Molecular Targets in Biliary Carcinogenesis and Implications for Therapy

The emerging role of targeted therapy in the treatment of biliary tract cancers was investigated. Findings from preclinical studies were reviewed and correlated with the outcomes of clinical trials undertaken to translate the laboratory discoveries.

Abstract

Biliary tract cancers (BTCs) encompass a group of invasive carcinomas, including cholangiocarcinoma (intrahepatic, perihilar, or extrahepatic), and gallbladder carcinoma. Approximately 90% of patients present with advanced, unresectable disease and have a poor prognosis. The latest recommendation is to treat advanced or metastatic disease with gemcitabine and cisplatin, although chemotherapy has recorded modest survival benefits. Comprehension of the molecular basis of biliary carcinogenesis has resulted in experimental trials of targeted therapies in BTCs, with promising results. This review addresses the emerging role of targeted therapy in the treatment of BTCs. Findings from preclinical studies were reviewed and correlated with the outcomes of clinical trials that were undertaken to translate the laboratory discoveries.

Implications for Practice:

Biliary tract cancers are rare. Approximately 90% of patients present with advanced, unresectable disease and have a poor prognosis. Median overall and progression-free survival are 12 and 8 months, respectively. Because chemotherapy has recorded modest survival benefits, targeted therapies are being explored for personalized treatment of these cancers. A comprehensive review of targeted therapies in biliary tract cancers was undertaken to present emerging evidence from laboratory and/or molecular studies as they translate to clinical trials and outcomes. The latest evidence on this topic is presented to clinicians and practitioners to guide decisions on treatment of this disease.

Introduction

Biliary tract cancers (BTCs) encompass a group of invasive carcinomas, including cholangiocarcinoma (CC), which refers to cancers arising in the intrahepatic, perihilar, or extrahepatic biliary tree; and gallbladder carcinoma (GBC) [1]. They originate from the epithelium of the gallbladder and the bile ducts [2]. More than 90% of BTCs are well-differentiated, mucin-producing adenocarcinomas, while squamous cell carcinoma and small-cell carcinoma occur less frequently [3].

The rate of incidence for BTCs in the Western world is 1–2 cases per 100,000. Conversely, these neoplasms occur more frequently in Asia and South America, with an incidence rate of 96 cases per 100,000 [4]. BTCs affect up to 12,000 people annually in the U.S., with a documented increase in the incidence of intrahepatic cholangiocarcinoma [5]. CC occurs more frequently in the seventh decade of life, with greater predilection for men. Women are more commonly affected by GBC; the median age at onset is 65 years. The difference in gender distribution correlates with the pattern of distribution of predisposing factors; for example, cholelithiasis is more common in women [3].

BTCs frequently stem from conditions that cause chronic inflammation, injury, and reparative biliary epithelial cell proliferation, such as primary sclerosing cholangitis, clonorchiasis, hepatolithiasis, or complicated fibropolycystic diseases. A close interplay among chronic inflammation, cellular injury within bile ducts, and partial biliary tract obstruction appears to be fundamental in the pathogenesis of cholangiocarcinoma [6–8].

Regional nodal and distant metastases tend to occur early in BTCs. Only 10% of patients present with early-stage, surgically resectable, and curable disease. The prognosis is poor for most patients with locally advanced or metastatic BTCs, with a median survival of <1 year [1].

A common denominator for this group of diseases is their anatomic origin in the biliary tract. Significant differences, however, are noticeable in disease course, molecular profiling, and response to treatment. GBC is known to be associated with worse survival compared with CC. Ironically, it exhibits greater response rates to chemotherapy. GBC tends to develop distant metastasis after surgical treatment, whereas CC is more likely to recur locally [9].

