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Cholangiocarcinoma (CCA) is a rare primary liver cancer associated with high mortality and few systemic chemotherapy options. The behavior of the immune system has come into focus as a potential treatment modality for many cancer types, but immunotherapy has yet to dramatically alter the treatment paradigm of CCA as it has for other diseases. Here, we review recent studies describing the relevance of the tumor immune microenvironment (TIME) in CCA. Various non-parenchymal cell types are critically important in controlling CCA progression, prognosis, and response to systemic therapy. Knowledge of the behavior of these leukocytes can assist in hypothesis generation for testing potential immune-directed therapies. Recently, an immunotherapy-containing combination was approved for the treatment of advanced stage CCA. However, the level 1 evidence demonstrating improved efficacy of this therapy still demonstrated suboptimal survival. In the current manuscript, we provide a comprehensive review of TIME in CCA, preclinical studies of immunotherapies against CCA, as well as ongoing clinical trials applying immunotherapies for the treatment of CCA. Particular emphasis is placed on microsatellite unstable tumors, a rare CCA subtype that demonstrates heightened sensitivity to approved checkpoint blockade immunotherapy. We also discuss the challenges involved in immunotherapies against CCA and the key roles of understanding the TIME with the goal to uncovering efficacy in future treatments for CCA.
XL and BG contributed to this paper with conception, literature review and writing. CX participated in literature review and revision. CL and XC participated in drafting, critical revision and editing. All the authors approved the final version of this manuscript.
Conflict of interest
The authors declare no conflict of interest.
Financial Support
This work is supported by NIH grant R01CA239251 to XC, the National Natural Science Foundation of China (82202981) to XL.
Accumulating data from clinical studies showed immunotherapy had manageable toxicity and safety in CCA patients.
2.
Currently the overall therapeutic benefit of immunotherapy for CCA is still very limited.
3.
Profiling the landscape of the CCA immune microenvironment will provide new insights into developing novel immune targeting therapy or combination therapy for CCA treatment.
4.
This review discusses the major challenges in CCA immunotherapies such as distinct anatomic sites of CCA, difficulties of clinical trials due to its low incident rate, and lack of adequate experimental models for basic and translational studies. The review also points to the major directions for future CCA research.
Introduction
Cholangiocarcinoma (CCA) is the second most common primary liver cancer type and an aggressive malignancy associated with poor prognosis.
. Most CCA patients are diagnosed at an advanced stage and with limited therapeutic options. The curative surgical treatment is limited to a small subset of patients with early-stage tumors. The first line therapy for unresectable CCA is either gemcitabine plus platin-based chemotherapy
though the efficacy and response rate from both of regimens are suboptimal. Several targeted therapeutic agents have been approved for a minority of cases at the second line setting, including pemigatinib and futibatinib for FGFR2-rearranged CCA as well as ivosidenib for IDH1 mutated CCA.
. Therefore, novel treatment strategies for this lethal tumor are in urgent need, since the overall prognosis for patients with CCA is very poor, with a median survival of less than 1 year.
Immunotherapy has been the major breakthrough of cancer research in the last decade, with many promising applications still being discovered. The ability of the immune system to recognize non-self tumor components is often inhibited by a variety of cancer intrinsic mechanisms that promote immune evasion. One prominent reason is the exhaustion of activated lymphocytes typified by upregulation of inhibitory markers, including programmed cell death protein 1 (PD1), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and T cell immunoglobulin domain and mucin domain-3 (TIM3). Tumor cells, as well as the surrounding stromal cells, often express or secrete the ligands of these inhibitory proteins, including programmed cell death 1 ligand 1 (PD-L1). Secreted inhibitory cytokines such as vascular endothelial growth factor (VEGF) or transforming growth factor beta (TGF−β) further inhibit the activation of lymphocytes. The principle of first-generation immune checkpoint inhibitors (ICIs) is to reinvigorate the potential of the host immune system to target and eradicate malignant cells. It has been proven to be an effective strategy used as single agents or a combination of different checkpoint inhibitors for multiple common epithelial tumor types, including NSCLC, colorectal adenocarcinoma, and, despite a generally immunosuppressed microenvironment, advanced hepatocellular carcinoma (HCC).
Numerous efforts have been made to profile the immune microenvironment landscape of CCA to identify potential targets for traditional immunotherapy. Additionally, accumulating evidence of promising preclinical studies and preliminary clinical data suggest that “second-generation” checkpoint inhibition or cellular-based immunotherapies might be effective against CCA.
. Here, we review the landscape of the CCA tumor immune microenvironment (TIME) and discuss the current and emerging progress of CCA immunotherapy.
The landscape of CCA tumor immune microenvironment
CCAs are adenocarcinomas arising from the biliary cells, although it has been reported that the tumors may also originate from hepatic stem cells or mature hepatocytes.
. A key histological feature of CCA is that tumor cells are often surrounded with dense desmoplasia populated by cancer-associated fibroblasts. It has been reported that the fibrotic tumor microenvironment, plus the infiltrated innate immune cells, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), facilitate the immunosuppressive TIME of the CCA (Figure 1 and table 1).
. Recent high-throughput genomic and transcriptomic analyses, as well as single cell RNA sequencing (scRNAseq) studies, have assisted to define a comprehensive landscape of the genetic, as well as the TIME, components of CCA.
