Mechanisms And Clinical Translation Of Intrahepatic Cholangiocarcinoma In The Era Of Precision Medicine

1Overview Of Intrahepatic Cholangiocarcinoma (ICC)

Cholangiocarcinoma can be classified based on its anatomical location into ICC, perihilar cholangiocarcinoma, and distal cholangiocarcinoma^[1]. ICC has a high incidence in East and Southeast Asian countries, with the highest incidence in Thailand, followed by China^[2]. Studies^[3] indicate that in recent years, the incidence and mortality rates of ICC have shown an upward trend globally. The exact etiology of ICC remains unclear, but potential risk factors include: hepatitis B, hepatitis C, intrahepatic bile duct stones, liver fluke disease, primary sclerosing cholangitis, etc.^[4]. Lymph node invasion is an independent risk factor affecting the prognosis of ICC^[5]. Early-stage ICC patients show no obvious clinical symptoms, and symptoms such as jaundice, fatigue, abdominal pain, and nausea appear only as the disease progresses. Therefore, most ICC patients are already in an advanced stage at the time of diagnosis, losing the opportunity for curative surgery. Even when curative surgery is possible, the 5-year survival rate remains only 30% to 40%, with a recurrence rate as high as 80%^[6]. For most ICC patients who cannot undergo surgical resection, palliative chemotherapy is typically administered. Currently, the first-line chemotherapy regimen for ICC mainly consists of a combination of gemcitabine and platinum-based drugs, but the 5-year survival rate is less than 5%.

2Mechanisms Of ICC

Bile duct obstruction and persistent biliary inflammation are recognized as classic causes of ICC. Chronic biliary inflammation can lead to abnormal signaling of bile acids, stimulate the secretion of growth factors, damage DNA repair functions, and suppress tumor suppressor genes, thereby creating a pro-cancer microenvironment. In this microenvironment, multiple key signaling pathways such as PI3K/AKT/mTOR are dysregulated, promoting unlimited replication of biliary epithelial cells, angiogenesis, invasion, and metastasis^[7].

It has long been believed that ICC originates from the epithelial cells of secondary bile ducts and above; however, in recent years, with the development of molecular biology, our understanding of the origins of ICC has deepened. Fan et al.^[8] utilized mouse models to discover that cholangiocarcinoma can originate from hepatocytes. Research by Sekiya and Suzuki^[9] indicated that hepatocytes can transdifferentiate into the biliary lineage mediated by the Notch signaling pathway, leading to ICC. Guest et al.^[10] employed cell tracking technology in mouse models to find that ICC can originate from intrahepatic bile duct epithelial cells. Additionally, related studies have found that peripheral glandular cells of the bile duct can also be a source of some ICC cells. Although there is still much debate regarding the origin of ICC, it suggests that ICC can arise from multiple cell lineages.

Large-scale genomic and transcriptomic studies^[11-12] have revealed key gene mutations and abnormal signaling pathways driving ICC, including TP53, KRAS, C1orf4 (ARID1A), IDH1/2 mutations, and FGFR gene fusions. In tissue samples from 122 ICC patients in China, 34% were found to have TP53 gene mutations, 25% had KRAS gene mutations, and 17% had ARID1A gene mutations^[13]. Common chromosomal arm changes in ICC include copy number amplifications of 8q, 17q, and 20q, as well as deletions of 4q, 17p, and 18q^[14]. The copy number variations of ICC may vary slightly by region.

