Biliary tract cancers (BTCs) are a heterogeneous group of adenocarcinomas that originate from the epithelial lining of the biliary tree. BTCs are characterized by presentation with advanced disease precluding curative surgery, rising global incidence, and a poor prognosis. Chemotherapy is the mainstay of the current treatment, which results in a median overall survival of less than one year, underscoring the need for novel therapeutic agents and strategies. Next-generation sequencing-based molecular profiling has shed light on the underpinnings of the complex pathophysiology of BTC and has uncovered numerous actionable targets, leading to the discovery of new therapies tailored to the molecular targets. Therapies targeting fibroblast growth factor receptor (FGFR) fusion, isocitrate dehydrogenase (IDH) mutations, the human epidermal growth factor receptor (HER) family, DNA damage repair (DDR) pathways, and BRAF mutations have produced early encouraging results in selected patients. Current clinical trials evaluating targeted therapies, as monotherapies and in combination with other agents, are paving the way for novel treatment options. Genomic profiling of cell-free circulating tumor DNA that can assist in the identification of an actionable target is another exciting area of development.

1. Introduction

1. Introduction

Biliary tract cancers (BTCs) are a heterogeneous group of adenocarcinomas that originate in the epithelial lining of the bile ducts and gallbladder. Based on the anatomical location, BTCs are classified into two major categories—cholangiocarcinoma (CCA), which arises from the bile ducts, and gall bladder carcinoma (GBC). CCAs are subdivided into intrahepatic CCA (iCCA) and extrahepatic CCA (eCCA), with the eCCA further split into perihilar (Klatskin’s tumor) and distal CCA ^[1][‎1]. Each of these anatomic subgroups have their unique natural history and treatment nuances ^[1][‎1].

BTCs, although relatively uncommon in the United States, with 23,000 new cases diagnosed annually ^[2][‎2], constitute approximately 3% of all gastrointestinal malignancies ^[3][‎3]. Advanced BTC has a dismal prognosis, with a five-year survival rate of less than 10% ^[4][‎4]. The majority of patients with BTC (>65%) present with unresectable disease, and patients who undergo potentially curative surgery experience a high rate of relapse ^[5][6][‎5,‎6], underscoring the importance of systemic therapy. The current standard therapy of advanced BTC primarily consists of systemic chemotherapy, which results in a median overall survival (OS) of approximately 12 months ^[7][‎7]. However, targeted therapies are emerging as a promising treatment option, encouraged by the recent Food and Drug Administration (FDA) approval of pemigatinib for treatment of adults with chemotherapy-refractory CCA, i.e., advanced CCA harboring a fibroblast growth factor receptor (FGFR) 2 fusion.

As the understanding of the genomic landscape of BTC is growing, therapies targeting actionable alterations are being evaluated in numerous clinical trials, often combined with chemotherapy, both in frontline and chemotherapy-refractory settings [‎8]. Herein, we review the recent advancements and the emerging novel targeted therapies for BTC that will likely improve patient outcomes in the near future.

2. Targeted Therapies

Studies utilizing NGS to elucidate the molecular profile of BTC have highlighted that BTCs are target-rich malignancies with therapeutically relevant genetic alterations identified in approximately half of the patients ^[8][9][‎25,‎26], which has led to a plethora of clinical trials with targeted therapies. Although no randomized study reported to date has confirmed the superiority of the targeted approach in advanced BTC, the MOSCATO-01 trial provides early supportive evidence ^[10][‎41]. In this trial, patients who received targeted therapy (18 out of 43 patients) had an improved median OS compared to those treated with unselected therapies (median OS 17 vs. 5 months; p = 0.008).

Currently, the most promising targets in advanced BTCs consist of IDH mutations, FGFR aberrations, BRAF mutations, the DNA damage repair (DDR) pathway, and the HER2 pathway. Table 1 summarizes the published study results of the targeted therapies in advanced BTC.

Table 1. Published study results of targeted therapies in patients with advanced biliary tract carcinoma (BTC).

Abbreviations: ORR, overall response rate; PFS, progression-free survival; OS, overall survival; CCA, cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma;BTC, biliary tract cancer.

