胆管癌靶向治疗进展

We present the first reported case of effective targeted treatment for extrahepatic cholangiocarcinoma with an Epidermal Growth Factor Receptor (EGFR) exon 19 deletion. A 36-year-old Taiwanese man initially presented with progressive jaundice 21 months ago. A computed tomography (CT) scan revealed a dilated biliary tree and gallbladder, along with a narrowed distal common bile duct (Figure S1). Initially, carcinoembryonic antigen (CEA) was at 1.14 ng/mL (normal range: ≤ 5 ng/mL) and carbohydrate antigen 19 – 9 (CA19-9) was at 19.4 U/mL (normal range: ≤ 27 U/ mL). He underwent a curative Whipple’s operation. Postoperative pathology revealed the distal common bile duct with infiltrative growth of dysplastic glands in desmoplastic stroma, consistent with moderately differentiated adenocarcinoma, pancreatobiliary subtype (Figure 1A,B). The tumour invaded into the adjacent pancreatic parenchyma, measuring 1.3 cm in size, with negative margins. All 16 lymph nodes were free of malignancy, resulting in an initial pathological stage of T2N0. However, after 20 months of active surveillance, a CT scan revealed infiltrative soft tissue surrounding the superior mesenteric artery and multiple lung tumours (Figure 2A). No definite colorectal lesion was found in the initial and subsequent CT scans. Laboratory tests also indicated an elevated CEA level to 6.09 ng/mL (Figure S2). A thoracoscopic wedge resection of the lingula was performed, and the pathologic examination demonstrated the nodule was composed of invasive

Abstract Purpose: FGFR2 fusions occur in 10% to 15% of patients with intrahepatic cholangiocarcinoma (iCCA), potentially benefiting from FGFR inhibitors (FGFRi). We aimed to assess the feasibility of detecting FGFR2 fusions in plasma and explore plasma biomarkers for managing FGFRi treatment. Experimental Design: We conducted a retrospective study in 18 patients with iCCA and known FGFR2 fusions previously identified in tissue samples from prior FGFRi treatment. Both tissue and synchronous plasma samples were analyzed using a custom hybrid capture gene panel with next-generation sequencing (VHIO-iCCA panel) and validated against commercial vendor results. Longitudinal plasma analysis during FGFRi was performed. Subsequently, we explored the correlation between plasma biomarkers, liver enzymes, tumor volume, and clinical outcomes. Results: Sixteen patients (88.9%) were positive for FGFR2 fusion events in plasma. Remarkably, the analysis of plasma suggests that lower levels of ctDNA are linked to clinical benefits from targeted therapy and result in improved progression-free survival and overall survival. Higher concentrations of cell-free DNA before FGFRi treatment were linked to worse overall survival, correlating with impaired liver function and indicating compromised cell-free DNA removal by the liver. Additionally, increased ctDNA or the emergence of resistance mutations allowed earlier detection of disease progression compared with standard radiologic imaging methods. Conclusions: VHIO-iCCA demonstrated accurate detection of FGFR2 fusions in plasma. The integration of information from various plasma biomarkers holds the potential to predict clinical outcomes and identify treatment failure prior to radiologic progression, offering valuable guidance for the clinical management of patients with iCCA.

Background: Cholangiocarcinoma (CCA) is a highly malignant tumor derived from the bile duct epithelium with increasing incidence worldwide. It divides into three subtypes: intrahepatic (iCCA), perihilar (pCCA), and extrahepatic (eCCA). Imaging techniques such as abdominal ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) can assist in diagnosing CCA. Gemcitabine combined with cisplatin is the first-line treatment for CCA. However, CCA often carries a poor prognosis due to advanced stage at time of diagnosis. Isocitrate dehydrogenase (IDH)1 mutations are the most common targetable alteration in CCA, occurring in 13% of iCCA cases. Recently, the FDA approved ivosidenib for the treatment of IDH1-mutated CCA. Our study aims to highlight the effect of IDH-1 alteration and its response to ivosedinib as compared to the presence of other genomic alterations. Methods: We used cBioPortal for Cancer Genomics server to retrospectively analyze and gather genomic data related to CCA from 2018 to 2024. Our search included five studies, from which we identified 699 cases of iCCA. The prevalence of genetic mutations such as IDH1, IDH2, FGFR2, ARID1A, BRAF, KRAS, and TP53 was obtained. Survival outcomes were extracted with a 95% confidence interval (CI) in patients who received ivosedinib therapy. We ensured to include overlapping groups to detect survival outcome superiority or inferiority based on the co-existence of genomic alterations. The Kaplan-Meier curves were conducted for an illustrated presentation of median overall survival (mOS) outcomes. Results: Among the studied cohort, IDH1 mutations were the most frequent, present in 16 cases. Combinations of mutations were also observed, with ARID1A (n=1), BAP1 (n=4), BRAF (n=1), FGFR2 (n=2), KRAS (n=2), TP53 (n=2), and PIK3CA (n=1) alongside IDH1. Survival outcomes varied based on mutation combinations. TP53 and IDH1 co-mutations (n=2) were associated with a mOS of 14.26 months. Co-mutations of ARID1A and IDH1 (n=4) were associated with a mOS of 21.85 months (95% CI: 12.55 - NA). Patients with IDH1 mutations alone (n=16) had a mOS of 46.75 months (95% CI: 20.87 - NA). While BAP1 and IDH1 co-mutations (n=4) demonstrated a mOS of 63.60 months (95% CI: 11.34 - NA). Conclusion: Our study showed that patients with IDH1 mutations, whether isolated or associated with other mutations such as BAP1, in patients who received ivosidenib as part of the treatment regimen, had superior mOS compared with other mutation types. More studies are needed to further elaborate on the effect of genomic alterations, both individually and in combination, on tumor response to therapy. Citation Format: Bayan Khasawneh, Waseem Abdelrahim, Abdullah Esmail, Ebtesam Al-Najjar, Ala Abudayyeh. Advancing early detection and targeted therapy: A comprehensive review of cholangiocarcinoma biomarkers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A042.

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Background: To improve the prognosis of advanced intrahepatic cholangiocarcinoma (iCCA), the authors retrospectively compared the effect and safety of combined hepatic arterial infusion chemotherapy (HAIC), targeted therapy, and immunotherapy with systemic chemotherapy (SC) in unresectable iCCA patients. Methods: The authors retrospectively enrolled 202 advanced iCCA patients treated with SC or targeted therapy, immunotherapy, and FOLFOX-HAIC combined between March 2015 and June 2023 at our institution. Two hundred two patients were divided into two groups based on the therapeutic regimens. Baseline characteristics and prognosis were reviewed and analyzed. Results: After 1-to-1 propensity score matching, 76 patients were included in each group. The triple combination therapy group demonstrated longer median overall survival (OS, 20.77 vs. 14.83 months, P=0.047), progression-free survival (PFS, 9.07 vs. 6.23 months, P<0.001), intrahepatic PFS (11.03 vs. 6.73 months, P<0.001), extrahepatic PFS (11.37 vs. 7.13 months, P=0.0064), and a higher objective response rate (35.5% vs. 14.5%, P=0.003) than the SC group. Fever, thrombocytopenia, elevated ALT, elevated AST, hypoalbuminemia, and hyperbilirubinemia were more common adverse events (AEs) in the triple combination therapy group, while fatigue and anemia were more prevalent in the SC group (P<0.05). For grades 3-4 AEs, the rates of elevated ALT were higher in the triple combination group (P=0.028). Conclusions: Compared with SC, triple combination therapy comprising HAIC, targeted therapy and immunotherapy appears to be an effective and safe treatment for advanced iCCA.

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Choledochal cysts (CCs) are rare cystic dilations of the intrahepatic and/or extrahepatic bile ducts. Malignancies arising during follow-up after excision of CCs have been reported in both children and adults, with no typical time frame for malignancy development. We present a case of a patient diagnosed with CCs 36 years ago, who underwent resection and subsequently developed cancer. The patient received chemotherapy, targeted therapy, and immunotherapy, with efficacy evaluation indicating a state of stable disease. Considering tumor resistance after continuous systemic therapy and an unresectable tumor, proton therapy was selected for the next treatment. To prevent gastrointestinal side effects after proton therapy, the bile-enteric anastomosis was dismantled, and a greater omentum strip was used to fill the subhepatic space, creating a barrier between the biliary duct and the intestine. The patient successfully underwent proton therapy without any gastrointestinal complications. As CC-associated malignancy poses a lifelong risk even with complete resection, surveillance should be maintained throughout the follow-up period. Comprehensive treatment should be adopted to improve prognosis in malignancy after CC resection.

Key Points Question What is the prevalence of BRAF variant subtypes and their association with disease characteristics, prognosis, and targeted therapy response in patients with intrahepatic cholangiocarcinoma? Findings In this cohort study including 1175 patients, a total of 20 different subtypes of BRAF somatic variants affecting 49 patients were identified, including V600E (27%), K601E (14%), D594G (12%), and N581S (6%). Patients with BRAF V600E variants were more likely to have larger tumor size, multiple tumors and more vascular/bile duct invasion. Meaning The findings of this cohort study suggest that there are broad differences among organoids with different BRAF variant subtypes in sensitivity to BRAF or MEK inhibitors.

Cholangiocarcinoma (CCA) is one of the cancers with the worst prognosis. Its incidence and mortality have increased in recent years, but treatment options are limited. Although various clinical trials have been conducted, there is no effective therapeutic agent yet. As molecular genetic profiling is progressing in CCA, the interest in targeted therapies is growing. To realize targeted therapy in practice, various clinical requirements must be met. A molecular biological test should be performed on a patient’s tissue or blood, a target should be determined using an appropriate analysis method, and there should be a targeted agent. Regarding CCA, genetic variation occupies an intermediate level. Accordingly, many new agents have been recently developed for molecular biological targets. The most common genetic aberrations show almost similar patterns in intrahepatic and extrahepatic CCA. However, HER2 gene amplification is more often observed in extrahepatic CCA, and FGFR2 fusion, IDH1 mutation, and RAS-RAF-MEK-ERK signaling system mutations are more frequently found in intrahepatic CCA. Phase 2 or 3 clinical trials are being conducted with zanidatamab for HER2 amplification, pemigatinib/infigratinib for FGFR fusion, ivosidenib for IDH1 mutation, and dabrafenib for BRAFV600E mutation. In addition, clinical trials for agents targeting the NTRK, BRCA, and ARID1A gene mutations are ongoing. These targeted therapies are expected to have a bright future as a new treatment for CCA.

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555 Background: BTCs are genomically diverse. TT have established antitumor activity in a subset of pts that harbour actionable alterations; however, most patients (pts) do not respond to or experience acquired resistance on TT. We sought to explore genomic mechanisms of response across the spectrum of BTCs. Methods: We leveraged a prospectively maintained cohort of pts with histologically confirmed BTC who underwent molecular profiling using an FDA authorized, targeted, NGS assay. Additional clinicopathologic information was retrospectively extracted with an aim to describe the genomic profile and clinical actionability using OncoKB, to explore outcomes for pts treated with TT, and to nominate innate and acquired genomic mechanisms of resistance. Results: N = 1254 pts had molecular profiling: 61.2% intrahepatic (iCCA), 16.8% extrahepatic cholangiocarcinoma (eCCA), 22.1% gallbladder cancer (GBC). MSI high was observed in 2.1% and the median TMB was 3.3 (range: 0-74.6 muts/mb). Among MSS tumors, the most frequently altered genes were TP53 (36%), CDKN2A (21%), ARID1A (18%), KRAS (17%), and IDH1/2 (16%). Genomic profiles differed significantly based on anatomic subsite. Oncogenic drivers were mostly clonal except for ERBB2 amplifications and mutations, which were subclonal in 19%, and 28% of cases respectively. OncoKB level 1/2 events observed in 32.2% (iCCA 40%, eCCA 15%, and GBC 22%). Progression-free survival (PFS) for pts with these oncogenic drivers: IDH , FGFR2 , ERBB2, BRAF V600E , MSI high , and TMB high from start of matched TT (N=164) is listed in the table. Clinical factors or co-occurring genomic alterations did not impact outcomes. Profiling of paired pre- and post-progression samples (N=25) nominated pathway reactivation in FGFR2 , BRAF , NTRK, and ERBB2 driven tumors with emergence of recurrent oncogenic FGFR2 , RAS , MEK , and MET alterations, respectively, as well as off target MYC amplification and CDKN2A loss. Loss of the oncogenic driver at progression was only observed for ERBB2 driven tumors. Conclusions: Acknowledging the limitations of the study design, matched TT in a real-world, prospectively maintained cohort offers meaningful PFS, most notably in those pts with MSI high BTC treated with immunotherapy. In those with acquired resistance to TT, alterations predicted to reactivate the primary oncogenic pathway were identified. ERBB2 heterogeneity and loss is predicted to be a mechanism of resistance to HER2 targeted therapy. Outcomes for BTC treated with matched TT. Target Median PFS, months (95% CI) IDH1/2 (N = 72) 4.2 (2.9-6.8) FGFR2 (N=37) 7.4 (6.5-9.4) ERBB2 (N =22) 8.2 (2.95-16.7) MSI high (N = 14) NR (10.15-NR) TMB high (N=12) 2.69 (1.64-NR) BRAF V600E (N=7) 16.6 (3.7-NR)

544 Background: Biliary tract cancer (BTC) is an aggressive malignancy with poor prognosis and limited treatment options. The prevalence of HER2 amplification in BTC has been reported to be 5-20%, and clinical trials suggested the clinical benefit of HER2-targeted treatment in this disease subpopulation. In a global collaboration, we investigated the comprehensive clinico-molecular characterization as well as the efficacy of anti-HER2 therapy in HER2-amplified BTC. Methods: Patients with advanced BTC who received systemic therapy were included from the GOZILA and MONSTAR studies in Japan and from the COLOMATE trial in the US. Genomic alterations were detected by tissue next-generation sequencing (NGS), including FoundationOne CDx and TempuslxT, and plasma circulating tumor DNA NGS including Guardant360 and FoundationOne Liquid CDx. The clinico-molecular characteristics were evaluated in an exploratory cohort comprised of patients from Japan, whereas the efficacy of anti-HER2 therapy was assessed in a biomarker selected cohort with patients from both Japan and the US. Results: Of 439 patients included in the exploratory cohort, 43 (10%) had HER2 amplification. BTC with primary tumor location in the gallbladder accounted for 58% of primary sites of patients with HER2 amplification. TP53 mutation (84% vs 61%, p=0.003) and BRAF amplification (9% vs 2%, p=0.030) were significantly more frequent in patients with HER2 amplification compared to those without, and no KRAS mutation co-occurred with HER2 amplification. There was no significant difference in overall survival (OS) between patients with and without HER2 amplification (17.7 months [M] vs 16.9 M, hazard ratio [HR] 0.95, 95% confidence interval [CI] 0.65-1.40, p=0.799). Of 58 patients with HER2-amplified BTC included in the biomarker selected cohort (43 Japan, 15 US), 27 (47%) received anti-HER2 therapy. The median OS of patients treated with anti-HER2 therapy was significantly longer than that of patients who did not receive anti-HER2 therapy (24.3 M vs. 12.1 M, HR 0.43, 95% CI 0.23-0.82, p=0.011). Multivariate analysis also identified anti-HER2 therapy as an independent prognostic factor for OS (HR 0.42, 95% CI 0.20-0.85, p=0.015). Conclusions: HER2 amplifications were found in 10% of advanced BTC and did not represent an independent predictive factor for OS. Of clinical significance, patients with HER2-amplified BTC derive a significant benefit from anti-HER2 therapy.

536 Background: The prognosis of advanced BTC remains poor. Although targeted agents have expanded late-line options, many patients (pts) become ineligible for these therapies after progressing on first-line treatment. Herein, we examined the real-world impact of targeted therapy on survival in advanced BTC. Methods: In this international collaborative study between the Mayo Clinic (US) and the National Cancer Center East (Japan), we included pts with advanced BTC with comprehensive tumor molecular profiling who had received at least 30 days of systemic therapy. Pts were categorized into 3 groups: 1) non-actionable, 2) pts with alterations who received matched targeted therapy at any point during their treatment (matched group), and 3) those with mutations who did not receive matched therapy (unmatched group). OS was calculated from the start date of first-line therapy until death and compared among three subgroups using Cox regression models. Results: Of 932 pts, 366 (39.3%) had an actionable alteration, and 135 of these received matched therapy. 566 (61.7%) did not have an actionable alteration (Table). The most common alterations were somatic BRCA1/2 (N=106, 11.4%), HER2 amplification (N=65, 7%), FGFR2 fusion (N=61, 6.5%), KRAS G12C/D (N=46, 4.9%), and IDH1 (N=38, 4.1%). 78.7% of FGFR2 fusions were found in Caucasian pts, whereas BRCA1/2 (97.5%), HER2 (78.5%), and KRAS G12C/D (67.4%) alterations were more prevalent in Asian pts. The median OS was significantly longer in the matched group (21.4 months; 95% CI 18.4-27.9) compared to the unmatched group (14.6 months; 95% CI 12.8-17.6) and the non-actionable group (17.2 months; 95% CI 15.5-19.0). The 36-month OS rate was 33% in the matched group vs. 7% in the unmatched group (p < 0.0001). After adjusting for age, gender, prior surgical resection, primary tumor location, and country of origin, matched targeted therapy remained a strong independent predictor for improved OS (HR 0.61; 95% CI 0.48-0.79). Conclusions: These real-world findings suggest that matched targeted therapies significantly improve survival in advanced BTC, underscoring the need to incorporate them earlier in the treatment course and address barriers to treatment access. The regional prevalence of actionable alterations should also be considered when developing new targeted therapies. Patient baseline characteristics stratified by actionable alterations and targeted therapy. Non-actionable(N=566) Unmatched group(N=231) Matched group(N=135) Site, n (%) Japan 409 (72%) 166 (72%) 50 (37%) US 157 (28%) 65 (28%) 85 (63%) Age at Dx > 65, n (%) 324 (57%) 122 (53%) 54 (40%) Female, n (%) 235 (42%) 92 (40%) 77 (57%) Primary tumor location (category), n (%) Intrahepatic 217 (43%) 115 (55%) 99 (76%) Extrahepatic 150 (30%) 39 (19%) 17 (13%) Gallbladder 142 (28%) 54 (26%) 14 (11%) Prior surgical resection, n (%) 220 (42%) 74 (34%) 48 (37%)

Background: Cholangiocarcinoma (CCA) remains a highly morbid cancer for which accurate models. Patient-derived cancer organoid (PCOs) have been generated in CCA, however their utility in clinical prediction has not been validated. Z’ factoring often fails in assessing CCA organoid response. Prior analysis for organoid response has shown an effect size by Glass’s Delta (GΔ) of ≥1.25 in growth inhibition is predictive of clinical response. Here, we present subclonal organoid response across a diverse set of CCA models in response to therapy by growth and organoid level viability response. Methods: PCOs were expanded from the NCI’s Patient-Derived Model Repository (PDMR), tissue biopsy, and rapid autopsy. Organoid response was tracked from growth using Z-stacked high content imaging (Cytation5) with endpoint at 144h and stained using established viability markers, Caspase-3/7 (C3/7) and ToPro3. Treatment groups included media control, positive control of cycloheximide 200uM continuous, gemcitabine (gem) 10uM 24h, cisplatin (cis) 5uM 48h, combination gem+cis, and FGFR2 inhibitor futibatinib (futi) 300nM continuous. Results: Across models, PCO growth directly correlated with Z’ factor thresholding (R= 0.868). NCI PDMR organoids, CK11523 and CK10519 are IDH1mutant (mt), and both showed superior response to gem when compared to cis by growth (gem GΔ 1.18 and 0.44 v. cis GΔ 0.11 and 0.23) and C3/7 (gem GΔ 3.25 and 2.34 v. cis GΔ 1.46 and 0.33). BRCA1mt CCA was found to achieve modest growth inhibition with gem and gem+cis (GΔ 1.09 and 1.17) and yet significant increase in C3/7 (gem GΔ 1.68 and gem+cis GΔ 1.36) and ToPro3 (gem GΔ 1.65 and gem+cis GΔ 1.82). Despite this result, the patient developed early clinical recurrence after neoadjuvant gem+cis chemotherapy (<3 months). FGFR2-HPGDS fusion CCA achieved no meaningful difference in growth response with gem+cis (GΔ 0.94) or futi (GΔ 0.36). These findings were consistent with the clinical outcome of primary chemotherapy resistance and progression on futi. However, we observed increase in C3/7 positivity with both gem+cis (GΔ 2.11) and futi (GΔ 1.37). Conclusions: Primary resistance to both targeted and chemotherapy remains a formidable barrier to therapeutic development in CCA. While growth inhibition remains a validated marker of clinical response, further work is needed to understand thresholds for C3/7 and ToPro3 across physiologic drug dosing. This is the first report of modeling the novel FGFR2-HPGDS fusion CCA in organoids. Ongoing work is evaluating therapeutic sensitivity across prospective specimens of CCA in support of establishing thresholding for response to apoptosis (C3/7) and necrosis (ToPro3). Citation Format: Eleanor Riedl, Austin Stram, Md Shahadat Hossan, Ethan Samuel Lin, Jamie Warner, Luke Koeppel, Jeremy D. Kratz. Thresholding response assessment in biliary tract cancer organoids to inform sensitivity to chemotherapy across molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 220.

PURPOSE An estimated 25% of patients with biliary tract cancer (BTC) do not undergo genotyping, representing a missed opportunity for therapeutic targeting. METHODS Cell-free DNA (cfDNA) and matched tumor sample from patients with BTC were analyzed using targeted next-generation sequencing (NGS) assay and compared. We sought to define the molecular profile of cancer-derived cfDNA, frequency of OncoKB level 1/2 alterations, plasma:tumor genotype concordance, the prognostic impact of cfDNA, and clonal evolution after targeted therapy progression. RESULTS cfDNA-based genotyping was performed on 297 blood samples from 170 patients with BTC. The most frequently altered genes were TP53 (29%), FGFR2 (16%), ARID1A (13%), CDKN2A (11%), and KRAS (11%); 25% of patients had OncoKB level 1/2 alterations and 36.2% of potentially actionable alterations were detected in plasma alone. The cfDNA:tissue concordance accuracy was high (96% IDH1, 98% BRAF, 92% KRAS mutations, 99% ERBB2 amplifications, and 96% FGFR2 fusions). Detectable tumor-derived cfDNA after resection did not predict recurrence. In treatment-naïve metastatic BTC, high variant allele fraction was associated with worse progression-free survival and overall survival. RAS alterations not detected in samples before treatment were identified at progression in 24% of patients who received BRAF-, FGFR-, or HER2-directed therapy, identifying RAS alterations as a convergent mechanism of targeted therapy resistance. CONCLUSION Molecular profiling of cfDNA from patients with BTC identified OncoKB level 1/2 gene alterations and putative genomic resistance mechanisms to targeted therapy. Concordance analysis suggests that cfDNA-based NGS is complementary to that of tissue-based sequencing in the identification of potentially actionable alterations.

