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Rational development of combination therapies for biliary tract cancers

Published:September 20, 2022DOI:https://doi.org/10.1016/j.jhep.2022.09.004

      Summary

      Biliary tract cancers are an uncommon set of gastrointestinal malignancies that are associated with high morbidity and mortality rates. Most patients present with incurable locally advanced or metastatic disease. The pathophysiology of biliary tract cancer can be exploited for direct therapeutic benefit, and indeed, chemotherapy, precision medicine, immunotherapy and combination treatments are now applied as both standard-of-care and investigational therapies. In the first-line setting, the immune-based chemotherapy combination of durvalumab plus gemcitabine and cisplatin has recently been shown to improve survival compared to chemotherapy alone. In the second-line, precision medicine can be employed in those with select genetic alterations in IDH1/2 (isocitrate dehydrogenase 1/2), FGFR2 (fibroblast growth factor receptor 2), KRAS, BRAF, ERBB2, NTRK (neurotrophic receptor tyrosine kinase), ROS, RET, and/or deficiencies in mismatch repair enzymes. In those patients without targetable genetic alterations, fluoropyridine doublets lead to modest improvements in outcomes. Next-generation sequencing is critical for direct patient care and to help elucidate genomic mechanisms of resistance in a research context. Currently, multiple clinical trials are ongoing – hence, this review seeks to provide an update on evolving standards of care and ongoing investigational agents, limitations to current treatments, and a framework for effective combination drug development for the future.

      Keywords

      Introduction

      Biliary tract cancers (BTCs) represent uncommon gastrointestinal cancers that are subdivided based on anatomic site into intrahepatic cholangiocarcinoma (iCCA), extrahepatic cholangiocarcinoma (eCCA), and gallbladder carcinoma (GBC).
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      Similar survival trends are observed globally, and it is critical to emphasise that outcomes in the setting of metastatic disease remain dismal, with just 2-5% of patients alive at 5 years from diagnosis.

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      • BTCs represent uncommon gastrointestinal cancers that are categorised based on anatomic site as intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder carcinoma.
      • The oncogenesis of BTC is a complex cellular process ultimately resulting in several vulnerabilities that can be exploited for therapeutic effect.
      • Historically, cytotoxic chemotherapy with gemcitabine and cisplatin has been the first-line standard-of-care for metastatic BTC based on the landmark ABC-02 study.
      • TOPAZ-1 was a multicentre, double-blind, randomised, phase III study which demonstrated that the addition of durvalumab to gemcitabine and cisplatin improved survival in treatment-naïve individuals with advanced BTC; the triplet regimen may displace gemcitabine and cisplatin as first-line standard-of-care.
      • Clinical grade next-generation sequencing is critical to guide treatment decisions and link patients to clinical trials.
      • After failure of front-line therapy, treatment decisions are driven by tumour genomics, and actionable alterations occur in IDH1/2, ERBB2, BRAF, KRAS, BRCA1/2; FGFR2, NTRK, ROS and RET, as well as in mismatch repair enzymes.
      • In those without a known driver alteration or for whom genomic analysis is incomplete, cytotoxic chemotherapy with a fluoropyridine doublet has shown modest anticancer activity and is a reasonable approach outside of a clinical trial.
      • Acquired resistance occurs in most patients on targeted therapies and, in the precision space, several genomic alterations have been identified at disease progression.
      • Novel agents are being developed to prevent or circumvent resistance, while rational combinations are being assessed in clinical trials for individuals with BTC.
      Table 1Clinical presentation of biliary tract cancers.
      Intrahepatic cholangiocarcinomaExtrahepatic cholangiocarcinomaGallbladder cancer
      Clinical presentationIncidental finding; Abdominal pain; Constitutional SymptomsObstructive jaundice; recurrent acute cholangitis; constitutional symptomsIncidental at routine cholecystectomy; Right upper pain; Constitutional Symptoms
      AetiologyPrimary sclerosing cholangitis; Caroli's disease; hepatolithiasis; liver fluke; cirrhosis; viral hepatitis; non-alcoholic fatty liver diseasePrimary sclerosing cholangitis; Caroli's disease; choledochal cyst; cholelithiasis; liver fluke; cirrhosis; viral hepatitis; chronic pancreatitisAdvanced age; female sex; obesity; cholelithiasis; structural abnormalities; primary sclerosing cholangitis; choledochal cysts; chronic Salmonella typhi or Helicobacter bilis;
      Global distributionChina, Korea, Taiwan, Vietnam, Thailand; Incidence increasing in Western countriesChina, Korea, Taiwan, Vietnam, ThailandChile, Northern India, Poland, Pakistan, Japan
      Natural historyIntrahepatic metastasis; Regional lymphadenopathyLocally invasive and recurrent, infiltrative disease in hilum; regional lymphadenopathyPeritoneal disease; Distant spread
      Common genomic driversIDH1/2, FGFR2, BRAF, ARID1AKRAS, TP53, SMAD4ERBB2
      ARID1A, AT-rich interaction domain 1A; FGFR2, fibroblast growth factor receptor 2; IDH1/2, isocitrate dehydrogenase 1/2.
      A deeper understanding of BTC biology has yielded important systemic treatment advances in the last two decades (Fig. 1). Cytotoxic chemotherapy has been the main bulwark against the disease, despite its modest impact on outcomes.
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      The advances in, and widespread availability of, gene sequencing technologies have enabled the identification and characterisation of aberrant oncogenes that have been proven to drive uncontrolled cellular growth of BTC and other solid tumours.
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      Genomic spectra of biliary tract cancer.
      In the clinic, when such alterations are detected with clinical-grade, next-generation sequencing (NGS) of tumour tissue or tumour-derived cell-free DNA (cfDNA), patients may be treated with precision medicines (multiple approaches have been approved by agencies worldwide), and/or investigational therapies.
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      As seen across all solid tumours, preclinical and translational studies indicate that BTC elicits and evades effective immune responses, and – even in the absence of the highly immune responsive microsatellite instability (MSI)-high/mismatch repair deficiency (MMR-D) cohort – there are patients for whom immune checkpoint inhibitors may be appropriate.
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      Genomic spectra of biliary tract cancer.
      Recently, the addition of durvalumab to gemcitabine and cisplatin has been shown to improve overall survival (OS) over cytotoxic chemotherapy in the metastatic setting — helping to clinically validate the immune synapse as an important drug target in BTC.
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      Durvalumab plus gemcitabine and cisplatin in advanced biliary tract cancer.
      ,
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      Phase II study assessing tolerability, efficacy, and biomarkers for durvalumab (D) ± tremelimumab (T) and gemcitabine/cisplatin (GemCis) in chemo-naïve advanced biliary tract cancer (aBTC).
      With the establishment of multiple effective therapeutic modalities, future research will seek to evaluate novel therapies and multiple new combinations. The current review provides an update on evolving standards of care and ongoing investigational agents, limitations to current treatments, and a framework for effective combination drug development for the future.
      Figure thumbnail gr1
      Fig. 1Timeline of drug development in biliary tract cancers.
      FGFR2, fibroblast growth factor receptor 2; FOLFOX, 5-FU and oxaliplatin; IDH1, isocitrate dehydrogenase 1; MSI, microsatellite instability; NTRK, neurotrophic receptor tyrosine kinase; TMB, tumour mutational burden.

      Therapeutic implications of oncogenesis in BTCs

      The oncogenesis of BTC is a complex cellular process ultimately resulting in several vulnerabilities that can be exploited for therapeutic effect (Fig. 2). Genomic alterations serve to sustain proliferative signalling and to evade growth suppression ultimately resulting in the development of BTCs.
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      Genomic spectra of biliary tract cancer.
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      Biliary cancer: utility of next-generation sequencing for clinical management.
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      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
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      Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma.
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      Genetic determinants of outcome in intrahepatic cholangiocarcinoma.
      Such alterations have been identified in cell cycle regulators, such as TP53, MDM2, CDK2NA; in receptor tyrosine kinases and cell signalling cascades including ERBB2, fibroblast growth factor receptor (FGFR), the MAPK pathway, the PI3K-AKT-TOR pathway, ROS, RET, and neurotrophic receptor tyrosine kinase (NTRK); in metabolomic pathways, such as isocitrate dehydrogenase 1/2 (IDH1/2); in epigenetic modifiers, such as BAP1 and ARID1; as well as in a set of DNA synthetic, DNA damage response (DDR), and homologous recombination pathways, such as POLD1, POLE, MSH2/6, MLH1, PMS2, BRCA1/2, PALB2, among others. It is estimated that up to 40% of individuals with BTC harbour at least one actionable or potentially actionable genetic alteration.
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      Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.
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      Biliary cancer: utility of next-generation sequencing for clinical management.
      ,
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      • Zehir A.
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      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
      Widespread epigenetic and metabolic changes are also observed in BTC and are implicated in malignant transformation and progression.
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      Global alterations of DNA methylation in cholangiocarcinoma target the Wnt signaling pathway.
      Along with these sweeping biochemical and cellular changes, the tumour microenvironment is impacted significantly, with prominent recruitment of cancer-associated fibroblasts endowed with a range of pro-tumorigenic functions. In the tumour microenvironment, cancer-associated fibroblasts are accompanied by effector T cells with an exhausted phenotype, tumour-associated macrophages, myeloid-derived suppressor cells, dysfunctional antigen-presenting cells, and regulatory T cells, leading to dysregulated cytokine production and abnormal deposition of extracellular matrix, which variably sustain an invasive phenotype of the tumour counterpart.
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      Single-cell landscape of immunocytes in patients with extrahepatic cholangiocarcinoma.
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      Tumor-associated neutrophils and macrophages interaction contributes to intrahepatic cholangiocarcinoma progression by activating STAT3.
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      CAFs shape myeloid-derived suppressor cells to promote stemness of intrahepatic cholangiocarcinoma through 5-lipoxygenase.
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      Targeting tumor-associated macrophages and granulocytic myeloid-derived suppressor cells augments PD-1 blockade in cholangiocarcinoma.
      Immune checkpoint molecules, such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) are expressed on immune effectors in the tumour microenvironment, and others such as programmed death ligand 1 (PD-L1) are aberrantly expressed by tumoural cholangiocytes and have the potential to be exploited as drug targets.
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      The immunogenomic landscape of resected intrahepatic cholangiocarcinoma.
      Although the value of PD-1/PD-L1 expression is still being defined, as detailed below, these preclinical and clinical data suggest that BTC are generally immunogenically ‘cold’, engendering a tumour microenvironment that leads to tumour-promoting inflammation and evasion of immune-mediated destruction. At a macroscopic level, several other hallmarks of cancer are implicated in BTC oncogenesis including recruitment of vasculature, in particular lymphatics, to sustain the primary tumour and condition metastatic niches and spread,
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      while a dysfunctional microbiome and exogenous viral particles or parasites may help to promote these cancers.
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      Figure thumbnail gr2
      Fig. 2Drug targets in biliary tract cancer based on oncogenesis.
      FGFR, fibroblast growth factor receptor; IDH, isocitrate dehydrogenase; NTRK, neurotrophic receptor tyrosine kinase.

