Emerging therapies for the treatment of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a cancer arising from the epithelium of intrahepatic or extrahepatic bile ducts. Cholangiocarcinoma often has a poor prognosis due to late diagnosis and the incidence and mortality rate of intrahepatic CCA appear to be increasing. Current therapies include surgical resection, orthotopic liver transplantation, chemotherapy/ chemoradiation and palliative care. Depending on the location, the 5-year survival for CCA ranges from 27–60%. Emerging new therapies are currently being developed for treating CCA include immunotherapy, altering the tumor microenvironment, targeting growth factor gene mutations and signal pathways and that control tumor growth, and targeting gene therapy. The objective of this paper is to summarize the research that is currently ongoing for treating this challenging disease.


INTRODUCTION Description
Cholangiocarcinoma (CCA) is a cancer arising from the epithelium of intrahepatic or extrahepatic bile ducts caused by the malignant transformation of hepatic biliary cholangiocytes [1,2]. Cholangiocarcinoma can occur anywhere from the small peripheral hepatic ducts to the distal common bile duct [2]. The three primary types of CCA and their relative frequency are intrahepatic (10%), distal (40%) and perihilar (50%), the latter being confined to the larger bile ducts in the hepatic hilum. Mixed hepatocellular cholangiocarcinomas have recently been described in which CCA and hepatocellular carcinoma are found in the same nodule [2]. Cholangiocarcinoma primarily arises from the biliary epithelium in the case of extrahepatic cholangiocarcinoma while hepatic progenitors are believed to play a role in intrahepatic CCA [3]. Cholangiocarcinomas have a dense stromal component that result from the recruitment of fibroblasts, remodeling of the extracellular matrix, altered immune cell migration, and angiogenesis. The tumor stroma surrounds the malignant ducts and glands and comprises most of the tumor mass [4].

Epidemiology
Cholangiocarcinoma is the second most common primary liver tumor with estimates ranging from 10-25% of all hepatobiliary malignancies [1]. The age-adjusted incidence of CCA ranges from a high of 2.8-3.3 per 100,000 among Hispanic and Asian populations to a low of 2.1 per 100,000 among non-Hispanic white and black people and is slightly more prevalent among men [2]. Several recent epidemiological studies have shown that the incidence and mortality rates of intrahepatic CCA are increasing [5].
In one series, five-year survival rates for intrahepatic, perihilar and distal cholangiocarcinoma were 60%, 30% and 27% respectively [6]. Median and five-year overall survival (OS) for intrahepatic CCA after surgical resection were 28 months (range: 9-53 months) and 30% (range: 5-56%), respectively [7]. Factors predicting shorter OS included large tumor size, multiple tumors, lymph node metastasis, and vascular invasion. Adjuvant chemotherapy or radiotherapy did not appear to be beneficial. With the exception of advanced patient age, factors associated with shorter OS are tumor-related and include larger tumor size, presence of multiple tumors, lymph node metastasis, vascular invasion, and poor tumor differentiation [7].

Diagnosis
The clinical presentation of CCA patients varies according to the clinical type. Patients with intrahepatic CCA present with abdominal pain, malaise, night sweats, weight loss and anorexia. Patients with extrahepatic CCA typically present with symptoms of obstructive jaundice and sometimes with complications such as cholangitis, which represents a major cause of morbidity in this population [18].
There are few approved serum biomarkers for detecting cancer and none are specific for CCA [19]. Fluorescence in situ hybridization (FISH) has high specificity for CCA and maybe useful for patients at high-risk for CCA, such as those with primary sclerosing cholangitis (PSC) [20,21]. However, this test has relatively poor specificity, especially in the presence of jaundice. Better biomarkers are needed for the early detection of CCA. Immunohistochemical methods can be used to distinguish intrahepatic cholangiocarcinoma and pancreatic ductal adenocarcinoma [22]. The measurement of volatile organic compounds in biliary fluid is emerging as a possible method for diagnosing CCA in patients with PSC [23]. The lack of early diagnostic biomarkers results in late diagnosis and poor prognosis. Thus, 80% patients with CCA will present with unresectable or metastatic disease with poor prognosis [24].
Ultrasonography, computed tomography (CT) scan and magnetic resonance imaging (MRI) scan are the most common non-invasive imaging modalities used in the diagnosis and staging of hilar cholangiocarcinoma [25]. Endoscopic ultrasound is emerging as a useful tool for the diagnosis and staging of CCA [26].

