Challenges and opportunities for treating intrahepatic cholangiocarcinoma: targeted therapy

Isocitrate dehydrogenase inhibitors

IDH gene mutations are frequently observed in approximately 13%-30% of iCCA cases, establishing them as the most prevalent targetable mutations in BTC^[8,9]. Extensive research has focused on the IDH gene in various cancers, particularly acute myeloid leukemia and other myeloid malignancies. Although the exact mechanisms underlying the oncogenic effects of IDH mutations remain unclear, it is believed that these mutations contribute to the excessive accumulation of metabolites associated with cancer development^[20]. Specifically, IDH1 mutations have been shown to enhance the conversion of α-ketoglutarate to 2-hydroxyglutarate (2-HG), leading to the accumulation of 2-HG and potentially impairing cell differentiation, as well as promoting the proliferation of tumor cells.

Ivosidenib, an oral IDH1 inhibitor, is currently recommended by the National Comprehensive Cancer Network (NCCN) as a second-line therapy for advanced, unresectable, and metastatic BTC harboring IDH1 mutations^[21]. The approval of ivosidenib by the Food and Drug Administration (FDA) was based on the compelling results of the phase III CLARIDHY study^[22]. This trial showed a statistically significant improvement in PFS for patients randomized to ivosidenib, with a HR of 0.37 (95%CI: 0.25-0.54; P < 0.000)^[9]. The most common treatment-related adverse events (TRAE) observed was ascites, which were observed in 9% of treated patients compared to 7% in the placebo arm, while the patients' quality of life did not appear to be significantly affected. The most common grade 3 or worse adverse event observed in both the ivosidenib and placebo groups was ascites, occurring in 7% of patients in each group. Furthermore, serious adverse events were reported in 30% of patients who received ivosidenib and in 22% of patients in the placebo group. A summary of targeted therapies for CCA is provided in [Table 2].

Several ongoing early-phase trials are assessing other IDH inhibitors in IDH mutant tumors including BTC such as LY3410738 (NCT04521686)^[23], HMPL-306 (NCT04762602)^[24], and IDH305 (NCT02381886)^[25]. Additionally, there is an ongoing study (NCT04088188) evaluating the addition of ivosidenib with gemcitabine and cisplatin as a treatment for advanced BTC patients^[26]. Although it is a phase I study, it holds the potential to provide valuable insights into the efficacy of combining targeted therapy and chemotherapy in the management of advanced BTC. A summary of ongoing targeted therapy trials with data is provided in [Table 3].

The promising data on IDH inhibitors in BTC hold potential, but challenges, especially regarding acquired resistance, must be addressed. Studies are investigating the mechanisms of secondary resistance to IDH inhibitors. However, the molecular underpinnings of this resistance are not yet fully elucidated. Several studies have found mutant IDH isoform switching as a potential mechanism of acquired resistance to IDH-targeted therapy^[31,32]. Nonetheless, additional research is necessary to gain a comprehensive grasp of these molecular processes.

Fibroblast growth factor receptor inhibitors

FGFR is a cell surface receptor that transmits signals from fibroblast growth factors, playing an essential role in regulating cell proliferation, tissue maturation, and cellular differentiation^[33]. Continuous activation of FGFRs can lead to the proliferation of cancer cells. Activation of FGFRs can occur via different mechanisms, including gene amplification, which results in the overexpression of receptors, activating mutations, or translocations that create activating gene fusions^[34]. Among all FGFR alterations, FGFR2 fusions exhibit the highest frequency in BTC, being detected in approximately 10%-15% of patients with iCCA, while being comparatively rare in other subtypes^[35,36]. Several pan-FGFR inhibitors and selective FGFR inhibitors have been undergoing investigation. The data obtained from registrational studies provide compelling evidence that FGFR2 fusions, as the most prevalent alterations in BTC, significantly contribute to the therapeutic efficacy observed in this disease^[37,38].

Pemigatinib and futibatinib are two FGFR inhibitors currently available for patients with pretreated BTC who have FGFR2 fusions or rearrangements^[39]. In April 2020, the FDA granted accelerated approval to an oral inhibitor of FGFR1-3, pemigatinib, as a therapy option for previously treated advanced BTC patients who tested positive for FGFR2 fusion or FGFR2 rearrangement, based on the findings of the phase II FLIGHT-202 study^[40,41]. The final analysis of the study, with a median follow-up duration of 45.4 months, showed a disease control rate (DCR) of 82.4% (95%CI: 73.9%-89.1%) and a median OS of 17.5 months (95%CI: 14.4-22.9 months)^[42]. Based on the promising data mentioned earlier, a phase III randomized study, FIGHT-302 (NCT03656536), is currently underway to evaluate pemigatinib as a frontline treatment for advanced BTC patients, comparing it with the combination of gemcitabine and cisplatin^[43].