MAITs are cytotoxic innate-like T cells, its infiltration in tumors positively correlates with favorable anti-tumor immune response and predicts long-term survival
To understand the potential for therapeutics that modulate certain components of the TIME, here we survey different pre-clinical studies on each immune cell type in the liver with and without CCA. In general, the liver is traditionally considered to be an immune-privileged organ.
. The immunosuppressive microenvironment in the liver is regulated by innate lymphoid cells, regulatory T cells (Tregs), dendritic cells (DCs), macrophages/Kupffer cells, and MDSCs, and pro-/anti-inflammatory cytokines, to prevent excessive immune response to pathogen-related and damage-related molecular patterns derived from microorganisms absorbed via the intestine.
. Although this immunosuppressive microenvironment is essential to maintain the dynamic balance of liver physiological functions, its intrinsic tolerogenic state during the initiation and progression of CCA must be fully considered for CCA immunotherapy, as evidenced by liver metastasis-specific acquired resistance of otherwise sensitive tumor subtypes.
Identification of Four Immune Subtypes Characterized by Distinct Composition and Functions of Tumor Microenvironment in Intrahepatic Cholangiocarcinoma.
the TIME can be divided into four distinct subtypes. 46% of CCAs belonged to the immune desert group which presents very weak immune signature expressions, while 13% of CCA tumors showed high infiltration of lymphocytes and strong activation of inflammatory and fibroblasts. The other two types were characterized by their low expression of lymphoid signatures (19%) and mesenchymal features of activated fibroblasts (22%). Notably, the inflamed subtype was associated with the longest survival, suggesting an important mechanism of tumor control played by the TIME.
T cells in CCA
T cells are a highly heterogeneous population of cells including CD8 cytotoxic T lymphocytes (CTL), CD4 helper T cells, and CD4+CD25+FOXP3+ Tregs. Both CD8 and CD4 helper T cells exhibit anti-tumor effects through a number of mechanisms and can be further divided into several sub-populations.
. The infiltration of CD3+ and CD8+ T cells in CCA tumors is associated with favorable survival and lower recurrence risk, while the infiltration of Tregs is a poor prognosis marker in resected CCA patients.
. Tregs were found in comparable quantities in HCC and CCA, but the prevalence of CTLs, which represent the anti-tumor response, was significantly reduced in the CCAs compared with HCCs.
Other T cells function primarily through innate-like mechanisms, including mucosal-associated invariant T cells (MAITs) and NKT cells. MAITs are highly enriched in the liver tissue and respond to MR1-restricted epitopes.
. NKT cells recognize CD1d-restricted epitopes and can have potent cytotoxic and immunomodulatory effects. Some CCA cell lines have been found to express CD1d and can stimulate NKT cells in vitro,
a property that has not yet been explored in detail but could potentially serve as a biomarker for CCAs with NKT immunoreactivity.
T cell penetration and expression of surface markers in CCA have particular mechanistic and therapeutic importance for ICI. Cancer cells have been found to express PD-L1 to escape attack from T cells via the PD-L1/PD1 axis by promoting TIL apoptosis.
. The elevated PD-L1 expression is correlated with tumor pTNM stage and poor overall survival (OS), and it is inversely correlated with CD8+ TILs in CCAs.
. In addition to PD-L1, the expression of HLA-I molecules may be associated with the infiltration of CTLs, and a positive correlation between HLA-I and CD8+ cells have been demonstrated in CCAs.
. Positive HLA-I expression combined with the negative PD-L1 expression, as well as high CD8+ T cell frequencies at the tumor border area, have both been associated with a favorable clinical outcome in CCA patients.
. The latter point may be underappreciated, as infiltration of CTLs and CD4 helper cells appears to be blocked spatially at the tumor margins. Finally, PD1 and CTLA-4 expression on the surface of T cells are found to be increased in lymphocytes within the CCA lesions, suggesting increased T cell exhaustion that may be amenable to targeting by ICI therapies.
. Like tumor cells, tumor-associated macrophages (TAMs) found in CCA may contribute to the immunosuppressive TIME via antigen presentation and expression of ligands for T cell exhaustion markers. In fact, TAMs are identified to be the main source of PD-L1 both in human and murine CCA tumors(34). The level of PD-L1 expressed on macrophages positively correlated with high PD1 expression CTLs, and it is a risk factor for CCA patient survival(35).
In addition, TAMs can polarize to promote either tumor progression (M2) or pro-inflammatory processes (M1). TAM polarization may be influenced by the cytokines IL-13, IL-34, and osteoactivin secreted by CCA tumor cells, which are strong differentiation factors for macrophage shaping toward to TAM-like features, contributing to tumor invasion both in vitro and in vivo.
. The M2 CD68+CD163+ macrophages may mediate their immunosuppressive effects indirectly through mechanisms such as the infiltration of Tregs and neovascularization in CCA tumors, correlating with poor survival.
. However, targeting TAMs in advanced CCA may not be straightforward. The infiltration of CD68+ macrophage cells appears to be significantly increased in locally advanced CCA primary tumors compared with metastatic sites, possibly related to the increased microvascular density within the primary tumors.
. Also paradoxically, inflammatory macrophages appear to be required for the WNT pathway activation in CCA tumors, as macrophage depletion or WNT signaling inhibition resulted CCA tumor regression.
. Thus, the decision to investigate macrophage depletion in CCA using newer targeted therapies or biologics maybe complicated by discrepancies between their phenotype and function in the TIME. Further research is needed to understand their intricate biology and predict the effects of TAM modulation.