Before results of the ABC02 randomized, controlled, phase III clinical trial were reported, scientific evidence for chemotherapy in advanced biliary tract cancers was from nonrandomized phase II trials, which were underpowered. The progression-free survival advantage demonstrated in the ABC01 trial triggered the ABC02 trial, which showed significant survival advantage with a combination of gemcitabine and cisplatin versus gemcitabine alone (median overall survival [OS] of 11.7 vs 8.1 months and progression-free survival [PFS] of 8.0 vs 5.0 months) [10]. A similar randomized study in the Japanese population posted a similar median OS advantage for the gemcitabine/cisplatin combination compared with gemcitabine alone (11.2 vs 7.7 months) [11]. While the ABC02 trial reported a tumor control rate of 81.4% for the combination regimen, the Japanese study documented a rate of 68.3% [10, 11].These two landmark trials established the gemcitabine/cisplatin combination as the standard of care for advanced biliary tract cancers. The infrequent incidence of BTCs and lack of randomized trials do not allow for definite conclusions regarding the role of radiation therapy. Molecular target-based cancer therapy holds the prospect of improved therapeutic efficacy, particularly in cases that are refractory to conventional chemotherapy [12].

The infrequent incidence of BTCs and lack of randomized trials do not allow for definite conclusions regarding the role of radiation therapy. Molecular target-based cancer therapy holds the prospect of improved therapeutic efficacy, particularly in cases that are refractory to conventional chemotherapy.

Genetic and Epigenetic Changes in Biliary Carcinogenesis

Complex changes in cholangiocyte genes that characterize transformation and progression to cholangiocarcinoma have been identified at the molecular level. Because these changes do not disrupt the DNA sequence of affected genes, they are not mutations. Rather, they oversee the mechanism of specific gene expression [13–16]. As such, they are better described as genetic/epigenetic changes. For the most part, these changes lead to silencing of genes involved in tumor suppression, cell cycle entry/ progression, apoptosis, and cell death [13, 15, 16]. Epigenetic changes have also been shown to impact regulation of genes involved in inflammation, DNA repair, cell adhesion, and invasion. Two key processes of genetic/epigenetic changes in biliary carcinogenesis are DNA methylation and regulation via microRNA.

DNA Methylation

The presence of abnormal DNA methylomes is a well-recognized event in carcinogenesis [17]. Sriraksa et al. were able to demonstrate that DNA methylation was a prevalent molecular abnormality in cholangiocarcinoma. They showed that the pattern of DNA methylation in primary CC cells was significantly different from those of surrounding normal tissues [18]. While hypermethylation was documented in the homeobox genes HOXA9 and HOXD9 (PCR2 targets) for CC cells, hypomethylation was observed at these sites in surrounding normal tissues. Thus, HOXA9 and HOXD9 hypermethylation has potential for use as a biomarker for early diagnosis of CC. A related observation is the overexpression of EZH2 (the catalytic subunit of PCR2 genes) in CC. This was reported by Sasaki et al. [19]. Hypermethylation of the promoter sequence of p16^INK4a results in its inactivation, which allows CDK4 to bind to cyclin D1 and subsequent unbridled entry into the S phase of the cell cycle [20]. Sasaki and coworkers also demonstrated the correlation between EZH2 levels and progression through the spectrum of low-grade dysplasia to invasive disease in cholangiocarcinogenesis [19]. Other cell cycle inhibitors implicated in cholangiocarcinogenesis that are suppressed via hypermethylation include p14^ARF [21], p16^INK4b [22], and 14-3-3 σ [23]. Tumor suppressor genes that have been shown to be inactivated via hypermethylation in cholangiocarcinomas include Semaphorin3B [24], RassF1A and p73 [22].

Studies have also shown an array of DNA repair genes that are suppressed in cholangiocarcinoma, through hypermethylation. These include hMLH1, O6-methylguanine-DNA methyltransferase, and glutathione S-transferase P1 (GSTP1) genes [22, 23].

Epigenetic silencing of SOCS-3 has also been documented in cholangiocarcinoma [25]. The result is overexpression of interleukin-6 (IL-6) [26–28], which translates to modulation of growth regulatory pathways like epidermal growth factor receptor (EGFR) in favor of tumor progression [29].