CURRENT THERAPIES Surgery
The treatment of choice for intrahepatic CCA is surgical resection. Surgical treatments are the only potentially curative therapeutic options for intrahepatic CCAs. Unfortunately, only a minority of patients qualify for surgical resection as most present with advanced unresectable disease [18]. Following surgical resection, the median time of disease-free survival is 26 months and five-year survival ranges from 30-60% [3]. There are no large randomized controlled trials demonstrating a survival benefit of combining neoadjuvant or adjuvant chemotherapy with surgical resection [18].

Orthotopic liver transplantation
Liver transplantation has not yet been shown to be a viable option for intrahepatic CCA as disease recurrence was reported to be as high as 70% within five years [27]. However, liver transplantation has been shown to result in significant clinical benefit for select patients with hilar CCA following neoadjuvant chemoradiation [28,29]. This modality typically involves preoperative chemoradiation and resulted in 75% five-year survival for these patients. The selection criteria for transplant as rigorous, however, and a few patients qualify for this therapy [30]. For early stage hilar CCA, surgical resection remains the standard of care and transplant is an option for selected small but unresectable cases of hilar CCA.

Chemotherapy
Chemotherapy may be considered for patients who are not candidates for surgical resection; however, there currently is no established adjuvant chemotherapy for CCA [31]. The combination of gemcitabine and cisplatin remains the standard therapy for advanced CCA [32,33]. No second line therapy has definitely demonstrated improved survival benefits [34]. Published studies for the treatment of CCA using a range of chemotherapeutic

Radiation
There is limited evidence supporting the use of radiotherapy alone although some series demonstrate superior five-year survival, especially in perihilar CCA. Adjuvant radiotherapy may improve overall survival in patients undergoing resection for extrahepatic biliary tract carcinomas [69].

Chemoradiotherapy
Chemoradiation can prolong survival in CCA, particularly in the cases of hilar CCA and massforming intrahepatic CCA. Capecitabine is commonly used concurrently with radiotherapy and the available modalities for radiation include photons, intensity modulated radiation therapy (IMRT), proton beams and stereotactic radiation [70]; however, a randomized clinical trial in this regard are lacking. Open-label At fourth month patients had unconfirmed PR (n = 6; 6%), SD (n = 10; 29%), PD (n = 18; 53%) and symptomatic deterioration leading to treatment discontinuation (n = 1). Three patients were not evaluable. Median PFS was two months and four-month PFS was 29%.

Unresectable or metastatic cholangiocarcinoma
Primary: Five-month PFS; Secondary: ORR and OS Open-label After a medial of seven cycles, 28 evaluable patients had CR (n = 2), PR (n = 9), SD (n = 15) for a disease-control rate of 74%. Two patients underwent surgical resection. The fivemonth PFS was 69%. The median PFS was 9.7 months and the median OS was 12.9 months.
Rubovszky et al. [44] Cetuximab, gemcitabine and capecitabine   Open-label Three patients had OR including confirmed PR (n = 3) and unconfirmed CR (n = 1) 17 patients (68%) had SD including 11 (44%) with SD duration ≥16 weeks and three (12%) with duration of more than one year. Median PFS was 3.7 months and median OS was 9.8 months.

Phase 1 Studies
Costello et al. [

Palliative care for unresectable Cholangiocarcinoma
Nearly half of patients with CCA have unresectable disease and are candidates for palliative care [71]. Endoscopic biliary drainage is the gold standard treatment in advanced or inoperable hilar cholangiocarcinoma [72]. The main goal is to provide biliary drainage with longterm relief from pruritus, cholangitis, pain and jaundice [73]. In patients with inoperable disease, drainage of 50% or more of the liver parenchyma via stenting can improve patient survival [2]. The management of biliary obstruction with stents is obligatory in perihilar CCA [2]. This may be done endoscopically or percutaneously [74]. Possible complications to stenting include infections [75] and stent migration causing injury [76]. Other palliative measures may include oral nutritional supplements or parenteral nutritional support for patients with cachexia [77].