MDSCs in CCA
Separate from TAMs, MDSCs are characterized by their immunosuppressive characteristics found in numerous malignancies. Overlapping tumor-promoting function with TAMs was suggested with the observation of a compensatory infiltration by MDSCs after the blockade of TAM in CCA models, resulting in impaired T cell response and immune escape.
. However, factors that cause MDSC recruitment may be dependent on the organ-specific context of the tumor’s residence, a quality that subcutaneous models of CCA do not capture. In an orthotopic mouse model of CCA established in the context of colitis, CXCR2+ polymorphonuclear MDSC (PMN-MDSC) recruitment within the liver was demonstrated to be dependent on CXCL1-secreting hepatocytes driven by gut microbial products.
. Such an indirect mechanism of tumor promotion by the compromised gut barrier is particularly relevant in western patients for whom IBD plays a causative role in carcinogenesis and points to an underappreciated role of the gut microbiome in the TIME of CCA. Thus, MDSCs represent a promising target for immunomodulation-based therapeutics for CCA.
NK cells in CCA
NK cells are potently cytotoxic lymphocytes with established roles in other tumor types, yet the studies of NK cells in CCA pathogenesis are quite limited. It has been shown that the high expression of CXCL9, induced by IFN-γ, is correlated with abundant NK cell infiltration in CCA tumors and prolongs survival outcomes.
. Furthermore, an antibody neutralizing MICA/B, the soluble NKG2D decoy shed from tumor cells, can increase IFN-γ secretion and degranulation of NK cells co-cultured with CCA tumor cells ex vivo.
. While NK cells hold promise for anti-tumor function, high-dimensional analysis suggests that their viability may be compromised in CCA (see below), making their relevance questionable.
Neutrophils in CCA
Neutrophils are a subtype of polymorphonuclear cells that act as first-responders in inflammatory processes through direct cytotoxicity and release of chromatin into the extracellular space. Multiple studies have demonstrated that neutrophils within CCA lesions are associated with poor prognosis and a high tumor recurrence rate.
. It has been shown that neutrophils can traffic into CCA lesions by the overexpressed chemokine CXCL5, a member of the CXC-type chemokine family, in CCA tumors via PI3K-Akt and extracellular signal-regulated kinase 1/2 signaling pathways.
. However, the precise roles of neutrophils during CCA pathogenesis remain to be determined.
b.
NGS data on TIME (High-resolution data)
ScRNASeq illuminates the transcriptomes of individual cells with unparalleled granularity and has been revolutionary in our understanding of tumor cells and the TIME. In the first scRNASeq study of the human liver, MacParland et al. analyzed the transcriptional profiles of 8,444 parenchymal and non-parenchymal cells.
. Two distinct of CD68+ macrophage populations were identified. One population was characterized as inflammatory with enriched expression of LYZ, CSTA, CD74, and the second population of macrophages was characterized as tolerogenic. In addition, three clusters of effector T cells were identified as tissue resident memory αβ T cells (CD8+CD69+), unconventional γδ T cells (T-bet+CD161+CD16+) and phosphoantigen-reactive γδ T cells in the liver. Furthermore, heterogeneity of NK and NKT cells in the human liver was identified by clustering three populations -- CD56+ NK cells, CD56-CD8A+ NKT, and CD56+CD8A+ NKT cells, which express different kinds of chemokine ligands, granzymes, and killer cell lectin-like receptors.
. This study provided the framework of physiological subsets of liver-resident immune cells, allowing for analysis of their alterations when compared with the context of CCA.
Ma et al. published the first scRNASeq analysis of human liver cancers for both HCC and CCA. It was found that VEGF may play an important role in the TIME reprogramming.
. Except for the malignant cells, VEGF was mainly expressed by TAMs within the tumor immune compartment. Furthermore, the infiltrated T cells showed significantly different expression profiles based on tumors’ transcriptomic diversity scores-an algorithm that estimates the correlation of gene expression and CNVs of each tumor sample.
. It was found the top-ranking genes in T cells derived from high diversity (above median diversity value) tumors, which have poor survival outcomes, were mainly enriched in the epithelial-mesenchymal transition and myogenesis process. However, T cells derived from low diversity (below median diversity value) tumors showed a better survival outcome than the highly diverse tumors and were mainly enriched in allograft rejection, oxidative phosphorylation, IFN-α/IFN-γ response, and proliferation pathways, indicating these cells may still have anti-tumor and/or cytotoxic activities.
. Although this study was not specific for CCA, it suggested an important link between the transcriptomic properties of primary tumor cells and T cell function in the liver which may have utility for investigation as a novel biomarker.
The major power of scRNAseq in cancer immunology lies in its ability to identify novel immune subsets and the factors/pathways on which they are dependent. In a subsequent scRNASeq study of 8 human CCAs, it was found that proliferating CD8 T cells in CCAs express exhaustion markers, such as lymphocyte-activation gene 3 protein, TIM3, and T cell immunoreceptor with Ig and ITIM domains (TIGIT), suggesting they are hyporeactive.
In addition, although NK cells in the tumor adjacent tissue appeared activated by expressing a high level of cytotoxic markers, the intratumoral NK cells had a transcriptional profile of hypoxia and apoptosis. Finally, Tregs in tumors were found to express inhibitory markers, including TIGIT, CTLA-4 and TNFR-related protein superfamily 18, indicating they could be highly immunosuppressive.
