Novel biomarkers and the future of targeted therapies in cholangiocarcinoma: a narrative review

Introduction

Cholangiocarcinoma (CCA) is a highly aggressive and diverse group of biliary malignancies arising from any site in the biliary tree and comprises 15% of all primary liver cancers (1). CCA can be classified into three distinct sub-types intrahepatic cholangiocarcinoma (iCCA), perihilar cholangiocarcinoma (pCCA), or distal cholangiocarcinoma (dCCA) based on anatomical location. The nature of the disease, challenging surrounding anatomy, and nonspecific presentation, particularly for iCCA, leads to late diagnosis and ultimately poor outcomes for patients. Since 2010, the standard of care in treating all advanced, inoperable biliary tract cancers has been combination gemcitabine and cisplatin (gem-cis). Valle et al. demonstrated in the phase III, randomized ABC-02 trial, a median overall survival (OS) of 11.7 months with this combination compared to 8.1 months with single-agent gemcitabine in patients with unresectable, recurrent, or metastatic biliary tract cancers. The rate of tumor control among patients in the gemcitabine-cisplatin group was significantly increased (81.4% vs. 71.8%, P=0.049) (2).

Unfortunately, since gem-cis was established as first-line therapy, progress in systemic therapies reaching clinical practice has been minimal. The reality is the 5-year survival for advanced CCA remains poor at 5–10% (3). There is a dire need for earlier diagnosis and improved treatment options for patients with CCA in both the first line and second-line settings, particularly given the known toxicity profile of gemcitabine-cisplatin (3). CCA has various etiologies and is clinically heterogenous, differing in presentation and complexity depending on tumor location, which presents a unique challenge (4,5). This has made development of universal treatments challenging, raising the question if targeted therapies will yield more promising results. Identifying clinically relevant biomarkers has come to the forefront of this discussion, with the hope for earlier diagnosis, prognostic information (i.e, overall survival or tumour recurrence), and ability to guide treatment decisions (6). Currently, carcinoembryonic antigen (CEA), CA 19-9, and CA 125 are the only biomarkers used in clinical practice with limited utility for diagnosis or monitoring of CCA given its poor sensitivity and specificity (1). However, the advent of next generation sequencing has started to elucidate the genetic landscape of CCA and helped to identify tumor biomarkers or “actionable mutations”, raising the possibility of novel treatment approaches with targeted therapies. This focused review of the literature highlights the role of NGS in CCA and identifies the clinically relevant tumor biomarkers that have become the focus of ongoing therapeutic development (Table 1). We present the following article in accordance with the Narrative Review reporting checklist (available at https://hbsn.amegroups.com/article/view/10.21037/hbsn-20-475/rc).

Section 1: the role of molecular profiling

Next-generation sequencing (NGS) in CCA

NGS involves several different modern sequencing technologies, allowing for the sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing (7). Significant advances in genomics and molecular biology are directly attributable to NGS, which has become increasingly reliable in terms of sequencing, chemical analyses, and data interpretation. In recent years, NGS has become increasingly useful in clinical practice with applications ranging from mutation detection in inherited cancer syndromes, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy (7). NGS has been central in advancing the understanding of cancer biology with the identification of genetic variances contributing to tumor growth, development and metastasis, driver genes or mutations and passenger mutations (7). This has added relevance in the clinical setting through improved patient classification, prognostication, targeted treatments, drug-resistance, and pharmacogenetics. Recent advances in the understanding of the pathogenesis of CCA are tied to molecular insights provided by NGS.

Jusakul et al. was one of the first to explore the molecular and epigenomic landscape in CCA, studying a cohort of nearly 500 CCAs from distinct geographical regions with NGS (8). Four distinct etiological clusters were recognized, classified by differences in gene expression, mutation and copy number changes, and epigenetic changes. The specific mutations reported were ERBB2 amplification, TP53 mutations, levels of PD-1/PD-L2 expression, IDH1/2 and BAP1 epigenetic mutations, and FGFR/PRKA related gene rearrangements (8). Classifying CCA’s by these driver genes, noncoding promoter mutations, and structural variants, revealed distinct molecular and clinicopathologic features. In other words, molecular profiling of these CCAs provided insight beyond the anatomical location of the tumor. Interestingly, CCAs in different anatomical locations did not differ in their survival trends, while CCA’s stratified by molecular clusters showed significant differences in survival were noted (8). This points to distinct cancer subtypes from the same organ arising from varying oncogenic processes. Given this, NGS offers the potential to identify disease subsets with differing prognostic and therapeutic implications, while further informing the development of targeted therapies.