      Investigational and established treatment landscape

      Chemotherapy

      Historically, cytotoxic chemotherapy with gemcitabine and cisplatin has been the first-line standard-of-care for metastatic BTC based on the ABC-02 study.
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      • et al.
      Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer.
      Cytotoxic chemotherapy’s mechanism of action, and the main driver of its anticancer effect, is its ability to damage DNA during cellular division. Increasingly, the immunogenic effect of cytotoxic treatment is becoming evident with the advent of effective immune checkpoint blockade.
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      Preclinical data regarding the effects of purine analogues and platinum on the tumour immune microenvironment are limited, though in the clinic, a phase I/II of gemcitabine and cisplatin with the addition of the anti-PD-L1 antibody durvalumab, led to a high objective response rate of 73.4% and favourable outcomes compared to historic controls – prompting the hypothesis that the addition of anti-PD-L1 therapy to gemcitabine and cisplatin would alter the natural history of the disease and improve OS.
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      • Bang J.-H.
      • Nam A.-R.
      • et al.
      Phase II study assessing tolerability, efficacy, and biomarkers for durvalumab (D) ± tremelimumab (T) and gemcitabine/cisplatin (GemCis) in chemo-naïve advanced biliary tract cancer (aBTC).
      The pivotal, TOPAZ-1 study was a multicentre, double-blind, randomised, phase III study that tested this hypothesis by evaluating gemcitabine and cisplatin with or without durvalumab in individuals with treatment-naïve, advanced, BTC.
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      • Vogel A.
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      Durvalumab plus gemcitabine and cisplatin in advanced biliary tract cancer.
      The study established this regimen as standard-of-care by randomising (1:1) participants with locally advanced or metastatic BTC to receive up to eight cycles of gemcitabine plus cisplatin with or without durvalumab. Durvalumab could be administered indefinitely as maintenance therapy until progression of disease. The primary endpoint of an improvement in OS was met, with the addition of durvalumab leading to a median OS of 12.8 (95% CI 11.1-14.0) months compared to 11.5 (95% CI 10.1-12.5) months in the group not receiving durvalumab (hazard ratio [HR] 0.8; 95% CI 0.66-0.97, p = 0.02). Importantly, the immune-based chemotherapy combination led to a pronounced tail of the curve, a known phenomenon for this class of agents (12-month OS: 54.1% vs. 48.0%, 18-month OS 35.1% vs. 25.6%, 24-month OS 25.0% vs. 10.4%). Secondary endpoints of objective response rate (ORR), duration of response, and progression-free survival (PFS) also favoured the triplet regimen.
      Caveats of the study and regimen must be acknowledged and include the lack of a validated predictive biomarker – the PD-L1 tumour area positivity score was tested in TOPAZ-1 but did not discriminate outcomes.
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      Durvalumab plus gemcitabine and cisplatin in advanced biliary tract cancer.
      Other proposed biomarkers are currently being evaluated (e.g., gene drivers, tumour mutational burden status, inflamed cytotoxic T-cell score, immune microenvironment composition, and host microbiome).
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      Other questions relate to the generalisability of the study, e.g. whether it applies to Western populations with BTC as 54% of participants came from Asia, and the degree to which aetiologic factors, or ethnic genomic diversity (i.e., HLA alleles, pharmacogenomics) may influence responses to immune checkpoint inhibitors.
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      There is also debate as to whether not continuing cytotoxic chemotherapy beyond 6 months in both arms may have led to a difference in outcomes, given that the observed survival benefit occurred after 6 months on study.
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      It should be noted that the original design of ABC-02 administered only 6 months of cytotoxic treatment followed by observation, and although many practitioners prescribe ‘maintenance’ chemotherapy – no prospective studies have evaluated this in the front-line setting.
      • Valle J.
      • Wasan H.
      • Palmer D.H.
      • Cunningham D.
      • Anthoney A.
      • Maraveyas A.
      • et al.
      Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer.
      The ongoing phase III study, Keynote-966 (NCT04003636) is evaluating a similar hypothesis with the addition of the anti-PD-1 antibody pembrolizumab to gemcitabine and cisplatin, though in contrast to TOPAZ-1, cytotoxic chemotherapy may be continued indefinitely; thus, the study may provide insights into the role of continued chemotherapy with anti-PD-1/L1 therapy. Finally, the late survival data was immature, owing to short follow-up, and the magnitude of the survival difference at 24 months observed in TOPAZ-1 may change with additional data. Despite these limitations, the observation that nearly 15% more patients are alive at 2 years argues for the early initiation of durvalumab in this disease. Taken together, these data support the antitumor efficacy of anti-PD-L1 antibodies when added to an established backbone of cytotoxic chemotherapy in BTC.
      Triple chemotherapy has also been evaluated in the first-line setting but is not yet widely accepted as a life-prolonging standard-of-care. The most studied triplet regimen is gemcitabine and cisplatin plus S-1, a novel oral compound comprised of tegafur, a prodrug of 5-fluorouracil, 5-chloro-2-4-dihydroxypyridine, which inhibits the activity of dihydropyridine dehydrogenase, and oxonic acid, which reduces gastrointestinal toxicity.
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      A modest survival advantage was demonstrated in a phase II study of cisplatin and gemcitabine plus S-1 in Japan.
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      • Miyamoto A.
      • et al.
      A multi-institution phase II study of gemcitabine/cisplatin/S-1 (GCS) combination chemotherapy for patients with advanced biliary tract cancer (KHBO 1002).
      A subsequent phase III trial of the triplet regimen compared to gemcitabine and cisplatin demonstrated an improvement in the median PFS (7.4 months vs. 5.5 months, respectively; HR 0.75; 95% CI 0.58-0.97; p = 0.0015) and median OS (13.5 months vs. 12.6 months, respectively; HR 0.79; 95% CI 0.60-1.04; p = 0.046).
      • Ioka T.
      • Kanai M.
      • Kobayashi S.
      • Sakai D.
      • Eguchi H.
      • Seo S.
      • et al.
      Randomized phase III study of gemcitabine, cisplatin plus S-1 (GCS) versus gemcitabine, cisplatin (GC) for advanced biliary tract cancer (KHBO1401-MITSUBA).
      ORR was 41.5% in the three-drug arm compared to 15% in those treated with gemcitabine plus cisplatin, though at the cost of higher rates of adverse events. Folinic acid, 5-FU, irinotecan and oxaliplatin (FOLFIRINOX) was also tested in the first-line setting against gemcitabine and cisplatin in an adaptive phase II/III study.
      • Phelip J.M.
      • Desrame J.
      • Edeline J.
      • Barbier E.
      • Terrebonne E.
      • Michel P.
      • et al.
      Modified FOLFIRINOX versus CISGEM chemotherapy for patients with advanced biliary tract cancer (PRODIGE 38 AMEBICA): a randomized phase II study.
      The study randomised 191 participants and did not meet a 6-month PFS endpoint required to expand to a formal phase III study. The median PFS was 6.2 months (95% CI 5.5-7.8) in the modified FOLFIRINOX arm and 7.4 months (95% CI 5.6-8.7) in the gemcitabine/cisplatin arm. Although not powered to assess survival – median OS was numerically worse in the modified FOLFIRINOX arm (11.7 months) than in the gemcitabine and cisplatin arm (13.8 months). In a subset analysis, gemcitabine and cisplatin were associated with better outcomes in individuals with iCCA – the largest anatomic subgroup in the study – while modified FOLFIRINOX was associated with better outcomes in all other subtypes. Finally, the combination of cisplatin, gemcitabine and nab-paclitaxel (known as the GAP regimen) has shown promising preliminary results, with an ORR of 45%, a median PFS of 11.8 months (95% CI 6-15.6) and a median OS of 19.2 months (95% CI 13.2 months to NE) in a small, single-arm, multicentre, phase II study.
      • Shroff R.T.
      • Javle M.M.
      • Xiao L.
      • Kaseb A.O.
      • Varadhachary G.R.
      • Wolff R.A.
      • et al.
      Gemcitabine, cisplatin, and nab-paclitaxel for the treatment of advanced biliary tract cancers: a phase 2 clinical trial.
      The triplet combination was associated with significant toxicity, with 16% of patients withdrawing due to treatment-associated adverse events and 58% of patients experiencing grade ≥3 events. SWOG-1815 is now investigating this triplet combination vs. gemcitabine/cisplatin in a phase III trial (NCT03768414).
      These data indicate that triplet-based cytotoxic regimens may offer a higher ORR, albeit with a greater risk of toxicity. However, the addition of S-1 to cisplatin and gemcitabine had only a modest effect on survival compared to traditional doublets in the first-line setting, while FOLFRINOX was possibly detrimental, and the effect of GAP is unknown. SWOG-1815 will provide needed clarity on the future role of treatment intensification in the metastatic space. Taken together, these observations reaffirm the urgent need for prospective comparative testing, prior to widespread application of new treatments.
      • Lee T.Y.
      • Bates S.E.
      • Abou-Alfa G.K.
      Equipoise, drug development, and biliary cancer.
      Although the impetus to treat patients with new therapies can be understood, it is our opinion that more data are required prior to adoption and routine use, and thus the best indication of novel approaches remains in the context of a clinical trial.
      After failure of gemcitabine and cisplatin, treatment is guided by tumour genomics.
      • Zehir A.
      • Benayed R.
      • Shah R.H.
      • Syed A.
      • Middha S.
      • Kim H.R.
      • et al.
      Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.
      ,
      • Javle M.
      • Bekaii-Saab T.
      • Jain A.
      • Wang Y.
      • Kelley R.K.
      • Wang K.
      • et al.
      Biliary cancer: utility of next-generation sequencing for clinical management.
      ,
      • Lowery M.A.
      • Ptashkin R.
      • Jordan E.
      • Berger M.F.
      • Zehir A.
      • Capanu M.
      • et al.
      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
      In those patients without a known driver alteration or in those for whom genomic analysis is incomplete, cytotoxic chemotherapy has shown modest anticancer activity and is a reasonable approach outside of a clinical trial.
      • Lamarca A.
      • Palmer D.H.
      • Wasan H.S.
      • Ross P.J.
      • Ma Y.T.
      • Arora A.
      • et al.
      Second-line FOLFOX chemotherapy versus active symptom control for advanced biliary tract cancer (ABC-06): a phase 3, open-label, randomised, controlled trial.
      ,
      • Yoo C.
      • Kim K.P.
      • Jeong J.H.
      • Kim I.
      • Kang M.J.
      • Cheon J.
      • et al.
      Liposomal irinotecan plus fluorouracil and leucovorin versus fluorouracil and leucovorin for metastatic biliary tract cancer after progression on gemcitabine plus cisplatin (NIFTY): a multicentre, open-label, randomised, phase 2b study.
      The ABC-06 study comparing 5-FU and oxaliplatin (FOLFOX) with active symptom control (ASC) vs. ASC alone in the second-line setting was the first trial to demonstrate a survival advantage for chemotherapy. The addition of FOLFOX to ASC improved median OS modestly from 5.3 months to 6.2 months (HR 0.69; 95% CI 0.50-0.97; p = 0.031). The sample size was not large enough to ascertain if prior platinum sensitivity impacted outcomes, nor was the proportion of homologous repair deficiency reported in the study population. In addition, standard practice in the United Kingdom, where this study was conducted, is to provide only a 6-month course of first-line gemcitabine and cisplatin; thus, it is unclear how to interpret these results in cases where first-line platinum is continued until progression of disease. Another fluoropyridine doublet, 5-FU plus nano-liposomal irinotecan, a modified version of irinotecan that enables enhanced tumour delivery, was assessed in the phase II NIFTY trial.
      • Yoo C.
      • Kim K.P.
      • Jeong J.H.
      • Kim I.
      • Kang M.J.
      • Cheon J.
      • et al.
      Liposomal irinotecan plus fluorouracil and leucovorin versus fluorouracil and leucovorin for metastatic biliary tract cancer after progression on gemcitabine plus cisplatin (NIFTY): a multicentre, open-label, randomised, phase 2b study.
      Following progression on gemcitabine/cisplatin, patients were randomised to either liposomal irinotecan/5-FU or 5-FU alone. ORR was higher in the arm receiving liposomal irinotecan (15% vs. 6%) as was PFS, the primary endpoint of the study (7.1 vs. 1.4 months; HR 0.56, 95% CI 0.39-0.81; p = 0.0019). Other attempts to chemically modify cytotoxic chemotherapy, for example with NUC-1031 (a phosphoramidate-modified gemcitabine derivative), showed early promise but did not demonstrate efficacy in a phase III trial.
      • McNamara M.G.
      • Bridgewater J.
      • Palmer D.H.
      • Faluyi O.
      • Wasan H.
      • Patel A.
      • et al.
      A phase ib study of NUC-1031 in combination with cisplatin for the first-line treatment of patients with advanced biliary tract cancer (ABC-08).