Futibatinib, an oral agent that inhibits FGFR 1-4, has received accelerated approval from the FDA in September 2022^[44]. This decision was based on the demonstrated efficacy in patients with previously treated advanced or metastatic iCCA with FGFR fusions or rearrangements, as shown in the data from the phase II FOENIX-CCA2 trial^[45]. An updated analysis data, with a median follow-up of 25 months, revealed an objective response rate (ORR) of 41.7% with a DCR of 82.5%^[46]. The median duration of response (DOR) was 9.5 months, while the median PFS was 8.9 months. Furthermore, the mature median OS was recorded as 20.0 months. Similarly to pemigatinib, a phase III clinical trial (FOENIX-CCA3, NCT04093362) is currently underway. This trial is evaluating futibatinib as a frontline option for locally advanced, metastatic, or recurrent unresectable iCCA with FGFR2 rearrangements. The trial aims to compare the efficacy of futibatinib with the doublet of gemcitabine and cisplatin.

Both studies evaluating pemigatinib and futibatinib showed a similar side effect profile. Hyperphosphatemia, alopecia, and diarrhea were the most frequent TRAEs noted with both drugs. In the case of pemigatinib, these side effects were mostly in grade 1 or 2. Among grade 3 or more severe adverse events, hypophosphatemia was the most frequently reported, occurring in 14.3% of cases^[42]. Similarly, for futibatinib, the most common grade 3 TRAE was hyperphosphatemia, seen in 30% of participants^[45]. Additionally, other notable grade 3 adverse events included increased aspartate aminotransferase levels (7%), stomatitis (6%), and fatigue (6%).

Several ongoing trials hold the potential to provide promising data in the near future. One of these is the phase II FIDES-01 study, investigating derazantinib, a pan-selective FGFR inhibitor, in patients with pretreated iCCA harboring FGFR2 fusion (FGFR2^F) or FGFR mutation/amplification (FGFR2^M/A). Data from the 2022 ESMO Congress showed a higher ORR of 21.4% (95%CI: 13.9%-30.5%) in FGFR2^F compared to 6.5% in FGFR2^M/A cohort (95%CI: 0.8%-21.4%)^[29]. The median OS was documented as 17.2 months (95%CI: 12.5%-22.4%) and the median PFS reached 8.0 months (95% CI: 5.5%-8.3%) in FGFR2^F patients.

In the ongoing ReFocus trial (NCT04526106), RLY-4008, a highly selective FGFR2 inhibitor, is being evaluated in advanced solid tumors, including FGFR inhibitor-naïve BTC. Preliminary data from the 2022 ESMO Congress showed a promising ORR of 88.2% (95%CI: 63.6%-98.5%) in 17 patients who received the recommended phase 2 doses^[30]. Notably, one patient achieved a near-complete response and underwent curative tumor resection. The encouraging findings underscore the significance of further monitoring the ongoing development of RLY-4008 as a potential treatment option for advanced stages and potentially in the neoadjuvant setting.

The encouraging data on FGFR inhibitors in BTC holds promise, but there are significant challenges to overcome, particularly concerning acquired resistance to these inhibitors. Numerous preclinical investigations have been conducted to identify the underlying mechanisms of resistance, including bypass signaling, epithelial-mesenchymal transition (EMT), and the development of secondary FGFR mutations, commonly referred to as gatekeeper mutations^[47]. However, it is vital to acknowledge that the majority of research in this area has primarily centered on urothelial, lung, and gastric cancer cell lines, potentially not encompassing the complete range of resistance mechanisms observed in BTC. Moreover, in vitro cell models may not entirely capture the heterogeneity and complexity of human disease, adding another layer of consideration in interpreting the findings and applying them to BTC treatment strategies.

BRAF inhibitors

The mitogen-activated protein kinase (MAPK) signaling pathway, which includes the RAS/RAF/MEK/ERK pathway, plays an essential role in cellular proliferation and survival^[48]. BRAF, an oncogene, activates the RAS/RAF/MEK pathway. BRAF mutations, particularly the V600E mutation, are commonly observed in various solid tumors, including colorectal malignancy, non-small cell lung cancer (NSCLC), and melanoma^[49,50]. The BRAF V600E mutation activates BRAF, resulting in tumor growth and metastasis^[51]. BRAF activating mutations are rare in BTC, comprising approximately 5.5% of cases, with 1.5% of BRAF V600E mutation in iCCA and 0.5% of BRAF V600E in GBC^[12].