NGS has facilitated the profiling and analysis of the tumor microenvironment in CCA and helped identify novel biomarkers ranging from extracellular vesicles, circulating nucleic acids, and circulating tumors cells (e.g, OPN, LOXL2, EPCAM) (4). Expression of non-coding RNA, including lncRNA, has shown some promising prognostic value in CCA (5). However, before these biomarkers can be utilized in the clinical setting, validation with large biopsy proven cohorts is needed (4). Accompanying this growing understanding of the molecular profile of CCA has prompted the search for actionable mutations to serve as targets for drug development. Among these potential biomarkers, the FGFR2 fusion and IDH1 and IDH2 mutations, which are the two most common genetic alterations seen in iCCA, have been identified as mutations holding considerable promise for development of targeted therapies (9).

Isocitrate dehydrogenase (IDH1/IDH2) mutations

IDH1 and IDH2 mutations were first discovered using NGS, and efforts to characterize IDH mutations have noted a higher mutation rate in iCCA, with mutations seen in about 20–25% of iCCA (8,9). However, the prognostic significance of IDH mutations remains unclear.

IDH1 and IDH2 are metabolic enzymes involved in the tricarboxylic acid cycle, catalyzing the interconversion of isocitrate and alpha-ketoglutarate. The mutant IDH loses its normal enzymatic activity and instead produces increased amounts of the oncometabolite 2-hydroxyglutarate (2-HG), which can be detected in increased amounts in both the tumor and blood of IDH mutated patients (9). 2-HG can competitively inhibit dioxygenases, which play a role in DNA demethylation. Furthermore, IDH and KRAS mutations can cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic iCCA (9).

Several highly specific IDH-inhibitors have been developed, working by blocking the function of mutant IDH1 or IDH2, and ultimately reducing the levels of oncometabolite 2-HG (10). One such inhibitor, Ivosidenib, an oral, reversible inhibitor of mutant IDH1 (mIDH1) was well tolerated among patients with advanced solid tumors with IDH1 mutations in a phase I trial (NCT02073994). Ivosidenib is currently approved in the US for the treatment of mIDH1 acute myeloid leukemia in newly diagnosed patients ineligible for intensive chemotherapy and patients with relapsed or refractory disease. Seventy-three patients with mIDH1-CCA were enrolled in the phase I study with Ivosidenib (9). These patients had a median of 2 prior treatments (range, 1–5), pointing to a refractory study population. The median progression-free survival was 3.8 months (95% CI: 3.6–7.3), 6-month progression-free survival was 40.1% (28.4–51.6%), and 12-month progression-free survival was 21.8% (12.3–33.0%) (10). Median overall survival was 13.8 months (95% CI: 11.1–29.3); however, data were censored for 48 patients (66%) (10). No dose-limiting toxicities were reported and maximum tolerated dose was not reached (10). Given these encouraging results, 500 mg daily dose of Ivosidenib was selected for expansion in a phase III trial (10).

The ClarIDHy study is the randomized phase 3 clinical trial that evaluated ivosidenib (IVO) versus placebo (PBO) in patients with previously treated nonresectable or metastatic mIDH1-CCA. As of January 2019, 185 patients were randomized to IVO (n=124) or PBO (n=61) (11). Crossover from placebo group to intervention (IVO) was permitted when progressive disease was documented. The primary endpoint was progression free survival, with IVO showing clear benefit with a hazard ratio (HR) 0.37 (95% CI: 0.25–0.54; P<0.001) (11). Initial results are promising, showing a PFS of 2.7 vs. 1.4 months (intervention vs. placebo). PFS rates at 6 and 12 months were 32.0% and 21.9% in IVO arm. On the other hand, no PBO patients were progression-free for ≥6 months at data cutoff (11). Ivosidenib’s efficacy was seen across all sub-groups. Intention to treat analysis showed median OS was 10.8 months for IVO compared to 9.7 months for placebo (HR 0.69; one-sided P=0.06) with 57% of PBO patients crossed over to IVO (10). The Rank Preserving structural failure time (RPSFT)-adjusted median OS was 6 months for PBO (HR 0.46; P=0.0008) (11). Grade ≥3 adverse events reported in 46% IVO vs. 36% PBO and there were no treatment-related deaths. This study demonstrated significant improvement in PFS with Ivosidenib and a favorable OS trend compared to placebo (11). This is the first pivotal study demonstrating the clinical benefit of targeting mIDH1 in patients with advanced mutant IDH1 CCA.

Several other IDH1 and IDH2 inhibitors are being tested in ongoing clinical trials (NCT02746081, NCT02273739, NCT02381886, NCT02481154) (9). The potential of these therapies targeting IDH1 and IDH2 mutations raises questions on how these treatments should be used—as first-line therapy in selected populations or whether these inhibitors should be combined with chemotherapy or other treatment modalities. Additionally, the utility of these inhibitors in the adjuvant and neoadjuvant settings for patients with resectable disease will need to be explored. Regardless, the potential of these inhibitors offers optimism for improved therapies and a direction for future drug development.