      Precision medicine

      In tumours with a potentially druggable oncogenic driver, precision medicine is a reasonable approach after failure of first-line cytotoxic therapy — although it must be noted that most approved agents are based on single-arm, phase II studies with a surrogate primary endpoint of ORR or on histology-agnostic basket studies that treated a limited number of individuals with BTC (Table 2). It is estimated that up to 40% of these oncogenic events are druggable and include mutations in IDH1, ERBB2, BRAF, KRAS, BRCA1/2; amplifications in ERBB2 and MDM2; fusions and other structural rearrangements in FGFR2, NTRK, ROS and RET; as well as inactivation in mismatch repair enzymes (MSI-high/MMR-D).
      • Zehir A.
      • Benayed R.
      • Shah R.H.
      • Syed A.
      • Middha S.
      • Kim H.R.
      • et al.
      Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.
      In this respect, three points should be emphasised i) most series on the frequency of genomic drivers in BTC are derived from tertiary centres and selection bias may inflate the true prevalence of actionable genes in this disease; ii) clinical grade genotyping is still an imperative to identify patients who might benefit from said therapy and is thus required for optimal clinic care; iii) acquired resistance to precision medicine is nearly ubiquitous and complex in nature.
      • Vasan N.
      • Baselga J.
      • Hyman D.M.
      A view on drug resistance in cancer.
      ,
      • Marin J.J.G.
      • Lozano E.
      • Herraez E.
      • Asensio M.
      • Di Giacomo S.
      • Romero M.R.
      • et al.
      Chemoresistance and chemosensitization in cholangiocarcinoma.
      Of note, extensive differences have been reported across the different CCA subtypes, since FGFR and IDH mutant-enriched subtypes are much more common in iCCA than in eCCA.
      • Banales J.M.
      • Marin J.J.G.
      • Lamarca A.
      • Rodrigues P.M.
      • Khan S.A.
      • Roberts L.R.
      • et al.
      Cholangiocarcinoma 2020: the next horizon in mechanisms and management.
      Table 2Available prospective data for precision medicine in biliary tract cancer.
      AgentPhasenORR (%)PFS (months)OS (Months)
      FGFR2
       PemigatinibII10735.56.921.1
       InfigratinibII10823.17.312.2
       FutibatinibII10341.77.820
       DerazantinibI/II2920.75.7
       ErdafitinibII12505.6
       Debio 1347I922
      IDH1
       IvosidenibIII18522.710.8
      BRAF V600E
       Dabrafenib + trametinibII33517.211.3
      HER2 (ERBB2)
       Trastuzumab + pertuzumabII3923410.9
       NeratenibII25162.85.4
      FGFR, fibroblast growth factor receptor; IDH1, isocitrate dehydrogenase 1.
      The quintessential example of the direct link between an oncogene to cellular energetics and ultimately to tumorigenesis, is observed with gain-of-function, single-nucleotide variants (SNVs) in IDH1/2.
      • Pirozzi C.J.
      • Yan H.
      The implications of IDH mutations for cancer development and therapy.
      Here, a ‘neo’- or ‘onco’-metabolite, 2-hydroxyglutarate (2-HG), is produced via NADPH-dependent reduction by the mutant protein; accumulation of 2-HG impairs cellular differentiation via direct effects on DNA methylation and chromatin structure. IDH1/2 mutations (IDH1 R132 and IDH2 R172 or 140) are observed in about 20-25% of iCCA.
      • Javle M.
      • Bekaii-Saab T.
      • Jain A.
      • Wang Y.
      • Kelley R.K.
      • Wang K.
      • et al.
      Biliary cancer: utility of next-generation sequencing for clinical management.
      ,
      • Lowery M.A.
      • Goff L.W.
      • Keenan B.P.
      • Jordan E.
      • Wang R.
      • Bocobo A.G.
      • et al.
      Second-line chemotherapy in advanced biliary cancers: a retrospective, multicenter analysis of outcomes.
      To date, targeting uncommon IDH2-altered tumours remains investigational. Ivosidenib (AG-120), an orally bioavailable selective IDH1 inhibitor, exhibits cytostatic anticancer activity in individuals with IDH1-mutant advanced iCCA.
      • Lowery M.A.
      • Burris 3rd, H.A.
      • Janku F.
      • Shroff R.T.
      • Cleary J.M.
      • Azad N.S.
      • et al.
      Safety and activity of ivosidenib in patients with IDH1-mutant advanced cholangiocarcinoma: a phase 1 study.
      ,
      • Fan B.
      • Mellinghoff I.K.
      • Wen P.Y.
      • Lowery M.A.
      • Goyal L.
      • Tap W.D.
      • et al.
      Clinical pharmacokinetics and pharmacodynamics of ivosidenib, an oral, targeted inhibitor of mutant IDH1, in patients with advanced solid tumors.
      The ClarIDHy study, a double-blind, placebo-controlled, phase III trial, was the pivotal trial that confirmed ivosedenib activity in individuals with advanced iCCA who failed prior gemcitabine-based treatment.
      • Abou-Alfa G.K.
      • Macarulla T.
      • Javle M.M.
      • Kelley R.K.
      • Lubner S.J.
      • Adeva J.
      • et al.
      Ivosidenib in IDH1-mutant, chemotherapy-refractory cholangiocarcinoma (ClarIDHy): a multicentre, randomised, double-blind, placebo-controlled, phase 3 study.
      When compared to placebo, the primary endpoint of PFS in the ivosidenib group was improved from 1.4 to 2.7 months (HR 0.37; 95% CI 0.25–0.54; p <0.0001). Importantly, evaluation of the Kaplan-Meier curves showed a tail of the curve indicating that a patient subset has prolonged benefit and disease control (6-month PFS of 32% vs. 0%). With extended follow-up on study and controlling for protocol allowed cross-over, OS also favoured active treatment.
      • Zhu A.X.
      • Macarulla T.
      • Javle M.M.
      • Kelley R.K.
      • Lubner S.J.
      • Adeva J.
      • et al.
      Final overall survival efficacy results of ivosidenib for patients with advanced cholangiocarcinoma with IDH1 mutation: the phase 3 randomized clinical ClarIDHy trial.
      Other selective IDH1 and IDH2 inhibitors, alone or in combination, are currently under investigation. LY3410738, a next-generation, selective, covalent IDH1/2 inhibitor, was designed to break established resistance mechanisms observed with ivosidenib (NCT04521686).
      • Harding J.J.
      • Lowery M.A.
      • Shih A.H.
      • Schvartzman J.M.
      • Hou S.
      • Famulare C.
      • et al.
      Isoform switching as a mechanism of acquired resistance to mutant isocitrate dehydrogenase inhibition.
      • Choe S.
      • Wang H.
      • DiNardo C.D.
      • Stein E.M.
      • de Botton S.
      • Roboz G.J.
      • et al.
      Molecular mechanisms mediating relapse following ivosidenib monotherapy in IDH1-mutant relapsed or refractory AML.
      • Pauff J.M.
      • Papadopoulos K.P.
      • Janku F.
      • Turk A.A.
      • Goyal L.
      • Shroff R.T.
      • et al.
      A phase I study of LY3410738, a first-in-class covalent inhibitor of mutant IDH1 in cholangiocarcinoma and other advanced solid tumors.
      Biologically, IDH alterations promote a BRCA-like phenotype and preclinical data have suggested synergy with poly (ADP-ribose) polymerase inhibitors, cytotoxic chemotherapy and radiotherapy.
      • Sulkowski P.L.
      • Corso C.D.
      • Robinson N.D.
      • Scanlon S.E.
      • Purshouse K.R.
      • Bai H.
      • et al.
      2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.
      ,
      • Lu Y.
      • Kwintkiewicz J.
      • Liu Y.
      • Tech K.
      • Frady L.N.
      • Su Y.T.
      • et al.
      Chemosensitivity of IDH1-mutated gliomas due to an impairment in PARP1-mediated DNA repair.
      Early-phase studies are now planned or underway examining such combinations with IDH1 inhibition (NCT03212274, NCT03878095, NCT03991832, NCT04521686). IDH1-mutant tumours also appear to engender an immunologically inactive tumour microenvironment.
      • Aguado-Fraile E.
      • Tassinari A.
      • Ishii Y.
      • Sigel C.
      • Lowery M.A.
      • Goyal L.
      • et al.
      Molecular and morphological changes induced by ivosidenib correlate with efficacy in mutant-IDH1 cholangiocarcinoma.
      In vitro, IDH1 inhibition reverses features of an immune suppressive microenvironment, stimulating cytotoxic T cells that can be further activated by immune checkpoint inhibitors, supporting IDH1 inhibition and immune checkpoint inhibitor combinations as a synergic strategy that warrants testing in the clinic (NCT04056910).
      • Wu M.J.
      • Shi L.
      • Dubrot J.
      • Merritt J.
      • Vijay V.
      • Wei T.Y.
      • et al.
      Mutant IDH inhibits IFNgamma-TET2 signaling to promote immunoevasion and tumor maintenance in cholangiocarcinoma.
      Several types of genomic alterations in FGFR have been identified in solid tumours, and preclinical models indicate that such alterations lead to aberrant signal transduction, through multiple signalling cascades, that drive uncontrolled cellular growth.
      • Babina I.S.
      • Turner N.C.
      Advances and challenges in targeting FGFR signalling in cancer.
      FGFR perturbations are more common in patients with iCCA, with FGFR2 fusions and rearrangements being the most common genomic events (occurring in 7–15% of patients).
      • Zehir A.
      • Benayed R.
      • Shah R.H.
      • Syed A.
      • Middha S.
      • Kim H.R.
      • et al.
      Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.
      ,
      • Javle M.
      • Bekaii-Saab T.
      • Jain A.
      • Wang Y.
      • Kelley R.K.
      • Wang K.
      • et al.
      Biliary cancer: utility of next-generation sequencing for clinical management.
      ,
      • Lowery M.A.
      • Ptashkin R.
      • Jordan E.
      • Berger M.F.
      • Zehir A.
      • Capanu M.
      • et al.
      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
      ,
      • Sia D.
      • Losic B.
      • Moeini A.
      • Cabellos L.
      • Hao K.
      • Revill K.
      • et al.
      Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma.
      Several FGFR inhibitors are now approved (pemigatinib and infigratinib) or are in active clinical development (futibatinib, derazantinib, erdafitinib). These agents appear safe and tolerable while leading to ORRs of 20-40% in iCCAs harbouring FGFR2 fusions or rearrangements. Pemigatinib, the first FGFR inhibitor approved by the FDA was tested in the multicentre, phase II, FIGHT-202 study.
      • Abou-Alfa G.K.
      • Sahai V.
      • Hollebecque A.
      • Vaccaro G.
      • Melisi D.
      • Al-Rajabi R.
      • et al.
      Pemigatinib for previously treated, locally advanced or metastatic cholangiocarcinoma: a multicentre, open-label, phase 2 study.
      In this study, 107 individuals with FGFR2 fusions/rearrangements attained an ORR of 35.5% with favourable median OS of 21.1 months. Importantly, responses were not observed for those patients with iCCA harbouring other FGFR alterations, or in those without such mutations — indicating selective activity for fusion events. Case series suggest FGFR2 indels may be targetable, and other FGFR inhibitors are being explored in individuals with non-fusion events.
      • Cleary J.M.
      • Raghavan S.
      • Wu Q.
      • Li Y.Y.
      • Spurr L.F.
      • Gupta H.V.
      • et al.
      FGFR2 extracellular domain in-frame deletions are therapeutically targetable genomic alterations that function as oncogenic drivers in cholangiocarcinoma.
      Infigratinib, another ATP-competitive FGFR inhibitor with comparable preclinical characteristics to pemigatinib, similarly led to an ORR of 30.1% in a pretreated iCCA population with FGFR2 fusions/translocations.
      • Javle M.
      • Roychowdhury S.
      • Kelley R.K.
      • Sadeghi S.
      • Macarulla T.
      • Weiss K.H.
      • et al.
      Infigratinib (BGJ398) in previously treated patients with advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements: mature results from a multicentre, open-label, single-arm, phase 2 study.
      ,
      • Javle M.
      • Lowery M.
      • Shroff R.T.
      • Weiss K.H.
      • Springfeld C.
      • Borad M.J.
      • et al.
      Phase II study of BGJ398 in patients with FGFR-altered advanced cholangiocarcinoma.
      Finally, futibatinib (TAS-120), a covalent and irreversible FGFR1-4 inhibitor, exhibits biochemical properties that allow the molecule to break established acquired resistance mechanisms, with a reported ORR of 25.4% in FGFR inhibitor-naïve iCCAs with FGFR2 fusions/rearrangements.
      • Meric-Bernstam F.
      • Bahleda R.
      • Hierro C.
      • Sanson M.
      • Bridgewater J.
      • Arkenau H.T.
      • et al.
      Futibatinib, an irreversible FGFR1-4 inhibitor, in patients with advanced solid tumors harboring FGF/FGFR aberrations: a phase I dose-expansion study.
      Given these clinical data, all three inhibitors are now under active investigation (vs. gemcitabine/cisplatin) in randomised, phase III studies in the first-line setting for individuals with iCCAs with FGFR gene fusions/rearrangements (NCT03656536, NCT03773302, NCT04093362).
      • Bekaii-Saab T.S.
      • Valle J.W.
      • Van Cutsem E.
      • Rimassa L.
      • Furuse J.
      • Ioka T.
      • et al.
      FIGHT-302: first-line pemigatinib vs. gemcitabine plus cisplatin for advanced cholangiocarcinoma with FGFR2 rearrangements.
      Challenges to study accrual include difficulties in timely genotyping and effective patient identification, the competitive landscape of FGFR inhibitor development, and the evolving landscape of the front-line space with the introduction of immune checkpoint inhibitors. This observation is supported by recent data indicating that FGFR inhibition induces loss of matrix MCL1 (myeloid cell leukaemia 1), a pro-survival member of the BCL2 protein family, resulting in failure of mitochondrial oxidative metabolism and cell necrosis. This process can enhance the antigenic burden of cancer cells, thereby stimulating the immune system and has implications for combination therapy using receptor inhibition with immunotherapy.
      • Kabashima A.
      • Hirsova P.
      • Bronk S.F.
      • Hernandez M.C.
      • Truty M.J.
      • Rizvi S.
      • et al.
      Fibroblast growth factor receptor inhibition induces loss of matrix MCL1 and necrosis in cholangiocarcinoma.
      Novel FGFR2 inhibitors that are designed to overcome established resistance mechanisms are in active clinical development. Although primary or innate resistance to FGFR inhibition is poorly understood, secondary or acquired resistance to therapy is well-documented and occurs as point mutations in the kinase domain of FGFR2 (i.e., gatekeeper mutations such as V565F, I or L and molecular brakes such as E566A, and N550D, H, K, or T).
      • Goyal L.
      • Saha S.K.
      • Liu L.Y.
      • Siravegna G.
      • Leshchiner I.
      • Ahronian L.G.
      • et al.
      Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in patients with FGFR2 fusion-positive cholangiocarcinoma.
      • Goyal L.
      • Shi L.
      • Liu L.Y.
      • Fece de la Cruz F.
      • Lennerz J.K.
      • Raghavan S.
      • et al.
      TAS-120 overcomes resistance to ATP-competitive FGFR inhibitors in patients with FGFR2 fusion-positive intrahepatic cholangiocarcinoma.
      • Varghese A.M.
      • Patel J.
      • Janjigian Y.Y.
      • Meng F.
      • Selcuklu S.D.
      • Iyer G.
      • et al.
      Noninvasive detection of polyclonal acquired resistance to FGFR inhibition in patients with cholangiocarcinoma harboring FGFR2 alterations.
      Such alterations create steric hindrance at the binding pocket of first-generation FGFR inhibitors or re-activate the kinase irrespective of drug binding, allowing for escape and neoplastic outgrowth despite continued FGFR inhibition. Indeed, as noted above, futibantinb, which binds to FGFR2 in a different manner and has in vitro activity against some established resistance alterations, led to a 17.9% (5 of 28 patients) response rate in patients who had progressed on first-generation FGFR inhibitors.
      • Meric-Bernstam F.
      • Bahleda R.
      • Hierro C.
      • Sanson M.
      • Bridgewater J.
      • Arkenau H.T.
      • et al.
      Futibatinib, an irreversible FGFR1-4 inhibitor, in patients with advanced solid tumors harboring FGF/FGFR aberrations: a phase I dose-expansion study.
      ,
      • Goyal L.
      • Shi L.
      • Liu L.Y.
      • Fece de la Cruz F.
      • Lennerz J.K.
      • Raghavan S.
      • et al.
      TAS-120 overcomes resistance to ATP-competitive FGFR inhibitors in patients with FGFR2 fusion-positive intrahepatic cholangiocarcinoma.
      There is preclinical evidence that newer more potent and selective inhibitors, such as RLY-4008 and KIN-3248 which are now in phase I/II development (NCT04526106, NCT05242822), can circumvent drug resistance mutations.
      • Schram A.M.
      • Kamath S.D.
      • El-Khoueiry A.B.
      • Borad M.J.
      • Mody K.
      • Mahipal A.
      • et al.
      First-in-human study of highly selective FGFR2 inhibitor, RLY-4008, in patients with intrahepatic cholangiocarcinoma and other advanced solid tumors.
      Efforts are also ongoing to ascertain the most appropriate position for FGFR inhibitors within combination strategies.
      • Wu Q.
      • Zhen Y.
      • Shi L.
      • Vu P.
      • Greninger P.
      • Adil R.
      • et al.
      EGFR inhibition potentiates FGFR inhibitor therapy and overcomes resistance in FGFR2 fusion-positive cholangiocarcinoma.
      ,
      • Kendre G.
      • Marhenke S.
      • Lorz G.
      • Becker D.
      • Reineke-Plaass T.
      • Poth T.
      • et al.
      The Co-mutational spectrum determines the therapeutic response in murine FGFR2 fusion-driven cholangiocarcinoma.
      The recently announced discontinuation of the PROOF-301 study, which was comparing infigratinib to gemcitabine plus cisplatin in the first-line setting for individuals with FGFR2 fusions (NCT03773302), is a clear reflection of the anticipated impact of adding durvalumab to gemcitabine plus cisplatin.
      SNVs as well as genomic amplification of ERBB2 have been documented in all subtypes of BTC, though they predominate in GBC.
      • Javle M.
      • Bekaii-Saab T.
      • Jain A.
      • Wang Y.
      • Kelley R.K.
      • Wang K.
      • et al.
      Biliary cancer: utility of next-generation sequencing for clinical management.
      ,
      • Lowery M.A.
      • Ptashkin R.
      • Jordan E.
      • Berger M.F.
      • Zehir A.
      • Capanu M.
      • et al.
      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
      ,
