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Cadamuro et al. Hepatoma Res 2022;8:11 https://dx.doi.org/10.20517/2394-5079.2021.140 Page 9 of 16

Furthermore, in eCCA, high PD-1 expression appears to correlate with increased lymphatic metastases and
[92]
lower patient survival . Only one study evaluated the role of CTLA-4 expression as a prognostic indicator
in CCA, demonstrating that, in eCCA, a high CTLA-4 H-score predicts better overall and disease-free
[93]
survival .

Immunosuppressive tumor microenvironment
It is becoming clear that TME can generate an immunosuppressive environment and confer to the tumor
cell a survival advantage by inducing tumor immune evasion. This is likely one of the main mechanisms
responsible for the still disappointing results of immunotherapy in CCA [3,12] . For example, secretion of CCL2
by cells populating the TME, such as CAFs and tumor cells, leads to the enrichment of Tregs and of
MDSCs. MDSCs in turn secrete ROS and other immunomodulatory compounds able to repress the activity
of cytotoxic T cells (in particular CD8 T cells) and NK cells. CAFs within the TRS of CCA secrete CXCL12,
+
which may prevent migration of T cells .
[94]

The effects of Tregs and their contribution to the pathogenesis of CCA, as well as their relevance as a
prognostic index of CCA, are still debated. Data from two cohorts of patient with eCCA indicate the
presence of Tregs as an indicator of poor outcome , while other papers identify their over-representation
[63]
as a positive prognostic factor . From a biological point of view, Tregs secrete immunosuppressive
[76]
mediators, such as IL10 and TGFβ, and further depress the antitumoral activity of CD8 T and NK cells [95,96] .
A subset of Tregs, forkhead box P3 (FoxP3) CD25 , bind IL-2, reducing the IL-2-mediated activation of the
+
+
[95]
immune milieu . The accumulation of FoxP3-positive Tregs is a distinctive trait of CCAs, showing worst
outcome and greater tendency to lymphocyte metastasis [63,66] . Moreover, the accumulation of FoxP3 Tregs is
+
accompanied by an increase in expression of CTLA-4, also an indicator of poor outcome .
[97]
IMMUNOTHERAPEUTIC STRATEGIES TO TREAT CCA
To date, only pembrolizumab (an anti-PD-1 monoclonal antibody) is an approved treatment in CCA.
Several other drugs and methods that exploit an immunotherapeutic approach are under evaluation. These
studies are summarized in Table 2.

Immune checkpoint inhibitors
Although the theoretical premises for the use of immune checkpoint inhibitors for the treatment of CCA
are solid, the expectations were not fulfilled. The first clinical trials gave encouraging results, especially in
patients with microsatellite instability due to mismatch repair deficiency. In these patients, the use of
pembrolizumab, a humanized anti-PD-1 antibody (NCT01876511), gave a good response, with one patient
in remission and three with stable disease . Moreover, the analysis of five single-arm open-label clinical
[98]
trials (KEYNOTE-012, -016, -028, -158, and -164) demonstrated an overall response rate (ORR) of 78%.
However, these results were not confirmed in a larger study on 104 patients, KEYNOTE-158
(NCT02628067), in which the ORR was 5.8%. A clinical trial using nivolumab, an anti-PD-1 antibody
(NCT02829918), failed to show favorable results . CTLA-4 inhibitors are little studied in CCA and mostly
[99]
in combined treatments in the hope to increase their efficacy. Some clinical trials have studied the
combination of treatment with nivolumab and ipilimumab, an anti-CTLA-4 antibody (NCT02834013 and
NCT02923934), in advanced solid tumors. Treatment of 39 patients showed an ORR of 23% and a disease
[100]
control rate (DCR) of 44%, but with an overall survival (OS) of only 5.7 months . The use of anti-CTLA-4
antibodies with anti-PD-1L antibodies is currently being evaluated in a phase II clinical trial
(NCT04634058) whose results are not yet available. Pembrolizumab was also used in combination with
levatinib, a tyrosine kinase inhibitor active on VEGFR1, VEGFR2, and VEGFR3, giving an ORR of 25% and
a DCR of 78% . Currently, a clinical trial for advanced CCA is in the recruitment phase (NCT04550624).
[101]