E-cadherin inhibits tumor cell invasion and metastasis [30–32]. Almost half of CC samples studied show methylation of the E-cadherin promoter, which indicates epigenetic suppression of the gene and consequent progression of the disease [22–24, 33].

MicroRNA

MicroRNAs are noncoding RNAs comprising approximately 22 nucleotides. They have regulatory roles in cellular processes like proliferation, death, apoptosis, fat metabolism, and differentiation [34–37].

Upregulation of miR-141, miR-200b, and miR-21 have been documented in CC. While miR-141 targets the CLOCK gene, which can act as a tumor suppressor, miR-200b dysregulates PTPN 12, thereby enhancing tumor cell survival and oncogenesis. miR-21 epigenetically silences PTEN, a tumor suppressor gene [38]. On the other hand, miR-29b and miR-370 were shown to be downregulated in CC. miR-29b downregulation may be related to overexpression of Mcl-1, an antiapoptotic protein, resulting in increased survival of CC cells [39, 40].

Upregulation of IL-6 in CC cells is associated with decreased expression of let-7a microRNA. This leads to downregulation of the NF2 gene, which is a known negative regulator of Stat-3. Activation of Stat-3 is reputed to play a role in many cancers [41]. Figure 1 presents a flowchart of DNA methylation and microRNA regulation of gene expression in BTC. Currently, there are no known DNA repair or cell adhesion genes involved in biliary carcinogenesis that are regulated by microRNAs.

Figure 1.

DNA methylation and microRNA regulation of gene expression in biliary tract carcinoma. Abnormal activation of major signal transduction pathways and molecular targeted inhibitors under clinic trials are summarized. MicroRNAs (miR-141, miR-200b, miR-21, miR-29b, and miR-370) regulate PI3K-associated effectors (Clock, PTEN, and Mcl-1), activating Akt/mTOR-mediated apoptosis resistance, survival, and angiogenesis [37–39]. Alterations of DNA methylation in the promoter regions of p16INKa/b, SOCS-3, Semaphorin-3B, and RassF1A, and changes of Let-7a miR levels upregulate CDK4/cyclin D, EGFR/HER2, and RAS/RAF, leading to MEK1/2 and ERK1/2-triggered cell cycle progression and tumor growth [19–28]. DNA methylation and modification of the E-cadherin HOXA9/D9 promoters and FAK hyperactivation contribute to aberrant transformation and differentiation [17, 18, 29–31]. Small molecular inhibitors and monoclonal antibodies have been developed to target the abnormal activity of the receptor or downstream kinases, preventing tumor proliferation and growth.

Altered Gene Expression in BTC

Aberrant levels of many transcripts and proteins in BTC have been observed using proteomic analysis, gene profiling, and immunodetection. The correlations between altered protein levels and BTC can be used as biomarkers to detect transformation, progress, and metastasis. The altered effectors are members of several signal transduction pathways that modulate inflammation, proliferation, anchorage dependency, and metabolism. Brief descriptions of some BTC-related proteins are shown in Table 1.

Table 1.

Altered gene expression in biliary tract cancer

Inflammation and Abnormal Growth

Persistent inflammation of the biliary tract is associated with cholangiocarcinoma. For example, IL-6 overexpression decreases the methylation of the EGFR promoter, leading to increased levels of EGFR mRNA and protein [29]. This suggests that IL-6-induced epigenetic regulation of EGFR can contribute to uncontrolled BTC growth.

Attachment and Survival

Transformation of the biliary tract epithelium leads to anchorage-independent growth. Decreased E-cadherin and increased N-cadherin levels are associated with epithelial to mesenchymal transition in prostate cancer [49]. Immunohistochemical analysis of tissues derived from 126 patients with gallbladder cancer has revealed a correlation between positive N- and P-cadherin expression with poor prognosis [48]. This indicates the involvement of cell adhesion modulation, including integrin-FAK signaling, in tumorigenesis of BTC.