2.1. FGFR Pathway

Targeting FGFR fusions, almost exclusively seen in iCCA, has shown clinically meaningful benefits in recent clinical trials. FGFR gene fusions are present in approximately 20% of patients with iCCAs . FGFRs consist of four transmembrane receptors (FGFR 1–4) with intracellular tyrosine kinase domains that regulate cell proliferation, migration, differentiation, and angiogenesis via binding to the ligands (fibroblast growth factors, FGFs) ^[24][‎54]. FGFR2 fusion is the most common FGFR aberration, which activates multiple oncogenic canonical signaling events downstream of FGFR ^[25][‎55]. FGFR2 fusions are therapeutically more relevant than FGFR mutations [‎21].

Among the FGFR tyrosine kinase inhibitors, Pemigatinib (INCB054828), Infigratinib (BGJ398), and Futibatinib (TAS-120) have shown encouraging results in phase I and II studies with manageable toxicity profiles (Table 1). These selective FGFR inhibitors are associated with an ORR of 15–35%, and a median PFS of around six months in previously treated patients with FGFR2 fusions. Pemigatinib, a selective oral inhibitor of FGFR1-3, has been evaluated in a phase II study (FIGHT-202) that enrolled chemotherapy-refractory patients with a variety of FGFR alterations [‎21]. This study reported an ORR of 35.5%, a median PFS of 6.9 months, and a preliminary OS of 21.1 months in patients with FGFR2 fusions. This study result led to the FDA approval of pemigatinib for the treatment of chemotherapy-refractory advanced iCCA, the first approval of a targeted agent for advanced BTC. Of note, pemigatinib had no significant activity in patients with FGFR amplification or mutations (ORR 0% and a median PFS of 2.1 months). Adverse events were manageable, with hyperphosphatemia being the most common any-grade adverse event (60%). Grade ≥ 3 adverse events were reported in 64% of patients, which included hypophosphatemia (12%), arthralgia (6%), stomatitis (5%), hyponatremia (5%), abdominal pain (5%), and fatigue (5%). Serious adverse events occurred in 45% of patients, but no treatment-related deaths were reported. Hyperphosphataemia appears to be related to the alteration of vitamin D and phosphorus metabolism mediated via fibroblast growth factor 23 (FGF23) ^[26][‎56]. Currently, Pemigatinib, infigratinib, and futibatinib are being investigated in treatment-naive patient cohorts in phase III trials against the gemcitabine and cisplatin combination (Table 2). ARQ087 (derazantinib), a nonselective multikinase inhibitor with activity on FGFR, has also recently entered phase III trial in pretreated patients after the publication of encouraging phase II data [‎45]. The study results of other FGFR inhibitors, including Debio 1347 and Erdafitinib, are summarized in Table 1.

Table 2. Selected ongoing and upcoming clinical trials evaluating targeted therapies in patients with advanced biliary tract cancer *.

Abbreviations: PFS, progression-free survival; MTD, maximal tolerated dose; ORR, overall response rate; RP2D, recommended phase II dose. * Information obtained from ClinicalTrials.gov (https://clinicaltrials.gov/), accessed between 17 May 2020, and 21 June 2020.

FGFR inhibitors are subject to a variety of resistance mechanisms, including polyclonal point mutation in the FGFR2 kinase domain [‎38] and the development of novel FGFR2 fusions ^[27][‎57]. Futibatinib (TAS-120), a highly selective pan-FGFR inhibitor, has activity against FGFR2 resistance mutations [‎43] and has shown promising clinical activity in FGFR-aberrant iCCA patients in an early-phase study, including in patients progressing on prior FGFR inhibitors. The phase II FOENIX-101 study with futibatinib is currently recruiting patients with iCCA-harboring FGFR2 gene rearrangements after progression on first-line treatment (Table 2).

2.2. Isocitrate Dehydrogenase (IDH) Mutations

Approximately 20% of patients with iCCA harbor IDH1 or IDH2 mutations (Table 1). IDH1 mutations are more frequent than IDH2 mutations and are generally found in iCCAs not related to hepatitis and fluke infection ^[28][‎58]. These somatic mutations cause an increase in IDH1/2 activity resulting in modulation of cell metabolism and accumulation of 2-hydroxyglutarate (2-HG), an oncometabolite that interferes with normal cell differentiation and promotes tumorigenesis ^[29][‎59]. IDH1 and IDH2 mutations do not have any prognostic implications, unlike FGFR alterations ^[30][‎60].