615 Background: Antagonism of Mouse double minute 2 homolog ( MDM2 ) can restore p53 activity and represents a novel therapeutic strategy in biliary tract cancer (BTC). We aimed to characterize the epidemiology of total population versus that harbouring MDM2 amplifications; potential associations of MDM2 to other genetic alterations and survival outcomes. Methods: We evaluated a large real-world cohort of patients (pt) diagnosed with BTC from 1st January 2017 to 31st December 2022 from the Spanish RETUD registry. Next generation sequencing (NGS) test was performed in all patients. Data collected included demographic and clinical characteristics, molecular profile, systemic oncologic treatment and effectiveness (best response and survival outcomes). Progression free survival (PFS) outcomes and best response were calculated for pts who received first line therapy. Descriptive statistic was used to analyse sociodemographic data, tumor characterization, molecular analysis, and best response. PFS and overall survival (OS) were estimated by the Kaplan-Meier method. Results: A total of 301 evaluable pt were included in 24 sites. MDM2 amplifications were reported in 19 pt (6.3%) and 2 pt (10.5%) had TP53 mutations. At primary diagnosis, in the MDM2 amplified population the tumour was mostly intrahepatic (63.2%); followed by gallbladder cancer (21.0%). Half of the pt had advanced disease at diagnosis (52.6%) and 21.0% resectable disease. No significant differences were observed in clinical characteristics (age, sex, tumor location, stage, ECOG performance status and metastasis location) and most common actionable genomic alterations (BRAF, HER2, IDH, FGFR2) in pt with MDM2 amplification compared to overall population. Median overall survival (95% Confidence Interval [CI]) in pt with MDM2 amplification was 18.4 months (12.3-19.9), and 17.8 months (95% CI, 12.3-19.9, p=0.247 in pt without amplification. Median PFS in first-line was 5.3 months (95% CI, 2.7-8.9) in MDM2 amplified group, as compared with 6.0 months in non- MDM2 amplified group (95% CI, 5.3-6.8; p=0.4233. The objective response rate in first-line was 21.4% in the MDM2 amplified group versus 29.6% in non-amplified group. The proportion of pt who received systemic therapy in first, second and third line was similar in both groups. The main first-line regimen in MDM2 amplified pt was cisplatin/gemcitabine (n=9, 47.4%), followed by clinical trial (n=3, 15.8%), GEMOX (n=2, 10.5%) and gemcitabine (n=2, 10.5%). The median OS and PFS of patients treated with cisplatin/gemcitabine in first line was similar in both groups. Conclusions: This analysis provides insights into the characterization of MDM2 amplified BTC pt. Similar incidence of MDM2 amplification was observed compared to other cohorts of BTC. No significant differences were observed in clinical characteristics, molecular profile and survival in MDM2 amplified pt compared to non-amplified pt.

Simple Summary Biliary tract cancer (BTC) carries a poor prognosis. Most patients with BTC will require ‘systemic’ therapy, treating cancer throughout the body to control the disease and extend life while maintaining or improving the quality of life, but not to cure. There is no standard third-line systemic therapy, and few patients remain fit to receive three lines of treatment. This study assessed 97 patients from three academic centres who received three lines of palliative systemic therapy for BTC. Median survival after starting third-line treatment was 6.4 months, and from first-line treatment it was 26.9 months. The region of the biliary tract in which the cancer originated did not significantly affect prognosis. The 10 patients with an identified molecular change ‘driving’ the cancer, who received third-line treatment targeting that change, survived longer (12.5 months, versus 5.9 months for all other included patients). This study provides a benchmark for future clinical trials within BTC. Abstract Phase 3 trials have established standard first-line (1L) and 2L systemic therapy options for patients with advanced biliary cancer (ABC). However, a standard 3L treatment remains undefined. Clinical practice and outcomes for 3L systemic therapy in patients with ABC were therefore evaluated from three academic centres. Included patients were identified using institutional registries; demographics, staging, treatment history, and clinical outcomes were collected. Kaplan–Meier methods were used to assess progression-free survival (PFS) and overall survival (OS). Ninety-seven patients, treated between 2006 and 2022, were included; 61.9% had intrahepatic cholangiocarcinoma. At the time of analysis, there had been 91 deaths. Median PFS from initiating 3L palliative systemic therapy (mPFS3) was 3.1 months (95%CI 2.0–4.1), while mOS3 was 6.4 months (95%CI 5.5–7.3); mOS1 was 26.9 months (95%CI 23.6–30.2). Among patients with a therapy-targeted molecular aberration (10.3%; n = 10; all received in 3L), mOS3 was significantly improved versus all other included patients (12.5 vs. 5.9 months; p = 0.02). No differences in OS1 were demonstrated between anatomical subtypes. Fourth-line systemic therapy was received by 19.6% of patients (n = 19). This international multicentre analysis documents systemic therapy use in this select patient group, and provides a benchmark of outcomes for future trial design.

Although durvalumab or pembrolizumab combined with gemcitabine plus cisplatin (GC) remains the standard first-line therapy, novel targeted agents and immunotherapies integrated with chemotherapy have demonstrated promising efficacy. However, the crosswise comparison between each regimen is rare. Therefore, we comparative the efficacy and safety of targeted therapeutics or immunotherapy agents combined with chemotherapy as first-line treatment for advanced biliary tract cancer. We included 18 randomized controlled trials (RCTs) meeting selection criteria to evaluate first-line targeted/immunotherapy-chemotherapy combinations for advanced BTC. Among them, 15 RCTs were analyzed via Bayesian network meta-analysis (NMA) for the average group, while 6 RCTs were used for pairwise meta-analyses of molecular subgroups, with three studies contributing to both analyses. Outcomes included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and serious adverse events (SAEs). Subgroup analyses were performed for KRAS wild-type and PD-L1-positive populations. Durvalumab (HR 0.76, 95% CI 0.64–0.91) and pembrolizumab (HR 0.83, 95% CI 0.72–0.95) combined with GC significantly improved OS versus chemotherapy alone, with comparable SAE rates. Sintilimab plus anlotinib and GC achieved the best PFS (HR 0.47, 95% CI 0.28–0.80), though OS benefits were nonsignificant. EGFR inhibitors prolonged PFS in KRAS wild-type patients (HR 0.72, 95% CI 0.52–0.98). Bintrafusp alfa and cetuximab showed higher SAE risks (OR 2.26 and 1.95, respectively). Our findings directly inform clinical guidelines, address gaps in current therapeutic decision-making. Durvalumab or pembrolizumab combined with GC are optimal first-line regimens for advanced BTC, balancing survival benefits and safety. Sintilimab plus anlotinib combined with GC demonstrates superior PFS but requires further validation. While EGFR inhibitors plus chemotherapy demonstrate potential in KRAS wild-type patients, confirmation in large-scale RCTs is required. PD-L1 expression may represent a promising predictive biomarker for response to PD-1 inhibitor therapy.

<jats:p> <jats:bold>628</jats:bold> </jats:p> <jats:p> <jats:bold>Background:</jats:bold> Biliary tract cancers (BTC) are aggressive malignancies with poor survival outcomes and limited treatment options. This study compared the epidemiological and molecular differences of advanced BTC patients through a global collaboration involving the Mayo Clinic and National Cancer Center Hospital East. <jats:bold>Methods:</jats:bold> We included patients with advanced BTC who underwent comprehensive tumor molecular profiling who received at least 30 days of systemic therapy. Patients were grouped based on specific alterations. Overall survival (OS) was calculated from the start of first-line therapy until death and compared among subgroups using Cox regression models. <jats:bold>Results:</jats:bold> 332 (36%) out of 932 patients had at least one actionable mutation <jats:italic>: somatic BRCA1/2</jats:italic> mutations N=106, <jats:italic>FGFR2</jats:italic> fusions N=61, <jats:italic>HER2</jats:italic> amplifications N=65, <jats:italic>KRAS G12C/D</jats:italic> mutations N=46, <jats:italic>IDH1</jats:italic> mutations N=38, and TMB >10 mut/Mb N=16. <jats:italic>BRCA1/2</jats:italic> -mutated BTC were more commonly found in older Asian males with intrahepatic tumors and often presented with peritoneal and liver metastases upon initial diagnosis. <jats:italic>FGFR2</jats:italic> fusions were more prevalent in younger White females with intrahepatic tumors, while <jats:italic>HER2</jats:italic> amplifications were mainly seen in Asian females with gallbladder tumors. <jats:italic>KRAS</jats:italic> mutations were primarily observed in Asian males, and <jats:italic>IDH1</jats:italic> mutations were common in White males with intrahepatic tumors, frequently presenting with liver metastasis. Survival outcomes varied significantly based on the alteration type: patients with <jats:italic>FGFR2</jats:italic> fusions had the longest median OS (mOS) at 23.3 months (mo) (95%CI 18.4-34.9), followed by those with <jats:italic>IDH1</jats:italic> mutations (19.3 mo; 95% CI 17.4-NE) and TMB >10 mut/Mb (17.8 mo; 95% CI 13.6-NE). <jats:italic>BRCA</jats:italic> mutations had a mOS of 16 mo (95% CI 14.6-19.8), while <jats:italic>HER2</jats:italic> amplifications and <jats:italic>KRAS</jats:italic> mutations had mOS of 15.5 (95% CI 11.9-20.2) and 11.6 mo (95% CI 10.0-20.9), respectively. <jats:bold>Conclusions:</jats:bold> These findings underscore the heterogeneity in clinical and genomic characteristics among BTC patients, highlighting the importance of tailored therapeutic approaches. <jats:table-wrap orientation="portrait" position="anchor"> <jats:caption content-type="mtgabstract-table-title"> <jats:p>Baseline patient characteristics stratified by mutation status.</jats:p> </jats:caption> <jats:table content-type="mtgabstract"> <jats:thead content-type="mtgabstract"> <jats:tr> <jats:th colspan="1" content-type="border-bottom align-left" rowspan="1"/> <jats:th colspan="1" content-type="border-bottom" rowspan="1"> <jats:italic>KRAS G12C/D</jats:italic> (N=46) </jats:th> <jats:th colspan="1" content-type="border-bottom" rowspan="1"> <jats:italic>BRCA 1/2</jats:italic> (N=106) </jats:th> <jats:th colspan="1" content-type="border-bottom" rowspan="1"> <jats:italic>IDH1</jats:italic> (N=38) </jats:th> <jats:th colspan="1" content-type="border-bottom" rowspan="1"> <jats:italic>HER2</jats:italic> amplification(N=65) </jats:th> <jats:th colspan="1" content-type="border-bottom" rowspan="1"> <jats:italic>FGFR2</jats:italic> fusion(N=61) </jats:th> <jats:th colspan="1" content-type="border-bottom" rowspan="1">TMB>10 mut/Mb(N=16)</jats:th> <jats:th colspan="1" content-type="border-bottom" rowspan="1">P-value</jats:th> </jats:tr> </jats:thead> <jats:tbody> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Median Age, year</jats:td> <jats:td colspan="1" rowspan="1">67</jats:td> <jats:td colspan="1" rowspan="1">68</jats:td> <jats:td colspan="1" rowspan="1">64</jats:td> <jats:td colspan="1" rowspan="1">66</jats:td> <jats:td colspan="1" rowspan="1">52</jats:td> <jats:td colspan="1" rowspan="1">73</jats:td> <jats:td colspan="1" rowspan="1"><0.001</jats:td> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Female, n (%)</jats:td> <jats:td colspan="1" rowspan="1">19 (41)</jats:td> <jats:td colspan="1" rowspan="1">34 (32)</jats:td> <jats:td colspan="1" rowspan="1">18(47)</jats:td> <jats:td colspan="1" rowspan="1">34 (52)</jats:td> <jats:td colspan="1" rowspan="1">45(74)</jats:td> <jats:td colspan="1" rowspan="1">6 (38)</jats:td> <jats:td colspan="1" rowspan="1"><0.001</jats:td> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Race, n (%)</jats:td> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"><0.001</jats:td> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Asian</jats:td> <jats:td colspan="1" rowspan="1">31 (67)</jats:td> <jats:td colspan="1" rowspan="1">97 (92)</jats:td> <jats:td colspan="1" rowspan="1">13 (34)</jats:td> <jats:td colspan="1" rowspan="1">51 (79)</jats:td> <jats:td colspan="1" rowspan="1">10 (16)</jats:td> <jats:td colspan="1" rowspan="1">6 (38)</jats:td> <jats:td colspan="1" rowspan="1"/> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">White</jats:td> <jats:td colspan="1" rowspan="1">14 (30)</jats:td> <jats:td colspan="1" rowspan="1">8 (8)</jats:td> <jats:td colspan="1" rowspan="1">24 (63)</jats:td> <jats:td colspan="1" rowspan="1">13 (20)</jats:td> <jats:td colspan="1" rowspan="1">48 (79)</jats:td> <jats:td colspan="1" rowspan="1">10 (63)</jats:td> <jats:td colspan="1" rowspan="1"/> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Primary tumor location, n (%)</jats:td> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"/> <jats:td colspan="1" rowspan="1"><0.001</jats:td> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Intrahepatic</jats:td> <jats:td colspan="1" rowspan="1">28 (68)</jats:td> <jats:td colspan="1" rowspan="1">45 (45)</jats:td> <jats:td colspan="1" rowspan="1">33 (100)</jats:td> <jats:td colspan="1" rowspan="1">19 (31)</jats:td> <jats:td colspan="1" rowspan="1">54(95)</jats:td> <jats:td colspan="1" rowspan="1">10 (67)</jats:td> <jats:td colspan="1" rowspan="1"/> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Extrahepatic</jats:td> <jats:td colspan="1" rowspan="1">9 (22)</jats:td> <jats:td colspan="1" rowspan="1">27(27)</jats:td> <jats:td colspan="1" rowspan="1">0 (0)</jats:td> <jats:td colspan="1" rowspan="1">10 (16)</jats:td> <jats:td colspan="1" rowspan="1">2 (4)</jats:td> <jats:td colspan="1" rowspan="1">4 (27)</jats:td> <jats:td colspan="1" rowspan="1"/> </jats:tr> <jats:tr> <jats:td colspan="1" content-type="row-heading" rowspan="1">Gallbladder</jats:td>

TPS2691 Background: TU2218 is a low molecular weight dual kinase inhibitor highly specific to TGFβR1 and VEGFR2 and has a potential to be an efficacious therapy against cancer growth. In vitro and in vivo nonclinical studies have shown that TU2218 reduced the growth and migration/invasion of tumor cells and increased antitumor effects in combination with anti-PD-1/anti PD-L1 antibodies. To investigate safety and tolerability of TU2218 Phase 1a trial was conducted with 6 dose level escalation (30mg/day → 60mg/day → 105mg/day → 150mg/day → 195mg/day → 270mg/day) of TU2218 alone, and it was confirmed that TU2218 was safe and tolerated in all dose levels. And to explore the synergistic effect of TU2218 in combination with Pembrolizumab and to decide RP2D Phase 1b trial was conducted with 3 dose level escalation (105mg/day → 150mg/day → 195mg/day) of TU2218 in combination with Pembrolizumab in patients with advanced solid tumors. The RP2D of TU2218 was established as 195mg/day in combination with Pembrolizumab, the total 19 patients received the treatment and most frequently observed TRAE was pruritus and proteinuria, and three Grade 3 TRAEs (Pruritus, Rash Maculo-Popular, Malaise) were observed. The MTD was not identified during dose escalation period. The ORR of overall dose levels demonstrated 19%, and DCR was about 63%. In particular, 80% DCR was observed in TU2218 195mg/day in combination with Pembrolizumab. The trial was expanded to the specific cancer types, Biliary Tract Cancer and Head and Neck Cancer using the established RP2D for Phase 2 trial. Methods: Locally advanced unresectable or metastatic biliary tract cancer (BTC) patient whose tumor has progressed on/after first line standard anticancer therapy and anti-PD-(L)1 agent-naïve metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC) patient whose tumor express PD - L1 (CPS ≥1) as determined by an FDA-approved test or recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy are eligible for this non-randomized, open-label multicenter trial. All patients are administered with TU2218 195mg/day (97.5mg BID) on a 2 weeks-on/1 week-off in combination with Pembrolizumab 200mg IV Q 3 weeks and will be evaluated by investigator-assessed objective response rate (ORR) defined as the proportion of patients with a best overall response of complete response (CR) or partial response (PR) according to RECIST version 1.1. If 2 or less patients out of 22 evaluable BTC patients are observed with CR/PR and 3 or less patients out of 22 evaluable HNSCC patients are observed with CR/PR, this suggests futility and the cohort may be stopped. Up to 40 BTC patients and up to 36 HNSCC patients are planned to be enrolled and a dropout rate of up to 10% is expected. As of this abstract submission date, 14 BTC patients and 8 HNSCC patients have been enrolled. Clinical trial information: NCT05784688 .

Background In patients with advanced biliary tract cancer (BTC), first-line chemotherapy plus immunotherapy has improved outcomes; however, second-line options that reflect the disease’s molecular heterogeneity are still needed. One emerging target is MDM2, amplified in ~5–8% of BTC cases. Methods This is a subset analysis of two ongoing Phase Ia/Ib trials assessing patients treated with brigimadlin (BI 907828; a highly potent, oral MDM2–p53 antagonist) ± ezabenlimab (PD-1 inhibitor) ± BI 754111 (anti-LAG-3; n = 1). Results Results from 12 patients with BTC are shown (monotherapy: n = 6/combination: n = 6). Six patients achieved partial response (monotherapy: n = 2/combination: n = 4), four had stable disease; responses were durable. Brigimadlin had a manageable safety profile. Seven patients had dose reductions due to adverse events, but no treatment-related adverse events led to treatment discontinuation. Conclusion Brigimadlin demonstrated anti-tumor activity in patients with advanced MDM2-amplified BTC, and warrants further investigation.

Abstract Background Biliary tract cancers (BTCs) are a diverse group of malignancies with varied genetic backgrounds. The prevalence of intrahepatic cholangiocarcinoma (iCC) is increasing, particularly in Western countries. Despite advancements in treatments, the prognosis for BTC remains poor. Recent molecular profiling has revealed that up to 40% of iCC cases have targetable genetic alterations. MET amplification, although rare, presents a significant target for therapy. Case Presentation A 25-year-old female with a history of ulcerative colitis presented with shoulder pain and a positron emission tomography–computed tomography (PET–CT) scan revealed an enlarged liver and multiple metastases. Histopathological analysis diagnosed poorly differentiated adenocarcinoma. First-line therapy with Cisplatin, Gemcitabine, and Durvalumab resulted in disease progression. Molecular profiling identified a TP53 mutation and MET amplification. Based on these findings, Tepotinib was initiated. Tepotinib treatment led to a significant reduction in tumor size and normalization of CA 19-9 levels within 2 months, achieving a complete metabolic remission lasting up to 17 months. The treatment was well tolerated with minimal side effects. Discussion MET-amplified BTCs are exceedingly rare, and evidence for targeted treatment is limited. This case demonstrates the efficacy of Tepotinib in a young patient with MET-amplified iCC, showing a long-term response and suggesting a potential new standard treatment option for this molecularly defined entity. This case also highlights the aggressive nature of MET-amplified tumors and the need for targeted second-line therapies. Conclusion Tepotinib showed remarkable efficacy in treating MET-amplified intrahepatic cholangiocarcinoma, underscoring the importance of molecular profiling in BTCs and suggesting a potential new therapeutic approach for this rare cancer subtype.

492 Background: A subset of biliary tract cancers (BTC) feature amplification (amp) of the human epidermal growth factor receptor 2 ( ERBB2) gene. The prognostic role and treatment in these patients (pts) are poorly understood. This study aims to characterize the clinical outcomes and molecular profiles of ERBB2-amp BTC. Methods: We conducted a retrospective analysis of clinical outcomes data and next-generation sequencing (NGS) at MD Anderson (MDA) and Foundation Medicine (FMI) from 2009-2023 in pts with ERBB2-amp BTC including all classes of genomic alterations (GA) in other genes. Progression-free survival (PFS) and overall survival (OS) were assessed by the log-rank test. Results: 80 pts (MDA) were identified with ERBB2-amp BTC with documented treatment and NGS data. Pts (Table) who received ERBB2-directed therapy (22.4 vs. 12.0 m, p=0.004), surgery (21.0 vs. 12.0 m, p=0.002), local therapy with RT/ablation/Y90 (excluding surgical pts, 20.6 vs. 11.6 m, p = 0.0001) had significantly longer OS. Median PFS (mPFS) without ERBB2-directed therapy for 1st, 2nd, and 3rd line systemic therapy was 4.1, 3, and 2.2 m. mPFS with all ERBB2-directed therapy was 4.1 m, but that of gemcitabine (gem)-based plus trastuzumab (tt) and 5FU-based plus tt was 7.8 and 3.8 m (p=0.018). The most common concurrent-GA at MDA, any co-amps (72.7%; 19.2 vs. 20.3 m, p=0.69) and TP53 inactivating mutation (70.4%; 16.6 vs. 14.9 m, p=0.89), had no OS association; 3rd most common co-GA found, CDK12 amp (46.4%), had a trend toward a better OS (19.2 vs. 14.3 m, p=0.089). FMI database showed ERBB2 amp in 3.2%, 5.4%, and 8.9% in intrahepatic cholangiocarcinoma (iCCA), extrahepatic CCA, and gallbladder cancer. The most common co-GA included TP53, CDKN2A/B, and CCNE1. FGFR2 fusions (iCCA) were mutually exclusive with ERBB2 amp (0%, MDA/FMI); IDH1 (iCCA) with ERBB2 amp, 5.7 and 4.3% (MDA and FMI). Conclusions: ERBB2-amp BTC has shorter mPFS or mOS without ERBB2-directed therapy, surgery, or local therapy, suggesting clinical benefit from multi-disciplinary care and targeted therapy. Co-GA with ERBB2-amp did not demonstrate a significant association with survival outcomes. [Table: see text]

503 Background: Biliary tract cancer (BTC) is a heterogeneous group of tumors, including cholangiocarcinoma (intra and extrahepatic) and gallbladder carcinoma (GC). We describe the epidemiology of BTC patients (pt) from the RETUD registry. Methods: We evaluated a large real-world cohort of pt diagnosed with BTC between January 1, 2017, and May 30, 2025. Analyses were based on first therapeutic approach as follows: resectable disease (RD) for patients with neoadjuvant/adjuvant therapy and/or radical treatment (surgery and/or locoregional therapy); and advanced disease (AD) for former patients who relapsed and those initially treated with first line therapy with/without other locoregional treatments. Analysis comprised demographic and clinical characteristics, tumor molecular profile, therapeutic procedures and efficacy outcomes (EO). Time to relapse (TTR) for RD, progression-free survival (PFS) for AD and overall survival (OS) for both were estimated using the Kaplan-Meier method. Results: A total of 1756 pt from 40 centers were classified as RD (695) or AD (1450). 389 (55,9%) pt were initially RD but progressed to AD. Median (m) age at diagnosis (dx) was 67.4 years (y), with 44.3% of women. Most frequent tumor locations at dx were intrahepatic (53.0%) and GC (15.5%). Distant metastasis in AD pt occurred primarily in liver (65.1%). 89.3% received chemotherapy, 10.7% were treated with immunotherapies, 6.3% received targeted therapies, and 7.7% of pt took part in at least one clinical trial. Biomarkers were determined in 553 pt (31.5%). ESCAT-I alterations were found in 176 pt (31.8%), most frequent ones are shown in table 1. RD pt: surgery was performed in 95.7%, locoregional therapy in 15.1% and 68.9% and 5.0% received adjuvant and neoadjuvant therapy respectively. With a m (p25, p75) follow-up of 23.9 months (mo) (14.4, 39.5), mOS was 33.0 mo (95% CI 30.2-38.9). mTTR was 19.4 mo (95% CI 17.3-22.2). AD pt: 1st and 2nd lines were administered to 96.4% and 42.6% pt. Most common schemes were CISGEM and FOLFOX, respectively. Thromboembolic events occurred in 17.3% of AD pt. With a m (p25, p75) follow-up of 13.2 mo (6.7, 22.9), mOS was 10.4 mo (95% CI 9.7-11.2). 1st line mPFS was 5.3 mo (95% CI 4.9-5.6). All pt: There were differences in mOS by age groups; 20.9 mo (95% CI 16.8-26.9) in <50 y pt vs 16.3 mo (95% CI 15.1-17.3) in ≥50 y pt (p=0.033); but not for sex; 16.3 mo (95% CI 14.9-17.9) in males vs 16.6 mo (95% CI 15.1-17.9) in females (p=0.444). Conclusions: This large multicenter real-world study provides a comprehensive characterization of BTC epidemiology in Spain. Treatment patterns observed, specifically those with IT and TT, reflect a progressive uptake of these innovative treatments in standard of care therapies. Most frequent ESCAT-I alterations. ESCAT-I Biomarker determination, N Alteration, N (%) IDH1 mutation 445 73 (16.4%) FGFR2 fusion 399 26 (6.5%) MSI 491 30 (6.1%) HER2 amplification/overexpression 448 24 (5.4%)

Abstract Cholangiocarcinoma (CCA) is an uncommon but morbid cancer arising from the intrahepatic or extrahepatic bile ducts. CCA is frequently asymptomatic at early stages and is often unresectable or metastatic at the time of initial diagnosis. While chemotherapy remains the mainstay of treatment for most patients with advanced disease, the addition of immunotherapy to frontline treatment has improved survival and provided an alternative to perpetual chemotherapy. Furthermore, a variety of targeted therapies have demonstrated benefit in patients with specific biomarkers including FGFR2 fusions, IDH1 mutations, HER2 overexpression, and tumor agnostic markers such as NTRK and RET fusions, among others. This review will summarize the established roles of immunotherapy, targeted therapies, and their combinations in CCA as well as treatment strategies that are under development with potential to impact clinical practice in the coming years.