Combining the RAF inhibitor, dabrafenib, with the MEK inhibitor, trametinib, has shown encouraging results in the treatment of BRAF-mutated BTC. The phase II ROAR trial investigated the effectiveness of combining dabrafenib and trametinib in patients with advanced solid tumors harboring the BRAFV600E mutation who had experienced the progression of disease after prior treatment^[52]. The trial showed a significant ORR of 51%, with a mean OS of 14 months and a mean PFS of 9 months. The dabrafenib and trametinib combination regimen demonstrated manageable side effects. In light of these results, the FDA approved dabrafenib plus trametinib as a subsequent line of therapy for BTC with BRAFV600E mutations^[53].

Ongoing phase 1 trials are exploring the response to selective BRAF inhibitors, such as ABM-1310 (NCT05501912, NCT04190628) and BGB-3245 (NCT04249843), in patients with BRAFV600-mutated solid tumors, including BTCs. Additionally, more studies are needed to evaluate the effectiveness of targeted therapies in patients with concurrent TP53 and BRAFV600E mutations, as initial findings suggest a more aggressive disease and reduced clinical benefits^[54].

Similar to other targeted therapies, the challenge of overcoming resistance to BRAF inhibitors persists. As evidenced by PFS data, a significant proportion of patients initially responsive to BRAF inhibitors eventually face resistance, necessitating adjustments in treatment strategies. While several potential mechanisms have been proposed, the majority of investigations have revolved around melanoma and a limited range of lung cancer cohorts. Nevertheless, a comprehensive analysis of these findings offers relevant insights that can be extrapolated to BTC patient populations. Notably, one of the potential mechanisms involves the reactivation of the MAPK pathway^[55-57], often occurring due to alterations affecting the drug target itself, such as BRAF copy number gains and MEK2 mutations^[58-60].

As discussed earlier, combination treatment involving BRAF inhibitors and MEK inhibitors has shown promising results. However, despite this encouraging observation, the development of acquired resistance to combination therapy remains an inevitable challenge. A study conducted within a melanoma cohort has highlighted that the mammalian target of rapamycin (mTOR) activation could potentially contribute to the acquired resistance seen with combined BRAF and MEK inhibition^[61]. The mTOR kinase plays an important role in cellular proliferation, and aberrant mTOR activation has been noted across various cancer types^[62,63]. Given that both the MAPK/ERK and PI3K/AKT signaling pathways converge into the mTOR complex 1, this pathway could hold significance for the effectiveness of targeted therapy in patients with BRAF-mutant melanoma^[61]. This potentially implies that the inclusion of an mTOR inhibitor might effectively restore sensitivity to BRAF or MEK inhibitors, prompting further exploration for potential therapeutic interventions.

Human epidermal growth factor receptor 2 inhibitors

HER2, encoded by the ERBB2 gene, is a receptor tyrosine kinase involved in crucial oncogenesis signaling pathways. Its dysregulation is well-documented in cancer development^[64]. Besides its well-known significance in breast cancer, emerging evidence suggests its involvement in BTC^[65]. HER2 expression shows ethnic variations, with a higher prevalence in Asian patients (28.4%) compared to Western patients (19.7%)^[66]. Additionally, HER2 positivity rates in BTC vary based on tumor location, with reported alteration rates of 2.2% in iCCA, 7.5% in eCCA, and 12.6% in GBC^[13]. Detection of HER2 amplification in BTC can be achieved through various testing modalities, including immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and NGS techniques, with NGS providing the advantage of simultaneously evaluating multiple molecular alterations, including HER2 activating mutations, particularly when diagnostic tissue is limited. However, IHC and FISH remain the more commonly employed methods^[67].

Pertuzumab and trastuzumab, two monoclonal antibodies targeting HER2, have shown promise in treating HER2-positive malignancies^[68]. In a phase II MyPathway study (NCT02091141) involving previously treated HER2-amplified and overexpressed metastatic BTC patients, the pertuzumab and trastuzumab combination demonstrated an ORR of 23% (95%CI: 11%-39%), a median PFS of 4.0 months (95%CI: 1.8-5.7 months), and a median DOR of 10.8 months (95%CI: 0.7-25.4 months)^[69]. Adverse events of grade 3 or higher were documented in 46% of patients, with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increases being the most common. Although the dual anti-HER2 therapy for HER2-positive BTC has not received FDA approval, it is included as an option in the NCCN guidelines for patients who have undergone prior treatment^[21]. To further confirm and establish the efficacy of the HER2-targeted treatment in subsequent lines of therapy for BTC, future randomized controlled trials are essential.