Fibroblast growth factor receptor 2 (FGFR2) fusions

The FGFR2 fusion, seen in about 15–20% of iCCA, is another actionable mutation showing considerable promise (9). Genome-wide structural analyses first revealed recurrent translocation events involving the FGFR2 locus (9). The mechanism by which FGFR2 fusions drive oncogenesis has yet to be fully understood, however, it is thought that genetic alterations of FGFR can alter FGFR kinase activity and result in constitutively active FGFR signaling that promotes cell proliferation and inhibits apoptosis (12). Jain et al. reported on clinical characteristics and treatment outcomes of biliary tract cancers with FGFR genetic alterations in a retrospective analysis and found this subtype to be associated with an indolent disease course and prolonged survival. It was additionally noted this subtype affected a disproportionate number of young women (12). Since the initial discovery of recurrent FGFR2 fusions being present in multiple tumor types and particularly in iCCA, it has become the focus of developing targeted therapies.

The oral agent infigratinib (BGJ398) had the earliest reported data of selective FGFR inhibition in CCA. Infigratinib is an orally bioavailable, selective pan-FGFR kinase inhibitor that has shown preliminary clinical activity against tumors with FGFR alterations (13). A multicenter, open-label, phase II study (NCT02150967) evaluated infigratinib antitumor activity in patients age ≥18 years with advanced or metastatic CCA containing FGFR2 fusions or other FGFR alterations whose disease progressed on prior therapy (13). Sixty-one patients (median age, 57 years) with FGFR2 fusion (n=48), mutation (n=8), or amplification (n=3) participated. At the prespecified data cutoff (June 30, 2016), 50 patients had discontinued treatment. All responsive tumors contained FGFR2 fusions. The overall response rate was 14.8% (18.8% in FGFR2 fusions population), disease control rate was 75.4% (83.3% FGFR2 fusions population) and estimated median progression-free survival was 5.8 months (95% CI: 4.3 to 7.6) (13). Infigratinib is a first-in-class FGFR kinase inhibitor with a manageable toxicity profile, showing meaningful clinical activity against refractory CCA containing FGFR2 fusions. This promising antitumor activity supports continued development of Infigratinib in this patient population and further suggests FGFR inhibitor therapy as a viable therapeutic option in advanced biliary tract malignancies. The PROOF trial is currently ongoing, evaluating the efficacy of Infigratinib vs. gem-cis in first line treatment of patients with unresectable locally advanced or metastatic CCA with FGFR2 fusions or translocations (NCT03773302). It is a multicenter, open-label, randomized, controlled phase III clinical trial aiming to enroll 384 patients (13). Patients will be randomized 2:1 to infigratinib versus standard of care, with the ability for patients who are unresponsive to gem-cis to crossover and receive infigratinib (14). In January 2020, Infigratinib (BGJ398) received fast-track and orphan drug designations by the FDA, highlighting the optimism surrounding the drug’s potential to treat CCA in this select patient population (14).

TAS-120 is another irreversible FGFR-inhibitor showing promise in clinical trials. A phase I study of TAS-120 (8–24 mg daily) in adult patients with advanced solid tumors (NCT02052778) found the maximum tolerated dose to be 20mg daily (15). Meric-Bernstam et al. specifically analyzed patients with CCA enrolled in this study. 45 patients with CCA (intrahepatic n=41) with FGF/FGFR aberrations were treated at 16 (n=24), 20 (n=14), and 24 mg (n=7) QD [median age 53 years (range, 29–73)]. Of note, 76% of the patients were female. Twenty-eight patients (62%) had tumor FGFR2 gene fusions, while 17 (38%) had other FGF/FGFR aberrations (15). All patients received prior systemic therapy, 28.9% with reversible FGFR-inhibitor, revealing a highly refractory patient population with prior FGFR inhibitor exposure. Of the 28 patients with FGFR2 gene fusions, 20 (71%) experienced tumor shrinkage and 7 achieved confirmed partial responses (cPRs) (15). The objective response rate was 25%. Fifteen of 28 (54%) patients had stable disease as best response (15). The disease control rate was 79%. Of the 17 patients with other FGF/FGFR aberrations, 3 had cPRs (all had FGFR2 rearrangements; 1 also had FGFR2 amplification). Median treatment time was 7.4 months. Of the 13 patients with prior FGFR-inhibitor treatment, 4 (3 with FGFR2 gene fusions, 1 with FGFR2 amplification) had cPRs (15). Grade ≥3 treatment related adverse events were reported in 23 of 45 (51%) patients, with the most common being hyperphosphatemia (22%) (15). TAS-120 showed notable clinical activity with manageable toxicities in patients with CCA and FGFR2 fusions. The drug further showed efficacy in patients who had progressed on prior FGFR inhibitors. A phase II study testing TAS-120 in patients with FGFR2 gene fusions is ongoing (16). Interim analysis as of May 2020, reported data for 67 patients with a minimum of 6 months of follow up and found the ORR was 37.3% (1 CR 1.5%; 24 PR 35.8%) (17). Median duration of response was 8.31 months. The most common treatment-related adverse events (all grades, grade 3) at the time of analysis were hyperphosphatemia (80.6%; 26.9%), diarrhea (37.3%; 0%), and dry mouth (32.8%; 0%) (17). There were no grade 4 treatment related adverse events. This initial anlaysis is encouraging that TAS-120 may having clinically meaningful benefit in patients with refractory iCCA with FGFR2 gene fusions.