      • Mondaca S.
      • Razavi P.
      • Xu C.
      • Offin M.
      • Myers M.
      • Scaltriti M.
      • et al.
      Genomic characterization of ERBB2-driven biliary cancer and a case of response to ado-trastuzumab emtansine.
      ERBB2 SNVs are less frequent than ERBB2 amplification or HER2 overexpression, though attempts to target this unique population have demonstrated only modest antitumor activity.
      • Hyman D.M.
      • Piha-Paul S.A.
      • Won H.
      • Rodon J.
      • Saura C.
      • Shapiro G.I.
      • et al.
      HER kinase inhibition in patients with HER2- and HER3-mutant cancers.
      Neratinib, an oral pan-HER inhibitor, was administered to 25 individuals with BTC in a cohort of the SUMMIT study — with an ORR of 16%.
      • Harding J.J.
      • Cleary J.M.
      • Quinn D.I.
      • Braña I.
      • Moreno V.
      • Borad M.J.
      • et al.
      Targeting HER2 (ERBB2) mutation-positive advanced biliary tract cancers with neratinib: results from the phase II SUMMIT ‘basket’ trial.
      Regarding HER2 overexpression, the MyPathway phase IIa basket study examined the dual combination of anti-HER2 antibodies pertuzumab and trastuzumab.
      • Javle M.
      • Borad M.J.
      • Azad N.S.
      • Kurzrock R.
      • Abou-Alfa G.K.
      • George B.
      • et al.
      Pertuzumab and trastuzumab for HER2-positive, metastatic biliary tract cancer (MyPathway): a multicentre, open-label, phase 2a, multiple basket study.
      Here, the ORR was 22% and individuals with GBC and iCCA appeared to have more favourable outcomes. Zanidatamab is a bispecific anti-HER2 antibody that binds two separate epitopes of HER2; it has shown preliminary antitumor activity and is now being investigated in a global single-arm phase II study (NCT03929666).
      • Meric-Bernstam F.
      • Hanna D.L.
      • El-Khoueiry A.B.
      • Kang Y.-K.
      • Oh D.-Y.
      • Chaves J.M.
      • et al.
      Zanidatamab (ZW25) in HER2-positive biliary tract cancers (BTCs): results from a phase I study.
      ,
      • Pant S.
      • Ducreux M.
      • Harding J.J.
      • Javle M.M.
      • Oh D.-Y.
      • Wasan H.S.
      • et al.
      A phase IIb, open-label, single-arm study of zanidatamab (ZW25) monotherapy in subjects with advanced or metastatic HER2-amplified biliary tract cancers.
      Beyond unmodified antibodies, antibody drug conjugates, such as trastuzumab-emtansine, trastuzumab-deruxtecan, and ZW49 (a combination of zanidatamab with a cleavable linker that delivers an auristatin cytotoxin to HER2-positive tumour cells) are all under active investigation in several basket studies (NCT02675829, NCT04482309, NCT04639219, NCT03821233).
      • Tsurutani J.
      • Iwata H.
      • Krop I.
      • Janne P.A.
      • Doi T.
      • Takahashi S.
      • et al.
      Targeting HER2 with trastuzumab deruxtecan: a dose-expansion, phase I study in multiple advanced solid tumors.
      The MAPK pathway is also amenable to targeting in the current era of precision medicine. Activating mutations in RAS occur in up to 40% of BTCs depending on the anatomical location, as they are more frequent in eCCA than iCCA.
      • Javle M.
      • Bekaii-Saab T.
      • Jain A.
      • Wang Y.
      • Kelley R.K.
      • Wang K.
      • et al.
      Biliary cancer: utility of next-generation sequencing for clinical management.
      ,
      • Lowery M.A.
      • Ptashkin R.
      • Jordan E.
      • Berger M.F.
      • Zehir A.
      • Capanu M.
      • et al.
      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
      Targeting RAS has been historically difficult and it was largely considered un-druggable — though exploiting the cysteine residue in KRAS G12C (occurring in <1% of BTCs) has led to the development of several covalent inhibitors of this subset that effectively lock KRAS G12C into its GDP inactive form.
      • Hong D.S.
      • Fakih M.G.
      • Strickler J.H.
      • Desai J.
      • Durm G.A.
      • Shapiro G.I.
      • et al.
      KRAS(G12C) inhibition with sotorasib in advanced solid tumors.
      There is anecdotal evidence that sotorasib, approved for KRAS G12C lung cancer, exhibits anticancer activity in BTCs.
      • Hong D.S.
      • Fakih M.G.
      • Strickler J.H.
      • Desai J.
      • Durm G.A.
      • Shapiro G.I.
      • et al.
      KRAS(G12C) inhibition with sotorasib in advanced solid tumors.
      Likewise, the phase II KRYSTAL-1 trial of adagrasib has documented partial responses in 4 of 8 (50%) participants to date.
      • Bekaii-Saab T.S.
      • Spira A.I.
      • Yaeger R.
      • Buchschacher G.L.
      • McRee A.J.
      • Sabari J.K.
      • et al.
      KRYSTAL-1: updated activity and safety of adagrasib (MRTX849) in patients (Pts) with unresectable or metastatic pancreatic cancer (PDAC) and other gastrointestinal (GI) tumors harboring a KRASG12C mutation.
      Currently, other small molecules (via direct or indirect actions on the MAPK pathway), vaccination programmes, as well as proteolysis-targeting chimera and molecular glues are currently being developed as strategies against more common RAS mutations.
      • Schneider M.
      • Radoux C.J.
      • Hercules A.
      • Ochoa D.
      • Dunham I.
      • Zalmas L.-P.
      • et al.
      The PROTACtable genome.
      ,
      • Moore A.R.
      • Rosenberg S.C.
      • McCormick F.
      • Malek S.
      RAS-targeted therapies: is the undruggable drugged?.
      Downstream of RAS, class 1 (BRAF V600E), 2 and 3 RAF alterations have all been identified but, to date, only the class 1 alterations are targetable with FDA approved agents. Evident anticancer activity is exemplified by the single-arm, phase II trial (ROAR) that tested dabrafenib and trametinib in 43 patients with BRAF V600E BTC, with an overall response rate of 51%, median PFS of 9 months and median OS of 14 months.
      • Subbiah V.
      • Lassen U.
      • Elez E.
      • Italiano A.
      • Curigliano G.
      • Javle M.
      • et al.
      Dabrafenib plus trametinib in patients with BRAF(V600E)-mutated biliary tract cancer (ROAR): a phase 2, open-label, single-arm, multicentre basket trial.
      Alterations in DDR/homologous recombination pathways including but not limited to ATM, ATR, CHEK1/2, BRCA1/2, and PALB2, have been identified in up to 20% of BTCs.
      • Ahn D.H.
      • Bekaii-Saab T.
      Biliary tract cancer and genomic alterations in homologous recombinant deficiency: exploiting synthetic lethality with PARP inhibitors.
      DDR/homologous recombination pathways are complex and determining their predictive and prognostic role — outside of BRCA1/2 inactivation — in response to chemotherapy is ongoing.
      • Chae H.
      • Kim D.
      • Yoo C.
      • Kim K.P.
      • Jeong J.H.
      • Chang H.M.
      • et al.
      Therapeutic relevance of targeted sequencing in management of patients with advanced biliary tract cancer: DNA damage repair gene mutations as a predictive biomarker.
      Several studies are now examining the efficacy of maintenance therapy with poly (ADP-ribose) polymerase inhibitors after platinum response, as well in combination with chemotherapy or immunotherapy (NCT03991832, NCT04895046, NCT04042831). Furthermore, some data suggest that these subtypes might be more sensitive to immune-based treatment, and such biomarkers are now being actively investigated.
      • Samstein R.M.
      • Krishna C.
      • Ma X.
      • Pei X.
      • Lee K.W.
      • Makarov V.
      • et al.
      Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.
      ,
      • Terrero G.
      • Datta J.
      • Dennison J.
      • Sussman D.A.
      • Lohse I.
      • Merchant N.B.
      • et al.
      Ipilimumab/nivolumab therapy in patients with metastatic pancreatic or biliary cancer with homologous recombination deficiency pathogenic germline variants.
      Other alterations in ROS, RET, and NTRK are extremely rare. Positive outcome anecdotes from “basket” clinical trials may help further support the tumour agnostic regulatory approval processes.
      • Drilon A.
      • Laetsch T.W.
      • Kummar S.
      • DuBois S.G.
      • Lassen U.N.
      • Demetri G.D.
      • et al.
      Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children.
      ,
      • Doebele R.C.
      • Drilon A.
      • Paz-Ares L.
      • Siena S.
      • Shaw A.T.
      • Farago A.F.
      • et al.
      Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials.
      Thus, it is likely that advances in this field will come from further tumour agnostic studies.
      Other targets, which were previously inaccessible, such as claudins, ALCAM, and other cell surface molecules, are now viable targets thanks to advances in protein chemistry and the application of antibody drug conjugates for targeted delivery of cytotoxic therapy.
      • Drago J.Z.
      • Modi S.
      • Chandarlapaty S.
      Unlocking the potential of antibody-drug conjugates for cancer therapy.
      ,
      • Boni V.
      • Fidler M.J.
      • Arkenau H.T.
      • Spira A.
      • Meric-Bernstam F.
      • Uboha N.
      • et al.
      Praluzatamab ravtansine, a CD166-targeting antibody-drug conjugate, in patients with advanced solid tumors: an open-label phase I/II trial.