Immunohistochemical analysis of tissues derived from 126 patients with gallbladder cancer has revealed a correlation between positive N- and P-cadherin expression with poor prognosis. This indicates the involvement of cell adhesion modulation, including integrin-FAK signaling, in tumorigenesis of BTC.

Oncogenic Metabolism

Comparison studies between normal and BTC tissues or cells, using different approaches, have revealed increased levels of several proteins important in glycolysis and the tricarboxylic acid cycle. The levels of Glut-1, Glut-2, enolase, IDH-1, and IDH-2 are correlated with BTC [43, 44, 50]. This suggests glucose oncometabolism in BTC and potential sites for targeted therapy.

Signal Transduction Pathways in BTC

Gene overexpression and deletion have been reported to define causal effectors that contribute to the development of BTC. Epigenetic, mutational, and transcriptional modulation promotes activation of several signaling pathways through effectors such as EGFR, human epidermal growth factor receptor 2 (HER2), VEGFR, IGF-1R, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Defining the cause-and-effect relationship of these signaling molecules and BTC can establish the molecular foundation of targeted therapy. Gene manipulation studies suggest several key players that contribute to transformation and/or progression of BTC; these are summarized in Table 2.

Table 2.

Major signal transduction pathways associated with BTC tumorigenesis

HER2/EGFR Signaling

The erbB2 (HER2) gene was constitutively expressed in the epidermis of transgenic mice to elucidate the role of HER/EGFR signaling in malignancy of the gallbladder epithelium [55]. All transgenic mice developed gallbladder adenocarcinoma and cholangiocarcinoma in the biliary tree by 3 months of age [55]. Hyperphosphorylation of HER2 and EGFR indicates their heterodimerization and activation in erbB2 transgenic mice. HER2 pathways can trigger activation of the mitogen-activated protein kinase (MAPK) ERK1/2, PI3K/Akt/mTOR, and STAT. Indeed, erbB2 overexpression leads to increased phosphorylation of MAPK [55].

K-ras/RAF Signaling

Tissue-specific activation of K-ras by a gain-of-function mutation G12D led to the development of intrahepatic cholangiocarcinoma in mice [50]. Combination of K-ras activation and p53 deletion shortens the mean survival time and causes widespread metastasis [51]. As an on-off switch, K-ras recruits and activates RAF and PI3K in cellular responses to growth factors such as VEGF and IGF-1. This is in agreement with the observations that mutations of K-ras, p53, and PIK3CA are common in specimens derived from patients with cholangiocarcinoma [55].

Pattern of Molecular Drivers of BTCs

Mutations in the TP53 gene are well documented for gallbladder cancer. Incidence rates for this mutation range from 44% to 47% in human gallbladder cancer series [57–59]. The incidence rate for this mutation in intrahepatic cholangiocarcinoma varies between 8.6% and 36% [59, 60], while for extrahepatic cholangiocarcinoma, it is as high as 17.5% [59].

KRAS was significantly mutated in a series of 51 gallbladder cancer cells matched with normal tissue. The mutation rate was 7.8% [58]. Two series that examined all BTCs showed much higher mutation rates for this gene in extrahepatic cholangiocarcinoma (23%–47%) compared with intrahepatic cholangiocarcinoma (5%–16%) and gallbladder cancer (4%–19%) [59].

Somatic mutations in IDH1 and IDH2 were documented for intrahepatic cholangiocarcinoma in different series of BTCs. Mutation rates are reported to be between 20% and 36%. More mutations were recorded in IDH1, with rates between 16% and 35%, compared with 3%–4% for IDH2 [57, 59, 60].

Several studies have reported on the role of FGFR fusions in intrahepatic cholangiocarcinoma. Among the fusions described are FGFR2-AHCYL1, FGFR2-BICC1, FGFR2-MGEA5, and FGFR2-TACC3. These fusions occur exclusively in intrahepatic cholangiocarcinoma at a rate of 13.6%. FGFR2 fusions result in cellular morphologic change that causes abnormal cell proliferation. In vitro and in vivo evaluations have also shown sensitivity of cholangiocarcinoma cells with FGFR2 mutations to FGFR inhibitors [61–63].