Pemigatinib is an orally bioavailable inhibitor of FGFR 1, 2, and 3 currently being investigated. The Fight 202 trial is a phase II, open-label, single-arm, multicenter study evaluating the efficacy of pemigatinib in patients with advanced or surgically unresectable CCA with FGFR2 translocation who have failed at least 1 previous treatment (NCT02924376). Patients were enrolled in three cohorts—those with FGFR2 translocations (A), those with other FGF/FGFR genetic alterations (B), or those with neither (C) (18). Each cohort received pemigatinib 13.5 mg daily on a 21-day cycle until disease progression or intolerable toxicity. Interim data presented in September 2019 showed an objective response rate (ORR) of 35.5% in cohort A (18). Median duration of response in this group was 7.5 months. No response was seen in cohort B or C. Median PFS was 6.9 months in cohort A compared with 2.1 months in cohort B and 1.7 months in cohort C (19). This data supports FGFR inhibition being a meaningful treatment for this subset of patients with CCA. Based on the data from FIGHT-202, a phase III study of pemigatinib versus gemcitabine plus cisplatin chemotherapy in first-line treatment of patients with unresectable or metastatic CCA with FGFR2 rearrangements is underway (NCT03656536). As of April 2020, pemigatinib became the first FDA approved targeted therapy for treatment of adults with previously treated, unresectable locally advanced or metastatic CCA with a fibroblast growth factor receptor 2 (FGFR2) fusion or other rearrangement (19).

Other selective FGFR inhibitors including Debio1347 (Debiopharm, NCT01948297) are currently being evaluated in early phase trials in patients with advanced solid tumors, including iCCA (9). A third non-selective tyrosine kinase inhibitor, ARQ-087 (Arqule, NCT01752920) has shown some encouraging anti-tumor activity in advanced iCCA with FGFR2 fusion as well (9). Overall, the preliminary data for FGFR inhibitors in advanced iCCA offers guarded hope. However, some debate exists regarding if these trends in survival can be attributed to these targeted therapies or to the natural history of the FGFR phenotype, given its favorable survival profile. Looking to the future, questions remain whether these drugs can evolve into front-line therapies or if they should be used in combination with chemotherapy or other treatment modalities.

Mechanisms of resistance

Initial clinical trials of targeted therapies in CCA have rightfully generated significant optimism. However, concerns of drug resistance have emerged as a real threat to the potential impact of these therapies (9). Mechanisms of resistance are not fully understood, and looking to the future, adequately addressing drug resistance will likely determine the durability of these targeted therapies.

Isoform switching is a mechanism for acquired resistance to IDH inhibitors that was first described in four clinical cases (20). Harding et al. describe a selective pressure of inhibiting mutant IDH activity in one subcellular compartment, which in turn provides a growth advantage for malignant subclones with unchecked mutant IDH activity in another subcellular compartment. In other words, by “isoform switching” from mutant IDH1 to mutant IDH2 or vice versa, IDH-mutant cancers can develop resistance to isoform-selective IDH inhibitors and restore the production of oncometabolite 2-HG, ultimately promoting tumor progression (20). The frequency of mutant IDH isoform switching as a mechanism of resistance to IDH inhibition is currently unknown.

The development of acquired resistance to FGFR kinase inhibitors has been even more common. Secondary FGFR2 kinase domain mutations has been observed in patients in the Infigratinib (BGJ398) clinical trials (13). Goyal et al. reported that the irreversible FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion-positive iCCAwho developed resistance to Infigratinib (BGJ398) or Debio 1347. After examining serial biopsies, circulating tumor DNA, and patient-derived iCCA cells it was found that TAS-120 was active against multiple acquired FGFR2 mutations which conferred resistance to BGJ398 or Debio 1347 (21). Functional assessment and modeling of the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor (21). These findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and circuluating tumor DNA (ctDNA) analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive iCCA. Moving forward, therapeutic strategies to prevent or overcome resistance will be pivotal to sustained success of these developing targeted therapies.

Section 2: smaller molecular targets in CCA

KRAS gene

KRAS is the proto-oncogene encoding for a small GTPase involved in several cell signaling pathways, including the MAPK-ERK pathway which regulates cell differentiation and proliferation. Mutations can disable the GTPase activity of KRAS, keeping it constitutively active in a GTP-bound state, which ultimately leads to continuous activation of downstream signal transduction pathways that drive tumor growth, remodeling, and migration (22).