Trastuzumab deruxtecan (T-DXd) has demonstrated encouraging anti-cancer efficacy in advanced solid tumors that are HER2-positive, including BTC^[70]. A phase I study involving advanced HER2 mutant solid tumors, excluding breast and gastric cancers, showed an ORR of 28.3%, and the median PFS reached 7.2 months (95%CI: 4.8-11.1 months). The HERB phase II study, which focused on BTCs, revealed an ORR of 36.4% (95%CI: 19.6%-56.1%) in patients with HER2-positive BTCs, along with a DCR of 81.8% (95%CI: 59.7%-94.8%). The study also revealed a median OS of 7.1 months (95%CI: 4.7-14.6 months) and a median PFS of 4.4 months (95%CI: 2.8-8.3 months)^[31]. However, the safety analysis revealed that 81.3% of patients had grade 3 or higher TRAEs, with cytopenia being common. Of particular concern, approximately 25% of patients developed interstitial lung disease (ILD), along with significant gastrointestinal and myelosuppression toxicities. This highlights the need for further investigation and follow-up.

Zanidatamab, a HER2-targeted bispecific antibody, underwent evaluation in the phase IIb HERIZON-BTC-01 study (NCT04466891) in patients with HER2-amplified, locally advanced, unresectable, or metastatic BTC. Among the 87 patients, 41.3% of participants had an objective response, with a DCR of 68.8% (95%CI: 57.4%-78.7%). The median PFS reached 5.5 months (95%CI: 3.7-7.2 months), and data for median OS were not yet mature at the time of data cutoff. However, the OS at 9 months reached 69.9% (95%CI: 57.8%-79.1%)^[32].

Despite remarkable advances in HER2-targeted therapy, resistance continues to pose challenges in HER2-positive BTCs. While resistance studies are primarily focused on breast and gastrointestinal cancers, they provide valuable insights into potential resistance mechanisms in BTC. Notably, the intrapatient and intertumor heterogeneity of HER2 expression is considered a critical factor contributing to primary resistance^[71,72]. Furthermore, HER2 positivity in BTC often coincides with other oncogenic drivers such as FGFR, MET, and KRAS alterations, known to impart resistance to anti-HER2 therapy in in vitro studies^[73].

Encouragingly, a small case series demonstrated the successful reversal of trastuzumab resistance by combining HER2 blockade with inhibition of the secondary driver mutation^[74]. Additionally, in studies involving gastric cancer patients, loss of HER2 has been linked to potential acquired resistance to anti-HER2 therapy^[75,76]. Notably, the SUMMIT study evaluated an irreversible pan-HER tyrosine kinase inhibitor, neratinib, in HER2-mutant advanced BTC, showing antitumor activity but not meeting the primary endpoint; in one GBC patient, loss of HER2 amplification and reduction in VAF of the original HER2 mutation were demonstrated through biopsy and NGS^[77]. Combinatorial approaches, continuous monitoring of HER2 alterations, and adaptive treatment regimens may offer potential solutions to improve clinical outcomes and enhance the efficacy of HER2-targeted therapy in the management of BTCs.

Tropomyosin receptor kinase inhibitors

The neurotrophic tropomyosin kinase receptors (NTRK or TRK) family is a group of transmembrane tyrosine kinases that hold significance in the development of neurons. These receptors are encoded by the genes NTRK1, NTRK2, and NTRK3. In 1986, the first identification of somatic fusions involving the NTRK genes was reported in a colorectal cancer patient. Subsequently, it was uncovered that mutations in NTRK genes, most commonly NTRK fusions, are associated with oncogenesis in various tumor types. The NTRK mutations are more common in patients with lung cancers and soft tissue tumors^[78]. The estimated prevalence of NTRK fusions in BTC is 0.75%^[15].

To date, the FDA has approved two first-generation NTRK inhibitors as first-line and subsequent line of therapy in patients with NTRK fusion-positive BTC: larotrectinib^[79] and entrectinib^[80]. In November 2018, the FDA granted accelerated approval to Larotrectinib based on results from three multicenter clinical trials: LOXO-TRK-14001, SCOUT, and NAVIGATE. A pooled analysis of the three studies showed an ORR of 69% (95%CI: 63-75), with more than one-fourth of the cases (26%) achieving a complete response (CR)^[81]. The response has shown considerable durability, with a median DOR of 32.9 months (95%CI: 27.3-41.7). The median PFS reached 29.4 months (95%CI: 19.3-34.3). The median was not reached at a median follow-up of 32.2 months. The TRAEs were primarily in grade 1 or 2, and 20% of patients had grade 3 or 4 adverse events, leading to treatment discontinuation in five cases.