BAP1, ARID1A, and PBRM1 are chromatin-remodeling genes that have demonstrated mutations in BTCs. Mutation involving ARID1A have been identified in intrahepatic cholangiocarcinoma (11%–36%), extrahepatic cholangiocarcinoma (12%), and gallbladder cancer (6%–11.5%). Studies have demonstrated BAP1 mutation rates for gallbladder cancer (4%–13%) and intrahepatic cholangiocarcinoma (14%–20%). PBRM1 mutations were also identified in gallbladder cancer (7.7%–25%), extrahepatic cholangiocarcinoma (3.5%), and intrahepatic cholangiocarcinoma (13%–14.3%). The implication for practice is that BTCs that have these mutations may be sensitive to therapeutic agents targeting chromatin-remodeling genes [57, 59, 60].

Molecular Targets

EGFR

EGFR belongs to the Erb B family of class I tyrosine kinases. It plays a key role in the proliferation of cancers and is overexpressed in several solid tumors. Receptor-specific ligands that belong to the EGF family of growth factors bind onto EGFR (Erb1), Erb3, and Erb4.

The EGFR inhibitor erlotinib was studied as monotherapy in a single-arm, phase II trial for patients with advanced or metastatic BTCs [64]. The overall response rate was 7%, and 81% of the assessable tumors demonstrated EGFR expression (Table 3). However, EGFR mutational status was not assessed. Subsequently, a randomized phase III trial evaluated the combination of gemcitabine and oxaliplatin with or without continuous dosing of erlotinib for unresectable BTCs [65]. While the overall response rate was significantly higher in the chemotherapy plus erlotinib group (30% vs 16%; p = .005), PFS and OS did not differ (Table 3). Due to the mechanism of erlotinib and potential cell cycle sequence-specific synergy of erlotinib with gemcitabine, a phase Ib study evaluated the combination of gemcitabine and oxaliplatin with intermittent dosing of erlotinib for advanced BTCs [66]. Preliminary results demonstrated a 24% overall response rate and 6-month PFS rate of 75% (Table 3). This study highlighted the potential importance of mechanistic-driven dosing of targeted therapies when combined with cytotoxic chemotherapies.

Table 3.

Clinical studies of EGFR inhibition in biliary tract carcinoma

Monoclonal antibodies targeting EGFR showed promising results in BTCs, particularly in combination with traditional cytotoxic drugs. Two phase II trials evaluated the efficacy of cetuximab with gemcitabine and oxaliplatin. Gruenberger et al. reported an objective response rate of 63% in a trial of 30 patients with BTC; 30% of patients underwent potentially curative resection after treatment (Table 3) [67]. Final analysis of the randomized phase II BINGO (Gemcitabine and Oxaliplatin With or Without Cetuximab in Advanced Biliary Tract Cancer) trial showed that the primary endpoint of 4-month PFS ≥60% was exceeded in the gemcitabine/oxaliplatin plus cetuximab arm, but median PFS and OS were similar in both arms (Table 1) [68]. Enrollment was not limited according to KRAS status in either of these trials, and given the proven importance of this biomarker in colorectal cancer, perhaps the efficacy of anti-EGFR antibodies in BTCs could be further improved by biomarker-driven patient selection. In contrast to the cetuximab trials, a phase II trial evaluating gemcitabine, oxaliplatin, capecitabine, and panitumumab enrolled patients with KRAS wild-type cholangiocarcinoma only, with a 71.6% 6-month PFS, response rate of 33%, and median OS of 9.8 months (Table 3) [69].

A phase III randomized trial compared the combination of gemcitabine and oxaliplatin (GEMOX) alone (arm A) with GEMOX plus erlotinib (arm B) in 268 Korean patients with BTCs (ampullary carcinomas were included). There was no difference in OS and PFS for both arms. Subgroup analysis, however, showed a PFS advantage for arm B in CC patients (5.9 vs. 3.0 months; p = .049) [70].

