Since 2010, immunotherapy has been one of the most important strategies in the treatment of malignancies, together with surgery, chemotherapy, radiotherapy, and targeted therapy, even if its efficacy is very variable, and only a percentage of patients obtain a durable response [36]. The mechanism of immunotherapy is to enhance the anti-tumor immune response, including both adaptative cells (B and T cells) and innate cells such as macrophages, neutrophils, natural killers. Immunotherapy includes immune checkpoint inhibitors (ICIs) targeting programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte antigen-4 (CTLA-4), cancer vaccines, and adoptive cell transfer (ACT). Several factors can influence the effect of immunotherapy-based treatments: the environment of tumor and immune cells, vascularization, extracellular matrix, and molecular signaling pathway [37]. Several therapeutic options in patients affected by biliary tract cancers are under investigation, such as immunotherapeutic strategies with checkpoint inhibitors, peptide- and dendritic cell-based vaccines, and adoptive cell therapy, in monotherapy or in combination with targeted therapy and/or chemotherapy. Nowadays, scientific evidence on the use of immunotherapy in CCA are limited, although different trials are currently investigating the role of anti-CTLA-4 monoclonal antibodies, the targeting of PD-L1 or its receptor, PD-1, and chimeric antigen receptor T (CAR-T) cell immunotherapy. Unfortunately, checkpoint inhibitor monotherapy has shown low efficacy in CCA patients. Indeed, Pembrolizumab, a PD-L1 inhibitor, demonstrated a median progression-free survival of 1.8 months in patients affected by CCA in the phase Ib basket trial KEYNOTE 028 [38]. Checkpoint inhibitors showed encouraging results in patients with microsatellite instability or DNA mismatch repair in the KEYNOTE 158 trial [39], even if only a small percentage of patients with a positive response to this kind of treatment reported a better clinical response [40]. Pembrolizumab demonstrated good efficacy in a recent Korean study that retrospectively analyzed 51 patients with PD-L1-positive CisGem-refractory biliary tract cancer. In PD-L1-positive patients, pembrolizumab showed durable efficacy, with a 9.8% response rate with manageable adverse events. Ongoing studies and clinical trials are currently exploring combined immunotherapeutic approaches targeting both the innate and the adaptive immune system, and/or combined strategies also involving chemotherapy or radiation.

Particularly, there are many ongoing phase I–III trials exploring the role of targeting PD-L1, its receptor PD-1, anti CTL-A4 with monoclonal antibodies in monotherapy or in combination with chemotherapy, targeted therapy, local ablative therapy, and the role of CAR-T cell immunotherapy in biliary tract cancer (Table 2 and Table 3). In particular, KEYNOTE-028 and KEYNOTE-158, two multicentric, non-randomized, open-label, phase IB and II trials, showed a durable antitumor activity of Pembrolizumab in 6–13% of patients with advanced BTC. In KEYNOTE-158, they observed a median progression free survival (PFS) of 2.0 months and a Median overall survival (OS) of 7.4 months; adverse events were mainly mild to moderate in severity [39]. Another immunotherapeutic agent, Nivolumab showed a response rate of 22% and a disease control rate of 59% in a Phase II multi-institutional study including 46 patients affected by advanced biliary tract cancer in second-line therapy [41].

The combination of immunotherapy and chemotherapy looks promising. Two Phase III trials are evaluating the efficacy and safety of KN035 plus Gemcitabine–Oxaliplatin compared to standard of care Gemcitabine–Oxaliplatin therapy (NCT03478488) and the association of Durvalumab and Gemcitabine plus cisplatin (NCT03875235). BilT-01, a multicenter randomized Phase II trial, described a prolonged PFS six months after the addition of nivolumab to gemcitabine and cisplatin (NCT02829918) [42]. LEAP 005 demonstrated a promising antitumor activity and manageable toxicity of Pembrolizumab in combination with Lenvatinib in 31 patients affected by BTC [43].

Regarding Adoptive Cell Therapy (ACT), a phase III, non-randomized trial is studying the role of cytokine-induced killer cells in association with radiofrequency ablation in 50 patients with CCA (NCT02482454).

Within the CCA clinical-pathological spectrum, the pattern of tumor growth has been correlated with specific histological features, e.g., small-bile duct iCCAs and cholangiolocarcinoma (CLC) showed a mass-forming growth pattern, while large-bile duct iCCAs showed both a mass-forming growth pattern and a combination of a mass-forming growth pattern with a periductal infiltrative growth pattern, the latter being the typical pattern of growth of pCCA [44]. Mass-forming iCCAs showed more heterogeneous clinical-pathological characteristics than other gross types [45]. Radiologically, at dynamic contrast-enhanced imaging, all large-bile duct iCCAs showed concentric filling at the venous phase, whereas small-bile duct iCCAs/CLCs showed washout in various patterns, in a clinical-pathological study including correlates with magnetic resonance imaging [44].

The USA Food and Drug Administration approved the use of pembrolizumab for patients with advanced solid tumors lacking the expression of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, and PMS2) or having high microsatellite instability (MSI-H) [46]. MMR proteins can be inactivated through somatic or germline mutations or they can be silenced through promoter hypermethylation, e.g., of the MLH1 gene [47]. These alterations culminate to hypermutation during DNA replication (MSI) and may lead to the development of malignancies [48]. Interestingly, such molecular alterations predispose to an increase of the neoantigen load of the tumor, promoting susceptibility to immunotherapies targeting the PD-1 pathway because of the increased inflammation surrounding these tumors [40].

Given the potential for immunotherapy in patients with CCA, authors studied the expression of PD-L1/PD-1 and evaluated the presence of associated genetic alterations. For example, in 652 biliary tract cancers that comprised 77 p/dCCA, 372 iCCA, and 203 gallbladder cancer (GBC), 8.6% tumors were PD-L1-positive [GBC 12.3% (25/203), iCCA 7.3% (27/372), and p/dCCA 5.2% (4/77)]. Interestingly, there was an increase in BRAF, BRCA2, RNF43, and TP53 mutations in the PD-L1-positive group with respect to the PD-L1-negative one. Furthermore, there was an association between PD-L1 expression and certain biomarkers (TOP2A, TMB high, MSI-H). As noted by the authors, the aforementioned combinations of molecular alterations might direct the use of rational combination strategies and clinical trial development [49]. On the same line, Ju et al. analyzed 96 cases of CCA for morphology using H&E staining and for mutations of MMR genes using immunohistochemical staining. The authors found that 6% of the samples showed MMR deficiency (MMR-d). Divided by location, 10% (3 of 31) of iCCA and 5% (3 of 65) of p/dCCA were MMR-d. The best predictive factor for MMR-d was a nontypical infiltrating pattern of invasion [50].

The increasing awareness of CCA heterogeneity at the morphological and molecular levels, together with the advent of radiomic, artificial intelligence (AI), and machine learning, has revitalized the study of radiological correlates. For example, it has been shown that the magnetic resonance imaging texture signature, including three wavelets and one 3D feature, has the ability to discriminate inflamed from non-inflamed immunophenotypes based on the density of CD8+ T cells. This may be a surrogate of the response to immune checkpoint blockade [51]. The preoperative prediction of PD-1/PD-L1 expression and outcome in iCCA patients using magnetic resonance biomarkers and a machine learning approach has been attempted [52]. Utilizing qualitative and quantitative imaging traits, reasonable accuracy in predicting tumor grade and higher AJCC stage in iCCA has been shown [53].