Cholangiocarcinoma (CCA) comprises intrahepatic (iCCA) and extrahepatic (eCCA) subtypes, each exhibiting distinct molecular characteristics. Understanding these differences is critical for identifying subtype-specific therapeutic targets and advancing precision medicine. Protein glycosylation, a key post-translational modification, regulates immune evasion and metastasis, yet the glycoproteomic difference between iCCA and eCCA remains unexplored. Here we presented the first comprehensive N-glycoproteomic profile of eCCA and compared it with iCCA using a publicly available dataset. Our N-glycoproteomic analysis of paired eCCA tumors and normal adjacent tissues (NATs) identified 8372 N-glycopeptides, 3467 N-glycosites, and 2627 N-glycoproteins. Comparative analysis revealed distinct N-glycosylation signature, with eCCA exhibiting higher fucosylated glycans and iCCA showing increased sialylation. Pathway enrichment analysis of N-glycoproteins revealed a more prominent lysosome-related enrichment in eCCA, whereas pathways related to immune modulation, cytoskeletal components, and the extracellular matrix were significantly enriched in both subtypes. Immune profiling revealed an immunosuppressive microenvironment in both eCCA and iCCA, characterized by reduced natural killer cell infiltration and subtype-specific fibroblast and endothelial cell remodeling. DPM1, a glycosylation enzyme highly expressed in eCCA, was associated with tumor-specific N-glycopeptides and reduced immune cell infiltration. Its knockdown impaired cell migration, and glycoproteomic analysis implicated DPM1 in regulating adhesion, proteostasis, and immune pathways, highlighting its potential as a therapeutic target in eCCA. Our findings provide insights into N-glycosylation alterations in CCA subtypes, underscoring N-glycosylation-related mechanisms as potential biomarkers and therapeutic targets, particularly in eCCA.

ABSTRACT Introduction Advances in precision medicine have expanded access to targeted therapies and demand for molecular profiling of cholangiocarcinoma (CCA) patients in routine clinical practice. However, pathologists face challenges in establishing a definitive intrahepatic CCA (iCCA) diagnosis while preserving sufficient tissue for molecular profiling. Additionally, they frequently face challenges in optimal tissue handling to preserve nucleic acid integrity. Areas covered This article first identifies the challenges in establishing a definitive diagnosis of iCCA in a lesional liver biopsy while preserving sufficient tissue for molecular profiling. Then, the authors explore the clinical value of molecular profiling, the basic principles of single gene and next-generation sequencing (NGS) techniques, and the challenges in tissue sampling for genomic testing. They also propose an algorithm for best practice in tissue management for molecular profiling of CCA. Expert opinion Several practical challenges face pathologists during tissue sampling and processing for molecular profiling. Optimized tissue processing, careful tissue handling, and selection of appropriate approaches to molecular testing are essential to ensure that the highest possible quality of diagnostic information is provided in the greatest proportion of cases.

ABSTRACT Molecular mechanisms behind potentially inferior prognosis of old cholangiocarcinoma (CCA) patients are unclear. Prevalence of interventional targets and the difference between young and old CCA patients are valuable for promising precision medicine. A total of 188 CCA patients with baseline tumor tissue samples were subgrouped into the young (≤45 years) and old (>45 years) sub-cohorts. Somatic and germline mutation profiles, differentially enriched genetic alterations, and actionable genetic alterations were compared. An external dataset was used for the validation of molecular features and the comparison of overall survival (OS). Compared to young patients, KRAS alterations were more common in old patients (P = .04), while FGFR2 fusions were less frequent (P = .05). TERT promoter mutations were exclusively detected in old patients. The external dataset (N = 392) revealed no significant difference in OS between young and old patients; however, old patient-enriched KRAS (hazard ratio [HR]: 1.96, 95% confidence interval [CI]: 1.37–2.80) and TERT alterations (HR: 2.03, 95% CI: 1.22–3.38) were associated with inferior OS. Approximately 38.3% of patients were identified of actionable oncogenic mutations indicative of a potential response to targeted therapy or immunotherapy. Actionable FGFR2 fusions (P = .01) and BRAFV600E (P = .04) mutations were more frequent in young females than old patients. The enrichment of KRAS/TERT alterations in CCA patients over 45 years resulted in inferior OS. Approximately one-third of CCA patients were eligible for targeted therapy or immunotherapy given the actionable mutations carried, especially young females.

BACKGROUND Cholangiocarcinoma (CCA) is a malignant tumor with an increasing incidence worldwide. Although radiation therapy has improved the therapeutic efficiency of CCA treatment, differential expression of genes among cholangiocarcinoma subtypes has been revealed through precise sequencing. However, no specific molecular therapeutic targets or biomarkers have been figured out for use in precision medicine, and the exact mechanism by which antitumorigenic effects occur is still unclear. Therefore, it is necessary to conduct further studies on the development and mechanisms associated with CCA. METHODS We examined the clinical data and pathological features of patients with cholangiocarcinomas. We investigated the associations between DNA Topoisomerase II Alpha (TOP2A) expression and patient outcomes, such as metastasis-free survival (MFS) and disease-specific survival (DSS), as well as clinical characteristics and pathological results. RESULTS TOP2A expression was shown to be upregulated in CCA tissue sections by immunohistochemistry staining and data mining. Moreover, we observed that the TOP2A expression correlated with clinical features, such as the primary tumor stage, histological variants, and patients with hepatitis. Furthermore, high expression of TOP2A was associated with worse survival outcomes in terms of the overall survival (p < 0.0001), disease-specific survival (p < 0.0001), and metastasis-free survival (p < 0.0001) compared with patients in the low TOP2A expression group. This indicates that a high level of TOP2A expression is related to an unfavorable prognosis. CONCLUSIONS Our results show that TOP2A is highly expressed in CCA tissues, and its upregulation is correlated with the primary disease stage and poor prognosis significantly. Consequently, TOP2A is a prognostic biomarker and a novel therapeutic target for the treatment of CCA.

Cholangiocarcinoma (CCA) is an aggressive malignancy with limited therapeutic options. Increasing evidence suggests that a subset of CCA patients can benefit from multiple tyrosine kinase inhibitors (mTKIs) such as surufatinib. Biomarker studies that can identify potential responders to mTKI therapy are crucial for improving treatment outcomes. In this study, we examined the expression of protein tyrosine phosphatase non-receptor type 9 (PTPN9) in CCA patients treated with surufatinib and analyzed its association with treatment response. Immunoprecipitation coupled with mass spectrometry (IP-MS) was performed to identify novel PTPN9 substrates. Orthotopic mouse models, biochemical assays, structural analyses, and functional experiments were conducted to validate these novel findings. Single-cell RNA sequencing, ELISA, and immunohistochemistry were utilized to investigate microenvironmental tumor-promoting factors. CCA patients with surufatinib non-response exhibited significantly decreased PTPN9 expression compared to that response to surufatinib. PTPN9 knockdown in orthotopic models led to reduced sensitivity to surufatinib. IGF1R was identified as a new substrate of PTPN9, which preferably to dephosphorylate IGF1R at Y1166. In clinical tissues, PTPN9 expression was inversely correlated with IGF1R Y1165/1166 phosphorylation. Establishment of a surufatinib-resistant CCA cell line further confirmed decreased PTPN9 expression and elevated IGF1R signaling. In vivo blockade of IGF1R signaling significantly enhanced surufatinib sensitivity. Mechanistically, crystal structure analysis revealed Tyr333 and Asp335 as key PTPN9 residues interacting with IGF1R; mutation of these residues restored IGF1R signaling and abolished the tumor-suppressive effect of PTPN9. Furthermore, cancer-associated fibroblasts (CAFs) were identified as the major source of IGF1 in CCA microenvironment, essential for IGF1R-driven tumor progression. In summary, the PTPN9–IGF1R axis plays a pivotal role in modulating mTKI sensitivity and tumor progression in CCA. This axis serves as a promising biomarker for identifying potential mTKI beneficiaries and represents a potential therapeutic target to enhance mTKI efficacy and overcome resistance.

e15190 Background: Cancer genomic diversity, with a median of ~5 pathogenic molecular alterations per tumor, presents a challenge in predicting clinical outcomes of investigational therapies. Precision medicine implies the ability of a drug/drug combination to address the unique molecular profile of an individual patient’s cancer. Methods: We describe an algorithmic solution developed by CureMatch, Inc. that evaluates drugs based on their ability to address a patient’s composite molecular profile from somatic next-generation sequencing (NGS) testing and other omics reports (Matching Score). The scoring system uses expert-curated knowledge base content within a digital reasoning framework, which assesses how well a given therapy option addresses a tumor’s biomarkers, with higher scores implying better molecular matching. Using TransThera’s phase 2 clinical trial results (NCT04919642) of tinengotinib, a potent small molecule multi-kinase inhibitor targeting FGFR, the CureMatch molecular Matching Scores were produced for all 37 patients with advanced cholangiocarcinoma and evaluable outcomes (includes cohorts with (i) FGFR2 fusion and primary progression on previous FGFR inhibitor (FGFRi) (ii) FGFR2 fusion(s) with progression after prior response to FGFRi; (iii) other FGFR alterations; and (iv) FGFR wild type. Evaluable patients had baseline tissue and/or liquid biopsy NGS (mostly performed by Foundation Medicine). Patients were considered to have better outcomes if they achieved stable disease (SD) > 5 months/complete or partial remission (CR/PR); vs. worse outcomes if best response was SD < 5 months/progressive disease (PD); patients with ongoing SD < 5 months were not evaluable. Results: Overall, 25 of 37 evaluable patients (68%) with cholangiocarcinoma who received tinengotinib achieved SD > 5 months/CR/PR; 29 patients (78% of 37) had > 1 FGFR alteration. Matching Score > 22% was found in 22 of 25 patients (88%) who achieved SD > 5 months/CR/PR vs. 4 of 12 patients (33%) with SD < 5 months/PD (p = 0.0004). Notably the Matching Score correctly predicted outcomes in 31 of 37 patients (84%), including in 6 of 8 patients (75%) without FGFR alterations and in 25 of 29 patients (86%) with FGFR alterations. Conclusions: The CureMatch Matching Score significantly correlated with outcomes for tinengotinib, a potent FGFR inhibitor given to patients with cholangiocarcinoma, with activity in both FGFR-altered and FGFR wild-type patients. Additional studies of this algorithmic Matching Score’s ability to predict outcome and therefore optimize treatment for a variety of pharmaceutical agents are warranted. Outcomes CureMatch Scores >22% CureMatch Scores <22% CureMatch Scores <22% P-Value SD>5 months/CR/PR N=23 N=2 Median = 40 (range, 0-99) 0.0004 SD<5 months/PD N=4 N=8 Median = 20 (range, 0-89)

Simple Summary Cholangiocarcinoma (CCA) accounts for approximately 15% of primary liver cancers. CCA has a poor prognosis and, thus, more effective systemic treatments are needed. We tested opaganib and upamostat, drugs that target sphingosine kinase 2 and multiple serine proteases, primarily trypsins, which are highly expressed in CCA tumors. This study demonstrates the results of inhibiting these novel targets with these drugs individually and in combination in a patient-derived CCA xenograft mouse model. Abstract Upamostat is an orally available small-molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2, trypsin 3 (PRSS1/2/3), and the urokinase-type plasminogen activator (uPA). These enzymes are expressed in many cancers, especially during tissue remodeling and subsequent tumor cell invasion. Opaganib (ABC294640), a novel, orally available small molecule is a selective inhibitor of the phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P) by sphingosine kinase 2 (SPHK2). Both sphingosine kinase 1 (SPHK1) and SPHK2 are known to regulate the proliferation-inducing compound S-1-P. However, SPHK2 is more critical in cancer pathogenesis. The goal of this project was to investigate the potential antitumor effects of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) xenografts in nude mice. PAX165, a patient-derived xenograft (PDX) from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2, and PRSS3. Four groups of 18 mice each were treated with upamostat, opaganib, both, or vehicle. Mouse weights and PAX165 tumor volumes were measured. Tumor volumes in the upamostat, opaganib, and upamostat plus opaganib groups were significantly decreased compared to the control group.

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) play essential roles in promoting cholangiocarcinoma (CCA) cell survival by mediating paracrine crosstalk between tumor and cancer-associated fibroblasts (CAFs), indicating the potential of PDGFR as a target for CCA treatment. Clinical trials evaluating PDGFR inhibitors for CCA treatment have shown limited efficacy. Furthermore, little is known about the role of PDGF/PDGFR expression and the mechanism underlying PDGFR inhibitors in CCA related to Opisthorchis viverrini (OV). Therefore, we examined the effect of PDGFR inhibitors in OV-related CCA cells and investigated the molecular mechanism involved. We found that the PDGF and PDGFR mRNAs were overexpressed in CCA tissues compared to resection margins. Notably, PDGFR-α showed high expression in CCA cells, while PDGFR-β was predominantly expressed in CAFs. The selective inhibitor CP-673451 induced CCA cell death by suppressing the PI3K/Akt/Nrf2 pathway, leading to a decreased expression of Nrf2-targeted antioxidant genes. Consequently, this led to an increase in ROS levels and the promotion of CCA apoptosis. CP-673451 is a promising PDGFR-targeted drug for CCA and supports the further clinical investigation of CP-673451 for CCA treatment, particularly in the context of OV-related cases.

e16118 Background: Patients (pts) with advanced cholangiocarcinoma (CCA) who progressed on or after 1L chemotherapy have few treatment options. FGFR2 fusions or rearrangements occur in 10–16% of pts in global and 6.14% in Chinese pts with intrahepatic cholangiocarcinoma (iCCA), and may predict tumor susceptibility to FGFR inhibitors. HMPL-453 is a novel, selective tyrosine kinase inhibitor against FGFR1, 2, and 3. Here, we report results from an open-label, multi-cohort, single-arm phase 2 study (NCT04353375). Methods: Pts with histologically or cytologically confirmed locally advanced or metastatic iCCA with FGFR2 fusions who were treated at least one previous line of systemic therapy were eligible and included in this analysis. They received 21-day cycles of HMPL-453 orally with 150 mg (QD, cohort 1) or 300 mg (QD, 2w on/1w off, cohort 2) until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) per RECIST 1.1. Results: At data cutoff (Sep 21, 2022), a total of 25 iCCA pts (12 in cohort 1, 13 in cohort 2) were enrolled and treated with HMPL-453. Median age was 51 yrs (range 28-76) and 12 (48%) were male. All pts had received ≥1 prior treatment line and 21 (84%) had received previous gemcitabine-based chemotherapy. The median follow-up duration was 12 months (mos) and 4.1 mos for cohort 1 and cohort 2, respectively. Of 22 evaluable pts (12 in cohort 1 and 10 in cohort 2) who had at least one post-baseline tumor assessment, the best overall response by investigator assessment was confirmed partial response in seven (31.8%) pts and stable disease in an additional 12 pts (54.5%), resulting in a disease control rate (DCR) of 86.4%. Specifically, in cohort 2 the confirmed ORR was 50% (95% CI, 18.7%–81.3%) and DCR was 90% (95% CI, 55.5%–99.7%). Duration of response was not reached in either cohort. Median progression-free survival was 5.7 (95% CI, 2.6, NR) mos in cohort 1 and not yet mature in cohort 2. Of all pts, 23 (92%) experienced ≥1 treatment-related adverse events (TRAEs). The most common TRAEs (any grade) were diarrhea (56%), dry mouth (44%), and blood phosphorus increased (44%). The common Gr ≥3 TRAEs (≥5% pts) were decreased neutrophil count (8%), nail toxicity (8%) and palmar-plantar erythrodysesthesia syndrome (8%). Compared to cohort 1, pts in cohort 2 experienced a lower incidence of Gr ≥3 TRAEs (23.1% vs 58.3%), less dose interruption/reduction (32.0% vs 41.7%) and less treatment discontinuation (0.0% vs 16.7%) due to TRAE. Based on the better safety profile and preliminary efficacy, 300mg, QD, 2w on/1w off was chosen as the recommended phase 2 dose (RP2D). Conclusions: HMPL-453 showed promising efficacy, particularly in RP2D regimen (300 mg, QD, 2w on/1w off) and acceptable toxicity in pts with previously-treated advanced iCCA and FGFR fusions. These results warrant further study in pts with advanced iCCA. Clinical trial information: NCT04353375 .

This research investigates the design and discovery of silmitasertib-based drugs as potential inhibitors of casein kinase II (CK2) for cholangiocarcinoma using hybrid in-silico methods. The study employs a two-fold approach, including ligand-based virtual screening and molecular docking, to assess the inhibitory potential of silmitasertib-based compounds. The ligand-based virtual screening involves the construction of a diverse compound library, followed by energy minimization and conformational analysis, revealing a set of compounds with varied similarities to silmitasertib. In parallel, the CK2 model undergoes meticulous selection, validation, and refinement through PDB REDO, showcasing significant improvements in model quality. Molecular docking results highlight promising candidates, with N-(2, 4, 6-trimethylphenyl) phenanthridin-6-amine (MCULE-1492185963-0) emerging as a lead compound with superior binding characteristics compared to silmitasertib. This hybrid in-silico approach demonstrates the potential of silmitasertib-based compounds as CK2 inhibitors for cholangiocarcinoma and identifies a lead compound for further experimental validation. The findings contribute valuable insights to the design of novel drugs for cholangiocarcinoma treatment, setting the stage for future experimental and clinical investigations

e16222 Background: Advanced metastatic ICC was characterized by poor survival and limited therapeutic options. Surufatinib, a selective tyrosine kinase inhibitor targeting VEGFR 1, 2, and 3, FGFR1, and CSF-1R, provides dual anti-tumor action via anti-angiogenesis and tumor microenvironment regulation. Surufatinib combined with immunotherapy and chemotherapy may enhances anti-cancer effects for advanced metastatic ICC. This study evaluates the effectiveness and safety of surufatinib combined with gemcitabine, cisplatin, and immune checkpoint inhibitor (ICI)as the first-line treatment for patients with unresectable locally advanced or metastatic intrahepatic cholangiocarcinoma (ICC). Methods: This is an open-label, single-arm, single -center trial. Eligible patients who were 18 -75 years old with histologically confirmed unresectable locally advanced or metastatic ICC were enrolled. Pts received surufatinib (250mg, orally, once daily), ICI(Zimberelimab or Toripalimab, 240mg, intravenous infusion,d1,q3w), and chemotherapy (gemcitabine 1000mg/m 2 intravenous infusion, 30min, d1,d8,q3w ; cisplatin 25mg/m 2 intravenous infusion, 2h, d1,d8,q3w) until disease progression, death, surgery, intolerable toxicity, or withdrawal of consent. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), disease control rate (DCR), overall survival (OS), conversion to surgical resection rate, pathological complete response rate (pCR), and safety. Results: By January 23, 2025, 20 pts were enrolled with median age 57.9 years (range: 43-70), male 50%, 85% ECOG PS 0-1, 100% stage IV. 12 pts were efficacy evaluable. 50% (6/12) pts achieved partial response (PR), 50% (6/12) pts achieved stable disease (SD). The confirmed ORR was 50%, DCR was 100%. The median PFS was 8.2m (95%Cl: 1.4-12.2 months),and the median OS had not yet matured .The most common TEAEs of all grades were myelosuppression (33.3%), abdominal pain (27.7%), hypertension (16.6%)and diarrhea (16.6%), No grade ≥ 3 TEAEs or new safety signals occurred. Conclusions: Surufatinib plus gemcitabine and cisplatin combined with immune checkpoint inhibitor, showed preliminary anti-tumor activity and manageable toxicity for the 1L treatment of ICC, providing an additional treatment option for pts with ICC. This ongoing study promises to finish updated results on efficacy in forthcoming reports. Clinical trial information: ChiCTR2400085526 .