VEGF

VEGF plays a prominent role in tumor-associated angiogenesis [71–73]. VEGFR-1 and VEGFR-2 are seldom observed in nondividing endothelial cells but are significantly expressed in association with tumor neovascularization [74–78].

VEGF expression in BTCs is associated with poor survival, metastasis, and disease recurrence. A phase II study of gemcitabine, oxaliplatin, and bevacizumab in advanced BTC reported a response rate of 40%, median PFS of 7 months, and OS of 12.7 months (Table 4) [79]. A single-arm phase II trial of erlotinib and bevacizumab without traditional cytotoxic chemotherapy in patients with advanced CC and gallbladder cancers demonstrated an 18.4% response rate, time to progression of 4.4 months, and OS of 9.9 months, with potential predictive signal for response seen from EGFR and KRAS status (Table 4) [80]. Another phase II trial combining bevacizumab with gemcitabine and capecitabine (NCT01007552) in patients with advanced BTCs is under way.

Table 4.

Clinical studies of VEGF inhibition in biliary tract carcinoma

Other antiangiogenic agents such as sorafenib and sunitinib have failed to show efficacy in this disease, either as single agents or in combination with gemcitabine, with response rates <10% and survival times less than that seen with other regimens [81–84]. Sorafenib is a multikinase inhibitor of VEGFR-2/-3, PDGFR-β, B-Raf, and C-Raf; it has shown some level of activity in preclinical models of cholangiocarcinoma. Based on this, in a single-arm, open-label, nonrandomized phase II clinical trial, Bengala et al. treated a total of 46 patients with advanced BTCs [81]. Twenty-six patients (56%) were pretreated with chemotherapy. A minimum of 45 days of treatment with sorafenib was completed by 36 patients. The objective response rate was 2% and the rate of stable disease at 12 weeks was 32.6%. For this study, PFS was documented as 2.3 months (range: 0–12 months) and the median OS was 4.4 months (range: 0–22 months). A significant association was noted between performance status and PFS, with median PFS values for Eastern Cooperative Oncology Group (ECOG) 0 and 1 being 5.7 and 2.1 months, respectively (p = .0002). Treatment toxicities included skin rash (35%) and fatigue (33%), which necessitated dose reduction in 22% of patients (Table 4) [81].

HER2

HER2 is also known as ErbB2, c-erbB2, or HER2/neu. It has an intracellular tyrosine kinase domain and an extracellular ligand binding domain. The major signaling pathways mediated by HER2 involve MAPK pathways and PI3K pathways.

The California Consortium conducted a phase II study using lapatinib (a dual tyrosine kinase inhibitor of HER2/neu and EGFR pathways) in patients with advanced BTC or hepatobiliary cancer [83]. In all 17 patients recruited into the study, no response was observed. However, HER2 status was not reported (Table 5). A phase I study by Siegel-Lakhai et al. using lapatinib in combination with oxaliplatin/fluorouracil/leucovorin documented partial response in 2 out of 34 patients (Table 5) [86]. This study also failed to document HER2 status. Another phase II clinical trial reported no response to lapatinib in nine patients with advanced, unresectable BTCs. There were no mutations in HER2/neu or HER2 overexpression in these patients [87].

Table 5.

Clinical studies of HER2 inhibition in biliary tract carcinoma

MEK/MAPK

MAP kinase signaling pathways feature in cell proliferation, differentiation, and migration, through the activation of protooncogenes such as JUN, FOS, MYC, and ELK1. There are four identified MAP kinase signaling pathways: the ERK1 pathway, the c-jun N-terminal-regulated kinase (JNK) pathway, the p38 pathway, and the ERK5 pathway [88]. JNK and p38 pathways are mainly stress activated by proinflammatory cytokines. ERK1 and ERK5 are induced by EGFR activation [89].