Adjuvant therapy
A recent phase 2 trial demonstrated gemcitabine and capecitabine followed by chemoradiotherapy with concurrent capecitabine is an effective and promising adjuvant regimen in extrahepatic cholangiocarcinoma and also gallbladder carcinoma [38]. Subjects with extrahepatic cholangiocarcinoma or gallbladder carcinoma and radical resection, stage pT2-4 or N+ or positive resection margins, M0, and performance status 0 to 1 were treated with four cycles of IV gemcitabine 1,000 mg/m 2 on day-1 and day-8 and daily capecitabine 1,500 mg/m 2 on days 1-14 followed by concurrent capecitabine 1,330 mg/m 2 daily and radiotherapy. With 80 evaluable patients, results would be promising if two-year survival 95% CI were >45% and R0 and R1 survival estimates were ≥65% and 45%, respectively. Among evaluable subjects (n = 79), the median overall survival was 35 months and disease-free survival at second year was 52%. Local, distant, and combined relapse occurred in 14, 24, and 9 patients, respectively.
The superiority of cisplatin and gemcitabine chemotherapy over gemcitabine alone for treating advanced biliary tract cancer (ABC) was demonstrated in two randomized trials (ABC02 and BT-22 studies) [78]. Combined data from these trials was used to investigate the derived neutrophil-to-lymphocyte ratio which may predict clinical outcomes in some solid tumors including ABC. A total of 462 individual patient records were analyzed, 328 with baseline derived neutrophil-tolymphocyte ratio <3 and 134 ≥3. All surviving patients (n = 19) had a derived neutrophil-to-lymphocyte ratio <3. There was strong evidence that derived neutrophilto-lymphocyte ratio was closely associated with both overall survival (hazard ratio 1.62; 95% CI 1.32-2.01) and progression-free survival (hazard ratio 1.40; 95% CI 1.13-1.72). There was significant evidence of an association between low baseline derived neutrophil-tolymphocyte ratio and long-term survival on a cisplatin and gemcitabine regimen.

Immunotherapy
Advances in cancer immunotherapy have encouraged the development of new treatment options. This type of treatment strengthens the patient's immune system by priming it against tumor-specific antigens. Immunotherapy is based on the observation that tumor infiltration by the cellular mediators of the adaptive immune response such as CD8+ and CD4+ cells is generally correlated with improved outcomes in biliary tract cancers [79,80]. Similarly, patients with higher total regulatory T lymphocyte counts have a significantly better prognosis when compared with those patients whose tumor tissues showed lower regulatory T-lymphocyte counts [81]. Such treatments are more selective against malignant cells and generally less toxic than traditional chemotherapy [82]. Conversely, antibodies against glycoprotein 2 are associated with more severe phenotype and poor survival due to cholangiocarcinoma [83].
Immunotherapy has recently made great advances in the field of oncology, such as the programmed death ligand 1 (PD-L1) inhibitors pembrolizumab for nonsmall cell lung cancer [84] and nivolumab for metastatic melanoma [85], non-small cell lung cancer [86] and renal cell carcinoma [87]. Elevated serum PD-L1 in patients with cholangiocarcinoma have been shown to have poorer overall survival [88], suggesting these checkpoint inhibitors may also be beneficial for this patient population. In one case report, a patient with extrahepatic cholangiocarcinoma demonstrated a strong and durable response to the immune checkpoint inhibitor pembrolizumab [89].

Orthotopic liver transplantation
Several ongoing clinical trials are assessing new methods for OLT. One prospective, open-label, randomized, study will compare the use of capecitabine and radiotherapy or neoadjuvant radiochemotherapy and liver transplantation versus conventional liver and bile duct resection (ClinicalTrials.gov Identifier: NCT02232932). An observational study is designed to validate results of a previous study performed at the Mayo Clinic [90] where patients were treated with combination chemotherapy and radiation and maintained on oral capecitabine until they can receive a liver transplant (ClinicalTrials.gov Identifier: NCT00301379).