      Multitargeted tyrosine kinase inhibitors

      Multitargeted tyrosine kinase inhibitors (TKIs) that target vascular endothelial growth factor receptor (VEGFR)1-3 and platelet-derived growth factor receptor (PDGFR), for example, have been evaluated in a molecularly unselected BTC population.
      • Kim R.D.
      • Sanoff H.K.
      • Poklepovic A.S.
      • Soares H.
      • Kim J.
      • Lyu J.
      • et al.
      A multi-institutional phase 2 trial of regorafenib in refractory advanced biliary tract cancer.
      A small, randomised, placebo-controlled, phase II trial (REACHIN) tested regorafenib in patients who progressed on gemcitabine and cisplatin.
      • Demols A.
      • Borbath I.
      • Van den Eynde M.
      • Houbiers G.
      • Peeters M.
      • Marechal R.
      • et al.
      Regorafenib after failure of gemcitabine and platinum-based chemotherapy for locally advanced/metastatic biliary tumors: REACHIN, a randomized, double-blind, phase II trial.
      No objective responses were observed and the improvement in PFS was modest (3.0 vs. 1.5 months, HR 0.49, 95% CI 0.29-0.81, p = 0.004). Further confirmatory testing is needed before this approach can be applied as standard-of-care. However, as described subsequently, multitargeted TKIs that block VEGFR and PDGFR, among others, may have clinical utility in augmenting the effects of immune-based treatments. It is conceivable that the reduced effect observed with anti-angiogenic therapies is due to marked hypovascularity, which is a distinctive feature of the tumour microenvironment in CCA.
      • Cadamuro M.
      • Brivio S.
      • Mertens J.
      • Vismara M.
      • Moncsek A.
      • Milani C.
      • et al.
      Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma.
      ,
      • Carpino G.
      • Cardinale V.
      • Di Giamberardino A.
      • Overi D.
      • Donsante S.
      • Colasanti T.
      • et al.
      Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.