Cholangiocarcinoma (CCA) patients (pts) harboring FGFR fusions treated with FGFR inhibitors (FGFRi) have shown clinical benefit, but often have progression in 5- 7 months. Secondary polyclonal mutations in the FGFR2 kinase domain (KD) may be a mechanism for acquired resistance to FGFRi. Tinengotinib, a spectrum-selective multi-kinase inhibitor with unique binding properties to FGFR2, potently inhibited FGFR2 KD mutations in pre-clinical models and exhibited antitumor activity in CCA pts. Here we present two cases of CCA pts treated with tinengotinib in a phase Ib/II trial. Biomarker samples were collected at baseline and C3D1 timepoints as an exploratory endpoint for pts receiving tinengotinib therapy. Efficacy was evaluated by RECIST v1.1. Tissue and blood samples were analyzed by FoundationOne® CDx and FoundationOne® Liquid CDx test, respectively, for FGFR alterations. Two CCA pts received oral tinengotinib 12mg once daily continuously for 28-day cycles and completed biomarker sampling at baseline and C3D1. Both pts were previously treated with chemotherapy and pemigatinib. Pt A is a 28yr female with CCA metastatic to liver and bone. Tinengotinib was initiated in August 2021. The pt achieved a partial response (PR) with tumor reductions of 29.2%, 36% and 41.6% at C3D1, C4D1 and C5D1, respectively. Pt A was treated for 210 days, discontinuing due to radiographic disease progression following a 17-day dose interruption for suspected grade 3 TSH increase. The progression-free survival (PFS) was 6.9 months. Baseline biomarker data showed presence of KD mutations including: E565G/A, N549H, K659M and I548T. At C3D1, E565G had reduced from 1.36% to 0.09%, and there was disappearance of all other FGFR2 KD mutations. Pt B is a 40yr female with CCA metastatic to liver, lung and lymph nodes. Tinengotinib was initiated in August 2021. The pt achieved a PR with tumor reductions of 23.4% and 48.6% at C3D1 and C5D1, respectively. Pt B was on treatment for 181 days and discontinued due to progression in non-target lesions at C6D1, following a 23-day dose interruption for unrelated grade 3 ALT increase. PFS was 5.9 months. Baseline biomarker data showed presence of KD mutations including: E565A/G/K, V564L/I/F, N549K/T An inverse correlation between the presence of KD mutations and the response to tinengotinib treatment was observed in these two FGFRi relapsed/refractory metastatic CCA pts. This suggests tinengotinib may overcome acquired resistance to FGFRi in CCA pts. An ongoing randomized phase III study is investigating tinengotinib vs. chemotherapy in FGFRi refractory/relapsed CCA (NCT05948475). Clinical trial information: NCT04742959 Sarina A. Piha-Paul, Sanjay Goel, Chih-Yi Liao, Nashat Gabrail, Farshid Dayyani, Syed Kazmi, Sayeh Lavasani, Katie Hennessy, Hui Wang, Caixia Sun, Peng Peng, Jean Fan, Milind Javle. Correlation of clinical and biomarker data in FGFR inhibitor failed metastatic cholangiocarcinoma patients with tumor response to tinengotinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 825.

593 Background: Advanced metastatic ICC, characterized by poor survival and limited therapeutic options, may benefit from the combined use of surufatinib—a selective tyrosine kinase inhibitor targeting VEGFR 1, 2, and 3, FGFR1, and CSF-1R—and ICIs, alongside locoregional treatments like TACE, DEB-TACE, and HAIC. Methods: Eligible pts who were 18 -75 years old with histologically confirmed unresectable or metastatic intrahepatic cholangiocarcinoma were enrolled. Pts received surufatinib (250mg, orally daily), ICI, and locoregional therapies until surgery, disease progression, death, intolerable toxicity, or withdrawal of consent. The primary endpoint was objective response rate (ORR). Secondary endpoints included progression-free survival (PFS), disease control rate (DCR), overall survival (OS), conversion to surgical resection rate and safety. Results: By July 31, 2024, 20 pts were enrolled and 10 pts were evaluated. Median age was 56.4 years (range: 36-69), with a majority being male (70%). 9 pts were pathologically confirmed as adenocarcinoma, 1patient was small bile tract type. Treatment included DEB-TACE (20%), TACE (30%), and HAIC (50%). 50% (5/10) pts achieved partial response (PR), 20% (2/10) pts accepted conversion surgery, 30% (3/10) pts achieved stable disease (SD), and the confirmed ORR was 50%, DCR was 80%. The median PFS (95%Cl: 6.9-NA months) had not yet matured, with no significant differences in survival benefits based on age or gender, though TACE showed potential benefits. The most common AEs of all grades were nausea (30%), dizziness (30%), and headache (10%), No grade ≥ 3 TEAEs or new safety signals occurred. Conclusions: Surufatinib plus immune checkpoint inhibitor, along with locoregional therapies showed preliminary anti-tumor activity and manageable toxicity for the 1L treatment of ICC, providing an additional treatment option for pts with ICC. Clinical trial information: NCT05236699 .

Src Family Kinases (SFKs) have a crucial role in tumor proliferation, survival, and metastasis. NXP900 is a highly selective SFK inhibitor a novel mechanism of action that locks SFKs in a closed, inhibited conformation, preventing interaction with substrate proteins, unlike other SFK inhibitors like dasatinib. A phase 1 study of NXP900 in patients with advanced solid tumors was recently initiated. Our preclinical studies showed therapeutic efficacy of NXP900 in cholangiocarcinoma (CCA) through decreased YAP, RAF-MAP and PI3K-AKT signaling. Here, we examined mechanisms of sensitivity and resistance to SFK inhibition. Secondary resistance was explored by generating a NXP900-resistant human cell line (HuCCT1-R) via prolonged exposure to NXP900, followed by multiomic profiling. Primary resistance was studied in vivo using nine heterogeneous patient-derived xenograft (PDX) tumors implanted in the flanks of NOD/SCID mice. Tumor-bearing mice were treated with vehicle or 40 mg/kg NXP900 daily for up to 4 weeks. To identify factors associated with response, a multiomic approach was used, integrating RNA-Seq, global proteomics, and phosphoproteomics. A hierarchical-all-against-all algorithm (HallA) assessed associations between multiomic features and drug response in tumor growth. Kinase enrichment analysis (KEA3) identified features linked to sensitivity or resistance. HuCCT1-R cells showed increased pTyr abundancy of TNK2, a protein known to increase PIP3 levels leading to AKT activation, and of DYRK3 and LAMTOR1, both of which can sustain AKT phosphorylation via mTORC, indicating acquired resistance through bypassing inhibited PI3K-AKT signaling to rescue AKT. NXP900 reduced tumor growth rates in 5 of 9 PDX models. Kinases associated with sensitivity in the PDX models included SFKs (SRC, LYN, and FYN), and AKT, PTK2, PRKCZ, EGFR, SYK, and GSK3B, suggesting involvement of the PI3K-AKT pathway in responsiveness to NXP900. Based on tumor inhibition responses in the tested models, we estimated NXP900 response for our additional PDX models, predicting 39% (11/28) of patient tumors may respond. This study identified molecular markers linked to response to SFK inhibition, which could be utilized for patient selection for NXP900 treatment. Further studies are validating these findings and exploring combination therapies to address resistance mechanisms. Hendrien Kuipers, Danielle M. Carlson, Jack W. Sample, Erik Jessen, Dong-Gi Mun, Aushinie M. Abeynayake, Jennifer L. Tomlinson, Amro M. Abdelrahman, Nathan W. Werneburg, Binbin Li, Mitesh J. Borad, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Multiomic profiling identified biomarkers of response to selective Src family kinase inhibition in preclinical models of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5595.

434 Background: Tinengotinib is a spectrum-selective multi-kinase inhibitor with unique binding properties to FGFR, potently inhibited FGFR2 fusion/rearrangement and acquired resistant mutations in pre-clinical models and in phase I trials that included cholangiocarcinoma (CCA) patients (pts). Here we present the efficacy and safety of tinengotinib in a phase II clinical trial. Methods: Eligible pts with advanced/metastatic CCA who had received ≥ 1 prior systemic chemotherapy therapy and ECOG PS 0 or 1 were treated with tinengotinib 10 mg QD. Four cohorts included: Cohort A1: FGFR2 fusion(s) with primary progression on previous FGFR inhibitor ( FGFRi), A2: FGFR2 fusion(s) with progression after prior response to FGFRi (acquired resistance); B: non-fusion FGFR alteration(s): C: FGFR wild-type ( FGFRwt). Primary endpoint was objective response rate (ORR) per RECIST v1.1. CTCAE V5.0 was used for safety assessments. Results: As of 7Aug2023, 48 pts with CCA were enrolled, 13 in Cohort A1, 10 in A2, 12 in B, 13 in C. Median age 61.5 [range 25-81] years old, 41.7% male, 58.3% had ≥ 3 lines of prior therapy. ECOG PS 0 in 47.9% pts. Among 35 pts with FGFR alterations, 80.0% had ≥ 1 prior FGFRi therapy, and 97.1% had prior chemotherapy. Forty (40) pts were efficacy evaluable. In A1, 1 out of 11 pts (9.1%) achieved PR with tumor reduction of 31.8%. In A2, 3 out of 8 pts (37.5%) achieved PR with tumor reduction of 40.7%, 47.0% and 54.6%. In B, 3 out of 9 pts (33.3%) achieved PR with tumor reduction of 36.5%, 48.6%, and 60.6%. No PR was observed in C. Overall DCR was 94.7% (18/19) in FGFR2 fusion/rearrangement pts (A1+A2), 88.9% (8/9) in other FGFR alterations pts (B), and 75% (9/12) in FGFRwt pts (C). Median progression-free survival (mPFS) was 5.26 months (95%CI, 2.86-9.10) in A1+A2, 5.98 months (95%CI, 1.87-NA) in B and 3.84 months (95% CI, 1.84-4.80) in C. Among 48 treated pts, treatment-related AEs (TRAEs) occurred in 45 (93.8%) pts, 14 (29.2%) in G1-2, 29 (60.4%) in G3 and 2 (4.2%) in G4. The most common G3 TRAEs were hypertension in 12 (25%), palmar-plantar erythrodysesthesia syndrome in 3 (6.3%), diarrhea in 3 (6.3%) and stomatitis in 3 (6.3%). One subject had G4 increased TSH and G4 increased lipase, and another subject had G4 posterior reversible encephalopathy syndrome. No G5 TRAE was observed. Conclusions: Tinengotinib has promising clinical benefit for FGFR2 fusion CCA after prior FGFRi and for non-fusion FGFR alterations. Tinengotinib-related toxicities were manageable. An ongoing randomized, controlled phase III study will evaluate the clinical efficacy, safety, and pharmacodynamic effect of Tinengotinib vs Physicians’ choice in subjects with FGFR2-altered refractory/relapsed CCA after prior chemotherapy and FGFRi therapy. Clinical trial information: NCT04919642 .

BACKGROUND & AIMS The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aim to evaluate the role of PTEN in the pathogenesis of eCCA and find novel therapies for this disease. METHODS The Pten gene in the biliary epithelial cells were genetically deleted using the Cre-loxp system. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry (IHC), RT-PCR, cell culture, and RNAseq. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. Experimental therapy was tested using an Aurka inhibitor. RESULTS We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as one month old. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated the disease progression, potentially through downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expressions of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum through an Aurka-dependent manner. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition (EMT), cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted the disease progression. This model shall be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.

TPS575 Background: Fibroblast growth factor (FGFR) alterations occur in 10-15% of adult patients with advanced intrahepatic cholangiocarcinoma (CCA) and 1-2% of adult patients with advanced extrahepatic cholangiocarcinoma. The first generation FGFR inhibitors (FGFRi) pemigatinib and futibatinib, have been approved for the treatment of advanced CCA with FGFR2 fusions or rearrangements after systemic chemotherapy. However, disease progression occurs within 6-9 months. Secondary polyclonal mutations in the FGFR kinase domain represent a prominent acquired resistance mechanism. Tinengotinib, a novel multi-kinase inhibitor with high potency against a variety of FGFR2 kinase domain mutations, has shown promising clinical benefit in subjects with FGFR-altered metastatic CCA who were heavily pretreated with chemotherapy and FGFRi(s) in phase I/II clinical trials. (NCT03654547, NCT04742959, NCT04919642). Methods: FIRST-308 is a phase III, randomized, global multicenter study to evaluate the efficacy and safety of oral tinengotinib versus Physician’s Choice in subjects with FGFR-altered, chemotherapy- and FGFRi-refractory/relapsed CCA. Approximately 200 subjects will be enrolled in US, Europe, and Asia. Key eligibility criteria include age ≥ 18 years, ECOG performance status 0 or 1, documentation of FGFR2 fusion/rearrangement gene status, prior treatment with at least one line of chemotherapy and exactly one prior FDA-approved FGFRi for unresectable or metastatic disease. The study includes Part A and Part B. The Part A is to select a dose for Part B. Eligible subjects will be randomized in a 2:2:1 ratio to receive tinengotinib 8 mg QD, tinengotinib 10 mg QD or Physician's Choice (FOLFOX or FOLFIRI) in Part A or 2:1 in Part B to receive the recommended Part B dose or Physician's Choice. The stratification factors at randomization include geographic region, prior lines of chemotherapy (1 or ≥ 2) and Physician’s choice. Tinengotinib will be administered orally QD in 28-day cycles. Subjects will continue to receive study treatment until confirmed disease progression, unacceptable toxicity, withdrawal of consent, or termination of study by Sponsor, whichever occurs first. For Part A, the primary endpoint is safety/tolerability; secondary endpoints include objective response rate (ORR), duration of response (DOR) and PK analysis. For Part B, the primary endpoint is progression-free survival (PFS); secondary endpoints include overall survival (OS), ORR, DOR, safety, quality of life and population PK. Exploratory endpoints will assess the correlations between baseline FGFR2 alterations by cfDNA and efficacy, and exposure-response in terms of efficacy and safety. Study is open for enrollment. Clinical trial information: NCT05948475 .

Background: Cholangiocarcinoma (CCA) is a lethal and heterogenous malignancy of the biliary tree characterized by perineural invasion. About 85% of CCA show activation of YAP signaling, which promotes proliferation, anti-apoptosis, and therapeutic resistance. YAP signaling is mediated by Src family kinases (SFK) by phosphorylation of YAP (pYAPy357). NXP900 is a first-in-class, highly selective, SFK inhibitor with a novel mechanism of action which locks SFK in its closed and inhibited conformation, in contrast to other Src inhibitors such as dasatinib. A phase 1a study of NXP900 in patients with advanced solid tumors was recently initiated. Here, we examined treatment responses of CCA to NXP900 in vitro and in vivo. Methods: Cell viability was determined in seven CCA cell lines by CellTiter-Glo. Cell death and apoptosis were assayed by PI/Hoechst and Caspase-Glo 3/7 assay. CalcuSyn software was used to determine synergistic drug effects. Immunoblot analysis, RT-PCR, and IF staining of YAP localization were used to evaluate YAP knockdown. Clones of human CCA cell lines resistant to NXP900 were generated through escalating exposure to NXP900 over 6 months. Five patient-derived xenograft (PDX) tumors were expanded into flanks of NOD/SCID mice. Tumor bearing mice were randomized 1:1 with 5 mice per arm and treated with vehicle or NXP900 (40 mg/kg) once daily for up to 4 weeks. To determine drivers of sensitivity and resistance, predicted sensitivity scores based on tumor growth were linked to multi-omics (RNA seq and [phospho-]proteomics) of our PDX models. Results: All cell lines were sensitive to NXP900 with IC50 values between 7nM-15µM; IC50’s of resistant clones were 1000 times higher. NXP900 induced more cell death compared to vehicle, which appeared to be through apoptosis. NXP900 inhibited pSrc, decreased pYAPy357, and upregulated inactive pYAPs127. Correspondingly, decreased YAP target gene (Cyr61, NUAK, and CTGF) levels and a nuclear-to-cytoplasmic translocation were observed. NXP900/GemCis combination therapy increased cell death demonstrated a synergistic effect (Combination Indices <1) at all concentrations. In our PDX models, treatment was associated with a significant decrease in tumor growth in 3 models (mean fold change 3.1 vs. 14.2; 1.0 vs. 7.6; 1.4 vs. 2.6). The major resistant signature was the TRK signaling network, involved in nerve growth factor binding activity, while drivers of sensitivity included the SRC network. Conclusion: NXP900 demonstrated therapeutic activity in vitro and in human PDX models. We are currently performing multi-omic approaches in NXP900 sensitive and resistant cell lines to unravel determinants of activity and resistance. Additional in vivo studies will be performed to determine effects of NXP900/GemCis and NXP900/anti-PDL-1 combination therapy. Citation Format: Hendrien Kuipers, Jennifer L. Tomlinson, Danielle M. Carlson, Amro M. Abdelrahman, Erik Jessen, Jack W. Sample, Nathan W. Werneburg, Hannah E. Stumpf, Mark J. Truty, Sumera I. Ilyas, Gregory J. Gores, Rory L. Smoot. Src family kinase inhibition demonstrates antitumor activity in vitro and in patient-derived xenograft models of human cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 614.

Deregulating fibroblast growth factor receptor (FGFR) signaling is a promising strategy for cancer therapy. Herein, we report the discovery of compound 5 (TAS-120, futibatinib), a potent and selective covalent inhibitor of FGFR1-4, starting from a unique dual inhibitor of mutant epidermal growth factor receptor and FGFR (compound 1). Compound 5 inhibited all four families of FGFRs in the single-digit nanomolar range and showed high selectivity for over 387 kinases. Binding site analysis revealed that compound 5 covalently bound to the cysteine 491 highly flexible glycine-rich loop region of the FGFR2 adenosine triphosphate pocket. Futibatinib is currently in Phase I-III trials for patients with oncogenically driven FGFR genomic aberrations. In September 2022, the U.S. Food & Drug Administration granted accelerated approval for futibatinib in the treatment of previously treated, unresectable, locally advanced, or metastatic intrahepatic cholangiocarcinoma harboring an FGFR2 gene fusion or other rearrangement.

No abstract available

Cholangiocarcinoma (CCA) is a highly heterogeneous and metastatic malignancy with a poor prognosis even after curative hepatectomy. Studies exploring its pathogenesis and identifying effective therapeutic targets are urgently needed. In this study, we found that TANK-binding kinase 1 (TBK1), a serine/threonine-protein kinase, showed a dynamic increase during the different stages of murine spontaneous CCA carcinogenesis (hyperplasia, dysplasia, and CCA). TBK1 was upregulated in human tissues, including intrahepatic ( n  = 182) and extrahepatic ( n  = 40) CCA tissues, compared with nontumor tissues, and the elevated expression of TBK1 was positively correlated with larger tumour diameter, lymph node metastasis, and advanced TNM stage. Functional studies indicated that TBK1 promoted CCA growth and metastasis both in vitro and in vivo. TBK1 directly interacts with β-catenin, promoting its phosphorylation at the S552 site and its nuclear translocation, which further activates EMT-related transcriptional reprogramming. GSK-8612, a TBK1 inhibitor or a kinase-inactivating mutation, effectively suppresses the above processes. In addition, we found that low-density lipoprotein receptor (LDLR), which mediates the endocytosis of cholesterol, was upregulated in CCA. Therefore, we designed a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting TBK1 (Cho-TBK1-HDO), which could accumulate in CCA cells via LDLR, reduce the TBK1 mRNA level and inhibit intrahepatic metastasis of CCA. Besides, in the experimental group of 182 ICC patients, high TBK1 expression combined with high nuclear β-catenin expression predicted a worse prognosis. In summary, TBK1 might serve as a potential prognostic biomarker and therapeutic target for patients with CCA.

No abstract available

Angiogenesis is critical in intrahepatic cholangiocarcinoma (ICC), a highly lethal cancer with limited treatment options. Sunitinib, a multi‐receptor tyrosine kinase inhibitor, has strong antiangiogenic and antitumor effects. We aimed to evaluate the efficacy and tolerability of sunitinib as a second‐line treatment in chemotherapy‐pretreated patients with advanced ICC.

Intrahepatic cholangiocarcinoma (ICC) is the second most prevalent liver cancer after hepatocellular carcinoma and is characterized by high malignancy and poor prognosis. Gemcitabine combined with cisplatin is the standard first-line therapy for metastatic or unresectable ICC. The combination of immunotherapy and targeted therapy represents a promising new direction for ICC treatment. Common genetic mutations in ICC include those in TP53, FGFR2, IDH1/2, and KRAS. MET alterations such as fusions and amplifications are rare in ICC. However, limited research has been conducted on the efficacy of specific MET inhibitors. We present two cases: the first with refractory ICC treated with a combination of immunotherapy and targeted therapy, harboring a ZKSCAN1-MET fusion and the second with a metastatic ICC with MET amplification. Both patients demonstrated a significant clinical response to crizotinib, a MET-specific tyrosine kinase inhibitor.

Cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) deletions are frequently identified in patients with biliary tract cancer; however, standard treatment options for this genetic alteration are lacking. Here, we present the case of a 64-year-old woman diagnosed with intrahepatic cholangiocarcinoma and hilar lymph node metastasis who underwent radical surgery. Postoperative pathology confirmed moderately differentiated adenocarcinoma. The tumor recurred during the second cycle of adjuvant chemotherapy following surgery, and the metastatic sites included the cranial region, right lung, and right adrenal gland. Genetic analysis revealed a CDKN2A/2B deletion, indicating palbociclib sensitivity. Subsequently, the patient received palbociclib plus lenvatinib as systemic therapy, along with stereotactic radiotherapy for the intracranial lesion. Notably, the right pulmonary metastasis significantly regressed after 12 months of treatment, with the complete disappearance of the intracranial tumor. However, the disease progressed at 32.2 months, with significant enlargement of the right adrenal gland metastasis and new metastasis in the right lung. The progression-free survival and overall survival were 32.2 months and 34.4 months, respectively. In conclusion, our case demonstrates that palbociclib plus lenvatinib is a promising chemotherapy-free second-line treatment for intrahepatic cholangiocarcinoma with a CDKN2A/2B deletion.

circular RNAs (circRNAs) have been reported to exert important effects in the progression of numerous cancers. However, the functions of circRNAs in intrahepatic cholangiocarcinoma (ICC) are still unclear. circPCNXL2 (has_circ_0016956) were identified in paired ICC by circRNA microarray. Then, we assessed the biological functions of circPCNXL2 by CCK8, EdU, clone formation, transwell, wound healing assays, and xenograft models. RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between cirrcPCNXL2 and serine-threonine kinase receptor-associated protein (STRAP). RNA pull-down, RIP and luciferase reporter assays were used to investigate the sponge functions of circPCNXL2. In the end, we explore the effects of circPCNXL2 and trametinib (a MEK1/2 inhibitor) in vivo. circPCNXL2 was upregulated in ICC tissues and cell lines, which promoted the proliferation and metastasis of ICC in vitro and in vivo. In terms of the mechanisms, circPCNXL2 could directly bind to STRAP and induce the interaction between STRAP and MEK1/2, resulting in the tumor promotion in ICC by activation of ERK/MAPK pathways. Besides, circPCNXL2 could regulate the expression of SRSF1 by sponging miR-766-3p and subsequently facilitated the growth of ICC. Finally, circPCNXL2 could partially inhibit the anti-tumor activity of trametinib in vivo. circPCNXL2 played a crucial role in the progression of ICC by interacting with STRAP to activate the ERK signaling pathway, as well as by modulating the miR-766-3p/SRSF1 axis. These findings suggest that circPCNXL2 may be a promising biomarker and therapeutic target for ICC.