Tumor microenvironment
Similar to other treatment-resistant cancers, intrahepatic CCA is characterized by the growth of fibrous or connective tissue, or desmoplasia, around the tumor [91]. This desmoplasia progresses during disease progression and includes stromal fibroblasts, immune cells, and excessive deposition of a complex extracellular matrix which is often rich in hyaluronan [92]. Proliferation of this tumor microenvironment forms an impediment to treating solid tumors such as CCA by increasing interstitial fluid pressure which compresses the surrounding vasculature and promotes tumor progression and the metastatic potential of cancer cells [93] and reduces the beneficial effects of systemic chemotherapeutic agents [91,94]. As hyaluronan is a major component of the tumor microenvironment, reducing tumor hyaluronan with PEGylated human recombinant hyaluronidase decreased interstitial fluid pressure, improved vascular perfusion and increased the effectiveness of docetaxel and liposomal doxorubicin in a murine model of prostate cancer [94].
Large amounts of hyaluronan also exist in the tumor microenvironment of intrahepatic CCA [95] and may represent a target for future therapies [96]. A phase 1 study assessed the efficacy of PEGylated human recombinant hyaluronidase in combination with gemcitabine in patients with untreated stage IV metastatic pancreatic ductal adenocarcinoma [97]. Among patients evaluated for pretreatment tissue hyaluronan levels, median progression-free survival and overall survival rates were 7.2 and 13.0 months, respectively, for patients with high hyaluronan levels versus and 3.5 and 5.7 months for patients with low hyaluronan levels.

Molecular targeted agents
Intrahepatic CCA exists as a range of genetic subtypes [98] with varying sensitivity to chemotherapeutics and molecular targeted agents [99]. Genetic studies of biliary tumors have identified known growth factor gene mutations [100,101] and signaling pathways [102] that control tumor growth and survival. Target-specific monoclonal antibodies and small molecule inhibitors directed against the signaling pathways that promote the growth and spread of cholangiocarcinoma are being developed [103]. Such targeted therapies are showing promise for treatment-resistant CCA.
Several ongoing clinical trials using targeted therapy for cholangiocarcinoma are described in Table 2. In one example, folic acid was used as a targeting agent in CCA cells expressing folic acid receptors [104]. When 5-fluorouracil and folic acid were linked to gold nanoparticles, its cytotoxicity was correlated with folic acid receptor expression, suggesting the use of folic acid as a targeted therapy.

Mutation profiling
Significant genetic differences between intrahepatic and extrahepatic CCA have been identified which may have implications for treatment and outcomes [105]. In one study, 75 samples undergoing next generation sequencing were from patients with intrahepatic (n = 55) and extrahepatic CCA (n = 20) [106]. Significant differences were found in these two groups with respect to the nature and frequency of the genetic aberrations. These are summarized in Table 3. IDH1 and DNA repair gene alterations occurred more frequently in intrahepatic CCA, while more ERBB2 genetic abnormalities occurred www.ijhpd.com Turbeville et al. 45 among extrahepatic. In intrahepatic CCA, KRAS, TP53 or MAPK/mTOR genetic abnormalities were significantly associated with a worse prognosis while FGFR genetic abnormalities were correlated with slow-growing tumors. Based on these mutational profiles, many patients were referred to phase 1 or 2 clinical trials with targeted therapy for enrollment [106].

Promoting the advancement of drug development
The cholangiocarcinoma Foundation (CF) was founded in 2006 with the mission of finding a cure and improving the quality of life for those affected by cholangiocarcinoma. In recent years, the CF has promoted the development of new treatments for CCA by sponsoring an annual conference where interested individuals come together to learn more about the latest advances in bile duct cancer research, treatment and care. Recognizing the unmet medical need for treating CCA, the CF is proactively working with the pharmaceutical and biotechnology companies to accelerate drug development by sponsoring an Industry Night at the annual conference. Representatives from industry are invited to present cholangiocarcinoma clinical trial concepts to a panel of more than 20 global biliary cancer experts. The objective is to accelerate drug development by providing immediate feedback at a considerable cost-savings. To date, this program has been very well received and will hopefully serve as a model for advancing the treatment of other challenging disorders.

CONCLUSION
Cholangiocarcinoma has long been a challenging disease with poor outcomes. The development of emerging new therapies is currently underway that may improve the treatment outcomes of this devastating disease. In addition to providing research grants, disease foundations have an opportunity to work co-operatively with the pharmaceutical and biotechnology companies to advance emerging new therapies. *********

Author Contributions
Sean Turbeville -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting of article, Revising it critically for important intellectual content, Final approval of the version to be published Carl S. Hornfeldt -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting of article, Revising it critically for important intellectual content, Final approval of the version to be published Milind Javle -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting of article, Revising it critically for important intellectual content, Final approval of the version to be published Eric Tran -Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting of article, Revising it critically for important