Recent studies have also found a certain correlation between the occurrence and development of ICC and the aberrant expression of non-coding RNAs in tumor cells. Non-coding RNAs are a class of RNA that do not have protein-coding capabilities; they play a role not only in tumorigenesis and the invasion and metastasis processes but also affect tumor cell proliferation and apoptosis by regulating gene expression. miRNAs are a type of non-coding RNA, and one study^[15] indicated that miR-370 in ICC can inhibit the proto-oncogene MAP3K8. Upregulation of miR-204 can inhibit epithelial-mesenchymal transition in ICC cells. Furthermore, miR-214, miR-21, and others can also affect ICC cell proliferation and metastasis. miRNAs can also serve as biomarkers for biliary tumors. Compared to healthy individuals or those with benign biliary diseases, miR-106a is significantly downregulated in the blood of ICC patients. Plieskatt et al.^[16] found that eight miRNAs were expressed in the blood of ICC patients, with lower expression in normal controls. Meng et al.^[17] discovered that two miRNAs (miR-21 and miR-200b) were associated with chemotherapy resistance in cholangiocarcinoma. Overall, as our understanding of non-coding RNAs deepens, it will provide new ideas for the treatment of ICC.

3The Tumor Microenvironment Of ICC

The typical characteristic of ICC is that tumor cells are embedded in a dense stroma, and the tumor microenvironment plays a crucial role in the progression and invasion of ICC. Cancer-associated fibroblasts (CAF) can induce ICC tumor growth and invasion by secreting mediators such as platelet-derived growth factor B and stromal cell-derived factor 1. There is heterogeneity of CAF in ICC. Zhang et al.^[18] discovered six CAF subpopulations in human ICC through single-cell sequencing technology, among which CD146-positive vascular-associated CAFs (vCAFs) account for the majority in ICC. vCAFs can induce EZH2 upregulation and enhance the malignancy of ICC through the IL-6/IL-6 receptor axis. Furthermore, exosomes from ICC tumor cells can carry miR-9-5p to upregulate IL-6 expression in vCAFs, thereby promoting the malignancy of ICC.

In the liver, neutrophils primarily participate in inflammatory responses and pathogen clearance. The ratio of neutrophils to lymphocytes in peripheral blood before surgery is significantly correlated with the survival and survival time of ICC patients^[19]. Zhou et al.^[20] indicated that CXCL5 promotes ICC metastasis and recurrence by recruiting tumor-associated neutrophils. There are two types of macrophage populations in the liver: bone marrow-derived macrophages and resident Kupffer cells, both of which can polarize into pro-inflammatory M1 or anti-inflammatory M2 subtypes. In ICC tissues, the high infiltration of tumor-associated macrophages (TAM) is associated with poor prognosis^[21]. TAM can promote the expression of programmed death ligand 1 (PD-L1) in ICC cells by activating hypoxia-inducible factor-1α, thereby inhibiting the anti-tumor functions of cytotoxic lymphocytes. The vascular endothelial growth factor A and angiopoietin secreted by TAM can promote angiogenesis, while Wnt proteins secreted can directly induce tumor cell growth^[22].

There are many regulatory T lymphocytes (Treg) in the ICC microenvironment, and these Tregs can inhibit the anti-tumor effects of natural killer cells and cytotoxic T lymphocytes by secreting IL-10 and TGFβ1^[23]. Tregs in the ICC microenvironment also express high levels of cytotoxic T lymphocyte-associated antigen 4 (CTLA4), and the high expression of the CTLA4/CD80 pathway promotes immune evasion and treatment resistance in ICC cells.

The microenvironment is crucial in the occurrence and development of primary liver cancer. Studies have shown that the microenvironment can influence the type of liver cancer produced. Seehawer et al.^[24] confirmed that cell death in the microenvironment caused by apoptosis can lead to hepatocellular carcinoma, whereas necrotic apoptosis can lead to a more aggressive ICC. The microenvironment in the liver is extremely complex; it not only determines the differentiation direction of liver cancer but also determines the therapeutic response to immunotherapy. For “hot” tumors with many immune cells in the microenvironment, immune checkpoint inhibitors are more effective. A large number of stromal cells and tumor cells together constitute the dynamic microenvironment of ICC, promoting ICC progression, and targeting the tumor microenvironment may be an effective treatment for ICC.