The inhibition of MEK or ERK carries significant potential as a therapeutic target for BTCs and other solid tumors, but is still being investigated. A phase II trial of selumetinib (MEK1/2 inhibitor) for patients with unresectable BTC recorded an objective response rate (ORR) of 12% and median OS of 9.8 months [90]. Although the ORR was low, 68% of patients had stable disease. Of these, 44% had stable disease for a minimum of 16 weeks; while 12% had stable disease for >1 year (Table 6). The treatment was well tolerated by the patients. Immunohistochemistry testing for KRAS/BRAF genotyping and phosphorylated ERK (pERK) and AKT (pAKT) was performed on all tumor tissues. Patients with short-lived stable disease had KRAS mutations, and absence of pERK staining was associated with no response. In another study, MEK 162 (a selective, ATP-uncompetitive inhibitor of MEK1/2) was used to treat 28 patients with advanced BTC. One complete response, 1 partial response, and 11 cases of stable disease were observed in 26 evaluable patients. The two responders had wild-type extrahepatic CC [91]. There is an ongoing randomized clinical trial (ClinicalTrials.gov identifier NCT02042443) studying trametinib (an MEK inhibitor) compared with chemotherapy with 5-fluorouracil or capecitabine in advanced BTCs.

Table 6.

Clinical studies in biliary tract carcinoma

IGF/IGFR

Autophosphorylation of the IGF-I receptor occurs when IGF-I and IGF-II bind to it. This interaction activates signaling pathways like ERK and the phosphatidylinositide 3-kinase/Akt-1 axis [92]. Other factors that play a role in the activation of IGF-I receptor are IGF-binding proteins and IGF-2 receptors [93–95]. Dysregulation of the IGF system contributes to the proliferation of several neoplasms [96, 97]. While mutations and chromosomal amplifications of IGF-IR are rare, the regulation of its expression is closely associated with the function of several oncogenes and tumor suppressor genes [97]. Elevated serum IGF-I level also increases the risk of developing several cancers [94]. Since IGF-IR signaling is crucial to processes like cell migration, angiogenesis, invasion, and metastasis, it plays a significant role in tumor dissemination [98].

The IGF pathway (more specifically, IGF-IR) is active in many malignancies [99], including colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian, and endometrial cancer, as well as sarcomas [100–109]. As such, novel therapeutics are evolving with monoclonal antibodies targeted at the IGF-IR. Inhibition of IGF-IR tyrosine kinase with small molecules has also been explored. However, therapeutic development has been approached very cautiously because of significant concerns for potential toxicity of inhibiting the IGF pathway. The IGF pathway is present in nearly all healthy and malignant cells alike. The IGF-IR also shares significant homology with the insulin receptor. Therefore, the potential for metabolic dysfunction like hyperglycemia is considerable and has been observed in human trials.

Successful in vivo and in vitro studies led to several phase I studies that subsequently evolved into phase II or III trials. Results regarding safety and efficacy have been satisfactory; however, long-term effects are still unknown. Details about dosing and scheduling are still being determined. Notably, when anti-IGF pathway treatment is combined with other targeted therapies or cytotoxic chemotherapeutics, toxicity increases.

To the best of our knowledge, we are not aware of any clinical trial specifically targeting IGF/IGFR in BTCs. However, we know of phase I/II trials directed at breast cancer, adrenocortical cancer, soft tissue sarcomas, Ewing sarcoma, lung cancer, and hepatocellular cancer [110–112].

PI3K/mTOR/AKT

PI3K signaling plays a critical role in cholangiocarcinogenesis, anticancer drug resistance, and autophagy (type II programmed cell-death regulation). Studies have shown disruption of the phosphatidylinositol 3-kinase/AKT/mTOR pathways to be a common occurrence in CC.

A prospective, single-arm protocol was used to assess tumor response to sirolimus (an mTOR inhibitor) in 21 patients with advanced HCC and 9 patients with CC. For the HCC patient group, one partial remission and five cases of stable disease were observed at 3 months, while the CC group had three cases of stable disease. The median survival for HCC patients treated under the protocol was 6.5 months (range: 0.2–36 months) and 7 months (range: 2.6–35 months) for CC patients (supplemental online Table 1) [113].