Entrectinib was approved by the FDA in the subsequent year for adults and pediatric patients who have solid tumors with NTRK gene fusion based on favoring data from three multicenter, single-arm clinical trials: ALKA, STARTRK-1 (NCT02097810) and STARTRK-2 (NCT02568267)^[82]. An updated pooled analysis data of the three phase 1-2 trials from the 2022 ASCO annual meeting has demonstrated meaningful clinical response and good tolerability^[83]. The ORR was 61.3% (95%CI: 53.1%-69.2%), with more than one-fourth of those who had responses achieving a CR. The median DOR reached 20.0 months (95%CI: 13.2-31.1 months) and the PFS reached 13.8 months (95%CI: 10.1-20.0 months). At a median survival follow-up of 30.6 months, the median OS reached 37.1 months (95%CI: 27.2-NE). Moreover, the TRAEs were mainly grade 1-2: dysgeusia (36.6%), diarrhea (29.8%), and weight gain (28.5%). A total of 7.2% of patients needed to discontinue the treatment due to adverse events.

While a significant portion of patients with NTRK fusion-positive cancers achieve enduring disease control, the emergence of resistance to TRK inhibition remains a substantial challenge^[84]. This emphasizes the critical need to develop second-generation TRK inhibitors and/or explore strategies to counter this resistance mechanism. A recent study examining post-progression tumor tissue through NGS from patients treated with first-generation TRK inhibitors found that the majority displayed on-target resistance (83%) as opposed to off-target resistance (11%) or an unidentifiable mechanism (6%)^[85]. Among patients exhibiting on-target resistance, mutations primarily affected the solvent front (87%) and, to a lesser extent, the gatekeeper region (13%). The sequential use of next-generation therapy appears to impact the pattern of mutation occurrence and development. At present, several next-generation TRK inhibitors, such as selitrectinib, repotrectinib, and taletrectinib, are undergoing evaluation in clinical trials due to their promising preclinical activity^[86]. These innovative agents hold considerable potential in addressing the resistance challenge that may arise with the use of first-generation TRK inhibitors.

RET inhibitors

For over three decades following the identification of the RET gene that encodes the receptor tyrosine kinase, mutations in the RET gene have been identified as actionable drivers of oncogenesis^[87]. Specific RET fusion proteins can initiate tumorigenesis and progression of cancer by stimulating subsequent signaling cascades, ultimately resulting in unregulated cell growth. While RET gene fusions are relatively rare in BTC, they are identified in around 1% to 2% of other types of cancer, such as NSCLC and thyroid cancers, indicating the promise of targeting RET kinase in therapeutic interventions^[88]. Activating aberrations in RET, including mutations, fusions/rearrangements, or amplifications, are found in about 1.8% of all tumors, with a favorable response observed in many cases to RET-directed therapy^[89]. Despite the overall rarity of RET aberrations, their significance in driving oncogenesis highlights the need for further research and understanding in order to explore potential therapeutic interventions.

Pralsetinib, a selective RET receptor tyrosine kinase inhibitor, has shown encouraging results in the phase 1/2 ARROW trial, where it was evaluated in patients with solid tumors carrying RET fusions, including BTC^[90]. The study showed an ORR of 57% (95%CI: 35%-77%), with a DOR of 11.7 months (95%CI: 5.5-19.0 months). The median PFS reached 7.4 months (95%CI: 5.1-13.6 months) and the median OS reached 13.6 months (95%CI: 7.5-NE) with a median follow-up of 23.5 months. Among the three BTC patients, two participants had objective responses. In the safety analysis of 29 eligible patients, 20 patients (69%) experienced grade ≥ 3 TRAEs, with the most frequent any-grade TRAEs being abnormal liver function tests. Based on these findings, pralsetinib was approved by the FDA for advanced or metastatic RET-fusion-positive lung and thyroid cancers^[91,92]. While pralsetinib has not received FDA approval as a tumor-agnostic drug, it is listed as a recommended option in the NCCN guidelines for both first-line and subsequent-line treatment of RET-positive BTC^[21].

