Cholangiocarcinoma (CC) is a malignant neoplasm originating from epithelial cells lining the biliary tract. CC is divided into intrahepatic CC (iCC) and extrahepatic CC (eCC) depending on its anatomical location. Over the past few decades, the overall incidence of CC, particularly iCC, has gradually increased worldwide [1]. CC is associated with a poor prognosis, with a 5-year survival rate of 10%–49% for iCC and 20%–40% for eCC [2]. However, the prognostic factors for CC remained poorly understood.

Tumor budding (TB) is defined as a single tumor cell or a cluster of fewer than 5 cells that are observed separately from the tumor mass exhibiting invasion [3]. TB is a histopathological phenomenon of the epithelial-mesenchymal transition (EMT), the process by which epithelial cells are transformed into mesenchymal stem cells. During this process, the cells tend to migrate and invade instead of losing cell polarity and intercellular adhesion. EMT is a crucial developmental process; however, in the case of solid tumors, it promotes tumor growth by increasing invasive and metastatic activity. Since Imai et al. [4] mentioned it first, TB has been actively investigated mainly in patient with colorectal cancer. TB is associated with lymph node metastasis, distant metastasis, local recurrence, and poor prognosis regardless of the TNM stage in patients with colorectal cancer [5]. In 2016, a consensus was reached to objectively and accurately measure TB in patients with colorectal cancer [6]. By introducing this concept, studies on the prognostic significance of TB in various cancer types, such as pancreatic, gallbladder, esophageal, and gastric cancers, have been conducted [7–10]. Although relevant studies have recently been performed to evaluate the importance of TB in CC, the number remains limited, and only 10 papers have been published to date (as of March 2023) [11–19].

Hence, we aimed to investigate the relationship between TB and clinicopathological parameters and the prognostic significance of TB in iCC and eCCs.

Our findings suggested that a higher TB was related to factors indicating aggressiveness, such as lymphatic invasion, higher tumor grade, and higher N category. In patients who underwent R0 resection, the higher TB group showed a shorter DFS compared with that of the lower TB group.

TB is an EMT phenotype. Anchorage-dependent cells undergo a programmed cell death process, called anoikis, when separated from the surrounding extracellular matrix. EMT provides the characteristics of stem cells, while destroying the intercellular junctions and avoiding anoikis in the tumor microenvironment, thus resulting in the acquisition of mobility and invasive phenotypes. TB cells in colorectal and gastric cancers overexpress tyrosine kinase receptor B (TrkB), an indicator of anoikis resistance, which allows TB cells to survive [21, 22]. Surviving TB cells are inevitably associated with invasiveness. Microscopically, TB cells are often observed near the lymphatic or blood vessels, which suggests the possibility of intravasation and spreading to distant tissues [22]. Lugli et al. suggested that TB is a good indicator of aggressiveness in rectal cancer, demonstrating that it is associated with the number of involved nodes, extramural spread, lymphocytic infiltration, and tumor differentiation [3]. Okudo et al. reported that TB occurs more frequently observed in the cases of poor differentiation at the invasive front of biliary tract cancer [16]. Agostini-Vulaj et al. also reported that TB is associated with high tumor grade, lymphovascular invasion, and perineural invasion in iCC and eCCs [11]. In this study, TB was significantly associated with lymphatic invasion, and higher tumor grade and N category. Higher TB was associated with the presence of venous and perineural invasion but did not show statistical significance in this study.

Okudo et al. are the first team to evaluate the prognostic impact of TB in the CC [16]. They demonstrated that the ≧5 TB foci were as an independent predictor in overall survival. Ogino et al. also reported that patients with perihilar and distal duct CC who had a high TB grade had a short postoperative overall survival [15]. Ito et al. reported that high TB was correlated with poor DSS in patients with perihilar CC; moreover, the DSS rate in the high TB group was similar to that of the group without resection [12]. In this study, DSS was not correlated with TB in the entire cohort. However, the DFS was significantly associated with TB in patients who underwent R0 resection.

All studies investigating TB in patients with CC published thus far graded the TB based on ITBCC scoring system [11–15, 17–19, 23]. Most of these studies classified TB into low and intermediate/high groups based on the cut-off value of 5 [11, 12, 14, 15, 18]. Conversely, Kosaka et al. reported a difference in the survival outcomes between low/intermediate and high TB groups, using a cut-off value of 10 in their multicenter study [13]. As in Kosaka’s study, we divided patients with TB into low/intermediate and high groups. Findings from previous studies and the present study suggest that the ITBCC method is appropriate for TB cell counting in cholangiocarcinoma. Higher TB levels were correlated with worse survival. However, few studies had revealed the correlation between TB and survival when patients with TB were divided into three groups according to the ITBCC scoring system. Previous studies had demonstrated a good correlation with survival when TB was divided into two groups based on a cut-off value of 5 or 10. Therefore, for CC, further investigation is required to confirm whether the ITBCC scoring system should be applied as it is or whether a revised system should be established.

Recently, Zlobec et al. proposed the revised ITBCC scoring system [24]. They reported that dividing TB into four categories, including “zero-buds,” is more informative on tumor behavior than the previous ITBCC scoring system. This study included 41 (18.7%) patients with zero buds. However, in this study, there was no difference between DFS and DSS in the zero-buds and lower-TB (1–9) groups (Supplementary Material S1).

Ogino et al. reported differences in prognostic stratification with TB in perihilar CC and distal CC [15]. These differences were in term of anatomical location and histological characteristics; for example, the connective tissue surrounding the proximal bile duct is less dense than the distal bile duct. On the contrary, Agostini-Vulaj et al. reported that TB occurred more in eCC than iCC [11]. In this study, there was no difference in the frequency of TB according to the anatomical location. Therefore, one cannot conclude on the difference in the frequency of TB.

This study has several limitations. First, we investigated the eCC and iCC, including large- and small-duct types. CCs may have varying carcinogenic mechanisms depending on their location. Particularly, in iCC, the large duct type is similar to perihilar CC, while the small duct type is similar to the adenocarcinoma component of combined hepatocellular-cholangiocarcinoma. However, considering these differences, the number of cases in each location was insufficient to conduct this study according to the tumor location. Second, patients who had incomplete resection accounted for over half of the cohort in this study. Therefore, the data used for analyzing the DFS were limited. Hence, further investigations are required to address these limitations.

In conclusion, our findings suggested that higher TB levels in patients with CC are associated with aggressiveness and worse DFS. Moreover, TB may have prognostic implications in CC.