The prevalence of KRAS mutations in iCCA is reported between 7–24%, with considerable variation between studies (23-26). However, despite the variable frequency of KRAS mutations, there is data suggestive that testing for KRAS mutations may provide prognostic value. Robertson et al. conducted DNA extraction and pyrosequencing of 54 iCCA cases and found 4 cases with KRAS mutations. The reported median OS was 13.5 months for KRAS mutant cases, compared to 37.3 months for wild type cases, suggesting a worse prognosis for KRAS mutant cases. Additionally, KRAS mutant cases were also associated with higher tumor stage and greater likelihood of lymph node involvement at time of resection (24). Javle et al. reported similar findings in a multicenter study using hybrid capture-based comprehensive genomic profiling of 412 iCCA samples. KRAS mutations were observed in 22% of iCCA samples, and among the 224 iCCA cases analyzed, KRAS mutated tumors were associated with poorer overall survival (P=0.048) in comparison with KRAS wild type tumors (27). Though these studies are suggestive of KRAS mutations having prognostic significance, specific treatments targeting this pathway are still in preliminary stages of development. An irreversible KRAS p.G12C inhibitor (AMG510) is currently being tested in Phase I/II trials (NCT03600883). However, the G12C mutation has not been frequently noted in CCA and AMG510 may have limited practical significance in the treatment of CCA presently.

B-raf gene

The B-raf proto-oncogene encodes a serine-threonine protein kinase which activates the MAPK/ERK proliferation signaling pathway. Mutations in B-raf, such as BRAF V600E, lead to constitutive activation of its downstream signaling molecules, resulting in cell cycle progression (28). The nucleotide sequence of the raf gene was first discovered from murine sarcoma virus 3611 (MSV-3611) in 1983 (29,30).

In 2002, researchers identified B-raf somatic missense mutations in 66% of malignant melanomas, bringing B-raf mutations to the forefront of the discussion on utilizing targeted therapies (31). Specifically, the BRAF V600E mutation, which accounted for 80% of these reported mutations, provided a promising new therapeutic target. BRAF inhibitors, dabrafenib, and vemurafenib, have significant results in BRAF V600E mutated metastatic melanoma, and initially there was hope for similar results in the treatment for BRAF-mutated CCA. However, the fact that B-raf mutations occur at a notably lower frequency (3–5%) in CCA than in melanoma have hampered these expectations (32-34). There are limited studies that evaluate the efficacy of Vemurafenib in BRAF-mutated CCA cases. In a phase II basket study of Vemurafenib in BRAF V600E-mutated non-melanoma cancers, one patient from a cohort of eight patients with CCA achieved a durable partial response of over 1 year (35). ROAR, a phase II trial investigating combination therapy with B-raf and MEK inhibition (Dabrafenib and Trametinib) in subjects with BRAF V600E- mutated rare cancers is ongoing (NCT02034110). Preliminary data for the ROAR trial was presented at the 2019 Gastrointestinal Cancers Symposium (36). Of the thirty-three biliary tract cancer patients enrolled in the study, thirty patients harbored BRAF V600E mutations. The objective response rate was 41% (13/32; 95% CI: 24–59%), with 6 of 13 responses ongoing at data cutoff, and 7 of 13 (54%) patients with a duration of response ≥6 months. The median progression-free survival was 7.2 months (95% CI: 4.6–10.1), and median overall survival was 11.3 months (95% CI: 7.3–17.6).

BRCA1 and BRCA2, PARP inhibition

The BRCA1 and BRCA2 tumor suppressor genes encode proteins that repair double-stranded DNA breaks (DSBs) through homologous recombination. BRCA1/2 mutated cells are unable to carry out homologous recombination and perform DNA repair by alternative error-prone mechanisms, predisposing them to genomic instability (37). Inhibiting both BRCA and poly ADP-ribose polymerase (PARP) proteins is one approach that has been utilized to target BRCA-mutated tumors. Normally, PARP enzymes repair single-stranded DNA breaks via base excision repair. However, in PARP-deficient cells, single-stranded DNA breaks remain unrepaired, and when encountered by a replication fork, progress into double-stranded breaks. BRCA-mutated cells are unable to repair these DSBs and overcome the collapsed replication fork (38). Essentially, PARP inhibition exploits the inherent vulnerability of BRCA-mutated tumors and other DNA repair deficient cells.

In a retrospective analysis of patients with BRCA-mutated CCA (n=18), one of four patients who received PARP inhibitors had a sustained disease response with a progression-free survival duration of 42.6 months (39). However, given the limited number of patients in this study, more research is needed to establish PARP inhibition as a viable treatment option for BRCA-mutated CCA. A Phase II trial of PARP inhibitor, Niraparib, is underway at the University of Florida (NCT03207347). A basket phase II trial of PARP inhibitor, Olaparib, is also ongoing and will include patients with metastatic solid tumors harboring IDH1 or IDH2 mutations (NCT03212274).