Ivosidenib (AG-120), an oral IDH1 inhibitor, has shown encouraging activity in patients with advanced IDH1-mutant, chemotherapy-refractory CCA  [‎48,‎49] (Table 1

). The phase I study [‎48] with ivosidenib in refractory patients reported an ORR of 5%, a median PFS of 3.8 months, and a median OS of 13.8 months. Although the response rate was low, a significant proportion of patients were progression-free at 12 months (21.8%), and the tolerability of ivosidenib was remarkable—only 5% of patients experienced grade 3 or higher toxicities, and there were no dose-limiting toxicities. The most frequent side effects, including fatigue and nausea, were manageable. Subsequently, a phase III, placebo-controlled trial (ClarIDHy) was conducted in patients with IDH1-mutant advanced iCCA who progressed on at least one line of chemotherapy [‎49]. The primary endpoint was PFS, and crossover from placebo to ivosidenib was permitted after progression. This study demonstrated a small median PFS improvement with ivosidenib over placebo, 2.7 months vs. 1.4 months (HR 0.37; 95% CI, 0.25–0.54; p < 0.0001). Although the short PFS improvement is a cause for concern, 32% of patients on the ivosidenib treatment arm had not progressed at six months, and 22% had not progressed at one year. Conversely, no patient on the placebo arm was progression-free at six months. The median OS in the ivosidenib arm was 10.8 months vs. 9.7 months in the placebo arm, which was not statistically significant. However, when adjusted for the crossover, the median OS in the placebo arm dropped to six months, and the OS difference was significant (HR 0.46; p = 0.0008). The toxicity profile was consistent with the previous report [‎48].

Given the modest activity of IDH inhibitors, novel strategies targeting IDH mutations are being explored in clinical trials. iCCA cell lines showed remarkable sensitivity to dasatinib, a multikinase inhibitor, in a preclinical study utilizing a high-throughput drug screening method, which was confirmed in a xenograft model ^[31][‎61]. Currently, dasatinib is being investigated in a phase II trial in patients of advanced iCCA harboring IDH-mutations (ClinicalTrials.gov Identifier: NCT02428855). Another preclinical study demonstrated that 2-HG produced as a result of IDH mutations suppresses homologous recombination and induces PARP (poly ADP ribose polymerase) inhibitor sensitivity (‘BRCAness’) ^[32][‎62], which led to a phase II trial with Olaparib in solid tumors, including CCA (ClinicalTrials.gov Identifier: NCT03212274). Furthermore, a phase I study is planned with a combination of ivosidenib and cisplatin/gemcitabine in patients with advanced CCA (ClinicalTrials.gov Identifier: NCT04088188).

2.3. Human Epidermal Growth Factor Receptor (HER) Pathway

The HER family receptors consist of four distinct receptors: epidermal growth factor receptor (EGFR) or HER1, HER-2, HER-3, and HER-4. In normal cells, binding of the ligands to the extracellular domain of these receptors leads to the dimerization of the receptors with eventual phosphorylation of the intracellular tyrosine kinase domain and activation of the downstream pathways, which include MAPK, PI3K/AKT/mTOR, and STAT pathways ^[33][‎63].

Therapies targeting HER2 have demonstrated modest activity thus far. Among the 5–15% of BTCs expressing HER2, most are gallbladder cancers or eCCA ^[34][‎64]. The combination of HER2 directed monoclonal antibodies pertuzumab and trastuzumab was investigated as a part of the MyPathway multi-basket study in 11 patients with refractory BTC ^[35][‎65]. At a median follow-up of 4.2 months, four patients had partial responses (PR), and three had stable disease (SD) for about four months. Neratinib, an oral tyrosine kinase inhibitor of EGFR, HER2, and HER4, was studied in HER2-mutant advanced solid tumors in the phase II SUMMIT basket trial [‎51]. An ORR of 10% was reported among the 20 patients with HER2-mutant BTC. Trastuzumab deruxtecan (DS-8201), an antibody-drug conjugate targeting HER2, is under evaluation in HER2-positive patients with advanced BTC in a clinical trial (Table 2).