. Another study utilized scRNAseq to identify the expression of transcription factor MEOX1 in Tregs as causing immunosuppression and correlating with survival in patients with CCA.
. While the number of studies is still limited, the wealth of data generated from scRNAseq has revealed a number of new transcriptional states and subtypes of cells within the TIME that hold great promise for future investigations into novel targets specific for CCA.
Preclinical CCA immunotherapy
The immunosuppressive mechanisms of the TIME in CCA support the investigation of immunotherapies against CCA. Due to the lack of adequate animal models of CCA, early studies typically employed in vitro co-culture techniques or xenograft models. For example, it was reported that the cytokine-induced killer cells cocultured with dendritic cells (DCs) suppressed the growth of human CCA cells in SCID mice.
. Another study showed that the combined treatment with cytokine-induced killer cells and cetuximab, an epidermal growth factor receptor inhibitor, demonstrated significant cytotoxicity to human CCA cells in vitro.
. Aspartate-β-hydroxylase is a type 2 transmembrane protein which is widely expressed in many cancer types, including CCA. Using a rat CCA model, Noda et al. showed that Aspartate-β-hydroxylase-exposed DCs had significant cytotoxicity against CCA cells and increased tumor infiltrating CD3+ T cells, leading to the inhibition of CCA growth and metastasis.
. A similar study found enhanced T cell cytotoxicity in a model using monocyte-derived DCs loaded with PRKAR1A, another protein that is overexpressed in CCA tumor cells, compared with conventional DCs.
. Neutralizing IL-10 and TGF-β increased the production of IFN-γ and enhanced the cytotoxicity of CTLs mediated by DC cells against CCA tumor cells in vitro.
. While these studies are useful as proof-of-concept investigations into CCA antigens and APCs, their design may not accurately reflect the complex interactions that occur in an in vivo system.
Recently, multiple models of mouse CCA have been developed, including cell lines
. These tools significantly facilitate the preclinical studies of immunotherapies against CCAs in immune-competent mice, allowing for relevant in vivo examination of the TIME. For example, using a syngeneic orthotopic mouse model of CCA, Loeuillard E et al. reported that the TAMs recruited from the bone were the main source of PD-L1 in CCA which had key roles during tumor progression. However, blockade of TAMs led to a compensatory accumulation of an immunosuppressive signature subset of Ly6CloLy6Ghi PMN-MDSC. This effect counteracted the anti-tumor effect of depleting TAMs in this CCA mouse model. Dual blockade of TAMs and PMN-MDSC facilitated the anti-tumor effect of anti-PD1 in CCA.
. Such treatment combinations and multi-subtype depletions demonstrate an important application of these newer immunocompetent mouse models of CCA, especially their utility in predicting compensatory effects in a plastic cell type such as TAMs. As noted above, TAM polarization oversimplifies the link between phenotype and function, which another group studied using an immunocompetent model of CCA. Establishing that TAMs were major immunosuppressive cells with CCA TIMEs, the authors showed that tumor cell-derived GM-CSF recruited and polarized TAMs, and blocking GM-CSF suppressed mouse CCA growth, leading to prolonged survival.
GM-CSF drives myelopoiesis, recruitment and polarisation of tumour-associated macrophages in cholangiocarcinoma and systemic blockade facilitates antitumour immunity.
. Because GM-CSF canonically promotes M1 macrophage differentiation which promotes tumor immune responses, while M-CSF promotes M2 macrophage differentiation which promotes tumor growth and metastasis and which is correlated with poor outcomes.
Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage.
Immunocompetent mouse CCA models are also being applied to investigations of combination therapies involving ICIs. With the recent progress of ICI based immunotherapy for cancer treatment, there are multiple studies on ICIs, expression of their targets, and possibly translating these molecules for CCA therapy. It was reported that, although an increased expression of PD-L1 is often observed in CCA tumors, CCA barely responds to anti-PD-L1 treatment,
suggesting an intrinsic resistance to ICI for CCA. However, ICI may be useful as a part of combination therapies to overcome resistance. For example, Diggs L et al. reported that activation of the antigen-presenting cells of macrophages and dendritic cells with anti-CD40 antibody showed a moderate response in murine CCA models, but a combination of anti-CD40 and anti-PD1 exhibited a significant anti-tumor effect in vivo.
. A recent study showed that trametinib, a mitogen-activated kinase inhibitor, upregulated the expression of PD-L1 on the CCA tumor cells. However, it also increased the immunogenicity of tumor cells by up-regulating their MHC-I expression. Combination of trametinib and anti-PD-1 inhibited the tumor growth of several CCA tumor models by increasing effector memory CD8+ and CD4+ T cells, as well as the CTLs in the liver.
In summary, the recent preclinical studies support the possible usefulness of immunotherapy, especially in the setting of combination therapy, against CCA.
CCA immunotherapy in clinical practice
Despite an increased understanding of the tumor microenvironment in CCA, the application of novel and repurposed immunotherapies has been challenging. The rarity and aggressiveness of CCA have caused progress to be slow and incremental, exemplified by the 12-year gap in-between the ABC-02 and TOPAZ-1 trials demonstrating an improved survival on the order of weeks. Here, we discuss select biologic-based immunotherapies in the treatment of CCA that are approved or show experimental promise. Cell-based immunotherapies for CCA are discussed elsewhere.