Currently, chemotherapy remains the primary treatment modality for advanced intrahepatic cholangiocarcinoma (iCCA). However, the efficacy of existing regimens in patients requiring later-line therapy is limited, with low objective response rates and considerable adverse effects. Therefore, the development of effective and safe novel therapeutic strategies is of critical importance. We report a case of unresectable metastatic iCCA in which first-line therapy with a PD-1 inhibitor combined with a tyrosine kinase inhibitor (TKI) failed. The patient subsequently received second-line treatment with lenvatinib in combination with trifluridine/tipiracil (TAS-102), which resulted in significant tumor shrinkage and a partial response (PR) upon evaluation, without the occurrence of grade 3 or higher adverse events. The progression-free survival (PFS) was 13.33 months. This case suggests that lenvatinib combined with TAS-102 may demonstrate favorable antitumor activity and may represent a promising therapeutic option for advanced iCCA in the later-line setting.

608 Background: Tinengotinib, a spectrum-selective multi-kinase inhibitor with unique binding properties to FGFR , potently inhibited FGFR2 fusion/rearrangement and acquired resistant/gatekeeper mutations in pre-clinical models and exhibited antitumor activity in cholangiocarcinoma (CCA) patients (pts) in phase 1/2 trials. Here we present the overall survival (OS) data and biomarker correlative analysis from a phase 2 clinical trial of tinengotinib in CCA. Methods: Eligible pts with advanced/metastatic CCA who had received ≥ 1 prior systemic chemotherapy with ECOG PS 0-1 were treated with tinengotinib 10 mg QD across four Cohorts: A1: FGFR2 fusion(s) with primary progression on previous FGFR inhibitor ( FGFR i); A2: FGFR2 fusion(s) with progression after prior response to FGFR i; B: FGFR alteration(s) without FGFR2 fusion(s); C: FGFR wild-type. Efficacy was evaluated per RECIST v1.1 and safety was assessed by CTCAE v5.0. Genomic alterations in circulating tumor DNA (ctDNA) were assessed by Foundation Medicine NGS panel at baseline, C3D1 and EOT. Planned correlative analyses of response (PR+SD>6 months) and PFS in pt subgroups based on ctDNA mutational status were performed. Results: 55 eligible pts were enrolled (18 in A1, 11 in A2, 13 in B, 13 in C) with median age 61.0 [range 24-81] years, ECOG PS 0 in 50.9% pts, 56.4% female, and 60.0% with ≥ 3 lines of prior therapy. Among 42 pts with FGFR alterations, 78.4% had ≥ 1 prior FGFR i, and 97.6% had prior chemotherapy. Median follow up time was 8.6 months (0.4-30.5). The median OS (months) was 17.1 (95%CI 7.5-19.5) in A1; not reached (95%CI 9.6, -) in A2; 18.0 (95%CI 9.6, -) in A1+A2; not reached (95%CI 8.0, -) in B; 6.5 (95%CI 4.8-16.4) in C. The OS rate in A, B and C was 100%, 91.7% and 75% at month 3; 83.7%, 83.3% and 66.7% at month 6, and 65.8%, 55.6% and 23.8% at month 12. Partial response was observed in 26% (10/39) of evaluable FGFR-altered pts (A1+A2+B). The safety profile was consistent with previous reports. The most common any-grade treatment-related AEs were hypertension (63.6%) and diarrhea (47.3%). Baseline ctDNA results were available in 46 pts, 35 in cohorts A+B and 11 in C. A positive correlation was shown in MED12 mutation vs response (P=0.03476). BCOR and ARID1A mutation were found to be negatively associated with PFS (P=0.01366 and P=0.0437). Acquired/secondary mutations at baseline were observed in cohort A, including V564F/I (3/24 and 5/24) and N549K/H (5/24 and 5/24); favorable anti-tumor activity was noted despite these resistant mutations, consistent with preclinical data. Conclusions: Tinengotinib has shown promising anti-tumor efficacy in CCA pts with FGFR fusion after prior FGFR i and in those with primary FGFR mutations. An ongoing randomized phase III study will investigate tinengotinib vs. chemotherapy in FGFR inhibitor refractory CCA (NCT05948475). Clinical trial information: NCT04919642 .

BACKGROUND AND AIMS Cholangiocarcinoma (CCA) is one of the most lethal cancers, characterized by molecular heterogeneity and treatment resistance. To uncover new biological signals and therapeutic opportunities, we employed multiomic characterization combined with machine learning. APPROACH AND RESULTS We profiled all anatomical CCA subtypes using whole exome sequencing, mRNA sequencing, and proteome/phosphoproteome analysis. Integrative dimensional reduction revealed RNA, protein, and phosphoprotein features driving tumor heterogeneity, enabling clustering. Machine learning algorithms identified molecular features for each cluster and mapped external datasets and patient-derived xenograft (PDX) models onto these clusters. Kinase enrichment analysis highlighted targetable kinases active in each cluster. In vivo validation was performed in cluster-specific PDX models using the selective TNK1 inhibitor, TP-5801. We identified three molecular clusters with distinct pathway characterization: immunomodulatory (cluster 1), metabolic (cluster 2), and gene regulation/cellular fate (cluster 3). Cluster assignment was independent of anatomic subtype but correlated with overall survival following curative-intent resection. We also identified multiomic features and pathways linked to overall survival and lymph node metastases, crucial for patient treatment selection. Kinase enrichment analysis pinpointed TNK1 as a highly active kinase in the metabolic cluster. Treatment with TP-5801 significantly reduced tumor growth in a metabolic PDX model, but not in models representing the other clusters. Combining internal data with publicly available datasets, we identified the immunomodulatory cluster as most responsive to gemcitabine/cisplatin therapy, confirmed in vivo using cluster-specific PDX models. CONCLUSIONS Integrated multiomic characterization provides translational insights by defining unique molecular subtypes associated both with therapeutic response and overall clinical outcomes. This approach identified TNK1 as a previously unrecognized therapeutic target in a defined subset of CCA tumors.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy that arises from the biliary tree, and often presents with advanced stage disease. For patients with disease arising from intrahepatic bile ducts, genomic analyses have revealed targetable mutations. Unfortunately, these mutations are exceedingly rare in extrahepatic disease (eCCA), limiting therapeutic options. It is therefore necessary to investigate the functional crosstalk resulting from different signal transduction pathways to identify new treatment options for this patient population. IHC analysis of eCCA tumors from 170 patients demonstrated intense Exportin-7 (XPO7) cytoplasmic staining in 30% of the samples, which correlated with abbreviated survival. With the co-immunoprecipitation, proteomic analysis and molecular docking, we demonstrated that XPO7 exists in a molecular complex with the Ste-20 like kinase (SLK) and beta-tubulin in the cytoplasm. A kinome screen identified tivozanib, a potent VEGFR2 inhibitor, as a moderate inhibitor of SLK kinase (IC50 - 36 nM @ 10 µM ATP or 2.2 µM @ 1 mM ATP) and cellular target engagement (NanoBRET) of 884 nM. The X-ray crystallography results confirmed that tivozanib binds to the SLK ATP-binding pocket, with the DGF motif in the inactive “DGF-out” conformation in the N-terminal region. In in vitro experiments, shRNA-mediated knockdown of SLK and tivozanib treatment in cholangiocarcinoma cell lines showed reduced spheroid forming capacity with downregulation of PI3K-AKT-mTOR pathway. This resulted in reduction in expression of phosphorylated mTOR (S2448), AKT (T308), and S6 (S235/236). SLK KD and tivozanib treatment also resulted in upregulation of ATM-dependent DNA damage pathway in cholangiocarcinoma cell lines with resulting downregulation of beta-tubulin and G2/M cell cycle arrest confirming its potential role in impacting the cellular processes. Moreover, tivozanib regressed tumors in established eCCA patient derived xenografts with cytoplasmic XPO7 expression. We further confirmed our results in our ex vivo system by treating eCCA patient tumor slice cultures with tivozanib. The IHC and Western blotting results confirmed the significant reduction of PI3K-AKT-mTOR pathway in high XPO7/SLK-expressing tumors (N=3) compared to the lower expression pattern (N=7). Lastly, tivozanib monotherapy demonstrated in vivo efficacy in the patients with high XPO7 expression with tumor regression and arrest of tumor progression (RECIST) in an ongoing clinical trial (NCT04645160). Priyanka Prakash Desai, Surajit Sinha, Emily Smith, Reed I. Ayabe, Pedro Torres-Ayuso, Ashley Rainey, Himanshu Verma, Kirsten Remmert, Tracey Pu, Craig Thomas, John Brognard, William J. Moore, Gregory Tawa, Thorkell Andresson, David Kleiner, Michael B. Yaffe, Jonathan M. Hernandez. Cytoplasmic complex of SLK-XPO7: A targetable player in extrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6915.

Background/Aim: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. Materials and Methods: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. Results: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. Conclusion: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Cholangiocarcinoma (CCA) is a lethal cancer originating from the epithelial cells within the bile duct and ranks as the second most prevalent form of liver cancer in Thailand. Polo-like kinase 1 (PLK1), a protein serine/threonine kinase, regulates a number of steps in cell mitosis and is upregulated in several types of cancer, including CCA. Our previous study identified PLK1 as a biomarker of the C1 subtype, correlating with poor prognosis in intrahepatic CCA. The present study aimed to examine the effect of PLK1 inhibition on CCA cells. Different CCA cell lines developed from Thai patients, HuCCA1, KKU055, KKU100 and KKU213A, were treated with two PLK1 inhibitors, BI2536 and BI6727, and were transfected with small interfering RNA, followed by analysis of cell proliferation, cell cycle distribution and cell apoptosis. It was discovered that BI2536 and BI6727 inhibited cell proliferation and caused G2/M-phase arrest in CCA cells. Furthermore, the number of total apoptotic cells was increased in PLK1 inhibitor-treated CCA cells. The expression levels of mitotic proteins, aurora kinase A, phosphorylated PLK1 (T210) and cyclin B1, were augmented in PLK1-inhibited CCA cells. Additionally, inhibition of PLK1 led to increased DNA damage, as determined by the upregulated levels of γH2AX and increased cleavage of poly (ADP-ribose) polymerase, an apoptotic marker. These results suggested that inhibiting PLK1 prolonged mitotic arrest and subsequently triggered cell apoptosis. Validation of the antiproliferative effects of PLK1 inhibition was accomplished through silencing of the PLK1 gene. In conclusion, targeting PLK1 provided promising results for further study as a potential candidate for targeted therapy in CCA.

No abstract available

Abstract Purpose: FGFR inhibitors are effective in FGFR2-altered cholangiocarcinoma, leading to approval of reversible FGFR inhibitors, pemigatinib and infigratinib, and an irreversible inhibitor, futibatinib. However, acquired resistance develops, limiting clinical benefit. Some mechanisms of resistance have been reported, including secondary FGFR2 kinase domain mutations. Here, we sought to establish the landscape of acquired resistance to FGFR inhibition and to validate findings in model systems. Experimental Design: We examined the spectrum of acquired resistance mechanisms detected in circulating tumor DNA or tumor tissue upon disease progression following FGFR inhibitor therapy in 82 FGFR2-altered cholangiocarcinoma patients from 12 published reports. Functional studies of candidate resistance alterations were performed. Results: Overall, 49 of 82 patients (60%) had one or more detectable secondary FGFR2 kinase domain mutations upon acquired resistance. N550 molecular brake and V565 gatekeeper mutations were most common, representing 63% and 47% of all FGFR2 kinase domain mutations, respectively. Functional studies showed different inhibitors displayed unique activity profiles against FGFR2 mutations. Interestingly, disruption of the cysteine residue covalently bound by futibatinib (FGFR2 C492) was rare, observed in 1 of 42 patients treated with this drug. FGFR2 C492 mutations were insensitive to inhibition by futibatinib but showed reduced signaling activity, potentially explaining their low frequency. Conclusions: These data support secondary FGFR2 kinase domain mutations as the primary mode of acquired resistance to FGFR inhibitors, most commonly N550 and V565 mutations. Thus, development of combination strategies and next-generation FGFR inhibitors targeting the full spectrum of FGFR2 resistance mutations will be critical.

4121 Background: Erdafitinib is an oral selective pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor approved in the US for the treatment of adult pts with locally advanced or metastatic urothelial carcinoma with susceptible FGFR3 alterations whose disease has progressed on or after ≥1 line of prior systemic therapy. Primary analysis of the RAGNAR study demonstrated tumor agnostic efficacy in patients with solid tumors harboring susceptible FGFR mutations or fusions (1). Results from the LUC2001 study in patients with cholangiocarcinoma were previously presented (2). Here we report on the pooled analysis of patients with cholangiocarcinoma treated in the RAGNAR and LUC2001 studies. Methods: RAGNAR (NCT04083976) and LUC2001 (NCT02699606) enrolled patients with advanced solid tumors after ≥1 prior lines of therapy. RAGNAR patients had exhausted standard of care therapies; LUC2001 enrolled patients only in China, Taiwan, and South Korea. Patients received erdafitinib (8 mg daily, optional up-titration) until disease progression or toxicity. Patients with cholangiocarcinoma and predefined FGFR alterations were pooled into an analysis of efficacy (objective response rate by Independent Review Committee [IRC], duration of response, progression free survival, overall survival) and safety. Results: At data cutoff, 78 patients had received erdafitinib (RAGNAR: 66; LUC2001: 12). Median efficacy follow-up was 15 months. Median (range) age was 56 years (24;77); 60% female, 47% White, 39% Asian. Patients had a median of 2 prior lines of therapy; 92% patients had visceral metastases, and 17% of patients responded to prior therapy. 94% of patients had FGFR2 alterations, and 91% of patients had fusions. Objective response rate by IRC was 55%. Responses were observed in patients with both, fusions or mutations. Median time to onset of response was 1.7 month; median duration of response, progression free survival, and overall survival were 6.9 (95% CI: 4.37, 8.61), 8.5 (95% CI: 6.83, 9.72), and 18.1 (95% CI: 13.40, 24.28) months, respectively. Most common treatment-emergent adverse events (TEAEs) were hyperphosphatemia (83%), stomatitis (72%), diarrhea (68%), dry mouth (51%), palmar-plantar erythrodysesthesia (51%); 42% of patients had serious TEAEs and 12% discontinued treatment due to an AE. No treatment-related deaths were observed. Conclusions: Data from pooled analysis of the RAGNAR and LUC2001 studies confirm robust efficacy of erdafitinib in a diverse population of patients with advanced or metastatic cholangiocarcinoma and prespecified FGFRfusions or mutations. Safety data were consistent with the erdafitinib safety profile. 1. Pant 2023. 2. Feng 2022. Clinical trial information: NCT04083976 and NCT02699606 .

Cholangiocarcinoma (CCA) is a cancer with a poor prognosis due to difficulties in diagnosis and limited treatment options, highlighting the urgent need for new targeted therapies. In a clinical setting, we found that leukotriene levels in bile were higher than in serum. Immunohistochemical analysis of surgically resected samples also revealed that CysLT receptor 1 (CysLTR1) was more highly expressed in CCA than in normal bile duct tissue, prompting us to investigate leukotriene as a potential therapeutic target in CCA. In vitro studies using CCA cell lines expressing CysLTR1 showed that leukotriene D4, a major ligand of CysLTR1, promoted cell proliferation, with increased phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Additionally, treatment with two clinically available anti-allergic drugs—zileuton, an inhibitor of CysLT formation, and montelukast, a CysLTR1 inhibitor—had inhibitory effects on cell proliferation and migratory capacity, accompanied by the reduced phosphorylation of AKT and ERK1/2. Furthermore, the simultaneous administration of both drugs synergistically enhanced the inhibitory effect on cell proliferation. Our study suggests that use of these drugs may represent a novel approach to treat CCA through drug repositioning.

Background Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver malignancy with a steadily increasing incidence worldwide. ICC has insidious onset, rapid progression, and poor prognosis. More multidisciplinary clinical studies are needed to continuously explore safer and more efficient diagnosis and treatment modes for ICC. Methods and results A 66-year-old female patient with ICC rapidly developed systemic multiple metastases after surgery, and the first-line two-drug combination chemotherapy was not effective. Due to cyclin-dependent kinase inhibitor 2A mutation and programmed cell death-ligand 1-positive, a partial response and progression-free survival of 9.5 months were achieved after a second-line treatment with cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) combined with immunotherapy. The patient developed thromboembolism 7 months after treatment and died due to disseminated intravascular coagulation. Conclusion The combination of targeted and immune therapy has revealed a potentially effective regimen for the effective treatment of patients with ICC, which needs to be observed in larger clinical studies. The thromboembolism rates in real-world patients treated with CDK4/6 inhibitors are higher than those reported in clinical trials, and the application of prophylactic anticoagulation in this patient population may be questionable.

Fibroblast growth factor receptors (FGFRs) are well-established oncology targets, with aberrant FGFR2 and FGFR3 activation implicated in multiple tumor types, including cholangiocarcinoma and urothelial carcinoma. Currently approved FGFR2/3-targeted therapies rely on pan-FGFR small-molecule kinase inhibitors, which often lead to off-target toxicities due to unintended inhibition of FGFR1 and FGFR4, as well as acquired resistance driven by gatekeeper mutations. Herein, we report the discovery of INCB126503, a highly potent, orally bioavailable FGFR2/3 inhibitor with excellent isoform selectivity and equipotent activity against gatekeeper mutants. INCB126503 effectively suppresses FGFR signaling in vivo without inducing hyperphosphatemia and demonstrates significant antitumor efficacy in xenograft models harboring FGFR3 genetic alterations.

Alterations in fibroblast growth factor receptors (FGFRs) are recognized as oncogenic drivers in numerous human cancers. Several FGFR inhibitors, with similar potencies against FGFR1-3 or FGFR1-4, have been approved and demonstrated modest clinical benefits in FGFR-altered urothelial carcinoma and bile duct cancer. The clinical benefit was limited partly due to FGFR1- and FGFR4-mediated side effects. In addition, FGFR2/3 acquired resistance mutations after initial response to pan-FGFR inhibitor limit the long-term clinical efficacy as well. 60% of patients develop these secondary FGFR2 kinase domain mutations in FGFR2-altered cholangiocarcinoma, and 13-25% of patients develop FGFR3 resistance mutations in urothelial carcinoma. ASN-8639 and ASN-7350 were designed to address these challenges in treating multiple solid tumors harboring FGFR2/3 alterations. ASN-7350 is a novel, highly selective, and potent non-covalent FGFR2/3 inhibitor (IC50 < 5 nM in kinase assays). It showed greater than 200-fold selectivity for FGFR3 over FGFR1/4 in cellular assays. The prodrug ASN-8639 is in preclinical developed to improve the pharmacokinetic profile of ASN-7350. ASN-8639 was efficiently converted to ASN-7350 and showed dose-proportional increases in exposure and clinically acceptable oral bioavailability across four different species. ASN-7350 was more potent in inhibiting FGFR3 wt/mut signaling than competitor compounds. Its cell killing potency was 10× stronger compared to a reference compound currently in clinical development in cell lines harboring FGFR3-TACC3 fusion or erdafitinib-resistant FGFR3-V555M mutation, and meanwhile with a better the cellular selectivity of FGFR3 over FGFR1 and FGFR4. ASN-8639 demonstrated significant anti-tumor efficacy in cancer model of RT112 with FGFR3-TACC3 fusion and erdafitinib-resistant mutations of FGFR3-V555M. ASN-7350 also exhibited high potency against FGFR2. It produced comparable activity to a reference FGFR2 inhibitor in clinical development in multiple FGFR2-altered cell lines. Notably, ASN-8639 was more efficacious than reference FGFR2 inhibitor in the endometrial tumor model with erdafitinib-resistant FGFR2-N549K mutation at the same dose. These findings support the further clinical development of ASN-8639 as a potential best-in-class FGFR2/3 dual inhibitor for the treatment of bladder, endometrial, and other solid tumors with FGFR2/3 aberrations. Qiaoling Sun, Jingmin Guan, Haifeng Song, Jing Lv, Peng Zeng, Mengyuan Wang, Yonghui Yu, Shuai Luo, Jinna Yu, Liang Wu, Tielin Wang, Alice Chen. ASN-7350, a highly selective FGFR2/3 dual inhibitor, for FGFR2/FGFR3 driven solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3014.

Abstract Purpose: Despite efficacy of approved FGFR inhibitors, emergence of polyclonal secondary mutations in the FGFR kinase domain leads to acquired resistance. KIN-3248 is a selective, irreversible, orally bioavailable, small-molecule inhibitor of FGFR1-4 that blocks both primary oncogenic and secondary kinase domain resistance FGFR alterations. Experimental Design: A first-in-human, phase I study of KIN-3248 was conducted in patients with advanced solid tumors harboring FGFR2 and/or FGFR3 gene alterations (NCT05242822). The primary objective was determination of MTD/recommended phase II dose (RP2D). Secondary and exploratory objectives included antitumor activity, pharmacokinetics, pharmacodynamics, and molecular response by circulating tumor DNA (ctDNA) clearance. Results: Fifty-four patients received doses ranging from 5 to 50 mg orally daily across six cohorts. Intrahepatic cholangiocarcinoma (48.1%), gastric (9.3%), and urothelial (7.4%) were the most common tumors. Tumors harbored FGFR2 (68.5%) or FGFR3 (31.5%) alterations—23 (42.6%) received prior FGFR inhibitors. One dose-limiting toxicity (hypersensitivity) occurred in cohort 1 (5 mg). Treatment-related, adverse events included hyperphosphatemia, diarrhea, and stomatitis. The MTD/RP2D was not established. Exposure was dose proportional and concordant with hyperphosphatemia. Five partial responses were observed; 4 in FGFR inhibitor naïve and 1 in FGFR pretreated patients. Pretreatment ctDNA profiling confirmed FGFR2/3 alterations in 63.3% of cases and clearance at cycle 2 associated with radiographic response. Conclusion: The trial was terminated early for commercial considerations; therefore, RP2D was not established. Preliminary clinical data suggest that KIN-3248 is a safe, oral FGFR1-4 inhibitor with favorable pharmacokinetic parameters, though further dose escalation was required to nominate the MTD/RP2D. Significance: KIN-3248 was a rationally designed, next generation selective FGFR inhibitor, that was effective in interfering with both FGFR wild-type and mutant signaling. Clinical data indicate that KIN-3248 is safe with a signal of antitumor activity. Translational science support the mechanism of action in that serum phosphate was proportional with exposure, paired biopsies suggested phospho-ERK inhibition (a downstream target of FGFR2/3), and ctDNA clearance may act as a RECIST response surrogate.

The oral kinase inhibitor futibatinib (Lytgobi – Taiho) has received accelerated approval from the FDA for treatment of adults with previously treated, unresectable, locally advanced or metastatic intrahepatic cholangiocarcinoma with fibroblast growth factor receptor 2 (FGFR2) fusions or other rearrangements. Accelerated approval was based on the overall response rate and duration of response. Futibatinib is the second drug to be approved for this indication; pemigatinib (Pemazyre) was approved earlier.