EGFR (HER1) overexpression is common among among patients with CCA and is associated with poor prognosis,particularly in iCCA ^[36][‎66]. A phase III study investigated the activity of an EGFR inhibitor, erlotinib, plus chemotherapy (gemcitabine and oxaliplatin) versus chemotherapy alone in therapy-naive patients with advanced BTC ^[37][‎67]. The combination of erlotinib and chemotherapy was associated with a significantly increased ORR (30% vs. 16%; p = 0.005), but an improvement in PFS or OS was not demonstrated. A post-hoc analysis of this study showed that the subgroup of patients with CCA had an improved median PFS with the combination therapy (5.9 months vs. 3.0 months; p = 0.049). Studies with a combination of chemotherapy and anti-EGFR antibodies cetuximab ^[38][‎68] and panitumumab ^[39][‎69] failed to show an improvement in ORR, PFS, or OS. Studies with other HER pathway inhibitors, including afatinib ^[40][‎70] and Lapatinib ^[41][‎71], also showed disappointing results. Conversely, varlitinib, a pan-HER inhibitor, demonstrated a somewhat encouraging result, with a partial response rate of 27%, stable disease of 43%, and a disease control rate of 70% in 37 patients with refractory BTC in a pooled analysis of three phase I studies ^[42][‎72]. Currently, varlitinib, in combination with gemcitabine and cisplatin, is being evaluated in patients with treatment-naive advanced BTC (ClinicalTrials.gov Identifier: NCT02992340) (Table 4).

2.4. DNA Damage Repair (DDR) Mechanisms and BAP1 Mutations

Somatic or germline mutations in BRCA are detected in about 3.5% of patients with BTC and result in an immunogenic tumor profile charact-herized by a higher TMB and a higher rate of microsatellite instability (MSI)-high tumors ^[43][‎73]. Furthermore, IDH mutations confer sensitivity to PARP inhibitors, as discussed above. DDR pathway mutations or IDH1 mutations were reported to be present in about half of patients with BTC ^[44][‎74]. Based on this rationale, a phase II study is underway to test the combination of rucaparib (a PARP inhibitor) and nivolumab (a PD-1 blocker) in a cohort of patients who progressed on chemotherapy (Table 2). Patients of CCA (mostly iCCA) with mutations in BAP1, a tumor suppressor gene involved in DNA double-strand break repair, are reported to have an aggressive disease and poor response to standard therapies ^[45][‎75]. A phase II basket trial is currently investigating the clinical benefit of the PARP inhibitor niraparib in patients with BAP1 mutations and other DDR-deficient solid tumors, including CCA (Table 2).

2.5. RAS/RAF/MEK/ERK Pathway

Although RAS mutations serve as oncogenic drivers in many different types of malignancies, targeting RAS has mostly been unsuccessful because of its intricate interactions with other signaling pathways. Consequently, inhibiting targets downstream of RAS, such as BRAF and MEK, has been attempted, but with limited clinical success thus far. Selumetinib, a MEK inhibitor, in combination with cisplatin/gemcitabine has been studied in a phase Ib study (ABC-04) in therapy-naive patients with advanced BTC ^[46][‎76]. Among eight evaluable patients, three had a partial response, and five had stable disease with a median PFS of 6.4 months. A phase II study with selumetinib, which included 39% of chemotherapy-refractory patients, showed an ORR of 12%, a median PFS of 3.7 months, and a median OS of 9.8 months with an acceptable safety profile ^[47][‎77].

BRAF mutations have been described in less than 5% of patients with BTC, primarily in the cohort with intrahepatic disease [‎23]. Dual targeting with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) within the ROAR trial, a phase II basket trial of 178 patients harboring the BRAF^V600E mutations, which included 33 patients with advanced refractory CCA, showed encouraging efficacy with an ORR of 41%, a median PFS of 7.2 months, and a median OS of 11.3 months [‎50]. PLX8394, a BRAF inhibitor, is currently being evaluated in patients with refractory solid tumors, including BTC (ClinicalTrials.gov Identifier: NCT02428712).

2.6. PI3K/AKT/mTOR Pathway

The PI3K/AKT/mTOR pathway regulates cellular proliferation and angiogenesis through its interactions with the RAS/RAF/MEK pathway and the mTOR signaling pathway ^[48][‎78] (Figure 1). Mutations in PIK3CA, resulting in upregulation of the PI3K/AKT/mTOR pathway, have been identified in many cancer types, including in BTC ^[49][‎79]. Everolimus, an mTOR inhibitor, has shown modest activity in patients with previously treated BTC in a phase II trial, which reported a median PFS of 3.2 months and a median OS of 7.7 months ^[50][‎80]. A phase II study with everolimus in the first-line setting demonstrated a PFS of 5.5 months and OS of 9.5 months ^[51][‎81]. A combination of cisplatin/gemcitabine and copanlisib, a pan-PI3K inhibitor, has shown modest activity in a phase I study with an ORR of 17.4% ^[52][‎82]. A phase II study with this combination in the first-line setting is planned (Table 2).