4Molecular Subtyping Of ICC

Different patients often respond very differently to drugs and prognosis, even though the same tumor may have similar pathological morphology. Traditional pathological classification based on histopathological features can no longer meet the needs of precision medicine. In recent years, molecular subtyping based on gene expression, mutations, proteomics, and other technologies has gradually been used to guide clinical decision-making. Currently, molecular subtyping has made significant progress in breast cancer, gliomas, etc. In ICC, there are also subtyping methods based on different omics. Job et al.^[25] proposed four immune subtypes based on the tumor microenvironment, which were associated with patient prognosis. Chaisaingmongkol et al.^[26] discovered common molecular subtypes between Asian hepatocellular carcinoma and ICC from genomic, transcriptomic, and metabolomic levels, where the shared C1 subtype is rich in mitotic checkpoint signaling pathways, and the C2 subtype is more enriched in immune-related pathways, with subtype C1 being associated with poor prognosis more frequently observed in Asians. Sia et al.^[27] identified two major biological categories of ICC through gene expression profiles, SNP chips, and mutation integration analysis: inflammatory type (38%) and proliferative type (62%). Although there has been some progress in the molecular subtyping of ICC, how to organically combine molecular subtyping with traditional clinical pathology and translate it into laboratory-operable testing methods to guide clinical treatment decisions and extend patient survival will be a key direction in the future.

5Early Diagnostic Markers For ICC

The clinical manifestations of cholangiocarcinoma are late, making diagnosis difficult, and confirmation relies solely on pathological and cytological diagnosis. Therefore, there is an urgent need for an in vitro marker to provide possibilities for the early diagnosis of cholangiocarcinoma. The main blood markers for ICC currently include: alpha-fetoprotein, carbohydrate antigen 19-9, carbohydrate antigen 125, and carcinoembryonic antigen, but these markers still lack specificity and sensitivity. Xu et al.^[28] discovered ten methylation sites through comparing ctDNA methylation in 2000 hepatocellular carcinoma patients and normal controls, which can be used for the early diagnosis of hepatocellular carcinoma, achieving an AUC of 96%. Below are some of the latest advances regarding non-invasive early diagnostic markers for ICC (Table 1).

Table 1: ICC Liquid Biopsy Markers

6Treatment Advances For ICC

Currently, treatment options for ICC include curative surgery, liver transplantation, and adjuvant radiochemotherapy. Early surgical resection is the only curative intervention available. Platinum-based drugs combined with gemcitabine are the first-line chemotherapy regimen for patients with advanced cholangiocarcinoma, but the efficacy still needs improvement and is prone to resistance. The application of neoadjuvant chemotherapy is of significant importance for reducing the original lesions and metastatic lymph nodes and achieving clinical translation. A large-sample Phase III clinical study of gemcitabine plus cisplatin published at ASCO 2019^[32] demonstrated that compared to gemcitabine alone, the combination therapy significantly prolonged overall survival (OS) and median progression-free survival (PFS) for patients with advanced cholangiocarcinoma. Chemotherapy combined with local treatment has also gradually shown advantages in neoadjuvant therapy for ICC. Continuous chemotherapy combined with hepatic artery infusion treatment for advanced ICC resulted in an objective response rate (ORR) of 67.6% and a disease control rate (DCR) of 89.2%.

Targeted drugs are an effective measure to ensure the prognosis of ICC patients. With the in-depth research of the mechanisms of ICC, the gradual popularization of second-generation sequencing technology, relevant gene mutations and pathogenic pathways have also been discovered. The development of targeted therapeutic drugs based on this is also in full swing. The FGFR2 fusion gene is an important cause of ICC, almost exclusively found in ICC, and can be detected in up to 20% of ICC patients. This year, the FGFR2-targeted drug Pemigatinib was officially approved by the FDA for the treatment of advanced or metastatic cholangiocarcinoma patients carrying FGFR2 gene fusions or rearrangements. This drug has an ORR of 35.5% and a DCR of 82% for second-line treatment of cholangiocarcinoma, marking a milestone in the treatment of ICC and other biliary tumors^[32-34]. Nearly 20% of ICC patients also carry IDH1 mutations; Ivosidenib is a highly selective targeted inhibitor of IDH1 mutations, and the Phase III clinical trial of ClarIDHy for advanced ICC patients with IDH1 mutations achieved a DCR of 53%, effectively prolonging OS and PFS^[35]. As clinical research deepens, more targeted drugs will be approved for market, benefiting more cholangiocarcinoma patients.