Simple Summary Cholangiocarcinoma (CCA) is an architecturally complex and highly heterogeneous tumour and is difficult to diagnose at early stages due to its late onset and asymptomatic nature. Recent studies have focused on the Fibroblast Growth Factor Receptors (FGFRs), a sub-family of Tyrosine Kinase Receptors (RTKs), as promising targets for therapy. Pemigatinib, a small-molecule inhibitor of FGFR, was the first FDA-approved targeted therapy drug for CCA patients with FGFR2 fusions who had failed first-line chemotherapy. However, only a limited cohort of patients benefit from this therapeutic strategy as the mutation does not necessarily correlate to the response. In this study, aberrant FGFR expression in CCA samples was found using a bioinformatics approach and further confirmed by immunohistochemistry. PD173074, a selective pan-FGFR inhibitor, was found to be sensitive to CCA cell lines with FGFR expression, suggesting that it can be used to suppress CCA cells even without the FGFR2 fusions. Moreover, correlation analysis of publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors and dual inhibition of PD173074 and erlotinib was found to be synergistic in CCA using cell lines and patient-derived complex models. Thus, this study suggests further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of CCA patients and novel therapeutic strategies involving dual inhibition of FGFRs and EGFR. Abstract Cholangiocarcinoma (CCA) is an architecturally complex tumour with high heterogeneity. Discovery at later stages makes treatment challenging. However, the lack of early detection methodologies and the asymptomatic nature of CCA make early diagnosis more difficult. Recent studies revealed the fusions in Fibroblast Growth Factor Receptors (FGFRs), a sub-family of RTKs, as promising targets for targeted therapy for CCA. Particularly, FGFR2 fusions have been of particular interest, as translocations have been found in approximately 13% of CCA patients. Pursuing this, Pemigatinib, a small-molecule inhibitor of FGFR, became the first targeted therapy drug to be granted accelerated approval by the FDA for treating CCA patients harbouring FGFR2 fusions who have failed first-line chemotherapy. However, despite the availability of Pemigatinib, a very limited group of patients benefit from this treatment. Moreover, as the underlying mechanism of FGFR signalling is poorly elucidated in CCA, therapeutic inhibitors designed to inhibit this pathway are prone to primary and acquired resistance, as witnessed amongst other Tyrosine Kinase Inhibitors (TKIs). While acknowledging the limited cohort that benefits from FGFR inhibitors, and the poorly elucidated mechanism of the FGFR pathway, we sought to characterise the potential of FGFR inhibitors in CCA patients without FGFR2 fusions. Here we demonstrate aberrant FGFR expression in CCA samples using bioinformatics and further confirm phosphorylated-FGFR expression in paraffinised CCA tissues using immunohistochemistry. Our results highlight p-FGFR as a biomarker to guide FGFR-targeted therapies. Furthermore, CCA cell lines with FGFR expression were sensitive to a selective pan-FGFR inhibitor, PD173074, suggesting that this drug can be used to suppress CCA cells irrespective of the FGFR2 fusions. Finally, the correlation analysis utilising publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors as they are significantly co-expressed. Accordingly, dual inhibition of FGFRs and EGFR by PD173074 and EGFR inhibitor erlotinib was synergistic in CCA. Hence, the findings from this study provide support for further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of patients. Altogether, this study shows for the first time the potential of FGFRs and the importance of dual inhibition as a novel therapeutic strategy in CCA.

Patients with intrahepatic cholangiocarcinoma (iCCA) harboring FGFR2 fusion/rearrangement benefit from targeted therapies, highlighting the need for reliable testing strategies to identify FGFR2 alterations. We assessed 226 iCCA cases using RNA-based NGS, DNA-based NGS, and break-apart FISH to evaluate the effectiveness of these methods in detecting FGFR2 fusion/rearrangement. The detection rates for FGFR2 fusion/rearrangement were 9.7% (22/226) for RNA-based NGS, 7.1% (16/226) for DNA-based NGS, and 10.2% (23/226) for FISH. Among the 26 FGFR2 fusion/rearrangement-positive cases identified by any method, only 15 (57.7%) were positive by all three techniques, yielding a concordance rate of 95.1% (215/226). RNA-based NGS confirmed oncogenic FGFR2 fusion in 81% (21/26) of positive cases and identified five novel oncogenic fusions. Thirty-five percent (6/17) of the partner genes were located on chromosome 10, with BICC1 being the most common fusion partner, while the rest were distributed across the other 9 chromosomes. FISH demonstrated a sensitivity of 95.2% and specificity of 98.5%, compared to oncogenic FGFR2 fusions confirmed by RNA-based NGS, while DNA-based NGS exhibited a sensitivity of 71.4% and specificity of 99.5%, identifying FGFR2 mutations in 4 cases. FGFR2-FISH positive cases displayed no significant heterogeneity in positive cell distribution. Oncogenic FGFR2 fusion/rearrangement was associated with small duct type iCCA, especially in cases with positive serum HBsAg and absent cholangiolocarcinoma components and peripheral liver steatosis. This study provides a comprehensive comparison of three assays for detecting FGFR2 fusion/rearrangement, along with clinicopathologic characterization of oncogenic FGFR2 fusion in iCCA.

Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and predict response to FGFR kinase inhibitors. However, response rates and durability are limited due to the emergence of resistance, typically involving FGFR2 kinase domain mutations, and to suboptimal dosing, relating to drug adverse effects. Here, we develop biparatopic antibodies targeting the FGFR2 extracellular domain (ECD) as candidate therapeutics. Biparatopic antibodies can overcome drawbacks of bivalent monospecific antibodies, which often show poor inhibitory or even agonist activity against oncogenic receptors. We show that oncogenic transformation by FGFR2 fusions requires an intact ECD. Moreover, by systematically generating biparatopic antibodies targeting distinct epitope pairs in FGFR2 ECD, we identified antibodies that effectively block signaling and malignant growth driven by FGFR2 fusions. Importantly, these antibodies demonstrate efficacy in vivo, synergy with FGFR inhibitors, and activity against FGFR2 fusions harboring kinase domain mutations. Thus, we believe that biparatopic antibodies may serve as an innovative treatment option for patients with FGFR2-altered cholangiocarcinoma.

Background/Aim: Cholangiocarcinoma (CCA) is an aggressive tumor with limited treatment options especially in 2nd line or later treatments. Targeting fibroblast growth factor receptor (FGFR) 2 has recently emerged as a promising treatment option for patients with CCA harboring FGFR2-fusion. This study investigated the antitumor activities of tasurgratinib as an orally available FGFR1-3 inhibitor, in preclinical FGFR2-driven CCA models. Materials and Methods: Antitumor activities of tasurgratinib were examined in vitro and in vivo using NIH/3T3 cells expressing FGFR2-fusion as FGFR2-driven CCA models, and in vivo using a CCA patient-derived xenograft model. The molecular mechanism of action of tasurgratinib was elucidated through co-crystal structure analysis with FGFR1, manual complex model analysis with FGFR2, and binding kinetics analysis with FGFR2. Furthermore, the cell-based inhibitory activities against acquired resistant FGFR2 mutations in patients with CCA treated with FGFR inhibitors were evaluated. Results: Tasurgratinib showed antitumor activity in preclinical FGFR2-driven CCA models by inhibiting the FGFR signaling pathway in vitro and in vivo. Furthermore, cell-based target engagement assays indicated that tasurgratinib had potent inhibitory activities against FGFR2 mutations, such as N549H/K, which are the major acquired mutations in CCA. We also confirmed that tasurgratinib exhibited fast association and slow dissociation kinetics with FGFR2, binding to the ATP-binding site and the neighboring region, and adopting an Asp-Phe-Gly (DFG)-“in” conformation. Conclusion: These data demonstrate the therapeutic potential of tasurgratinib in FGFR2-driven CCA and provide molecular mechanistic insights into its unique inhibitory profile against secondary FGFR2 resistance mutations in patients with CCA treated with FGFR inhibitors.

Abstract Increasing evidence highlights that fibroblast growth factor receptor 2 (FGFR2) fusion/rearrangement shows important therapeutic value for patients with intrahepatic cholangiocarcinoma (ICC). This study aims to explore the association of FGFR2 status with the prognosis and immune cell infiltration profiles of patients with ICC. A total of 226 ICC tissue samples from patients who received surgery at the Department of Liver Surgery at Zhongshan Hospital, Fudan University, were collected retrospectively and assigned to a primary cohort (n = 152) and validation cohort (n = 74) group. Fluorescence in situ hybridization was performed to determine FGFR2 status. Multiplex immunofluorescence (mIF) staining and immunohistochemistry were performed to identify immune cells. Thirty-two (14.2%) ICC tissues presented with FGFR2 fusion/rearrangement. FGFR2 fusion/rearrangement was associated with low levels of carcinoembryonic antigen (CEA, P = .026) and gamma glutamyl transferase (γ-GGT, P = .003), low TNM (P = .012), CNLC (P = .008) staging as well as low tumor cell differentiation (P = .016). Multivariate COX regression analyses revealed that FGFR2 fusion/rearrangement was an independent protective factor for both overall survival (OS) and relapse-free survival in patients with ICC. Furthermore, correlation analysis revealed that an FGFR2 fusion/rearrangement was associated with low levels of Tregs and N2 neutrophils and high levels of N1 neutrophils infiltrating into tumors but not with CD8+ T-cell or macrophage tumor infiltration. FGFR2 fusion/rearrangement may exert a profound impact on the prognosis of ICC patients and reprogram the tumor microenvironment to be an immune-activated state. FGFR2 status may be used for ICC prognostic stratification and as an immunotherapeutic target in patients with ICC.

Abstract Multiple FGFR inhibitors have demonstrated significant activity in pretreated advanced FGFR2 fusion–positive intrahepatic cholangiocarcinoma. The irreversible pan-FGFR inhibitor futibatinib has the potential to overcome acquired resistance to ATP-competitive FGFR inhibitors in a subset of patients. We present a case of prolonged clinical benefit using FGFR inhibitors sequentially, initially an ATP-competitive inhibitor followed by futibatinib upon progression, for a total of 36 months of FGFR-targeting therapy. This case supports sequential FGFR-targeting therapies for FGFR2 fusion–positive cholangiocarcinoma, with futibatinib acting as rescue therapy after failure of ATP-competitive inhibitors.

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BACKGROUND & AIMS There is a knowledge gap in understanding mechanisms of resistance to FGFR inhibitors (FGFRi) and a need for novel therapeutic strategies to overcome it. We investigated mechanisms of acquired resistance to FGFRi in FGFR2-fusion-positive cholangiocarcinoma (CCA) patients. METHODS A retrospective analysis of patients, who received FGFR inhibitor therapy and had tumor and/or cfDNA analysis before and after treatment, was performed. Longitudinal ctDNA samples from a cohort of patients in the phase I trial of futibatinib (NCT02052778) were assessed. FGFR2-BICC1 fusion cell lines were developed to evaluate the sensitivity of FGFR inhibitors in vitro followed by introduction of secondary acquired resistance mutations in the MAPK pathway. RESULTS On retrospective analysis of 17 patients with repeat sequencing following FGFRi treatment, 11 (64.7%) had new FGFR2 mutations detected, 9 (52.9%) had new alterations in MAPK pathway genes, 7 of these patients developing new alterations in both FGFR2 and MAPK pathway. In serially collected plasma samples, a patient treated with an irreversible FGFRi tested positive for previously undetected BRAF V600E, NRAS Q61 K, NRAS G12C, NRAS G13D and KRAS G12 K mutations upon progression. Introduction of FGFR2-BICC1 into biliary tract cells in vitro sensitized the cells to FGFRi, while concomitant KRAS G12D or BRAF V600E conferred resistance. MEK inhibition (MEKi) was synergistic with FGFRi in vitro. In an in vivo animal model, the combination had antitumor activity in FGFR2 fusions but was not able overcome KRAS-mediated FGFRi resistance. CONCLUSIONS These findings suggest convergent genomic evolution in the MAPK pathway may be a potential mechanism of acquired resistance to FGFRi. IMPACT AND IMPLICATIONS We evaluated tumors and plasma from patients who previously received inhibitors of Fibroblast Growth Factor Receptor (FGFR), an important receptor that plays a role in cancer cell growth, especially in tumors with abnormalities in this gene such as FGFR fusions, where the FGFR gene is fused to another gene, leading to activation of cancer cell growth. We found that patients treated with FGFR inhibitors may develop mutations in other genes such as KRAS, and this can confer resistance to FGFR inhibitors. CLINICAL TRIAL NUMBER NCT02052778.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA), an extremely rare and underinvestigated subtype of primary liver cancer in children, generally has a poor prognosis and greater aggressiveness. Histological diagnosis of cHCC-CCA is difficult because of its diverse components, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). cHCC-CCA shares some genetic alterations with HCC and CCA. However, only a few studies on genetic alterations in fibroblast growth factor receptor 2 (FGFR2) in cHCC-CCAs have been reported in adults. Therapeutic strategies for cHCC-CCAs are limited, and surgical resection is the only standard of care. No standard systemic treatment has been established for unresectable cHCC-CCAs. Herein, we report a rare case of a 14-year-old female patient diagnosed with unresectable cHCC-CCA with multiple liver masses and metastases to the lungs, lymph nodes and peritoneum. Next-generation sequencing (NGS) has identified an FGFR2-PRDM16 fusion, which has not been previously reported as a common FGFR2 fusion. The blood tumour markers alpha-fetoprotein (AFP) and carbohydrate antigen 19 - 9 (CA19 - 9) were both elevated. The patient was treated with pemigatinib (a selective FGFR inhibitor) in combination with Gemcitabine and Cisplatin at our hospital. After three cycles of the combination therapy, the patient achieved a partial response and normalization of tumor markers. After seven cycles of combination therapy, the patient achieved stable disease with the best response. Subsequently, the patient was administered received pemigatinib and gemcitabine. As of the last follow-up date, the patient has survived for 26 months. To the best of our knowledge, this is the first reported rare case of unresectable cHCC-CCA with FGFR2-PRDM16 fusion in a child successfully treated with a combination of pemigatinib and chemotherapy as a first-line regimen. This treatment combination may be effective and safe for patients with unresectable cHCC-CCAs.

Background FGFR2 fusions or rearrangements occur in 13%–20% of patients with intrahepatic cholangiocarcinoma (iCCA). Pemigatinib, a representative FGFR inhibitor, is commonly used for targeted therapy in such patients. Additionally, brain metastasis (BM) is extremely rare in advanced iCCA, and there is currently no standard treatment strategy for advanced iCCA patients with BM. Stereotactic body radiation therapy (SBRT) combined with immune checkpoint inhibitors (ICIs) may exhibit synergistic antitumor effects, presenting a promising approach for advanced iCCA. Case presentation The patient, a 58-year-old male, experienced a recurrence of iCCA following surgery and chemotherapy, with multiple metastases in the liver, lungs, and brain. Genetic testing revealed FGFR2-TXLNG-fusion, and the patient was treated with pemigatinib in combination with tislelizumab and SBRT for the BM, resulting in significant tumor shrinkage. Adverse events (AEs) such as liver dysfunction, nail loss, and dry mouth were observed during treatment, which were considered to be related to pemigatinib. These AEs were significantly alleviated after dose reduction and symptomatic treatment. Conclusion This case presented a rare occurrence of FGFR2 fusion-positive iCCA with BM, with extremely limited data on treatment options and survival outcomes in such patients. Our study was the first to report the application of the treatment strategy combining pemigatinib with ICI and SBRT in this specific case. The combined therapy proved effective and well-tolerated, providing new insights for future treatment considerations.

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The liquid CGP was useful for detecting FGFR2 fusion and the patient experienced typical side effects (nail disorders, hyperphosphatemia, and taste disorders) of pemigatinib that required treatment.

Background Cholangiocarcinoma during postpartum or pregnancy is a rare presentation. There are limited cases reported in the literature. Diagnosis can be delayed as presenting signs and symptoms may be attributed to pregnancy or postpartum state. Case Description We present the case of a 33-year-old postpartum woman with intrahepatic cholangiocarcinoma with severe hyperbilirubinemia who was found to have fibroblast growth factor receptor 2 (FGFR2)-adenosylhomocysteinase like 1 (AHCYL1) fusion on next-generation sequencing (NGS). She initially was treated with two doses of gemcitabine and cisplatin with increasing hyperbilirubinemia requiring hold of further chemotherapy. NGS showed FGFR2-AHCYL1 fusion, and she was started on the FGFR inhibitor pemigatinib, with dramatically decreasing bilirubin within 10 days. She eventually normalized her bilirubin values and had partial response on follow-up imaging. Conclusions This is the first report, to our knowledge of response to an FGFR inhibitor in the postpartum setting, as well to show response in the setting of life-threatening hyperbilirubinemia. Our patient did not tolerate standard chemotherapy, likely due to liver dysfunction, but responded to pemigatinib, suggesting that the liver dysfunction was driven by her disease. This case underscores the need to include NGS as part of initial workup to identify important therapeutic targets and increase available lines of therapy, including those patients who are postpartum or pregnant.

Background: Pemigatinib is a selective FGFR inhibitor that showed effectiveness and tolerability in patients with cholangiocarcinoma (CAA) with FGFR2 fusion or rearrangement. Data from prior data cutoffs (primary: Jan 29th, 2021; initial update: Dec 20th, 2021) showed that patients receiving pemigatinib had durable responses. Confirmed objective response rate (ORR) (60%) met the primary endpoint, median duration of response (DOR) was 8.3 months, median progression-free survival (PFS) was 9.1 months at the initial update (G-M. Shi et al. ASCO 2022). Safety results were also consistent with previously reported data on pemigatinib. Here we report updated overall survival (OS) results. Methods: Patients aged 18 years or older with recurrent or metastatic CCA that failed at least one prior systemic therapy were enrolled. Thirty-one subjects with documented FGFR2 fusion or rearrangement received 13.5 mg pemigatinib. The primary efficacy endpoint was ORR assessed by the independent radiological review committee (IRRC) per RECIST V1.1. And the second endpoints included disease control rate (DCR), DOR, PFS, and OS. Updated OS were evaluated by Cox proportional hazards model and summarized using Kaplan-Meier methods. Results: At data cutoff (December 28th, 2022), a total of 30 patients were assessed (1 participant excluded due to inadequate FGFR2 aberrant frequency). Median age was 56 years (range, 28-68).With a median OS follow-up of 25.6 months (95% CI, 23.0-25.8), the median OS was 23.9 months (95% CI, 15.2-NC), with 16 (53.3%) OS events. Estimated OS rate at 12 month, 18 month and 24 month were 73.3% (95% CI, 53.7%-85.7%), 66.5% (95% CI, 46.7%-80.4%), and 41.4% (95% CI, 22.4%-59.4%), respectively. No new safety signals were observed. Conclusions: These updated results demonstrated encouraging and durable survival benefit of pemigatinib in Chinese patients with recurrent or metastatic cholangiocarcinoma with FGFR2 fusion or rearrangement. Clinical trial identification: NCT04256980. Editorial acknowledgment: Guoming Shi and Xiaoyong Huang contributed equally to this work. Jian Zhou is the corresponding author. Citation Format: Guoming Shi, Xiaoyong Huang, Tianfu Wen, Tianqiang Song, Ming Kuang, Haibo Mou, Lequn Bao, Haitao Zhao, Hong Zhao, Xielin Feng, Bixiang Zhang, Tao Peng, Yubao Zhang, Xiangcheng Li, Hongsheng Yu, Yu Cao, Yang Luo, Ye Chen, Mingxia Chen, Jia Fan, Jian Zhou. Pemigatinib in Chinese patients with advanced/metastatic or surgically unresectable cholangiocarcinoma Including FGFR2 fusion or rearrangement: Updated overall survival from an open-label, single-arm, multicenter Phase II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT153.

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FGFR2 fusions occur in up to 14% of patients with intrahepatic cholangiocarcinoma (iCCA) and have been considered as therapeutic target for FGFR inhibitors (FGFRi). However, response to targeted treatment may be limited due to the emergence of various resistance mechanisms. We report a case of recurrent iCCA in a 43-year-old patient with a FGFR2 fusion, who was treated with Lenvatinib. Next-generation sequencing (NGS) of tumor-normal DNA and tumor RNA under Lenvatinib treatment confirmed the FGFR2 fusion, however no further molecular resistance mutation was observed. After failure of FGFRi treatment (Lenvatinib and Infigratinib) ten months later, repeated NGS analysis revealed a new gain-of-function mutation in PIK3CA and a homozygous deletion of CDKN2A/B, potentially representing an acquired resistance mechanism. The emerging acquired resistance to FGFR inhibitor treatment has implications for subsequent treatment strategies.

516 Background: Infigratinib (BGJ398), an oral inhibitor of fibroblast growth factor receptor (FGFR)1-3 with demonstrated clinical activity and a manageable adverse event profile in a phase 2 study in patients with previously treated, unresectable or metastatic CCA with an FGFR2 fusion or other rearrangement, represented one of the few targeted treatment options after progression on first-line therapy, as per FDA conditional approval. The confirmatory phase III trial PROOF 301 of infigratinib versus gem/cis as frontline treatment of FGFR2 Gene Fusions/Translocations CCA was early discontinued. Here we report the efficacy and safety in the patients accrued prior to termination. Methods: Eligible patients were randomized 2:1 to infigratinib 125 mg on days 1-21 of a 28-day cycle vs intravenous gemcitabine (1000 mg/m2) + cisplatin (25 mg/m2) on days 1 and 8 of a 21-day cycle. The primary endpoint was progression-free survival (PFS), confirmed by blinded independent central review (BICR). Secondary endpoints included overall survival (OS), investigator determined PFS, overall response rate (ORR), best overall response (BOR), disease control rate (DCR), duration of response (DOR), and safety and tolerability. Crossover to the investigational arm was permitted at progression. Results: Over 40 months, a total of 1127 patients were pre-screened at 120 sites and 48 enrolled. In a 2:1 randomization, 29 patients received infigratinib and 19 received gem/cis. Upon progression, 2 patients crossed over from gem/cis to infigratinib. Median PFS (95% CI) by BICR was 7.4 (5.4, not evaluable [NE]) and 8.0 (3.4, NE) months with infigratinib and chemotherapy, respectively. The ORRs by BICR were 37.9 % with infigratinib and 15.8 % with gem/cis. Grade 3–4 adverse events occurred in 79.3% and 58.8% patients treated with infigratinib and chemotherapy. Conclusions: Infigratinib showed a preliminary signal of efficacy in the first-line treatment of FGFR2-rearranged CCA, but due to early termination of the study, the power is insufficient to draw conclusions regarding its efficacy in comparison to gem/cis. This study highlights the challenge of performing confirmatory studies in biomarker-selected subpopulations of rare tumors and the importance of exploring alternative approaches to delivering confirmatory data for regulatory purposes. Clinical trial information: NCT03773302 .