Her2/neu gene

The Her2/neu gene encodes for the Her2 (human epidermal growth factor receptor-2) protein, a transmembrane receptor with tyrosine kinase activity which is an upstream driver of multiple signal transduction pathways that promote tumorigenesis. The Her2/neu gene was first discovered when a series of neuroglioblastomas from carcinogen treated rats were found to have the same oncogene (40). Her2 overexpression has notably been used as a predictive marker in breast and gastric cancers. However, the prevalence of Her2 overexpression in biliary tract cancers was previously undetermined.

Galdy et al. were the first to delve into this question, performing a systematic review and meta-analysis in 2017 of 40 studies (including 3,839 patients in total) which revealed that extrahepatic biliary tumors have higher Her2 overexpression compared to iCCA: 19.9% (95% CI: 12.8–27.1%) vs. 4.8% (95% CI: 0–14.5%), respectively (41). In addition to the low prevalence of Her2 mutations in iCCA, previous Phase II trials of Lapatinib (n=17), a dual inhibitor of EGFR and Her2/neu, have shown poor results with a reported median progression-free survival duration of 1.8 months with a zero percent response rate (42). A similar phase II study of Lapatinib in nine biliary tract cancer patients reported zero responses and a median progression-free survival duration of 2.6 months (42,43). In comparison to Lapatinib, monoclonal antibodies such as Trastuzumab may be a better alternative for Her2 overexpressing biliary tract cancers. Varlitinib, a small molecule Her-1/2/4 inhibitor, is also being investigated in a phase II/III study in combination with Capecitabine as second line treatment for unselected advanced biliary tract cancers (44). Preliminary results from an ongoing clinical trial (NCT02091141) has shown that pertuzumab plus trastuzumab has activity in HER2 mutated biliary cancers, further supporting HER2 as a potential therapeutic target for these cancers. Additionally, data from a Phase I study showed single agent ZW25, a bispecific antibody, induced anti-tumor activity in heavily pretreated patients with a variety of HER2-expressing cancers, including CCA (45). Phase II clinical trials are currently underway testing ZW25 in breast, gastric, and other Her2 expressing cancers (Table 2).

Section 3: role of immunotherapy in CCA

Immunotherapy is a burgeoning field within oncology, particularly gaining popularity as a viable treatment option for cancers such as melanoma and colorectal cancer. Studies investigating the role of immunotherapy in CCA are also emerging. Immunotherapies such as programmed cell death protein 1 (PD-1) inhibitors enable tumor recognition by blocking the PD-1 and programmed cell death protein 1 ligand (PD-L1) interaction that allows tumor cells to evade T-cell recognition. The presence of mismatch repair (MMR) deficiency further enhances tumor recognition since MMR deficiency results in a high mutation burden with subsequent creation of neoantigens and microsatellite instability. As a result, tumors with MMR deficiency have been shown to be especially sensitive to PD-1 inhibitors such as Pembrolizumab (46). Unfortunately the prevalence of MMR deficiency in CCA is low as Goyal et al. report a 9% rate of MMR protein loss, with only 4.5% patients being microsatellite instability-high (MSI-H) (47).

The phase Ib KEYNOTE-028 trial evaluated 24 patients with biliary tract cancers, all of whom were PD-L1 positive but negative for MSI-H. The reported overall response rate (ORR) to pembrolizumab was 13.0% (3/23, all partial response; 95% CI: 2.8‒33.6%) and median duration of response was not reached (range, 21.5 to 29.4+ months). Median overall survival and progression-free survival were 6.2 months (95% CI: 3.8‒10.3) and 1.8 months (95% CI: 1.4‒3.7), respectively (48). In the larger KEYNOTE-158 Phase II basket trial, 104 patients were enrolled in the biliary tract cancer cohort and treated with Pembrolizumab; 58% were PD-L1 positive and none harbored MSI-H. Here the ORR was disappointing at only 5.8% (6/104, all partial response; 95% CI: 2.1–12.1%) though median duration of response was not reached (range, 6.2 to 23.2+ months). Results from these trials, especially the low overall response rate in the larger KEYNOTE-158 trial, suggest that pembrolizumab is not an effective single-agent treatment option for CCA. Although there is no existing literature for immunotherapy combination regimens in CCA, combination therapy with Atezolizumab and Cobimetinib is being studied in the treatment of metastatic colorectal cancer. Results from a phase III, randomized controlled trial with 363 metastatic colorectal cancer patients showed a median overall survival of 8.8 months (95% CI: 7.00–10.61) with Atezolizumab plus Cobimetinib, compared to 7.1 months (95% CI: 6.05–10.05) with Atezolizumab, and 8.5 months (95% CI: 6.41–10.71) with Regorafenib (49). Although these results do not demonstrate that immunotherapy combinations are superior, this trial presents the possibilities for studying immunotherapy combinations. The first randomized trial of immunotherapy in BTC was the phase II trial of Atezolizumab plus Cobimetinib in metastatic, unresectable CCA in 2017. Eighty-six patients were enrolled in 23 centers across the Unites States (NCT03201458). Results were notable for a median PFS of 3.65 months (cobimetinib + atezolizumab) vs. 1.87 months (atezolizumab monotherapy) (P=0.027) (50). OS data was still pending at the time of analysis. Treatment was well tolerated in both groups as grade 3–4 treatment-related adverse events were similar in both arms, and there were no treatment-related deaths (50). Ultimately, the combination of atezolizumab + cobimetinib significantly prolonged PFS as compared to atezolizumab monotherapy in BTC without significant toxicity, warranting further investigation in treatment of biliary tract cancers.