Immune checkpoint inhibitors have shown good efficacy in melanoma, renal cell carcinoma, and other tumors. However, in cholangiocarcinoma, many clinical trial results indicate that the efficacy of immune inhibitors alone is poor. Among them, the most mature clinical trial is KEYNOTE-028^[36], which explored the efficacy of Pembrolizumab in biliary cancer patients. The results showed that Pembrolizumab monotherapy yielded an ORR of 17%, a DCR of 34%, a PFS of 1.9 months, and an OS of 9.7 months. Many reports indicate that targeted combination immunotherapy for ICC is significantly effective. In 2018, Professor Zhao Haitao’s team from Peking Union Medical College Hospital investigated the clinical efficacy and safety of Pembrolizumab or Nivolumab combined with Lenvatinib for treating advanced ICC in a small sample. The results showed that the Lenvatinib combined with PD-1 inhibitor regimen had a certain therapeutic effect. Among 14 patients receiving treatment, 3 achieved partial remission, with an ORR of 21.4% and a DCR of 93.0%, demonstrating a significant survival advantage. Similarly, in 2019, Medicine reported the efficacy of a patient with recurrent and metastatic ICC receiving Opdivo combined with Lenvatinib, showing a good response and effectively controlling distal metastasis^[38]. Lenvatinib is an FDA-approved multi-kinase targeted inhibitor, with FGFR being one of its targets. At the ASCO conference in 2019, Professor Qin Shukui from the PLA 81 Hospital announced the mid-term results of a Phase II clinical trial of Carrelizumab combined with FOLFOX4 or GEMOX for treating biliary cancer, with an ORR of 9.4% and a DCR of 90.6%^[39]. Although many drug combinations have significantly extended the survival of advanced ICC patients, their mechanisms still need to be explored and studied to determine the beneficiary populations. This year at ASCO, a study reported the evaluation of PD-L1 monoclonal antibodies combined with CTLA-4 monoclonal antibodies, gemcitabine/cisplatin in advanced biliary cancer, showing that the combined ORR reached over 70% with good tolerance^[40]. Another study^[41] showed that Nivolumab combined with gemcitabine/cisplatin or Ipilimumab as first-line treatment for unresectable biliary tumors also achieved good PFS. These findings suggest that immune combination chemotherapy and combinations have great therapeutic prospects for ICC.

7Conclusion

ICC, as a highly invasive liver malignancy, has an unclear pathogenesis. Curative surgery is currently the only possible way to cure ICC. With the rapid development of next-generation sequencing technology and the improvement of accompanying analytical algorithms, multi-omics integration is becoming mainstream in tumor research and precision medicine. Liquid biopsies and molecular subtyping based on genomics, transcriptomics, proteomics, and metabolomics provide possibilities for the early diagnosis and precise treatment of ICC. As the molecular biology research of ICC continues to deepen, some key molecular targets of ICC, such as FGFR, will be translated into clinically usable intervention drugs, effectively improving the rate of curative surgery for ICC, alleviating its poor prognosis, and extending patient survival. It is believed that with the continuous advancement of science and technology, exploring the biological mechanisms of ICC and promoting the clinical translation of results will bring significant developments in the diagnosis and treatment of ICC.

Yu Wenhua, Liang Yuan, Lü Ling. Mechanisms And Clinical Translation Of Intrahepatic Cholangiocarcinoma In The Era Of Precision Medicine. Journal Of Clinical Hepatobiliary Diseases, 2021, 37(4): 935-938.

Edited by: Liu Xiaohong

Public Account Editor: Xing Xiangyu