BACKGROUND Alterations in fibroblast growth factor receptor 2 (FGFR2) have emerged as promising drug targets for intrahepatic cholangiocarcinoma, a rare cancer with a poor prognosis. Futibatinib, a next-generation, covalently binding FGFR1-4 inhibitor, has been shown to have both antitumor activity in patients with FGFR-altered tumors and strong preclinical activity against acquired resistance mutations associated with ATP-competitive FGFR inhibitors. METHODS In this multinational, open-label, single-group, phase 2 study, we enrolled patients with unresectable or metastatic FGFR2 fusion-positive or FGFR2 rearrangement-positive intrahepatic cholangiocarcinoma and disease progression after one or more previous lines of systemic therapy (excluding FGFR inhibitors). The patients received oral futibatinib at a dose of 20 mg once daily in a continuous regimen. The primary end point was objective response (partial or complete response), as assessed by independent central review. Secondary end points included the response duration, progression-free and overall survival, safety, and patient-reported outcomes. RESULTS Between April 16, 2018, and November 29, 2019, a total of 103 patients were enrolled and received futibatinib. A total of 43 of 103 patients (42%; 95% confidence interval, 32 to 52) had a response, and the median duration of response was 9.7 months. Responses were consistent across patient subgroups, including patients with heavily pretreated disease, older adults, and patients who had co-occurring TP53 mutations. At a median follow-up of 17.1 months, the median progression-free survival was 9.0 months and overall survival was 21.7 months. Common treatment-related grade 3 adverse events were hyperphosphatemia (in 30% of the patients), an increased aspartate aminotransferase level (in 7%), stomatitis (in 6%), and fatigue (in 6%). Treatment-related adverse events led to permanent discontinuation of futibatinib in 2% of the patients. No treatment-related deaths occurred. Quality of life was maintained throughout treatment. CONCLUSIONS In previously treated patients with FGFR2 fusion or rearrangement-positive intrahepatic cholangiocarcinoma, the use of futibatinib, a covalent FGFR inhibitor, led to measurable clinical benefit. (Funded by Taiho Oncology and Taiho Pharmaceutical; FOENIX-CCA2 ClinicalTrials.gov number, NCT02052778.).

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Aim To investigate the clinicopathological features and prognostic factors of intrahepatic and extrahepatic cholangiocarcinoma. Methods Clinicopathological and follow-up data of 328 cholangiocarcinoma patients treated at Shanxi Cancer Hospital from November 7, 2016, to August 11, 2021, were retrospectively reviewed. All samples were tested for Fibroblast growth factor receptor 2 (FGFR2) fusion by FISH. The expression of the proliferative marker Ki67 in patients with intrahepatic cholangiocarcinomas (iCCA) was evaluated by immunohistochemistry. All patients were followed up from the date of surgery to the time of death or August 31, 2023. Pathological specimens from patients with recurrence were collected and FGFR2 was tested again. Results The positivity rates for FGFR2 fusion in intrahepatic, perihilar, and distal cholangiocarcinomas were 15%, 2.73%, and 1.69%, respectively. The chi-square test showed that tumor diameter, perineural invasion, complications, and FGFR2 fusion were statistically significant. Immunohistochemistry showed that patients with low expression of Ki67 accounted for 30% of iCCA, low expression of Ki67 and FGFR2 fusion was statistically significant. Relapse specimens were collected from 13 patients, and FISH showed that the expression of FGFR2 was consistent with that of the primary lesion. Multivariate analysis showed that lymph node metastasis was an independent factor for the prognosis of cholangiocarcinoma (P<0.05). Conclusion CCA is an aggressive tumor with high mortality and low survival rates, especially for perihilar cholangiocarcinoma (pCCA). Therefore, it is necessary to understand the clinicopathological features and prognostic factors of iCCA, pCCA and distal cholangiocarcinoma (dCCA). In addition, lymph node status is likely to be an independent and important prognostic factor.

638 Background: Fibroblast growth factor receptor 2 (FGFR2) fusion is a well-established mutation in intrahepatic cholangiocarcinoma (iCCA), accounting for approximately 10% of cases. Current treatments, including pemigatinib, infigratinib, and futibatinib, have shown promising results with response rates of less than 40%. Most patients experience progression within 6-8 months, and reliable biomarkers to predict treatment response are still lacking. This study aimed to assess the clinical outcomes of FGFR inhibitors in patients (pts) with FGFR2-fused iCCA, while investigating tumor vascularity as a potential predictor of treatment efficacy. Methods: We retrospectively analyzed data from 134 pts treated at MD Anderson Cancer Center between 2009 and 2024, focusing on 73 pts with comprehensive clinical data. The cohort included pts treated with futibatinib (n=16), infigratinib (n=16), pemigatinib (n=36), and derazantinib (n=5). Tumor vascularity was assessed via Hounsfield units (HU) from CT images, including a mathematical parameter to calculate a ratio of hypervascular tumor rim thickness to tumor diameter. Transcriptomic analysis of 11 pts was performed to explore mRNA expression related to tumor vasculature. Kaplan-Meier analysis for progression-free survival (PFS) and ANOVA for statistical comparisons were applied. Results: The median age was 60 years, and 41 pts were female. For FGFR2-fused iCCA, median PFS for first-line gemcitabine-based chemotherapy with or without immunotherapy was 4.07 months [3.0-5.1]. Among FGFR inhibitors, mPFS was 7.07 months [95% CI: 5.10-8.16], with no significant difference in outcomes between FGFR inhibitors. 59 pts had comparable CT images and were grouped into 3 groups based on K-means clustering: Group 1 (n=30) had mPFS of 3.07 months [2.03-4.10]; Group 2 (n=22), mPFS of 9.10 months [8.2-11.2]; Group 3 (n=7), mPFS of 34.03 months [18.2-37.6]. Group 1 had the most hypovascular tumors (the lowest HU in the periphery of tumors), while Group 3 had the most hypervascular tumors (p=0.017). The HU ratio of the hypervascular tumor rim thickness to tumor diameter showed a trend, with the highest ratio in Group 3 and the lowest in Group 1 (p=0.09). Transcriptomic analysis revealed a trend toward higher VEGF/VEGFR gene expression in hypervascular tumors. Eight pts received second-line FGFR inhibitors, with mPFS of 5.0 months [2.1-8.3]. Conclusions: Pts with FGFR2-fused iCCA have a shorter PFS on first-line chemotherapy, and PFS outcomes for FGFR inhibitors were consistent with existing data. Tumor vascularity, as assessed by CT imaging, may be a valuable predictor of response to FGFR inhibitors. This study suggests that hypervascular tumors may have better outcomes, warranting further investigation in a larger cohort to confirm these findings and establish tumor vascularity as a predictive biomarker for FGFR inhibitor therapy.

Fibroblast growth factor receptor (FGFR)−2 can be inhibited by FGFR-selective or non-selective tyrosine kinase inhibitors (TKIs). Selective TKIs are approved for cholangiocarcinoma (CCA) with FGFR2 fusions; however, their application is limited by a characteristic pattern of adverse events or evocation of kinase domain mutations. A comprehensive characterization of a patient cohort treated with the non-selective TKI lenvatinib reveals promising efficacy in FGFR2-driven CCA. In a bed-to-bench approach, we investigate FGFR2 fusion proteins bearing critical tumor-relevant point mutations. These mutations confer growth advantage of tumor cells and increased resistance to selective TKIs but remain intriguingly sensitive to lenvatinib. In line with clinical observations, in-silico analyses reveal a more favorable interaction pattern of lenvatinib with FGFR2, including an increased flexibility and ligand efficacy, compared to FGFR-selective TKIs. Finally, the treatment of a patient with progressive disease and a newly developed kinase mutation during therapy with a selective inhibitor results in a striking response to lenvatinib. Our in vitro, in silico, and clinical data suggest that lenvatinib is a promising treatment option for FGFR2-driven CCA, especially when insurmountable adverse reactions of selective TKIs or acquired kinase mutations occur.

471 Background: CCA represents ~3% of all gastrointestinal malignancies globally; it is particularly prevalent in Asian countries. Additionally, FGFR2 gene fusions occur in ~13% of intrahepatic CCA cases. Tasurgratinib (E7090) is an orally available selective inhibitor of FGFR 1–3; the recommended dose is 140 mg per day per the dose-escalation part of a first-in-human phase 1 study. In this pivotal phase 2 study, this regimen was evaluated in patients (pts) with FGFR2 fusion-positive CCA. Methods: Japanese and Chinese pts with surgically unresectable advanced or metastatic CCA were treated with tasurgratinib 140 mg PO daily. FGFR2 gene fusion was confirmed by fluorescence in situ hybridization performed in central laboratories; ≥ 1 prior chemotherapy regimen including a gemcitabine-based combination was required; pts treated with FGFR2 inhibitors were excluded. The primary endpoint was objective response rate (ORR; complete response [CR] + partial response [PR]). Secondary endpoints included duration of response (DOR), time to response (TTR), disease control rate (DCR; CR + PR + stable disease [SD]), clinical benefit rate (CBR; CR + PR + SD lasting ≥ 23 weeks), progression-free survival (PFS), overall survival (OS), and safety. The study was considered successful if the lower limit of the ORR 90% CI was > 15% in a planned population. Tumor responses were measured every 8 weeks by RECIST v1.1 per independent imaging review. Adverse event (AE) severity was measured per CTCAE v4.03. Results: 63 Pts (Japanese: n = 28; Chinese: n = 35) were treated (median age: 55 years); 23 pts (37%) had received 1 prior regimen, all others had received ≥ 2. By the data cutoff date (March 15, 2023), 55 pts discontinued treatment (disease progression, n = 48; adverse events, n = 4; pt choice, n = 2; loss of clinical benefit, n = 1). 19 Pts (30%) had a PR; 31 pts (49%) had SD and 13 (21%) had SD for ≥ 23 weeks. The ORR was 30% (2-sided 90% CI 20.7–41.0; 95% CI 19.2–43.0); the DCR was 79% (95% CI 67.3–88.5); the CBR was 51% (95% CI 37.9–63.6). The median [m]TTR / DOR for responders were 1.87 mos (IQR 1.77–2.10; range 1.6–14.7) / 5.6 mos (95% CI 3.7–9.3; range 1.0+–14.8+). The mPFS / OS were 5.4 mos (95% CI 3.7–5.6) / 13.1 mos (95% CI 10.8–17.4). 61 Pts (97%) had ≥ 1 treatment-related AE (TRAE), most commonly hyperphosphatemia (n = 51; 81%); 18 pts (29%) had ≥ 1 grade ≥ 3 TRAE, most commonly lipase increased (n = 4; 6%). 34 Pts (54%) had a dose reduction and 18 (29%) had treatment interruption due to TRAEs. 4 Pts (6%) had a fatal AE, none related to treatment. Conclusions: Tasurgratinib had promising antitumor activity in pts with CCA harboring FGFR2 gene fusion and who received ≥ 1 prior chemotherapy regimen. The primary endpoint (ORR) met the study’s predefined success criteria. Tasurgratinib had a manageable safety profile consistent with previous reports and with the known pharmacological profile of FGFR inhibitors. Clinical trial information: NCT04238715 .

FGFR2 alterations are infrequent but currently represent the most common targetable mutation in cholangiocarcinoma. We performed a large-scale genomic analysis to assess associations between FGFR2 and other driver gene alterations in cholangiocarcinoma with patient demographics. Clinical and molecular data for patients with intrahepatic cholangiocarcinoma were abstracted from the Foundation Medicine (FMI) and Genomics, Evidence, Neoplasia, Information, Exchange (GENIE) genomic databases. The Chi-squared test was used to assess the association between sex and alterations in driver genes. FGFR2 fusion partners were analyzed using descriptive statistics. Across both FMI (N = 7,859) and GENIE (N = 1,395) cohorts, FGFR2 rearrangements were more frequent in female than male patients with cholangiocarcinoma (odds ratio, 1.69 and 2.45). By contrast, FGFR2 missense mutations, TP53 mutations, and KRAS mutations were not associated with sex. Stratifying patients by age groups showed that FGFR2 rearrangements were specifically enriched in younger ages (<45 years). Although the most common FGFR2 fusion partners were shared across both sexes, a subset of fusion partners was recurrent and uniquely associated with female or male patients. FGFR2 rearrangements but not other driver alterations in cholangiocarcinoma are concentrated in young female patients, where the prevalence reached nearly 20% across two large patient cohorts. These results suggest that the molecular basis of FGFR2 rearrangements may be distinct between sexes. These results indicate the importance of genetic testing in cholangiocarcinoma, particularly for younger patients, and suggest that patient recruitment strategies in trials for FGFR2-targeting therapies should seek to reduce barriers to enhance the enrollment of young female patients.

No abstract available

Abstract Background Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease. Methods We describe an integrated whole‐exome sequencing and transcriptomic study of 37 iCCAs patients in Germany. Results We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke‐associated Asian iCCAs. Conclusions By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

Patients with advanced intrahepatic cholangiocarcinoma (iCCA) have a poor prognosis. Recent advances in molecular profiling offer hope by enabling targeted treatment for those with specific mutations, potentially increasing survival rates. Pemigatinib targets fibroblast growth factor receptor 2 (FGFR2) fusions or rearrangements, providing a promising avenue for treatment. Here, we report the case of a 70-year-old woman diagnosed with advanced iCCA, characterized by FGFR2-Bicaudal family RNA binding protein 1 (BICC1) fusion, who received pemigatinib therapy. Despite adverse effects including mucositis and nail changes, she continued treatment following dose adjustments for over 18 months. This report highlights the importance of dose optimization in maintaining patients on therapy and preventing treatment discontinuation. Real-world patients are often more fragile than those in clinical trials, necessitating tailored dose adjustments. However, the literature on alternative dose modifications remains limited. This case represents the longest documented successful treatment of advanced iCCA with pemigatinib in Taiwan, emphasizing the potential efficacy of pemigatinib treatment in a real-world setting.

Intrahepatic cholangiocarcinoma harbours druggable genetic lesions including FGFR2 gene fusions. Reliable and accurate detection of these fusions is becoming a critical component of the molecular work‐up, but real‐world data on the performance of fluorescence in situ hybridisation (FISH) and targeted RNA‐based next‐generation sequencing (NGS) are very limited. Bridging this gap, we report results of the first round robin test for FGFR2 fusions in cholangiocarcinoma and contextualise test data with genomic architecture. A cohort of 10 cholangiocarcinoma (4 fusion positive and 6 fusion negative) was tested by the Institute of Pathology, University Hospital Heidelberg, Germany. Data were validated by four academic pathology departments in Germany. Fusion‐positive cases comprised FGFR2::BICC1, FGFR2::DBP, FGFR2::TRIM8, and FGFR2::ATE1 fusions. In a second step, a round robin test involving 21 academic and non‐academic centres testing with RNA‐based NGS approaches was carried out; five participants performed FISH testing in addition. Thirteen of 16 (81%) centres successfully passed the NGS only and 3 of 5 (60%) centres passed the combined NGS + FISH round robin test. Identified obstacles were bioinformatic pipelines not optimised for the detection of FGFR2 fusions and assays not capable of detecting unknown fusion partners. This study shows the benefit of targeted RNA‐NGS for the detection of FGFR2 gene fusions. Due to the marked heterogeneity of the genomic architecture of these fusions, fusion partner agnostic (i.e. open) methodological approaches that are capable of identifying yet unknown fusion partners are superior. Furthermore, we highlight pitfalls in subsequent bioinformatic analysis and limitations of FISH‐based tests.

Translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) are found in up to 45% of Intrahepatic Cholangiocarcinoma (ICC). Treatment of patients bearing FGFR2 fusions with FGFR kinase inhibitors induces tumor regressions, though response rate and durability of response appear limited due to the emergence of resistance or to sub-optimal dosing which is limited by drug adverse effects. Thus, therapeutics that drive activity specifically towards FGFR2 are needed to enhance drug efficacy and reduce adverse effects. We first asked whether the extracellular domain (ECD) of FGFR2-BICC1 fusion is functional and important for receptor dimerization and signaling using NanoBiT system and western blotting. FGFR ECD consists of 3 Ig-fold domains D1, D2, and D3. Using focus transformation and viability assays, we found that deletions in D1, D2, D3, and D2-3 ECD FGFR2-BICC1 decreased growth and transformation of FGFR2 fusion driven cells. Next, we sought to investigate if antibodies against the ECD could perturb the growth of FGFR2 fusion transformed cells. We identified six FGFR2 specific antibodies that bind to epitopes in the ECD and confirmed the ligand blocking activity of these bivalent antibodies. In the absence of ligand, however, most of the bivalent antibodies were relatively inactive. As standard bivalent antibodies engage two receptors at a time, they are at risk for agonism or the maintenance of tonic signaling. Thus, to enhance efficacy and prevent the potential agonist or tonic activity of bivalent antibodies, all possible biparatopic antibodies (15), recognizing two distinct epitopes on the FGFR2 ECD, were engineered from the six parental antibodies. The biparatopic antibodies exhibited enhanced efficacies in FGFR2-fusion driven NIH3T3/BaF3 and ICC13-7 cancer cell lines and significantly improved binding affinities (~10-fold) as analyzed by Octet, MSD-SET, and SPR. Two biparatopic antibodies (BpAb-1, BpAb-2) showed the highest antiproliferative activity and induced apoptosis in FGFR2 fusion driven cells. BpAb-1, BpAb-2 were superior to their parental antibodies in reducing FGFR2-fusion driven growth, transformation and downstream signaling (pFGFR2, pFRS) in vitro and in reducing tumor growth in xenograft models. Importantly, BpAb-1 and BpAb-2 significantly induced FGFR2 internalization and lysosomal-mediated degradation as compared to their parental antibodies. Moreover, BpAb-1 and BpAb-2 had anti-growth activities against ICC patient-derived oncogenic FGFR2 ECD in-frame deletions, and FGFR kinase resistance mutations, including the gatekeeper mutation V565F. BpAb-1 and BpAb-2 were also active in FGFR2 WT amplified cells SNU-16. These results suggest that these FGFR2 biparatopic antibodies are a novel treatment option for ICC patients with tumors harboring FGFR2 fusions. Citation Format: Saireudee Chaturantabut, Sydney Oliver, John Kim, Dennie Frederick, Foxy Robinson, Alessandro Sinopoli, Diego J. Rodriguez, Liang Chang, Nabeel Bardeesy, William Sellers. Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2647.

Significance Existing targeted therapies for solid tumors harboring FGFR2 alterations include pan-FGFR inhibitors, which often cannot be dosed to maximum efficacy due to FGFR1- and FGFR4-mediated toxicities. The structural similarity among FGFR family members has thwarted conventional approaches to structure-based design of FGFR2-selective inhibitors, so we used long-timescale molecular dynamics simulations to identify differential motions of FGFR2 and FGFR1 that could be leveraged to design FGFR2-selective inhibitors. Our efforts led to lirafugratinib (RLY-4008), an FGFR2 inhibitor exhibiting substantial selectivity over other FGFRs. Lirafugratinib was reported to have a 73% objective response rate in early clinical studies in FGFR-inhibitor naive, FGFR2 fusion-positive intrahepatic cholangiocarcinoma patients treated orally (once daily doses ≥70 mg) without inducing clinically significant adverse effects by inhibiting off-targets.

Genetic abnormalities of the fibroblast growth factor receptor 2 (FGFR2) gene, including amplification, fusions, and mutations, have been reported in various solid tumors. While molecular targeted therapies against FGFR2 fusion have been proved to be useful in cholangiocarcinoma, the therapeutic significance of FGFR2 inhibitors remains unclear in other various solid cancers. Genomic and clinical information from solid tumor cancer gene panel testing cases is consolidated in the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database in Japan. This study aimed to utilize the C-CAT database to clarify the clinical–pathological significance of FGFR2 abnormalities. A total of 101,231 patients with solid cancer have been registered in the C-CAT database between June 2019 and June 2025. Of the 101,231 cases, 1312 cases with FGFR2 gene abnormalities were analyzed. FGFR2 alterations included amplification in 515 cases, fusion in 280 cases, and mutations in 568 cases. They were detected most frequently in the biliary tract (271 cases), esophagus/stomach (231 cases), and breast (211 cases). Amplification was frequent in the esophagus/stomach (205 cases) and breast (105 cases). Mutations were frequent in the uterus (111 cases), breast (89 cases), and biliary tract (86 cases). Among 515 FGFR2 alteration cases, FGFR2 inhibitors were administered in 85 cases. Of the 85 cases, disease control was achieved in 49 cases, 44 cases of which were biliary tract cancer. FGFR2 might be a promising therapeutic target not only for cholangiocarcinoma with fusion but also for esophagus/stomach cancer and breast cancer with FGFR2 alterations.

FGFR2 and FGFR3 alterations are oncogenic events across many solid tumors, often through chromosomal fusion or activating mutations. FGFR2 translocations are prevalent in intrahepatic cholangiocarcinoma, while FGFR3 mutations and fusions are prevalent in bladder cancer. Both indications have approved pan-FGFR inhibitors that demonstrate responses in patients that harbor these genetic alterations, however, responses are short-lived or show reduced activity in patients with on-target resistance mutations. In addition, efficacy of these agents has been hampered by inhibition of FGFR1, which leads to dose-limiting hyperphosphatemia. To address these issues, we present our clinical candidate CGT4859, a novel reversible FGFR2/3 inhibitor with activity against clinically relevant mutations and >140-fold selectivity over FGFR1. In vivo characterization of CGT4859 demonstrates robust efficacy in target-altered models at tolerated doses. Together, these data demonstrate CGT4859 as a potential best-in-class FGFR1-sparing pan-mutant FGFR2/3 inhibitor. John Fischer, Karyn Bouhana, Richard Brizendine, Mark Chicarelli, Brad Fell, Jennifer Fulton, Anna Guarnieri, Leyla Haygood, Ravi Jalluri, Amber Johnson, Keith Koch, Erika Koslov-Davino, Macedonio J. Mejia, Rob Rieger, John Robinson, Mareli Rodriguez, Francis Sullivan, Yang Wang, Shannon Winski, Silas Wood, Yeyun Zhou. The reversible and selective FGFR2/3 inhibitor CGT4859 has superior target coverage of resistance mutations missed by leading FGFR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4729.

Background/aim Fibroblast Growth Factor Receptor (FGFR) gene fusions are recognized as pivotal oncogenic drivers, contributing to cancer initiation and progression across diverse malignancies. These fusions often represent significant therapeutic targets, particularly in challenging malignancies like cholangiocarcinoma. This study aimed to characterize the novel FGFR2::SHTN1 fusion, identify it as a de novo chimeric protein, and elucidate its precise oncogenic mechanism. Materials and methods FGFR2::SHTN1 fusions were identified via cancer genomics databases and modeled using AlphaFold and HADDOCK. SHTN1 variants were expressed in Neuro-2a cells for coimmunoprecipitation, purification, and native polyacrylamide gel electrophoresis to assess oligomerization. Structural modeling included membrane embedding with Chemistry at HARvard Macromolecular Mechanics–Graphical User Interface (CHARMM–GUI). Results We found that FGFR2::SHTN1 is an in-frame fusion formed by the joining of upstream FGFR2 exons 1–17 with downstream SHTN1 exons 7–17 in human, resulting in a chimeric protein retaining the intact FGFR2 tyrosine kinase domain. Our analyses revealed that Shootin1 inherently forms oligomers through its coiled–coil domains, which, within the fusion, mediate ligand-independent dimerization and constitutive activation of FGFR2. Conclusion Our findings establish FGFR2::SHTN1 as a potent oncogenic driver in various cancers, particularly in cholangiocarcinoma, highlighting a unique mechanism of constitutive activation mediated by Shootin1’s CCD-II domain. This study represents the first molecular characterization of the FGFR2::SHTN1 fusion, advances understanding of FGFR2 fusion biology, and identifies a particular target for future diagnostic and therapeutic strategies in relevant malignancies.