      Immunotherapy

      Immune checkpoint inhibitors, as well as other immunotherapies, will continue to be developed in BTC. After failure of front-line chemotherapy, MSI-high/MMR-D is a predictive biomarker of response to single-agent anti-PD-1 therapy. KEYNOTE-158, a phase II study of pembrolizumab in patients with advanced, pre-treated, non-colorectal MSI-high/MMR-D tumours included 22 patients with BTC, of whom two had a complete response and seven had a partial response (ORR 40.9%). Median PFS was 4.2 months (95% CI 2.1-not reached) and median OS was 24.3 months (95% CI 6.5-not reached).
      • Marabelle A.
      • Le D.T.
      • Ascierto P.A.
      • Di Giacomo A.M.
      • De Jesus-Acosta A.
      • Delord J.P.
      • et al.
      Efficacy of pembrolizumab in patients with noncolorectal high microsatellite instability/mismatch repair-deficient cancer: results from the phase II KEYNOTE-158 study.
      Importantly, emerging data in other MSI-high tumours indicate that the means by which MSI-high status arises may have important implications for response.
      • Rousseau B.
      • Foote M.B.
      • Maron S.B.
      • Diplas B.H.
      • Lu S.
      • Argiles G.
      • et al.
      The spectrum of benefit from checkpoint blockade in hypermutated tumors.
      How the implementation of the TOPAZ-1 regimen in the first-line setting will affect the use of second-line immune-based therapy in this molecular subset remains to be seen.
      Outside of this rare genomic subset, immune checkpoint inhibitors have only limited activity, with ORRs ranging from 3%-13% and OS ranging from 5.2 to 8.1 months in a variety of small single-arm studies
      • Piha-Paul S.A.
      • Oh D.Y.
      • Ueno M.
      • Malka D.
      • Chung H.C.
      • Nagrial A.
      • et al.
      Efficacy and safety of pembrolizumab for the treatment of advanced biliary cancer: results from the KEYNOTE-158 and KEYNOTE-028 studies.
      ,
      • Kim R.D.
      • Chung V.
      • Alese O.B.
      • El-Rayes B.F.
      • Li D.
      • Al-Toubah T.E.
      • et al.
      A phase 2 multi-institutional study of nivolumab for patients with advanced refractory biliary tract cancer.
      (Table 3). As noted, only a small BTC subset exhibits features of immune activation, characterised by lymphocyte infiltration and overexpression of PD-1/PD-L1 (about 10% in both iCCA and eCCA),
      • Martin-Serrano M.A.
      • Kepecs B.
      • Torres-Martin M.
      • Bramel E.R.
      • Haber P.K.
      • Merritt E.
      • et al.
      Novel microenvironment-based classification of intrahepatic cholangiocarcinoma with therapeutic implications.
      ,
      • Montal R.
      • Sia D.
      • Montironi C.
      • Leow W.Q.
      • Esteban-Fabro R.
      • Pinyol R.
      • et al.
      Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma.
      and the consensus is that BTC is an immunologically ‘cold’ tumour. Another option might be the use of rational combination strategies, like those for FGFR inhibitors, to enhance an immune response. Preclinical data support several novel combinations such as chemotherapy or precision medicine (as discussed above), as well as checkpoint antagonists (e.g., CTLA-4, TIM-3, LAG-3, TIGIT) and agonists (e.g., 4-1BB and OX40), antiangiogenics/TKIs, and cytokines.
      Table 3Available prospective data for checkpoint inhibitors in biliary tract cancer.
      MechanismAgentPhasePatient populationnORR (%)PFS (months)OS (Months)
      Anti-PD-1PembrolizumabIIChemotherapy refractory microsatellite instability-high solid tumours2240.94.224.3
      Anti-PD-1PembrolizumabIIChemotherapy refractory Microsatellite stable solid tumours1045.827.4
      Anti-PD-1NivolumabIIChemotherapy refractory biliary tract cancer54223.6814.2
      Anti-PD-L1DurvalumabIChemotherapy refractory biliary tract cancer4258.1
      Anti-PD-1 + anti-CTLA4Nivolumab + ipilimumabIIChemotherapy refractory biliary tract cancer39232.95.7
      Anti-PD-L1 + anti-CTLA4Durvalumab + tremelimumabIChemotherapy refractory biliary tract cancer6210.810.1
      Chemotherapy + anti-PD-L1Gemcitabine/cisplatin + durvalumabIIFirst-line treatment of biliary tract cancer4573.41118.1
      Bifunctional fusion protein against PD-L1 and TGFBR2Bintrafusp alphaIChemotherapy refractory biliary tract cancer3023.312.7
      CTLA-4, cytotoxic T lymphocyte-associated protein 4; PD-1, programmed death 1; PD-L1, programmed death ligand 1; TGFBR2, transforming growth factor beta receptor 2.
      To date, anti-PD-1/L1-and anti-CTLA-4-based combinations have been evaluated. Nivolumab and ipilimumab led to an ORR of 23% while the combination of durvalumab and tremelimumab demonstrated an ORR of 7.1% with a median OS of 10.1 months in small phase II studies.
      • Klein O.
      • Kee D.
      • Nagrial A.
      • Markman B.
      • Underhill C.
      • Michael M.
      • et al.
      Evaluation of combination nivolumab and ipilimumab immunotherapy in patients with advanced biliary tract cancers: subgroup Analysis of a phase 2 nonrandomized clinical trial.
      ,
      • Ioka T.
      • Ueno M.
      • Oh D.-Y.
      • Fujiwara Y.
      • Chen J.-S.
      • Doki Y.
      • et al.
      Evaluation of safety and tolerability of durvalumab (D) with or without tremelimumab (T) in patients (pts) with biliary tract cancer (BTC).
      Likewise, multitargeted TKIs and VEGF inhibition, which may promote T-cell activation and reduce immunosuppressive regulatory T cells in tumours in vivo, may enhance the effect of anti-PD-1 therapy.
      • Zhang Q.
      • Liu X.
      • Wei S.
      • Zhang L.
      • Tian Y.
      • Gao Z.
      • et al.
      Lenvatinib plus PD-1 inhibitors as first-line treatment in patients with unresectable biliary tract cancer: a single-arm, open-label, phase II study.
      Thirty-two individuals with BTC were treated with the combination of lenvatinib and pembrolizumab in a recent phase II study following progression on standard chemotherapy, with a reported median PFS and OS of 4.9 months and 11.0 months, respectively.
      • Lin J.
      • Yang X.
      • Long J.
      • Zhao S.
      • Mao J.
      • Wang D.
      • et al.
      Pembrolizumab combined with lenvatinib as non-first-line therapy in patients with refractory biliary tract carcinoma.
      Larger studies are ongoing to define the operating characteristics of such TKI-based combinations in BTC (NCT04550624, NCT03895970). Addressing the importance of cytokines released in the tumour microenvironment as a synergic strategy in BTC treatment, a bifunctional fusion protein targeting PD-L1 and transforming growth factor-β, bintrafusp alpha (M7824), was tested in advanced BTC as monotherapy in the second-line setting.
      • Yoo C.
      • Oh D.Y.
      • Choi H.J.
      • Kudo M.
      • Ueno M.
      • Kondo S.
      • et al.
      Phase I study of bintrafusp alfa, a bifunctional fusion protein targeting TGF-beta and PD-L1, in patients with pretreated biliary tract cancer.
      Long-term follow-up of the phase I study reported a median OS of 12.7 months with a 24-month OS rate of 27.7%. Following this approach, a phase II/III first-line study of M7842 in combination with chemotherapy was initiated (NCT04066491). More recently, the study sponsor discontinued the study, citing futility for the primary endpoint of improving OS. Other means of eliciting an immune response through bispecific T-cell engagers, vaccines, oncolytic virus, and adoptive cellular therapy are ongoing with anecdotal evidence of activity.
      • Tran E.
      • Turcotte S.
      • Gros A.
      • Robbins P.F.
      • Lu Y.C.
      • Dudley M.E.
      • et al.
      Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer.
      Based on the protocol of TOPAZ-1, a patient subset will now receive anti-PD-L1 maintenance therapy. This unique therapeutic space may allow for novel clinical trials that seek to prolong disease control and may be a ripe field for exploring agents designed to further enhance antitumor immunity, such as vaccines or cytokines.