Two well-characterized molecular subtypes within various tumors, including CCAs, are tumor mutation burden high (TMB-H) and microsatellite instability high (MSI-H). Both TMB-H and MSI-H are associated with an increase of tumor-specific neoantigens,
. A comprehensive genomic analysis of 260 biliary tract cancers found that 14 cases (5.9%) were classified as hypermutated, and only 5 of these harbored inactivating mutations in mismatch-repair genes.
. In a cohort study of 352 CCA samples analyzed by next generation sequencing, 2.0% of tumors were identified as MSI-H, while 4.0% were classified as TMB-H based on a cutoff of 17 somatic missense mutations per Mb.
Despite their rarity, several cases reported that CCA tumors harboring TMB-H treated with anti-PD-1 antibody immunotherapy experienced significant ongoing anti-tumor response.
Successful response to the combination of immunotherapy and chemotherapy in cholangiocarcinoma with high tumour mutational burden and PD-L1 expression: a case report.
Two cases of intrahepatic cholangiocellular carcinoma with high insertion-deletion ratios that achieved a complete response following chemotherapy combined with PD-1 blockade.
. In another advanced-stage patient with MSI-H CCA, although the expression of PD-L1 and the infiltrated CTLs were not elevated, there was a strong and durable response to Pembrolizumab therapy.
Results from a phase 2 study (NCT01876511) of evaluating anti-PD1 immunotherapy for progressive metastatic carcinomas included four ampullary or CCA patients. Surprisingly, the response rates of MSI-H colorectal cancer patients were similar to that of noncolorectal cancer patients including CCA.
. Based on these promising results, the trial was expanded to further evaluate the efficacy of anti-PD1 immunotherapy in 12 different tumor types with advanced mismatch repair deficiency. It was reported that three of the four enrolled CCA patients experienced stable disease, while another experienced a complete response. These results suggest that neoantigens generated by cancer cells caused by MSI-H genomes lead to the enhanced sensitivity of CCA to PD1-blockade in a manner similar to other cancer types.
. These promising results accelerated the approval of anti-PD1 for the immunotherapy of adult and pediatric patients with unresectable or metastatic solid tumors, including CCA, that harbor MSI-H and have progressed following prior treatment.
KEYNOTE-158, a larger trial, evaluated the efficiency of pembrolizumab for 233 patients with MSI-H advanced noncolorectal cancer who failed to prior therapy, including 22 CCAs. The combined ORR was 34.3%, the mPFS was 4.1 months, and mOS was 23.5 months. Specifically, in the 22 CCAs cohort, 2 patients had a CR and 7 patients had a partial response (PR). The ORR of 40.9% for CCA was similar to other cancers, and a similar mPFS (4.2 months) and mOS (24.3 months) were observed.
Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study.
. These results were remarkable but not unexpected based on previous smaller studies of single-agent nivolumab, in which all responders were found to have a MSI-H profile.
. Together, these promising results in the MSI-H/TMB-H subset of CCA have significantly altered the prognosis for this unique population that responds to ICI favorably, opening up the possibility for further application of immunotherapy to patients lacking these biomarkers.
b.
PD-L1 as a biomarker for ICI immunotherapies
Unfortunately, the results from ICI monotherapy for TMB-L/MSI-L CCAs have been unencouraging, and there are no approved immunotherapy-alone regimens for CCA. Some investigations have focused on finding biomarkers in CCA that correlate with response to ICI (Table 2). PD-L1 expression within the tumor is such a marker for the prediction of anti-tumor response to ICI therapy across multiple tumor types.
suggesting the potential response to anti-PD1 or anti-PD-L1 immunotherapy. In a phase Ib trial (Keynote 028) evaluating the anti-tumor efficacy of Pembrolizumab in PD-L1 positive (IHC≥1%) CCA tumors, a 13% ORR was observed in 24 patients.
T-Cell-Inflamed Gene-Expression Profile, Programmed Death Ligand 1 Expression, and Tumor Mutational Burden Predict Efficacy in Patients Treated With Pembrolizumab Across 20 Cancers: KEYNOTE-028.
. In a larger trial of 104 enrolled CCA patients (Keynote 158), a total ORR of 5.5% was reported, of which the ORR was 6.6% and 2.9% in PD-L1 positive (n = 61) and PD-L1 (n = 34) negative patients, respectively.
. In a phase II multi-institutional trial of evaluating Nivolumab, a PD-L1 antagonist, it was found the positive expression of PD-L1 in tumors was associated with significantly prolonged progression-free survival when compared to the PD-L1 negative tumors.
. Despite PD-L1 expression correlating with response, these results suggest that both Pembrolizumab and Nivolumab monotherapy showed only modest efficacy for CCA patients, and intrinsic tolerance mechanisms need to be overcome in order to unlock the efficacy of ICI.
c.