A single-center, prospective cohort study, conducted between May 2018 and February 2019 in Korea, evaluated the efficacy and safety of pembrolizumab in patients who were positive for PD-L1 and progressed on first-line gemcitabine plus cisplatin. A total of 40 patients were enrolled and Pembrolizumab 200 mg was administered intravenously every 3 weeks. The objective response rate was 10% and 12.5% by Response Evaluation Criteria in Solid Tumor (RECIST) v1.1 and immune- modified RECIST (imRECIST) and median duration of response was 6.3 months (51). The median PFS and OS were 1.5 months (95% CI, 0.0 to 3.0) and 4.3 months (95% CI: 3.5 to 5.1), respectively, and objective response per imRECIST was significantly associated with PFS (P<0.001) and OS (P=0.001) (51). The data presented in this study suggests Pembrolizumab having modest anti-tumor activity in heavily pretreated PD-L1-positive BTC patients, with a durable response seen in patients who showed objective response.

In a recent phase II trial, Kim et al. investigated the efficacy of nivolumab, a PD-1 inhibitor in refractory biliary tract cancers. Fifty-four patients were enrolled in this multicenter study between October 2016 and December 2018 (52). Of these patients, 46 were examined for objective response via radiologic imaging. Investigator assessed objective responses (all partial responses) were observed in 10 (22%) of 46 patients. Additionally, it was noted 17 (37%) patients had stable disease, yielding DCR of 59%. Of note, all responses were observed in patients with mismatch repair protein-proficient tumors. Among all 54 patients, median PFS was 3.68 months and median OS was 14.24 months (52). Patients with PD-L1 positive tumors had significantly prolonged PFS (median 10.4 vs. 2.3 months, HR 0.23, P<0.001) and nonsignificantly prolonged overall survival (median not reached vs. 10.8 months, P=0.19) vs. patients with PD-L1 negative tumors (52). This study found nivolumab was well tolerated and showed modest efficacy and durable response with patients with refractory BTC. The median OS of 14.24 months was impressive given the refractory patient population. Additionally, the ORR of 22% was a strikingly higher ORR than what has been previously reported with other checkpoint inhibitors, such as pembrolizumab. However, it is important to note centrally reviewed ORR was lower at 11% and more in line with previously published results. Interestingly, although all the patients were noted to be mismatch repair–proficient, there appeared to be a correlation between PD-L1 positivity and response. Moving forward, further studies to both verify these findings and to evaluate biomarkers for improved treatment selection are needed.

One such ongoing study is a randomized, multi-institutional, phase 2 study investigating the role of combinational immunotherapy, using nivolumab with chemotherapy (gemcitabine/cisplatin) or as dual immunotherapy (nivolumab and ipilimumab) in patients with advanced BTC (NCT03101566). The primary objective is to evaluate the PFS rate at 6 months. Secondary objectives include evaluation of ORR, median PFS and OS and safety. Exploratory objectives include identification of biomarker predictors of response and mechanisms of resistance through serial biopsies and blood collection, including sequential whole exome/transcriptomic analysis and immune cell analysis of tissue and blood (53). Patient accrual was completed as of January 2020 and final data analysis is pending. These results will not only provide information regarding efficacy of nivolumab in combination with gem-cis and ipilumamab but may also elucidate the role of NGS data in guiding treatment selection for patients (Table 3).

Conclusion: the future of treatment in CCA

The discovery of prognostic and predictive biomarkers in CCA has expanded the understanding of CCA as well as informed the development of novel therapies. Identifying actionable mutations suited for targeted therapies has been a point of emphasis of recent research in CCA. Since Gemcitabine-cisplatin combination therapy was established as first-line therapy in the treatment of advanced biliary cancers in 2010, advances in systemic therapies extending to standard clinical practice have been limited. However, a step forward was made with recent data showing the addition of nab-paclitaxel with gem-cis notably prolonging median progression-free survival when compared to historical control of gem-cis alone in patients with advanced biliary tract cancers (3). These findings are being tested in a phase III randomized clinical trial (S1815 Study, NCT03768414).