Selpercatinib, a highly selective RET kinase inhibitor, was approved by the FDA for the treatment of any RET-mutated solid tumors following promising data from the LIBRETTO-001 trial, a phase 1/2 basket trial assessing its efficacy in solid tumors with RET fusion, regardless of their primary location^[93,94]. Among 41 efficacy-evaluable patients, the ORR reached 43.9% (95%CI: 28.5-60.36), with a DOR of 24.5 months (95%CI: 9.2-NE), including two BTC patients, one of whom had a response. The PFS was 13.2 months (95%CI: 7.4-26.6) and the OS reached 18.0 months (95%CI: 10.7-NE). 38% of patients had grade ≥ 3 TRAEs, with hypertension and abnormal liver function tests being frequently observed as grade 3 TRAEs. Selpercatinib is currently recommended in the NCCN guidelines for both progressive and first-line treatment of RET-positive BTC^[21]. Furthermore, its CNS activity highlights its potential in treating locally advanced or metastatic solid tumors harboring RET fusions.

RET fusions exhibit diverse patterns, and the impact of fusion partners and breakpoints on drug response is not fully understood^[95]. Despite an initial positive response, acquired resistance to RET inhibition treatment inevitably emerges^[96]. While data on acquired resistance to RET inhibitors is growing, a comprehensive understanding is not yet matured. Recent data in lung and thyroid cancer patients suggest that primary and acquired resistance mechanisms converge on MAPK pathway activation^[97]. Therefore, employing a sequential approach to RET-targeted therapy could require a combination of treatments involving inhibitors that target distinct MAPK pathways. Furthermore, it appears necessary to reevaluate the characterization of tumors treated with RET inhibitors when they show progression under RET inhibitor therapy.

High tumor mutational burden

Programmed cell death ligand-1(PD-L1) is an immune checkpoint ligand that allows cancer cells to evade immune recognition^[98]. Immunotherapy targeting PD-L1 or its receptor, programmed cell death 1 (PD-1), has shown promising results in various cancers. Hypermutated tumors with a high tumor mutational burden (TMB) are particularly responsive to immune checkpoint inhibitors^[99,100]. TMB is the number of somatic gene mutations present in a tumor and it serves as a biomarker for predicting the response to immunotherapy^[101]. TMB-H, with a threshold of ≥ 10 mutations per megabase, indicates a high probability of neoantigen formation and has been associated with improved outcomes in several cancer types. Approximately 4% of all subtypes of BTCs are estimated to have TMB-H^[19].

Pembrolizumab, an immune checkpoint inhibitor targeting PD-1 receptors, was approved by the FDA for the treatment of TMB-H tumors^[102]. The KEYNOTE-158 trial assessed pembrolizumab in TMB-H tumors across various sites^[103]. The study revealed a 29% ORR (95%CI: 21%-39%). Additionally, 57% of participants achieved a DOR lasting 12 months or longer, and 50% attained a DOR of 24 months or longer. Although there were 63 BTC patients in the KEYNOTE-158 trial, none of them had high TMB. However, two out of the 63 patients had objective responses.

Recent data from the phase II CheckMate-848 study (NCT03668119) demonstrated promising results using nivolumab and ipilimumab in patients with previously treated advanced or metastatic TMB-H solid tumors^[104]. The ORR was 35.5% (95%CI: 24.1%-47.8%) in the group with tissue TMB-H and 22.5% (95%CI: 13.9%-33.2%) in the group with blood TMB-H. Based on the above findings, the current NCCN guidelines list nivolumab and ipilimumab combination therapy as frontline and subsequent-line treatment for TMB-H BTCs, and pembrolizumab is listed as a subsequent-line treatment for TMB-H BTCs^[21].

High microsatellite instability and mismatch repair deficient

Tumors with mismatch repair deficiency (dMMR) or high microsatellite instability (MSI-H) levels show increased neoantigen production and CD8+ T cell infiltration^[105]. MSI-H/dMMR leads to difficulties in repairing DNA replication errors, causing mutations. The MSI-H/dMMR is identified in about 2.06% of iCCA patients^[17].

Pembrolizumab has received full FDA approval for the treatment of adult and pediatric patients with unresectable or metastatic MSI-H or dMMR solid tumors, who have progressed after prior treatment and have limited alternative options^[106]. The approval is based on data from multiple trials, including KEYNOTE-158 (NCT02628067), KEYNOTE-164 (NCT02460198), and KEYNOTE-051 (NCT02332668), involving 504 patients with over 30 types of tumors. An integrated analysis of these studies revealed that, at a median follow-up of 20.1 months, pembrolizumab achieved an ORR of 33.3% (95%CI: 29.2%-37.6%), comprising a CR rate of 10.3% and a partial response rate of 23.0%. According to the NCCN guidelines, pembrolizumab is currently listed as both a first-line and subsequent-line treatment option for MSI-H/dMMR tumors^[21].