ICI based combination therapy for CCA
Table 2Summary of completed and ongoing clinical trials of ICI based CCA immunotherapy
These clinical trials (https://www.clinicaltrials.gov/) were included from their first start date until March 20, 2022. A search strategy was developed in combination with the Medical Subject Headings, Emtree and text terms, include ‘liver cancer’, ‘liver tumor’, ‘biliary cancer’, ‘biliary tumor’, ‘biliary tract cancer’, ‘biliary carcinoma’, ‘cholangiocarcinoma’, ‘intrahepatic cholangiocarcinoma’, ‘ICC’, ‘iCCA’, ‘CCA’, ‘immunotherapy’, ‘immune checkpoint blockade’, ‘immune checkpoint inhibitor’, ‘anti-PD1’, ‘anti-PD-L1’, ‘anti-CTLA4’, ‘anti-TIM3’. According the retrieved results, Camrelizumab, Cemiplimab, Keytruda, MK-3475, Nivolumab, Pembrolizumab, SHR-1210, Sintilimab, Sym021, Tislelizumab and Toripalimab, were classified as anti-PD1; Atezolizumab, Durvalumab, KN035, MEDI4736 were classified as anti-PD-L1; Tremelimumab, Ipilimumab were classified as anti-CTLA4. Abbreviation: CCA, cholangiocarcinoma; ICI, immune checkpoint inhibitor; GEMOX: Gemcitabine and Oxaliplatin; GEMCIS: Gemcitabine and Cisplatin; XELOX: Oxaliplatin and Capecitabine; FOLFOX4: Oxaliplatin, Folinic Acid and 5-Fluorouracil; HAIC: Hepatic artery infusion chemotherapy; TACE, Transarterial chemoembolization.
Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study.
Ramucirumab Plus Pembrolizumab in Patients with Previously Treated Advanced or Metastatic Biliary Tract Cancer: Nonrandomized, Open-Label, Phase I Trial (JVDF).
Triplet combination of durvalumab, tremelimumab, and paclitaxel in biliary tract carcinomas: Safety run-in results of the randomized IMMUNOBIL PRODIGE 57 phase II trial.
Efficacy and biomarker analysis of nivolumab plus gemcitabine and cisplatin in patients with unresectable or metastatic biliary tract cancers: results from a phase II study.
A Phase II Study of Pembrolizumab in Combination with Capecitabine and Oxaliplatin with Molecular Profiling in Patients with Advanced Biliary Tract Carcinoma.
Camrelizumab in combination with apatinib in second-line or above therapy for advanced primary liver cancer: cohort A report in a multicenter phase Ib/II trial.
Gemcitabine and cisplatin plus durvalumab with or without tremelimumab in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, single-centre, phase 2 study.
∗ These clinical trials (https://www.clinicaltrials.gov/) were included from their first start date until March 20, 2022. A search strategy was developed in combination with the Medical Subject Headings, Emtree and text terms, include ‘liver cancer’, ‘liver tumor’, ‘biliary cancer’, ‘biliary tumor’, ‘biliary tract cancer’, ‘biliary carcinoma’, ‘cholangiocarcinoma’, ‘intrahepatic cholangiocarcinoma’, ‘ICC’, ‘iCCA’, ‘CCA’, ‘immunotherapy’, ‘immune checkpoint blockade’, ‘immune checkpoint inhibitor’, ‘anti-PD1’, ‘anti-PD-L1’, ‘anti-CTLA4’, ‘anti-TIM3’. According the retrieved results, Camrelizumab, Cemiplimab, Keytruda, MK-3475, Nivolumab, Pembrolizumab, SHR-1210, Sintilimab, Sym021, Tislelizumab and Toripalimab, were classified as anti-PD1; Atezolizumab, Durvalumab, KN035, MEDI4736 were classified as anti-PD-L1; Tremelimumab, Ipilimumab were classified as anti-CTLA4. Abbreviation: CCA, cholangiocarcinoma; ICI, immune checkpoint inhibitor; GEMOX: Gemcitabine and Oxaliplatin; GEMCIS: Gemcitabine and Cisplatin; XELOX: Oxaliplatin and Capecitabine; FOLFOX4: Oxaliplatin, Folinic Acid and 5-Fluorouracil; HAIC: Hepatic artery infusion chemotherapy; TACE, Transarterial chemoembolization.
Based on the results of both pre-clinical and clinical data of ICIs having limited efficacy in CCAs, many clinical trials have attempted to combine ICI with other ICIs, chemotherapy, locoregional therapy, or targeted therapies to improve the CCA immunotherapy response rate (Table 2).
The ABC-02 trial demonstrated gemcitabine plus platin-based chemotherapy superiority to gemcitabine monotherapy.
. Thus, there was rationale to combine ICI with the standard of care gemcitabine and cisplatin (Table 2). In a phase II study of accessing the Nivolumab in combination with gemcitabine and cisplatin chemotherapy, 15 patients achieved an objective response in 27 response-evaluable patients, of whom 5 patients (18.6%) had CR, and the disease control rate was 92.6%. Meanwhile, an encouraging objective response of 61.9% was achieved in the 21 chemotherapy-naive patients. The mPFS in this study was 6.1 months and the mOS was 8.5 months, respectively, and the toxicity profile of the Nivolumab in combination with chemotherapy was acceptable.
Efficacy and biomarker analysis of nivolumab plus gemcitabine and cisplatin in patients with unresectable or metastatic biliary tract cancers: results from a phase II study.
More recently, results from the phase 3 TOPAZ-1 trial demonstrated an improvement in overall survival for patients with CCA treated with durvalumab (an anti-PDL1 antibody) in combination with gemcitabine and cisplatin,
the first since the ABC-02 trial. mOS was 12.8 months in the Durvalumab combination group and 11.5 months in the placebo treatment group, and rates of grade 3/4 adverse events were comparable. On post-hoc analysis, only modest survival effects were seen in subgroups defined by PD-L1 expression, and over 50% patients had an unknown MSI status. Nonetheless, this big achievement emphasized the promise of combined CCA immunotherapy with chemotherapy that led to the recent approval of this combination therapy in CCA in the USA.
