis virus status, hilar invasion, and intrahepatic metastasis were also included. These factors might be important to develop an accurate prognosis of the ICC patients.16–18 Intraductal spread was included in the evaluation of ECC because carcinoma in situ is sometimes met at the surgical stump of ECC.19 As regards perihilar cholangiocarcinoma and ECC, liver tissue was not available in 32 cases (hepatectomy was not performed in these cases). Among hepatectomy cases, no cirrhosis was found. Consequently, we did not include the cirrhosis factor in the statistical analyses in perihilar cholangiocarcinoma and ECC. The factors of multivariate Cox regression analyses were selected in reference to the following items: clinicopathologic significance, the value of hazard ratio of univariate analysis, confounding statistics, and the number of events (number of factors=number of events/10).
Tumor budding was evaluated in the entire cholangiocarcinoma tumor area (Figs. 1A, B). The frequency of tumor-budding–positive cases was lowest in ICC (Fig. 1C): 40.2% in ICC (43/107), 70.4% in perihilar cholangiocarcinoma (38/54), and 60.0% in ECC (24/40) (ICC vs. perihilar cholangiocarcinoma, P<0.001, ICC vs. ECC, P=0.029).
Clinicopathologic Features of Tumor-budding–positive ICC
To assess the clinical significance of tumor budding, we compared the clinicopathologic features of tumor-budding–positive and tumor-budding–negative cases of ICC (Table 1).
The ICC cases with tumor budding showed significant association with many clinicopathologic factors. Tumor budding was associated with advanced UICC stage (P=0.007). Microscopically, tumor-budding–positive cases more frequently showed poorly differentiated histology (P=0.016). Significant correlation was also observed with pathologic factors such as hepatic hilar connective tissue invasion (P=0.008), major vascular invasion (P=0.012), and lymph node metastasis (P=0.001). More cases of noncurative resection occurred among tumor-budding–positive cases (P=0.010).
Prognostic Significance of Tumor Budding in ICC
Results of survival analyses using Kaplan-Meier curves are depicted in Figure 2 and univariate and multivariate Cox regression analyses were conducted to identify prognostic factors of ICC.
Tumor-budding–positive cases were associated with shorter RFS and OS than tumor-budding–negative cases (P<0.05). The median times to recurrence in budding-positive and budding-negative cases were 10.26 and 35.57 months, and the median survival times were 18.90 and 106.03 months, respectively (P<0.001). No significance was found between intermediate-grade budding-positive cases and high-grade budding-positive cases (Supplementary Fig. 1, Supplemental Digital Content 1, http://links.lww.com/PAS/A819).
The results of univariate and multivariate analyses are presented in Table 2. Significant prognostic factors for RFS by univariate Cox regression analyses were tumor budding, tumor size, advanced UICC stage, hepatic hilar invasion, major vascular invasion, intrahepatic metastasis, and lymph node metastasis. By multivariate analyses, tumor budding was significant for RFS (hazard ratio [HR]: 3.038; 95% confidence interval [CI]: 1.591-5.973) and major vascular invasion (HR: 5.698; 95% CI: 1.665-19.490). Significant prognostic factors for OS were tumor budding, advanced UICC stage, hepatic hilar invasion, poorly differentiated tumor histology, major vascular invasion, intrahepatic metastasis, lymph node metastasis, and noncurative surgical margin by univariate analyses. Multivariate analysis demonstrated significant factors to be tumor budding (HR: 4.547; 95% CI: 2.348-8.805), advanced UICC stage (HR: 3.591; 95% CI: 1.419-9.083), intrahepatic metastasis (HR: 3.593; 95% CI: 1.838-7.026), and noncurative surgical margin (HR: 2.311; 95% CI: 1.039-5.141).
Tumor Budding Meaning Based on Anatomic Tumor Location
Because the frequencies of tumor budding for ICC, perihilar cholangiocarcinoma, and ECC differ greatly, we evaluated the clinicopathologic and prognostic significance of tumor budding occurring with perihilar cholangiocarcinoma and ECC. Compared with ICC, the correlative clinicopathologic factors were limited in both perihilar cholangiocarcinoma and ECC (Table 3). For perihilar cholangiocarcinoma, advanced UICC stage and lymphatic vessel invasion were observed more frequently in tumor-budding–positive cases (P=0.049 and 0.001, respectively). For ECC, only vascular invasion was correlated with tumor budding (P=0.020).
Tumor budding was an important prognostic factor for perihilar cholangiocarcinoma, but not for ECC (Fig. 2,Tables 4, 5). For perihilar cholangiocarcinoma, tumor budding and lymph node metastasis were prognostically significant for RFS by univariate analyses, and tumor budding (HR: 7.410; 95% CI: 1.656-33.150) and lymph node metastasis (HR: 3.687; 95% CI: 1.430-9.507) by multivariate analyses. The prognostic factors for OS were tumor budding, lymphatic vessel invasion, and lymph node metastasis by univariate analyses, and tumor budding (HR: 5.833; 95% CI: 2.486-13.690), lymph node metastasis (HR: 3.453; 95% CI: 1.675-7.115), and noncurative surgical margin (HR: 2.356; 95% CI: 1.160-4.783) were significant by multivariate analyses.
For ECC, tumor-budding–positive cases were not associated with worse RFS (Table 5). The prognostic factors for RFS were lymph-vessel invasion and lymph node metastasis by univariate analysis. Lymph node metastasis was only prognostically significant for RFS (HR: 6.692; 95% CI: 2.071-21.620) by multivariate analysis. Advanced UICC stage and lymph node metastasis were important for OS by univariate analysis, and tumor budding did not show statistical significance for OS (P=0.05035). Lymph node metastasis was the only significant prognostic factor (HR: 9.729; 95% CI: 2.150-44.020) by multivariate analysis.