2.7. Vascular Endothelial Growth Factor (VEGF)

Vascular endothelial growth factor (VEGF) overexpression in BTC is a poor prognostic factor, particularly in iCCA [‎66]. However, targeting VEGF has not been a successful strategy so far. A meta-analysis of seven randomized trials involving 964 patients reported a lack of PFS or OS benefit with anti-VEGF therapy in combination with chemotherapy compared to chemotherapy alone ^[53][‎83]. Cediranib, a potent VEGF receptor tyrosine kinase inhibitor, did not show a survival benefit in therapy-naive patients when combined with gemcitabine/cisplatin in comparison to gemcitabine/cisplatin plus placebo in the randomized phase II ABC-03 trial ^[54][‎84]. Studies with other multikinase VEGF receptor inhibitors, including sorafenib ^[55][‎85], lenvatinib ^[56][‎86], vandetinib ^[57][‎87], and regorafenib ^[58][‎88], have also been disappointing. Alternative strategies, for example, a combination of VEGF inhibition (lenvatinib) and immune checkpoint inhibition (pembrolizumab), have reported encouraging results (detailed in Section 5.9).

2.8. Miscellaneous Pathways

NTRK (neurotropic tyrosine kinase receptor) fusions, identified in 3.5% of patients with iCCA ^[59][‎89], are targetable with currently approved first-generation TRK (tropomyosin receptor kinase) inhibitors larotrectinib and entrectinib, which have produced an impressive ORR of 57% to 75% in advanced solid tumors harboring NTRK fusions ^[60][61][‎90,‎91]. Of note, TRK inhibitors have activity against ROS1 and ALK fusions, which are reported to be present in 0–8.7% and 2.7% of patients with CCA, respectively ^[62][‎89,‎92].

c-MET regulates cell proliferation, migration, and invasion ^[63][‎93]. A phase II study with cabozantinib, an oral MET inhibitor, reported a median PFS of 1.8 months and a median OS of 5.2 months in patients with previously-treated advanced CCA ^[64][‎94]. A phase I study of an oral c-MET inhibitor, tivantinib, in combination with gemcitabine, demonstrated a modest activity with an ORR of 19% ^[65][‎95]. Merestinib, a small molecule inhibitor of MET, did not improve ORR, PFS, or OS when added to gemcitabine and cisplatin in a recently reported randomized phase II trial ^[66][‎96].

2.9. Targeted Therapy and Immunotherapy Combinations

Although a detailed account of immunotherapy in BTC is beyond the scope of this article, a discussion of novel immunotherapy/targeted therapy combinations is relevant. The modest activity of single-agent immunotherapeutic agents in advanced BTC, reporting an ORR of 5%–20% ^[67][‎97], led to the exploration of a variety of combination strategies, including immunotherapy/targeted therapy combinations. The combination of pembrolizumab (a PD-1 inhibitor) and ramucirumab (a VEGF receptor 2 inhibitor) investigated in a phase I trial in patients with previously treated advanced BTC reported a modest median PFS and median OS of 1.6 and 6.4 months, respectively ^[68][‎98]. A promising result has been reported with the combination of a multikinase inhibitor, lenvatinib, and pembrolizumab in 32 patients who had received at least two prior anticancer therapies ^[69][‎99]. This study reported an ORR of 25%, a median PFS of 4.9 months, and a median OS of 11 months. A multicenter randomized phase II trial (n = 86) of atezolizumab (a PD-L1 blocker) as monotherapy or in combination with cobimetinib (a MEK inhibitor) in refractory advanced BTC has recently reported superior median PFS with the atezolizumab/cobimetinib combination—3.65 months vs. 1.87 months (p = 0.027); OS data are not mature at this time ^[70][‎100]. A combination of PD-1 inhibitor and DNA repair modulators, as described in section 5.4, is another area of exploration. These early results suggest that the immunotherapy/targeted therapy combinations will likely be a new frontier for further exploration.