. A similar phase I study was performed in Japan, where there have already been relatively favorable results of combining Nivolumab with cisplatin plus gemcitabine chemotherapy with a reported median overall survival of 15.4 months and a median progression-free survival of 4.2 months. In the combination group, 11 of 30 patients had an objective response compared with only 1 of 30 patients in the Nivolumab monotherapy group with a 5.2 month mOS and 1.4-month mPFS.
Nivolumab alone or in combination with cisplatin plus gemcitabine in Japanese patients with unresectable or recurrent biliary tract cancer: a non-randomised, multicentre, open-label, phase 1 study.
The Lancet Gastroenterology & Hepatology.2019; 4: 611-621
Currently, there are over 25 ICI combination based clinical trials for CCA treatment (Table 2). For example, A phase II study combining Nivolumab with Ipilimumab for advanced biliary tract cancer enrolled 39 patients (20 men and 19 women) that all received prior chemotherapy and had no MSI. The mPFS and OS were 2.9 months and 5.7 months, respectively. This combination therapy showed a more favorable efficacy when compared with results from a separate trial using anti-PD1 monotherapy.
Evaluation of Combination Nivolumab and Ipilimumab Immunotherapy in Patients With Advanced Biliary Tract Cancers: Subgroup Analysis of a Phase 2 Nonrandomized Clinical Trial.
. In a Phase 1 study of evaluating durvalumab (anti-PD-L1) combined with tremelimumab (anti-CTLA-4) in Asian CCA patients, durvalumab monotherapy group (n=42) had a median OS of 8.1 months, and the combination group (n=65) had a median OS of 10.1 months.
. While the treatment-related adverse events were comparable between the two groups, the combination group had one treatment-related death, pointing to the difficulty of combining immunotherapy regimens. Another promising phase II trial was terminated before reaching endpoint due to an unexpected increase of anaphylactic adverse events from combining durvalumab, tremelimumab, and paclitaxel. The dose-limiting toxicities were observed in 5 patients in the combination group (n=10).
Triplet combination of durvalumab, tremelimumab, and paclitaxel in biliary tract carcinomas: Safety run-in results of the randomized IMMUNOBIL PRODIGE 57 phase II trial.
Further efforts are testing enhanced ICI blockade of established targets. A phase 2 trial of evaluating first-line combination Camrelizumab, a humanized high-affinity PD-1 IgG4 monoclonal antibody, plus oxaliplatin-based chemotherapy for advanced biliary tract cancer, enrolled 92 patients: 29 received Camrelizumab plus 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) while 63 received Camrelizumab plus gemcitabine and oxaliplatin (GEMOX). A combined 16.3% objective response rate, a 5.3 month mPFS, and a 12.4 month OS were reported.
. In a similar study, 37 advanced biliary tract cancer patients were recruited to evaluate the efficacy and safety of Camrelizumab plus gemcitabine and oxaliplatin as the first line treatment. 54% of patients (20/37) experienced an objective response, and a 6.1 month mPFS and an 11.8 month mOS with a manageable safety profile of the combination therapy were reported.
. In a cohort study of comparing the efficacy and safety of PD-1 inhibitors plus chemotherapy (n=75) and chemotherapy alone (n=59) as first-line treatment for advanced CCA patients, though no significant differences were found in the ORR and disease control rate between the two groups, a significantly longer mPFS was observed in the combination group (5.8 months VS 3.2 months, P = 0.004).
PD-1 Inhibitors Could Improve the Efficacy of Chemotherapy as First-Line Treatment in Biliary Tract Cancers: A Propensity Score Matching Based Analysis.
In summary, multiple clinical trials are in progress examining the therapeutic efficacy of ICI based combination therapy against CCA. Most of the trials are still in early phases. Nevertheless, we expect that during the next several years, the results from these ongoing clinical trials may provide novel therapeutic options for the treatment of this deadly malignancy.
Future directions and challenges
Patients suffering from CCA are in urgent need of new systemic therapies. Despite the established efficacy of ICI monotherapy for the minority of patients whose CCAs carry TMB-H or MSI-H genotypes, the introduction of immunotherapy into treatment regimens for CCA broadly has been slow for several reasons. First, unlike HCC, clinical trials for CCA are challenging to perform due to low incidence, making efficacy of any new therapy difficult to demonstrate or disprove prospectively without the coordination of an international clinical trial. Second, cholangiocarcinoma cells and the overall liver microenvironment demonstrate particularly strong resistance to immunotherapies that are otherwise effective in other cancer types/sites, making treatment combinations necessary. Third, the lack of identifiable biomarkers necessitates treating the majority of CCA the same despite divergence in driver mutations and anatomic site amongst patients.
Fortunately, the diversity of CCA based on molecular landscape is actively being addressed. In addition to approved targeted therapies for known driver mutations of intrahepatic CCA, the preclinical studies reviewed above demonstrate unique mechanistic attributes that may explain the relative resistance to therapy. Some of these molecular features are being addressed by second-generation ICIs (Table 2), including TIGIT, LAG3, or TGFβ targeting, however, further identification of biomarkers will be critical to this effort. The introduction of large scRNAseq studies in CCA patients have already identified different various immune cell types and tumor cell states that may serve as suitable biomarkers for future therapeutics. The results should be combined with other omics studies, including whole-exome sequencing, copy number variations, proteomics and metabolomics. These integrated studies will provide the comprehensive pictures of CCAs and its immune-microenvironment. The results will also be critical to develop novel immunotherapies or combined immunotherapies and targeted therapies for CCA treatment.