The Multi-Institutional Phase II Study of the Akt Inhibitor MK-2206 in Refractory Biliary Cancers (ClinicalTrials.gov identifier NCT-01425879) is an ongoing, multi-institutional phase II study of the Akt inhibitor MK-2206 in refractory biliary cancers. This trial is designed to show how effective MD2206 is in treating patients with advanced refractory biliary cancer that cannot be removed by surgery. The findings of another clinical trial (NCT01859182) assessing the response of unresectable gallbladder cancer and bile duct cancer to combination therapy with selumetinib (MEK inhibitor) and Akt inhibitor MK-2206 are also pending.

Conclusion

While the prognosis is unfavorable in most cases of BTC, progress is being made in the systemic management of this disease. There is no substitution for early detection and improvements in surgical techniques for treating BTC, but opportunities to improve the systemic treatment of this disease abound and should be explored. However, the particular challenges of developing drug-like small molecules and antibody-based inhibitors for BTC treatment include (a) targeting DNA methylation and microRNA-related signaling, (b) interactions of multiple signaling pathways, and (c) lack of clinical trials on inhibition of transformation and metastasis.

The known alterations of gene expression in BTC are often related to hyper- or hypomethylation of promoters and/or microRNA. Current investigations mainly are focusing on receptors such as EGFR, HER2, VEGFR, and downstream effectors such as sirolimus and selumetinib inhibition of mTOR and MEK/ERK. The specificity of these drugs targeting downstream events may be low, since the major mediators of BTC are likely related to epigenetics and transcriptional events. Development of inhibitors targeting BTC-related DNA methylation and microRNA modulation is expected to overcome this obstacle.

Epigenetic and transcriptional modulation often impacts multiple signal transduction pathways. Indeed, abnormalities of transformation, cell cycle control, survival, and invasion have been observed in BTC. Targeting a single pathway may not prevent BTC progression. Rational combinations of targeted therapies and targeted therapy with conventional chemotherapy are crucial to future success in treatment.

Current clinical trials of inhibitors for BTC treatment are aimed at blocking PI3K/Akt- and MEK/ERK-mediated proliferation and tumor growth. DNA methylation or microRNA-altered cadherin, HOX, and FAK/SRC expression play a critical role in tumorigenesis and metastasis. Therefore, it could be beneficial for BTC patients if specific small molecules or antibodies are developed that attenuate hyperactivation of these molecules associated with transformation and invasion.

In this era of personalized medicine, identification of predictive markers, including well-characterized genetic mutations, will help to select specific populations of BTCs for targeted therapy.

See http://www.TheOncologist.com for supplemental material available online.

This article is available for continuing medical education credit at CME.TheOncologist.com.

Author Contributions

Conception/Design: Tolutope Oyasiji, Jianliang Zhang, Boris Kuvshinoff, Renuka Iyer, Steven N. Hochwald

Provision of study material or patients: Tolutope Oyasiji, Jianliang Zhang, Boris Kuvshinoff, Renuka Iyer, Steven N. Hochwald

Collection and/or assembly of data: Tolutope Oyasiji, Jianliang Zhang, Boris Kuvshinoff, Renuka Iyer, Steven N. Hochwald

Data analysis and interpretation: Tolutope Oyasiji, Jianliang Zhang, Boris Kuvshinoff, Renuka Iyer, Steven N. Hochwald

Manuscript writing: Tolutope Oyasiji, Jianliang Zhang, Boris Kuvshinoff, Renuka Iyer, Steven N. Hochwald

Final approval of manuscript: Tolutope Oyasiji, Jianliang Zhang, Boris Kuvshinoff, Renuka Iyer, Steven N. Hochwald

Disclosures

Renuka Iyer: Genentech (RF); Steven N. Hochwald: Ethicon Endosurgery (C/A). The other authors indicated no financial relationships.

(C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board