Dostarlimab, another anti-PD-1 monoclonal antibody, was assessed in the phase I GARNET trial. This study included 106 patients who had advanced solid tumors, and two of them had MSI-H/dMMR BTCs^[107]. The trial showed that an ORR at 38.7% (95%CI: 29.4%-48.6%) in patients with dMMR and CR was around 7.5%. A median DOR was not reached with a median follow-up of 12.4 months. About 8.3% of patients experienced at least one grade 3 or higher adverse event, with the most common being an elevated lipase level. Based on this, the FDA approved dostarlimab for recurrent or advanced solid tumors with MSI-H/dMMR, which have progressed after previous lines of therapy and do not qualify for suitable alternative treatment options^[108].

Adoptive immunotherapy

While immune checkpoint inhibitors have made notable progress, their effectiveness in treating BTC remains unsatisfactory. Another promising approach in the field of BTC immunotherapy is Chimeric Antigen Receptor (CAR) T-cell therapy, which is currently being evaluated. CAR T-cell therapy has demonstrated exceptional efficacy in the treatment of hematologic cancers, having received regulatory approval for conditions such as B-cell lymphoma. However, expanding the use of CAR T-cell treatment to solid tumors, such as BTC, presents unique challenges due to these tumors' ability to exclude T cells from the tumor microenvironment and avoid immune system recognition.

Researchers have been actively exploring the potential of introducing highly potent cancer-specific T cells into solid tumors as an innovative strategy. Encouraging findings have been observed, particularly with fourth-generation anti-CD133 CAR T-cells, which have demonstrated significant effectiveness against BTC cells expressing CD133^[109]. Additionally, the combination of CAR T therapies targeting both EGFR and CD133 in a cocktail immunotherapy approach has produced promising outcomes in advanced BTC patients^[110]. Furthermore, a phase I study (NCT01869166) involving EGFR-specific CAR T cells for EGFR-mutated advanced BTC has shown favorable results, including a 5.8% CR rate and stable disease in 58.8% of patients^[111].

Moreover, at the 2023 ASCO meeting, results from a phase I trial using gavocabtagene autoleucel, an autologous genetically engineered anti-mesothelin T cell receptor fusion construct cell therapy, were presented^[112]. This trial focused on patients with refractory mesothelioma and other mesothelin-expressing solid tumors, including BTC. The results were promising, with an ORR of 21% and a DCR of 77% among the 32 patients who participated in the study, including one patient with BTC. Furthermore, the 6-month OS rate reached 70.2%. These findings suggest a potential avenue for effective treatment in patients with mesothelioma patients and other mesothelin-expressing solid tumors, offering hope in the fight against these challenging malignancies. Nevertheless, concerns about the toxicity of CAR T-cell therapy in BTC persist, mainly due to potential toxicity and endothelial damage, emphasizing the imperative for further investigation and the conduct of clinical trials.

In conclusion, while CAR T-cell therapy has significantly advanced the treatment of hematologic malignancies, its application for solid tumors, including BTC, brings forth a landscape filled with both promise and hurdles. Research involving CAR T cells targeting specific antigens such as CD133 and EGFR has demonstrated effectiveness in both preclinical and clinical contexts, instilling hope for patients with conditions like BTC. Nonetheless, persisting safety concerns and the imperative for more extensive research underscore the continuous endeavors to enhance the potential of CAR T-cell therapies for solid tumors, with the ultimate goal of improving their efficacy while mitigating potential adverse effects.

Sequencing of cancer genomics

The evolution of targeted therapy is closely intertwined with the advancements in DNA sequencing technology used in cancer cells. Cancer, being a genomic disease, is often influenced by both somatic and germline mutations, highlighting the crucial need to comprehend cancer's genomic sequencing in order to uncover its origins and develop personalized treatment approaches. Recent progress and cost reductions in NGS have greatly accelerated the transformation of cancer genomic data into practical clinical applications. It allows simultaneous analysis of hundreds or thousands of genes, empowering the implementation of molecularly targeted therapy based on the insights gained from the results.

NGS technology has enabled the identification of genomic profiles for individual cancer cells, paving the way for personalized treatment approaches. However, the diversity of tumor genomic sub-categories identified by NGS makes it exceedingly difficult to conduct randomized controlled trials with an adequate number of patients for each specific sub-category^[113]. Furthermore, the intratumoral environment consists of diverse and heterogeneous cells, resulting in variations in genomic profiles and mutation data among these cells. As a consequence, there are concerns about the feasibility of relying solely on sequencing results to determine the most suitable targeted therapy^[114].