Next generation sequencing has been central in elucidating the pathogenesis of CCA from a molecular level, bringing hope of identifying actionable mutations for the development of targeted therapies. The paradigm that “one-size fits all” for the treatment of CCA is likely obsolete, given the varying etiologies and heterogeneity of the disease. Looking to the future, next generation sequencing will likely become essential to clinical and therapeutic decision-making. Novel treatments for CCA are closer than ever to entering clinical practice, with the IDH1 inhibitor, ivosidenib showing considerable promise in the ClarIDHy study. Similarly, FGFR inhibitors BGJ-398, TAS-120, and pemigatinib have also shown meaningful activity in phase II clinical trials, with pemigatinib becoming the first FDA approved therapy for CCA patients with FGFR2 fusions, as of April 2020. The expectation is that more targeted therapies will follow pemigatinib’s lead and enter the front-line of care in the near future.

The search to identify more actionable mutations is ongoing, with molecular targets such as B-raf and BRCA touted as potential targets. The obvious challenge is that many of these molecular targets occur at lower frequencies in CCA than IDH1/IDH2 mutations and the FGFR2 Fusion. Nevertheless, these targets may still have a role in the treatment of CCA, given several of these targets have approved therapies in other cancer types, raising the question whether these agents have a therapeutic role in select subsets of CCA patients. Further investigation is needed to evaluate the efficacy of targeting these mutations in CCA, which has proven challenging given the rarity of these cancers. One practical approach to increasing these investigations is through basket trials, which allow for evaluation of rare tumor subsets with shared controls. Introducing more basket trials could provide researchers with more opportunities to study some of the less common mutations seen in rare cancers such as CCA.

While immunotherapy has shown promising results for malignancies such as melanoma and colorectal cancer, results for pembrolizumab treatment in CCA have been mixed. Kang et al. (51) reported modest efficacy of pembrolizumab in PD-L1 positive patients, but the applicability of this study remains unknown given all patients were enrolled were Asian. Additionally, the results of Kim et al.’s (52) study are intriguing given reported ORR 22%, which was notably higher than prior studies, namely the large KEYNOTE-158 study. The data was certainly suggestive of durable response in patients who responded to nivolumab. However, it is important to note no molecular profiling data was reported in patients to this study. Looking to the future, identifying biomarkers beyond microsatellite instability or PD-L1 and integrating this information into clinical trials will be essential to elucidating the role of immunotherapy in the treatment of CCA.

The application and integration of these novel therapies and targeted approach in patients with resectable disease represents an opportunity for investigation. The difficulty in obtaining adequate tissue for diagnosis and NGS preoperatively in patients with hilar or dCCA poses an obstacle for neoadjuvant trials for these two disease sites. iCCA, on the other hand, is amenable to core biopsy and the prevalence of actionable mutations makes this ripe for neoadjuvant trials to assess the feasibility and efficacy of introducing targeted therapies in the preoperative space. Adjuvant trials for CCA utilizing these novel therapies are under development as well. Additioannly, the field of transplant oncology within CCA may benefit from advances in this targeted approach to therapy, but requires further investigation.

The development of targeted therapies has generated significant optimism for expanded treatment options for patients with CCA, however, a new set of challenges accompany these novel treatments. Questions remain on the ideal use of targeted therapies—specifically whether they should be used in combination with systemic therpies, surgery, or other treatment modalities. Furthermore, issues with resistance to IDH and FGFR inhibitors in clinical trials have emerged as a real challenge, threatening the potential impact of targeted therapies. Dedicated research is needed to better understand mechanisms of resistance and to ultimately address concerns of resistance threatening the durability of these therapies. Additionally, approaching CCA from a molecular level has made it clear that not all CCAs are created equal. However, the ideal means of stratifying CCA, likely with prognostic and therapeutic implications, remains a mystery. Despite the unknowns that remain in the treatment of CCA, the clinical use of tumor biomarkers and the development of targeted therapies have provided a tangible hope of a future in which patients with CCA will have improved survival.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://hbsn.amegroups.com/article/view/10.21037/hbsn-20-475/rc

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://hbsn.amegroups.com/article/view/10.21037/hbsn-20-475/coif). SKM serves as an unpaid editorial board member of Hepatobiliary Surgery and Nutrition. RTS reports grants from Merck, non-financial support from Seattle Genetics, grants from Exelixis Pharmaceuticals, non-financial support from QED, non-financial support from Debiopharm, non-financial support from Agios, non-financial support from Clovis, grants from Haloyzme, grants from Pieris, grants from Taiho, non-financial support from Incyte, outside the submitted work. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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