Type 1 and Type 2 Subgroups of ICC
When ICC was divided into 2 subtypes, type 1 and type 2, the frequencies of tumor-budding–positive cases were 65.9% in type 1 (29/44) and 17.2% in type 2 (10/58). Tumor budding was found to be a strong prognostic factor for OS in type 2 ICC by univariate analysis (HR: 6.139; 95% CI: 2.600-14.490) and by multivariate analysis (HR: 3.216; 95% CI: 1.092-9474) (Table 6). However, the prognostic impact of tumor budding was not significant in type 1 ICC by univariate or multivariate analyses.
The present study is the first one to analyze the association between tumor budding and clinicopathologic features in cholangiocarcinoma, with a special focus on ICC. The frequency and the prognostic significance of tumor budding were found to be dependent on the biliary tract tumor location. Tumor budding was present in about 40% of ICC, 70% of perihilar cholangiocarcinoma, and 60% of ECC cases and was a powerful prognostic factor for ICC and perihilar cholangiocarcinoma, but not for ECC.
For ICC, tumor budding was a powerful and the strongest prognostic factor for OS (HR: 4.547). Other factors were advanced UICC stage, intrahepatic metastasis, and noncurative surgical margin. Tumor budding in ICC showed correlation with some factors associated with local invasion, such as advanced UICC stage, hilar invasion, major vascular invasion, and surgical margin. These facts suggest a possibility that tumor-budding affects the behavior of ICC, like perihilar cholangiocarcinoma, through invasion to the hilar region, which consists of important landmarks, such as a large hepatic artery, portal vein, and peripheral nerve bundle. In the present comparative study, tumor budding was the strongest prognostic factor in RFS (HR: 7.410) and OS (HR: 5.833) in perihilar cholangiocarcinoma. Recently, several research groups, including ours, proposed dividing ICC into 2 subtypes, such as large duct type/small duct type, type 1/type 2, perihilar type/peripheral type, and bile duct type/cholangiolar type, because these 2 subtypes showed different clinicopathologic behavior.14,20–22 Moreover, some reports have described that histologic and molecular characterization between large duct type and perihilar cholangiocarcinoma are apparently similar.23,24 However, in the present study, the significance of tumor budding was different between type 1 ICC, and perihilar cholangiocarcinoma, suggesting that different mechanisms determine the worse prognosis by tumor budding in each type of carcinoma.
It is noteworthy that intrahepatic metastasis, independent of tumor budding, was the second strong prognostic factor for OS of ICC. Tumor budding is linked to the presence of distant metastasis in colorectal cancer,25,26 but not in pancreatic carcinoma.27 Tumor budding is believed to be based partially on epithelial-mesenchymal transition.28,29 However, to establish the metastatic site, mesenchymal-epithelial transition is necessary in addition to epithelial-mesenchymal transition. Vaeteewoottacharn et al30 demonstrated that attenuation of CD47-SIRPa signal suppresses intrahepatic metastasis of cholangiocarcinoma through activation of tumor-associated macrophage-mediated phagocytosis. As an inflammatory microenvironment might be important for intrahepatic metastasis of ICC,29 further studies must be conducted to clarify the mechanisms underlying both tumor budding and intrahepatic metastasis in ICC.
Unlike ICC, tumor budding seemed not to be a reliable prognostic factor in ECC cases. No obvious anatomic structure, except for the duodenum and pancreas, is shown near the distal bile duct in ECC. For that reason, tumor budding cannot be a powerful prognostic factor. Moreover, in perihilar cholangiocarcinoma and ECC, tumor budding is not associated with lymph node metastasis, which is a prognostic factor. These facts might reflect biological differences between perihilar cholangiocarcinoma/ECC and ICC. Ogino et al31 described recently that higher tumor-budding grade was associated with adverse prognosis among 195 perihilar cholangiocarcinoma cases and 115 ECC cases. However, their P-value of multivariate analysis of ECC was 0.046, which requires further verification. The rate of tumor-budding–positive cases was higher in Ogino cohort (92/115, 82.6%) than in ours (60%). Moreover, the cohort of Ogino study is apparently less advanced than our cohort. This difference of cohorts might have engendered different results. The ECC cases examined in our study were few. Therefore, further validation might be necessary to infer the effects of tumor budding in perihilar cholangiocarcinoma and ECC.
Tumor budding is an independent adverse prognostic factor in several cancers, especially in colorectal cancer; however, a wide variety of different methods have been applied to count tumor budding. The primary objective of the ITBCC took place in Bern in April 2016.5 For this study, we evaluated tumor budding on the basis of the ITBCC method, with the exception that we adopted the whole tumor area for the evaluation. Intratumoral budding was present in colorectal cancers,32,33 in pancreatic cancer,12,34,35 and in breast cancer.36 They defined intratumor budding using the same criteria as for tumor budding at the invasive front. Lugli et al37 showed intratumoral budding as an independent prognostic factor in mismatch repair-proficient and mismatch repair-deficient colorectal cancer patients. Giger et al26 demonstrated that intratumoral budding is useful for the prediction of metastasis in the preoperative biopsy sample of nonrectal colon cancer.
This study has several limitations. Our cases did not include intraductal growth–type ICC and cancer associated with IPN-B, and the number of cases were relatively small, as described above.
In summary, we clarified that tumor budding was associated with poor outcomes in ICC. We also were able to ascertain the different characteristics of cholangiocarcinoma by tumor location. Tumor budding is an easy-to-use, cost-effective pathologic evaluation for ICC.
The authors thank all members of the Department of Pathology of The University of Tokyo, especially Kei Sakuma.
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cholangiocarcinoma; intrahepatic; tumor budding; prognosis; type
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