Resistance and potential overcome strategies

The iCCA develops resistance to treatments, which is closely associated with cancer-related mortality, although the precise mechanisms underlying this resistance are not fully understood. During cancer treatment, adaptive changes lead to acquired resistance, involving mechanisms such as genetic alterations, changes in the tumor's microenvironment, metabolic reprogramming, and loss of the treatment target^[115,116].

One of the investigational methods to elucidate the development of acquired resistance in cancer, as observed in targeted therapy, involves analyzing the molecular differences between tumor samples before and after acquiring resistance^[117]. By comparing the resistant tumor sample with the pre-treatment tumor sample at the molecular level, we can identify whether the resistance is due to changes in previously actionable mutations or the emergence of new mutations. This comprehensive analysis can potentially provide insights into the underlying causes of resistance and guide the modification of treatment strategies accordingly.

Furthermore, to overcome acquired resistance driven by tumor genetic alterations, exploring the next generation of inhibitors and researching strategies for combination treatments appears to be essential. By focusing on these areas of investigation, we can aim to develop more effective approaches to combat acquired resistance and enhance the overall efficacy of cancer therapies.

Determining the sequence of treatment

We have thoroughly reviewed various systemic treatment options for advanced iCCA, along with the foundational research data. While we are aware of the several options available, the next consideration and decision-making process involve the prioritization and sequence of these treatment modalities. As previously mentioned, any systemic treatment may initially demonstrate a favorable response but ultimately develop acquired resistance due to various factors, necessitating a change in treatment.

Given the relatively recent use of targeted therapies and the rarity of actionable mutations among the iCCA patient population, conducting head-to-head comparing trials between targeted therapy and chemotherapy and/or immunotherapy is challenging. Consequently, selecting treatment strategies tailored to individual patient situations is essential. Elements shaping these individualized decisions encompass factors such as the extent of tumor burden, the urgency of treatment initiation takes into account the time required for sequencing the tumor genome, the patient's medical history, drug accessibility, financial aspects, anticipated toxicity, patient performance status, and anticipated treatment response.

In many cases, combining chemotherapy with immunotherapy would be a preferable first-line treatment while awaiting tumor genomic information. However, as the tumor progresses, established guidelines for selecting second-line therapy remain absent due to limited supportive data. Tailored to the unique context of individual patients, second-line treatment might encompass targeted therapy, immunotherapy, or FOLFOX, with reference to the ABC-06 trial findings, alongside the consideration of other cytotoxic chemotherapy treatments. Although direct comparative prospective study data could be lacking, using targeted therapy upfront and reserving FOLFOX could be a reasonable approach if the patient has any indication for targeted therapy. Targeted therapies are generally perceived as more tolerable and can exhibit considerable effectiveness, particularly when administered early, potentially yielding enhanced patient outcomes. The investigation into the sequencing of treatment strategies demands further exploration.

Potential role as neoadjuvant and adjuvant therapy

In patients with iCCA, complete resection is regarded as the sole definitive curative option, although its feasibility is limited for many due to the advanced disease at diagnosis. While not conclusively established, based on current data, hepatic resection with negative margins is considered the preferred surgical goal^[118]. The available research data on neoadjuvant and adjuvant chemotherapy in resectable iCCA presents challenges in drawing definitive conclusions. A study result by Buettner et al. showed similar OS and PFS between patients with perioperative chemotherapy and those without, although this needs to be cautiously interpreted due to low neoadjuvant treatment patient numbers and possible selection bias effect^[119]. Conversely, more recent research indicates that neoadjuvant therapy, particularly in stage II-III disease, may lead to enhanced OS^[120-122]. Moreover, the optimal adjuvant treatment strategy for resected iCCA remains uncertain due to limited clinical trial data supporting a standard regimen.

The efficacy of targeted therapy as neoadjuvant or adjuvant treatment for resectable disease remains an unexplored area of research. The challenges of limited early-stage detections and infrequent actionable mutations hinder the conduct of studies in this domain. Moreover, the effects of targeted therapy on tumor downstaging, survival benefits, recurrence-free survival, and potential adverse events leading to unfavorable outcomes require further elucidation.

In an attempt to address this, a clinical trial (NCT05514912) is underway to evaluate chemo-targeted therapy for resectable intrahepatic cholangiocarcinoma using infigratinib in combination with chemotherapy in FGFR2 fusion-positive iCCA^[123]. The study aims to provide valuable insights into the treatment efficacy for this specific patient subset. To our knowledge, there are currently no ongoing clinical trials investigating the efficacy of targeted therapy or combination treatments with targeted therapy in an adjuvant setting. Additional research is needed to address these uncertainties and shed light on potential treatment strategies.