      Practical concerns in drug development and patient care

      Practical concerns that must be accounted for in drug development relate to design/endpoints, patient selection, and correlative science to establish resistance mechanisms and propose new therapies.
      • Lee T.Y.
      • Bates S.E.
      • Abou-Alfa G.K.
      Equipoise, drug development, and biliary cancer.
      A drug’s mechanism of action will continue to dictate trial endpoints. Some agents — such as IDH1 inhibitors and immune-based therapies — may require additional efficacy evaluation by landmark analysis for both PFS and OS (to capture the importance of the tail of the curve for instance) in the context of randomised clinical trials. For example, IDH1 inhibition generally results in tumour cell differentiation without a corresponding reduction in tumour biomass,
      • Aguado-Fraile E.
      • Tassinari A.
      • Ishii Y.
      • Sigel C.
      • Lowery M.A.
      • Goyal L.
      • et al.
      Molecular and morphological changes induced by ivosidenib correlate with efficacy in mutant-IDH1 cholangiocarcinoma.
      while some immune-based therapies require time to generate an antitumor response.
      • Monge C.
      • Pehrsson E.C.
      • Xie C.
      • Duffy A.G.
      • Mabry D.
      • Wood B.J.
      • et al.
      A phase II study of pembrolizumab in combination with capecitabine and oxaliplatin with molecular profiling in patients with advanced biliary tract carcinoma.
      In contrast, future comparative designs in the precision space might seek to be smaller in size, as well as attempt to validate surrogates — such as cfDNA response, RECIST response rate, PFS – that might limit the duration of study follow-up in rare genomic subsets.
      • Lee T.Y.
      • Bates S.E.
      • Abou-Alfa G.K.
      Equipoise, drug development, and biliary cancer.
      First and foremost, rapid and accurate clinical grade NGS is another critical tool for trial design and direct patient care, as a means to identify currently available treatments in a disease for which there are limited options. Although these genotyping efforts are ongoing, several reports indicate that genotyping is not performed or fails (owing to inadequate tissue samples) in a subset of individuals with BTC.
      • Lowery M.A.
      • Ptashkin R.
      • Jordan E.
      • Berger M.F.
      • Zehir A.
      • Capanu M.
      • et al.
      Comprehensive molecular profiling of intrahepatic and extrahepatic cholangiocarcinomas: potential targets for intervention.
      Even in the presence of an actionable driver, patients are not always connected to either an FDA-approved agent or an investigational clinical trial for many reasons, including lack of prescriber knowledge, organ dysfunction, clinical performance status or access to drug. Genotyping is also critical for trial identification and, importantly, to understanding how specific genomic alterations may have prognostic significance in the disease.
      • Boerner T.
      • Drill E.
      • Pak L.M.
      • Nguyen B.
      • Sigel C.S.
      • Doussot A.
      • et al.
      Genetic determinants of outcome in intrahepatic cholangiocarcinoma.
      Although availability of tumour samples would be critical to meet this need, cfDNA may provide a valid alternative to identify driver alterations, and emerging data indicate that NGS of both tumour tissue and cfDNA may indeed be complimentary.
      • Rose Brannon A.
      • Jayakumaran G.
      • Diosdado M.
      • Patel J.
      • Razumova A.
      • Hu Y.
      • et al.
      Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.
      False negatives, due to the limits of detection of current assays or lack of cfDNA production, and false positives may occur in the setting of clonal haematopoiesis or germ-line alterations.
      • Rose Brannon A.
      • Jayakumaran G.
      • Diosdado M.
      • Patel J.
      • Razumova A.
      • Hu Y.
      • et al.
      Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.
      ,
      • Ettrich T.J.
      • Schwerdel D.
      • Dolnik A.
      • Beuter F.
      • Blatte T.J.
      • Schmidt S.A.
      • et al.
      Genotyping of circulating tumor DNA in cholangiocarcinoma reveals diagnostic and prognostic information.
      Acquired resistance occurs in most patients on targeted therapies and, in the precision space, several genomic alterations have been identified at disease progression (e.g., IDH2 isoform ‘switching’ in response to selective IDH1 inhibitors; FGFR SNVs in response to FGFR2 inhibition, ERBB2 loss in the setting of HER2 inhibition).
      • Harding J.J.
      • Lowery M.A.
      • Shih A.H.
      • Schvartzman J.M.
      • Hou S.
      • Famulare C.
      • et al.
      Isoform switching as a mechanism of acquired resistance to mutant isocitrate dehydrogenase inhibition.
      ,
      • Goyal L.
      • Saha S.K.
      • Liu L.Y.
      • Siravegna G.
      • Leshchiner I.
      • Ahronian L.G.
      • et al.
      Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in patients with FGFR2 fusion-positive cholangiocarcinoma.
      ,
      • Goyal L.
      • Shi L.
      • Liu L.Y.
      • Fece de la Cruz F.
      • Lennerz J.K.
      • Raghavan S.
      • et al.
      TAS-120 overcomes resistance to ATP-competitive FGFR inhibitors in patients with FGFR2 fusion-positive intrahepatic cholangiocarcinoma.
      ,
      • Harding J.J.
      • Cleary J.M.
      • Quinn D.I.
      • Braña I.
      • Moreno V.
      • Borad M.J.
      • et al.
      Targeting HER2 (ERBB2) mutation-positive advanced biliary tract cancers with neratinib: results from the phase II SUMMIT ‘basket’ trial.
      Investigational tumour biopsy, warm autopsies, and acquisition of cfDNA at progression will better delineate the frequency and intertumoral molecular heterogeneity of such events. It will not be possible to elucidate the functional mechanisms of resistance using these techniques, so developing tumour organoids, patient-derived xenografts, and other ex vivo models, as we are doing, will be key to uncovering novel strategies to overcome or delay resistance.
      • Vasan N.
      • Baselga J.
      • Hyman D.M.
      A view on drug resistance in cancer.
      Furthermore, ongoing efforts employing mathematical modelling have the potential to elucidate the complexity of acquired resistance.
      As new treatments are developed in the metastatic setting it will be critical to devise improved treatments for earlier stages of disease. With the exception of single-agent fluoropyridines whose benefit is modest, most large phase III studies have failed to show a benefit in this space.
      • Primrose J.N.
      • Fox R.P.
      • Palmer D.H.
      • Malik H.Z.
      • Prasad R.
      • Mirza D.
      • et al.
      Capecitabine compared with observation in resected biliary tract cancer (BILCAP): a randomised, controlled, multicentre, phase 3 study.
      ,
      • Ebata T.
      • Hirano S.
      • Konishi M.
      • Uesaka K.
      • Tsuchiya Y.
      • Ohtsuka M.
      • et al.
      Randomized clinical trial of adjuvant gemcitabine chemotherapy versus observation in resected bile duct cancer.
      • Edeline J.
      • Benabdelghani M.
      • Bertaut A.
      • Watelet J.
      • Hammel P.
      • Joly J.P.
      • et al.
      Gemcitabine and oxaliplatin chemotherapy or surveillance in resected biliary tract cancer (PRODIGE 12-ACCORD 18-UNICANCER GI): a randomized phase III study.
      • Ikeda M.
      • Nakachi K.
      • Konishi M.
      • Nomura S.
      • Katayama H.
      • Kataoka T.
      • et al.
      Adjuvant S-1 versus observation in curatively resected biliary tract cancer: a phase III trial (JCOG1202: ASCOT).
      Traditional adjuvant studies, such as ACTICCA-1 – evaluating gemcitabine plus cisplatin vs. capecitabine (NCT02170090), are ongoing. The field may consider the application of these agents as neoadjuvant or perioperative approaches. Perioperative or neoadjuvant approaches, which are now in development, may serve to treat micro-metastatic disease at earlier stages, activate a tumour-directed immune response (i.e., making tumours amenable to immunotherapy), prevent unnecessary surgery, and provide ample tissue for correlative science that may ultimately help to validate surrogates or understand acquired and innate resistance as discussed earlier. Unfortunately, early detection of BTC remains challenging and limits the number of individuals available for specific studies in this context.

      Conclusions

      BTC remains a worrisome group of diseases that are often unrelenting in their natural history. Clinical trial-based work has established that gemcitabine and cisplatin plus durvalumab is a reasonable standard-of-care in the first-line setting, while the application of fluoropyridine-based doublets in a molecularly unselected patient population or precision medicine based on genotype can be pursued in the second-line setting. Intense investigation is now ongoing to explore new therapies and novel combinations in the metastatic setting and at earlier stages of disease. Early surrogates of OS need to be validated to help make trial design more efficient and the time to definitive study outcomes shorter. Furthermore, as immunotherapy will possibly play a greater role in treatment of this disease, a deeper understanding of the immunobiology of BTCs is required to raise the tail of the survival curve higher in the years ahead. In this respect, studies aimed at unravelling the complex interplay between the multiple cell types populating the tumour microenvironment are sorely needed to unveil innovative therapeutic targets that may be harnessed as part of combination strategies.

      Abbreviations

      ASC, active symptom control; BTC, biliary tract cancer; cfDNA, cell-free DNA; CTLA-4, cytotoxic T lymphocyte-associated protein 4; DDR, DNA damage response; eCCA, extrahepatic cholangiocarcinoma; FGFR2, fibroblast growth factor receptor 2; FOLFOX, 5-FU and oxaliplatin; FOLFIRINOX, folinic acid, 5-FU, irinotecan and oxaliplatin; GBC, gallbladder cancer; HR, hazard ratio; iCCA, intrahepatic cholangiocarcinoma; IDH1/2, isocitrate dehydrogenase 1/2; MMR-D, mismatch repair deficiency; MSI, microsatellite instability; NGS, next-generation sequencing; NTRK, neurotrophic receptor tyrosine kinase; ORR, objective response rate; OS, overall survival; PD-1, programmed death 1; PDGFR, platelet-derived growth factor receptor; PD-L1, programmed death ligand 1; PFS, progression-free survival; SNVs, single-nucleotide variants; TKIs, tyrosine kinase inhibitors; VEGF(R), vascular endothelial growth factor (receptor).

      Financial support

      James J. Harding, Danny N. Khalil, and Ghassan K. Abou-Alfa are supported by NCI Institutiobal grant 5P30CA008748-46 (P30) . James J. Harding is supported by NCI U01 CA238444-03.

      Authors' contributions

      All authors helped develop the outline, contributed to writing, final review, and approval.

      Conflict of interest

      James J. Harding reports grant research support from BI, Bristol Myers Squibb, Calithera, CytomX, Eli Lilly, Genoscience, Incyte, Novartis, Polaris, Yiviam and Zymeworks; consulting supprot from Adaptimmune, Bristol Myers Squibb, Eisai, Elevar, Exelixis, Hepion, Medivir, and Merck; and honoraria from HCC Connect, and NCCN. Danny N. Khalil reports royalty for intellectual property rights associated with Merck Sharp & Dohme; consulting fees from AbbVie and Psioxus; and patents EP3331612A1, CA3042867A1 for which he holds intellectual property interests related to CD40, nanoparticle therapeutics, and in situ vaccination. Luca Fabris reports consulting fees from L.E.K. Consulting and MedPanel; spport for attending European Association for the Study of the Liver (EASL) Liver Cancer Summit (2020), Prague, CZE, and European Network for the Study of Cholangiocarcinoma (ENS-CCA) Biennal Meeting (2022), Edinburgh, Scotland, ledership fiudicary role in International PSC Study Group, Working Group Malignancy (ongoing) (unpaid), Member Expert Panel, National Science Centre, Poland - Narodowe Centrum Nauki (NCN), Life Science section NZ5, Krakow (2019 e 2020), and external Reviewer of Doctoral Thesis for International Assessment. Invited by the Rabdoud University, The Netherlands (2022). Ghassan K. Abou-Alfa reports grant research support from Arcus, Astra Zeneca, BioNtech, BMS, Celgene, Flatiron, Genentech/Roche, Genoscience, Incyte, Polaris, Puma, QED, Silenseed, Yiviva, and consulting supprot from Adicet, Alnylam, Astra Zeneca, Autem, Beigene, Berry Genomics, Boehringer Ingelheim, Celgene, Cend, CytomX, Eisai, Eli Lilly, Exelixis, Flatiron, Genentech/Roche, Genoscience, Helio, Helsinn, Incyte, Ipsen, Merck, Nerviano, Newbridge, Novartis, QED, Redhill, Rafael, Servier, Silenseed, Sobi, Vector, Yiviva.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

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