However, significant challenges remain. One of the major challenges is that CCA is a heterogenous disease in multiple levels. Anatomically, CCAs consist of three subsets, namely, intrahepatic (iCCA), perihilar (pCCA), and distal cholangiocarcinoma (dCCA). They have distinct driver mutations, histological features
and possible distinct responsiveness to immunotherapies. Indeed, based on TOPAZ-1 clinical trial, it appears that durvalumab/gemcitabine/cisplatin combination therapy is much more effective against iCCA than extrahepatic CCA (which include pCCA and dCCA).
. This issue has not been adequately addressed in clinical and preclinical studies.
In addition, the success of these future directions will depend on access to preclinical testing in CCA, and until recent years, mouse models for CCA have been lacking. For CCA cell lines, few of them are commercially available. In most cases, iCCA, dCCA, and gall bladder cancer cell lines are used interchangeably.
. Mouse CCA models include chemically induced CCA, such as TAA-induced CCA, as well as genetically engineered mouse CCA models. The latter includes transgenic/knock-out mouse models, as well as mouse CCAs produced by hydrodynamic injections. All of these models have been used to investigate therapeutic efficacy for immunotherapies. Most of these murine CCA models are iCCA models and few pCCA or dCCA models exist. Clearly, additional efforts are required to develop clinically relevant mouse CCA models with various genetic alterations seen in human CCAs, especially for pCCA and dCCA.
Although immunotherapy has been in the arena for advanced CCA treatment in combination with chemotherapy in the first line setting, the response rate and clinical outcome are still suboptimal. It would be of great significance to explore the biomarkers with predictive or prognostic value in order to screen the patients with the most beneficial outcome. Clinical biospecimens, including blood, urine, tumor samples and radiographs, collected during the trials will be valuable for this purpose by dissect tumoral response and dynamic immune landscape of CCA using current cutting-edge omics technologies. The findings will be significantly helpful to guide the design of combination of different chemotherapy regimen with various immune checkpoint blockades and improve the outcome. Additionally, drug resistance has been one of the challenges related to failure of targeted and immunotherapies, and illumination of drug resistance mechanism will be helpful for the exploration of next generation of immunotherapies or combination therapies. As multiple modalities are on the table for the treatment of advanced CCA, selection and sequencing of therapies for individual patients will become an important consideration for personalizing the treatment plan and optimizing patient outcomes.
In summary, immunotherapy against CCA represents exciting opportunities as well as unique challenges. It requires the combined effects from basic scientists, translational researchers and clinicians to advance this field. The key issues that we need to address is to better understand the molecular mechanisms underlying CCA pathogenesis, develop better and representative small animal models for CCA, identify biomarkers for patient selection and international collaborative clinical trials.
References
Author names in bold designate shared co-first authorship
Identification of Four Immune Subtypes Characterized by Distinct Composition and Functions of Tumor Microenvironment in Intrahepatic Cholangiocarcinoma.
GM-CSF drives myelopoiesis, recruitment and polarisation of tumour-associated macrophages in cholangiocarcinoma and systemic blockade facilitates antitumour immunity.
Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage.
Successful response to the combination of immunotherapy and chemotherapy in cholangiocarcinoma with high tumour mutational burden and PD-L1 expression: a case report.
Two cases of intrahepatic cholangiocellular carcinoma with high insertion-deletion ratios that achieved a complete response following chemotherapy combined with PD-1 blockade.
Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study.
T-Cell-Inflamed Gene-Expression Profile, Programmed Death Ligand 1 Expression, and Tumor Mutational Burden Predict Efficacy in Patients Treated With Pembrolizumab Across 20 Cancers: KEYNOTE-028.
Efficacy and biomarker analysis of nivolumab plus gemcitabine and cisplatin in patients with unresectable or metastatic biliary tract cancers: results from a phase II study.
Nivolumab alone or in combination with cisplatin plus gemcitabine in Japanese patients with unresectable or recurrent biliary tract cancer: a non-randomised, multicentre, open-label, phase 1 study.
The Lancet Gastroenterology & Hepatology.2019; 4: 611-621
Evaluation of Combination Nivolumab and Ipilimumab Immunotherapy in Patients With Advanced Biliary Tract Cancers: Subgroup Analysis of a Phase 2 Nonrandomized Clinical Trial.
Triplet combination of durvalumab, tremelimumab, and paclitaxel in biliary tract carcinomas: Safety run-in results of the randomized IMMUNOBIL PRODIGE 57 phase II trial.
PD-1 Inhibitors Could Improve the Efficacy of Chemotherapy as First-Line Treatment in Biliary Tract Cancers: A Propensity Score Matching Based Analysis.
Ramucirumab Plus Pembrolizumab in Patients with Previously Treated Advanced or Metastatic Biliary Tract Cancer: Nonrandomized, Open-Label, Phase I Trial (JVDF).
A Phase II Study of Pembrolizumab in Combination with Capecitabine and Oxaliplatin with Molecular Profiling in Patients with Advanced Biliary Tract Carcinoma.
Camrelizumab in combination with apatinib in second-line or above therapy for advanced primary liver cancer: cohort A report in a multicenter phase Ib/II trial.
Gemcitabine and cisplatin plus durvalumab with or without tremelimumab in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, single-centre, phase 2 study.
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