548 Background: Comprehensive genomic profiling (CGP) is recommended for patients (pts) with metastatic BTC (mBTC) given its aggressive, heterogeneous disease profile. Liquid biopsy is a rapid, non-invasive option with high concordance to tissue-based CGP. First-line (1L) therapy in mBTC is gemcitabine-cisplatin (GemCis) +/- durvalumab or pembrolizumab (IO) regardless of CGP results. However, data suggests pts with targetable alterations may have worse outcomes with addition of IO compared to all comers. We examined outcomes for mBTC pts receiving 1L GemCis vs. GemCis+IO following ctDNA-detected IDH1 mutation ( IDH1 +) or FGFR2 fusion ( FGFR2 +). Methods: Real-world data was sourced from GuardantINFORM, which comprises aggregated commercial payer health claims and de-identified records from pts with clinical ctDNA testing via Guardant360 (G360). Pts with mBTC and >1 treatment claim after G360 results, and IDH+ or FGFR2+ between April 2019 and June 2024 were analyzed. Only pts treated with 1L GemCis +/- IO were included. Outcomes were assessed via real-world time to treatment discontinuation (rwTTD), real-world time to next treatment (rwTTNT), and real-world overall survival (rwOS), all in months with 95% confidence intervals. Log-rank test was used to compare Kaplan-Meier survival curves. Results: 412 pts were IDH1+ and 154 were FGFR2+ . In the IDH1 + cohort, 184 pts were treated with GemCis and 106 pts with GemCis+IO. Pts receiving GemCis demonstrated improved rwOS vs. GemCis+IO [27.2 (23.6-37.8) vs. 16 (13.6-19.9) mo.; HR=0.4 (95CI 0.27-0.59), p<0.001]; there was no difference in rwTTNT [13 (10.8-14.3) vs. 10 (8.4-12.3); HR=0.85(95CI 0.6-1.22), p=0.4] or rwTTD [4.8 (4.1-5.5) vs. 6.6 (4.7-8.1); HR=1.17 (95CI 0.9-1.5), p=0.25]. Similarly, in the FGFR2 + cohort, pts receiving GemCis + IO (n=44) had worse rwOS than pts on GemCis (n=70) [NR (15.6-NR) vs. 43 (34.2-2.1); HR=0.37(95CI 0.18-0.74), p=0.005], with no difference in rwTTNT [8.9 (4.89-NR) vs. 13.78 (7.42-NR); HR=0.63 (95CI 0.36-1.1), p=0.1] or rwTTD [5.0 (3.1-7.8) vs. 4.2 (3.5-5.5); HR=1.08 (95CI 0.72-1.6), p=0.7]. Among all mBTC pts receiving GemCis+IO (n=1113), non- IDH1 + pts (n=1007) had numerically better rwOS compared to IDH1 + pts (n=106) [18.6 (17.2-21.3) vs. 16.0 (13.6-19.);HR=0.76 (95CI 0.6-1.03), p=0.075]. No survival difference was observed between IDH1+ (n=184) and non- IDH1 + (n=2333) pts receiving GemCis [27.2 (23.6-37.8) vs. 26.2 (24.5-28.2); HR=1.08 (95CI 0.88-1.32), p=0.4]. Conclusions: Addition of IO to GemCis in IDH1 + and FGFR2 + mBTC pts resulted in decreased rwOS. This data supports ctDNA CGP to inform clinical decision-making prior to 1L as well as at progression. Further studies in clinical cohorts are needed to confirm these findings.

500 Background: Isocitrate dehydrogenase 1 mutations (IDH1mut) define a distinct molecular subtype of biliary tract cancer (BTC). We characterized the epidemiology of a cohort of BTC patients (pt) harboring pathogenic IDH1mut, treatment patterns and efficacy outcomes (EO). Methods: We evaluated a real-world pt cohort from the Spanish RETUD registry, diagnosed with BTC between January 1st, 2017, and May 30th, 2025. IDH1 status was assessed by next-generation sequencing (NGS), polymerase chain reaction (PCR), immunohistochemistry (IHQ), or pyrosequencing (PSQ). Data included demographic and clinical characteristics, molecular profile, therapeutic procedures, and EO (objective response rate [ORR], overall survival [OS] and progression-free survival [PFS]). PFS and OS were estimated using the Kaplan-Meier method. Results: IDH1 status was determined in 445 pt from 31 centers. Among them, 73 (16.4%) presented pathogenic IDH1mut, with R132C mutation present in 53.4%. For IDH1mut pt, median (m) age at diagnosis (dx) was 64.5 years (y), 65.7% were women and the most frequent tumor location at dx was intrahepatic (91.8%). Among all pt with intrahepatic tumors, 24.2% harbored an IDH1mut. Metastatic disease occurred in 77.8% of all patients (78.1% in IDH1mut). Surgery was performed in 33.0% (21.9% IDH1mut) and locoregional therapy in 11.2% (9.6% IDH1mut). Systemic treatment was administered to 98.7% (100.0% IDH1mut), and 20.0% received immunotherapy. Among IDH1mut pt, 27.4% received anti-IDH1 therapy. Most frequent schemes for first and second line were CISGEM (71.6%) and FOLFOX (41.2%) respectively. With a m (min, max) follow-up time of 16.7 (0.9, 81.2) months (mo), mOS in IDH1mut vs IDH1 wild type (wt) were 20.2 mo (95% CI 17.8-28.6) and 18.4 mo (95% CI 16.5–20.4) (p=0.270). mOS in IDH1mut pt not treated with anti-IDH1 therapies was 20.0 mo (95% CI 17.1-28.6) compared to 18.4 mo (95% CI 16.5-20.4) in IDH1wt pt (p=0.450) and 26.1 mo (95% CI 17.8-54.0) in IDH1mut treated pt (p=0.530). First line mPFS and ORR were 8.3 mo (95% CI 7.7-9.5) and 22.4% in IDH1mut vs 6.1 mo (95% CI 5.4-7.3) and 28.9% in IDH1wt, respectively. A total of 49 (67.1%) IDH1mut patients presented co-expression with other biomarkers, 8 of them ESCAT-I. The most frequent co-mutations were CDKN2A (20.4%), CDKN2B (16.3%) and ARID1A (16.3%). Mutations of genes involved in RAS/MAPK signaling pathway like KRAS, BRAF and ERBB2 were less frequent in IDH1mut pt than in IDH1wt pt (10.2% vs 23.8%, 6.1% vs 6.7% and 4.1% vs 9.2% respectively). Regarding TP53/RB pathway, frequency was lower in IDH1mut pt for TP53 (10.2% vs 36.5%) and MDM2 (4.1% vs 4.4%) but not for CDKN2A/B (36.7% vs 34.2%). Conclusions: This analysis provides insights into the characterization of real-world IDH1 BTC pt. Patients receiving anti-IDH1 therapies showed a trend towards improved survival.

BACKGROUND Biliary tract cancers (BTCs), including gallbladder cancer (GBC) and cholangiocarcinoma (CCA), are rare but aggressive malignancies with distinct molecular landscapes and poor prognosis. Genomic profiling has revealed significant molecular alterations, but the genomic landscape of BTC in the Indian population remains underexplored. This study aims to comprehensively characterize the mutation landscape of BTC in the Indian population. METHODS A total of 154 BTC cases, including 69 CCA and 85 GBC, were retrospectively analyzed using data collected from various targeted sequencing panels. Somatic mutations, copy number variations (CNVs), and gene fusions in key oncogenic and tumor suppressor genes were identified from these panel reports. Downstream analyses were performed to derive key biological insights, including pathway enrichment and mutual exclusivity and co-occurrence analyses of genomic alterations. RESULTS TP53 was the most frequently mutated gene (53%), followed by KRAS (18%), ARID1A (9%), IDH1 (7%), and PIK3CA (7%). Recurrent amplifications were observed in MYC (12%) and ERBB2 (9%). Pathway enrichment analysis revealed significant dysregulation in the PI3K-AKT-mTOR, Notch, and Wnt/β-catenin signaling pathways. Notably, IDH1 mutations were primarily observed in CCA, while STK11 mutations were exclusive to GBC, highlighting distinct molecular characteristics between the two subtypes. PD-L1-negative tumors exhibited distinct genomic alterations, notably SMAD4 mutations, which were associated with reduced PD-L1 expression. This loss of SMAD4, involved in TGF-β signaling, could impair immune response regulation and facilitate immune evasion. CONCLUSIONS This study provides a comprehensive molecular profiling of BTCs in the Indian population, revealing key genomic alterations, subtype-specific differences, and associations with immune features. The findings underscore the importance of molecular profiling in guiding personalized treatment strategies.

Intrahepatic cholangiocarcinoma (CCA) is an aggressive biliary tract cancer that carries unfavorable prognosis. 10-20% of intrahepatic CCAs harbor a mutation in IDH1 that can be targeted with mutant IDH1-specific inhibitors. However, objective and durable responses to treatment with mutant IDH1 inhibitors are rare in CCA. Mutant IDH1 has neomorphic enzymatic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG promotes biliary tumor formation through cancer cell-intrinsic mechanisms but is also a paracrine factor in the tumor microenvironment (TME). Our group and others have identified that IDH1-mutant CCAs are not responsive to immunotherapy treatment approaches in part due to decreased CD8+ T cell infiltration and an increase in immunosuppressive macrophage cells in the TME. To better understand the immunosuppressive features of the IDH1-mutant CCA TME, we generated an isogenic cell line panel of IDH1-mutant (MUT) and IDH1-wild type (WT) mouse cholangiocarcinoma cells in the SB1 cell line background through a CRISPR homology directed repair approach. Compared to IDH1-WT cells, IDH1-MUT cells produce high levels of D-2-HG and both cell line panels readily form tumors in immunocompetent C57BL/6 mice. Tumors formed from IDH1-mut cells have reduced CD8+ T cell infiltration compared to IDH1-WT tumors, a finding that correlates with analysis of human CCA samples. Using cytometry by time of flight (CyTOF), we identified that CD8+ T cells in IDH1-MUT tumors have decreased expression of key regulators of the antitumor immune response including EOMES, TBET, and GZMB compared to those isolated from IDH1-WT tumors. Analysis of secreted factors from these IDH1-MUT and IDH1-WT cells identified that IDH1-MUT cells release increased levels of CCL2, a chemokine shown to diminish the antitumor immune response across multiple cancer types, compared to IDH1-WT cells. Further, IDH1-MUT tumors grown in syngeneic, immunocompetent mice have increased CCL2 staining by IHC compared to IDH1-WT tumors. CCL2 expression is also increased in a preliminary analysis of IDH1-mutant human CCA primary tumors. Treatment of mice harboring orthotopic liver tumors formed from IDH1-MUT cells with a CCL2 neutralizing antibody resulted in decreased tumor size compared to isotype control. Together, our data suggest that CCL2 is a mediator of immunosuppression in the IDH1-mutant CCA TME and that modulation of CCL2 activity may improve outcomes in this rare disease subtype. Further work to understand the mechanisms through which IDH1 mutations regulate CCL2 levels in these tumors is underway. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Kiyoko Yoshima, Daniel J. Zabransky, Mark Yarchoan. Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6819.

No abstract available

Background: Biliary tract cancer (BTC) management has undergone tremendous changes, benefiting from the identification of highly actionable molecular alterations. Among these, IDH1 mutations and FGFR2 fusions are the most common alterations detected and are classified as ESCAT tier 1 in BTC. However, their prognostic value in real-world settings remains uncertain. Objective: To explore overall survival (OS) in patients harbouring locally advanced or metastatic BTC (mBTC) with IDH1 or FGFR2 alterations, compared to those with wild-type tumours. Methods: This retrospective, multicentre study included patients with mBTC treated between 2020 and 2023 across five French centres. Patients were categorized into two cohorts based on molecular profiling: those with IDH1 or FGFR2 alterations, and those with wild-type tumours (WT-mBTC). Results: 119 consecutive patients were included. 18 were classified as altered (IDH1 = 13; FGFR2 = 5). Sixty-four pts underwent no molecular testing. The median OS of the entire cohort was 11.9 months (10.3–14.3). The median OS was 24.2 months (12.3–NA) versus 10.8 months (7.9–12.9), p = 0.02, in the altered and WT-mBTC cohorts, respectively. The Cox regression model conducted depicted an HR for death of 0.46 (CI95%, 0.2–0.9) for IDH1 or FGFR2 alterations. There were no diffence in PFS for first-line. Conclusions: Our cohort suggests that IDH1 or FGFR2 alterations may be associated with prognostic differences in patients with metastatic BTC, although they do not appear to influence outcomes under first-line treatment. These findings are consistent with trends observed in clinical trials. Whether improved survival is solely attributable to targeted therapies remains questionable. In line with ESMO recommendations, systematic molecular profiling should be considered in patients with mBTC.

TPS4219 Background: Cholangiocarcinomas (CCAs) are often advanced and incurable at the time of diagnosis. The phase 3 TOPAZ-1 trial showed improved OS and ORR with gemcitabine/cisplatin (GEM/CIS) and durvalumab (DURVA) vs GEM/CIS in unresectable advanced or metastatic biliary tract cancers. The phase 3 ClarIDHY trial demonstrated that the mIDH1 inhibitor ivosidenib (IVO) improved progression-free survival in CCA patients who have progressed from first or second-line chemotherapy and who have activating mutations in isocitrate dehydrogenase-1 (mIDH1). Additionally, mIDH1 suppresses key immune-related genes, with reversal of this effect when mIDH1 inhibitors are administered in preclinical CCA models. Finally, encouraging activity has been observed in treatment-naive mIDH1 patients administered with the mIDH1 inhibitor LY3410738 in combination with GEM/CIS. Given the ability of ivosidenib to stabilize advanced CCA, ability of IDH1 inhibition to restore immune activity, promising clinical activity of an mIDH1 inhibitor in combination with GEM/CIS, and the limited overlapping toxicities of these treatments, this study seeks to explore safety and preliminary activity of the quadruplet combination. Methods: This is a phase 1b/2, multicenter, safety lead-in and dose expansion, open-label study of IVO in combination with DURVA/GEM/CIS in first-line therapy of locally advanced, unresectable, or metastatic CCA with mIDH1. Treatment with up to one cycle of DURVA/GEM/CIS is permitted before initiation of study treatment. Key eligibility criteria include: a histopathological diagnosis; tumor mIDH1 based on local or centralized tissue testing (local testing by plasma ctDNA may be used); at least 1 measurable lesion as defined by RECIST v1.1; and adequate bone marrow, hepatic, and renal function. The study has a safety lead-in phase where IVO will be administered orally to the first 6 patients at a starting dose of 500 mg QD on every day of the 21-day cycle, plus DURVA 1500 mg IV infusion every 3 weeks for up to 8 cycles, plus GEM 1000 mg/m2 IV and CIS 25 mg/m2 IV on days 1 and 8 of each 21-day cycle, followed by IVO 500 mg QD and DURVA 1500 mg every 4 weeks of a 28-day cycle. Dose-limiting toxicities (DLTs) will be evaluated during the first cycle of quadruplet treatment. 6 additional patients may be enrolled to evaluate an alternative reduced dose of IVO 250 mg QD. The primary objective is to evaluate the safety and tolerability of the quadruplet combination, and to determine the recommended combination dose (RCD). The expansion phase will enroll approximately 40 patients who will be treated with the RCD, with the primary objective being to assess the clinical activity of the combination, as determined by a primary endpoint of confirmed complete or partial response using RECIST v1.1 criteria. Clinical trial information: NCT06501625 .

Abstract Biliary tract cancers (BTC) represent a heterogeneous group of malignancies with a poor prognosis and rising incidence. Oncogenic FGFR2 fusions are one of several actionable molecular alterations. In this context, selective FGFR tyrosine kinase inhibitors have demonstrated promising and durable response rates and are now approved and included in clinical guidelines. However, secondary kinase mutations frequently arise over time, leading to resistance against these drugs. We present the case of a 41-year-old male patient with metastatic BTC who underwent molecular analysis after disease progression to various established chemotherapy combinations. Testing identified an oncogenic FGFR2 fusion (FGFR2::BICC1). The patient was treated with pemigatinib for 14 months. Upon disease progression, the resistance-associated FGFR2 p. E565A variant was detected in a follow-up biopsy. Treatment was switched to futibatinib, resulting in rapid disease progression. Lacking other therapeutic options, the patient was treated with lenvatinib, supported by previously published data suggesting a potential benefit in similar settings. The treatment was well tolerated, with only a mild increase in transaminases, and the patient remained on treatment with noteworthy effects for 15 months to date. With a growing incidence of BTC and growing use of targeted therapies for FGFR2 alterations, the emergence of secondary resistance-causing point mutations following treatment with approved inhibitors is becoming increasingly challenging. Beyond selective inhibitors, lenvatinib may represent a viable therapeutic option.

No abstract available

Cholangiocarcinomas are highly aggressive malignancies with a poor prognosis. The global incidence of cholangiocarcinoma patients appears to be on the rise. While surgical resection remains the curative option for cholangiocarcinoma patients, the majority of cases are diagnosed at an advanced stage, resulting in poor outcomes. Patients with inoperable cholangiocarcinomas typically undergo chemotherapy regimens, including Gemcitabine, but their efficacy is limited, leading to low 5-year survival rates. Recent advancements in organoid culture technology allow for the in vitro replication of tissue structures by culturing tissue stem cells in three dimensions, offering great promise for personalized medicine. Utilizing patient-derived organoids has proven effective in predicting the outcomes of therapeutic drugs. We have successfully established organoids using cancer tissue derived from cholangiocarcinoma patients, and stably culturing and maintaining them, promoting their application to personalized medicine (Saito Y et al. Cell Rep. 27, 1265, 2019, Saito Y et al. Cancer Cell 40, 226, 2022). IDH1 mutations have been identified in approximately 15% of intrahepatic cholangiocarcinomas and are known to convert α-ketoglutarate (α-KG) in the TCA cycle to produce 2-hydroxyglutarate (2-HG), which is observed in gliomas and acute myeloid leukemia. 2-HG acts as an antagonist to α-KG-dependent dioxygenases, promoting cancer development. This study aims to develop a novel treatment for cholangiocarcinomas with IDH1 mutations. Metabolomic analysis of patient-derived cholangiocarcinoma organoids revealed 2-HG accumulation and reduced ATP production in IDH1-mutated cholangiocarcinoma organoids compared to IDH1 wild-type cholangiocarcinoma organoids. This may suppress the protein synthesis of MCL-1 and mTOR signaling through the activation of AMPK kinase, suggesting that further inhibition of MCL-1 could be lethal in IDH1-mutated cholangiocarcinoma organoids. Indeed, exposure to S63845, an MCL-1 inhibitor, significantly inhibited cell proliferation in IDH1-mutated cholangiocarcinoma organoids, unlike in IDH1 wild-type cholangiocarcinoma organoids. Furthermore, the combination of MCL-1 inhibitor and BCL-XL inhibitor demonstrated a synergistic and potent growth inhibitory effect on cholangiocarcinoma organoids. These findings indicate that the MCL-1 inhibitor holds promise as a new therapeutic agent for cholangiocarcinoma patients with IDH1 mutations. Citation Format: Shiho Suzuki, Juntaro Matsuzaki, Toshihide Muramatsu, Yae Kanai, Yoshimasa Saito. Effect of MCL-1 inhibitor on organoids derived from cholangiocarcinoma patients with IDH1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 226.

No abstract available

558 Background: Cholangiocarcinomas (CCAs) are rare cancers that are often advanced and incurable at diagnosis. Durvalumab (DURVA) + gemcitabine/cisplatin (GEM/CIS) is approved for first-line treatment for CCA. This study (NCT06501625) evaluates the safety/tolerability of ivosidenib (IVO) + DURVA/GEM/CIS to determine the recommended combination dose (RCD) (safety lead-in phase, SLI) and the preliminary activity of this combination (expansion phase). Here we present the SLI results. Methods: Patients (pts) with locally advanced unresectable or metastatic mIDH1 CCA with ECOG 0 or 1 and at least 1 measurable lesion (RECIST v1.1) were dosed in the SLI. Treatment with up to 1 cycle of DURVA/GEM/CIS was permitted prior to study treatment initiation. Pts received IVO 500 mg QD + DURVA 1500 mg IV infusion every 3 weeks for up to 8 cycles + GEM 1000 mg/m 2 IV and CIS 25 mg/m 2 IV on days 1 and 8 of each cycle, followed by IVO 500 mg QD and DURVA 1500 mg every 4 weeks of each cycle. DLTs were evaluated during the first cycle of study treatment. Results: Seven pts with locally advanced or metastatic mIDH1 CCA (based on local or central testing) were enrolled in the SLI: age range, 37-75 years, 3 (43%) female, and 3 (43%) received 1 cycle of DURVA/GEM/CIS prior to study treatment. Through the data cut off of July 10, 2025, pts received 1–5 cycles with 6 (86%) ongoing. All pts reported a treatment-emergent adverse event (AE); 4 pts (57%) required a dose interruption and 1 (14%) required a dose reduction. One pt (14%) discontinued study treatment due to an adverse event (the only DLT), which was an AE of drug-induced liver injury, with LFT and bilirubin elevation that resolved after discontinuation of the quadruplet. Six pts were DLT evaluable, and 1 was not DLT evaluable, due to receiving <75% of the planned dose of IVO during the first cycle. This pt experienced an adverse event of special interest (AESI) of grade 3 QT prolongation requiring IVO dose reduction, but with re-escalation to full dose IVO 500 mg + DURVA/GEM/CIS during the second cycle after optimization of electrolytes and resolution of QT prolongation within 7 days, not assessed as a DLT. No other pts experienced AESIs. All pts experienced an AE, and 6 (86%) had a grade ≥3 AE. Two pts (29%) had a serious AE — 1 was the DLT of drug-associated liver injury and 1 was a non-treatment related cholangitis that recovered with resumption of IVO at full. One pt had a partial response and 6 had stable disease at the time of the data cut-off. IVO 500 mg + DURVA/GEM/CIS was confirmed as the RCD and the expansion phase was initiated. Conclusions: IVO at 500 mg QD + DURVA /GEM/CIS demonstrated a safety profile similar to DURVA/GEM/CIS. This dose will be evaluated further during the expansion phase, which will enroll ~40 pts. Clinical trial information: NCT06501625 .

625 Background: In the TOPAZ-1 study (NCT03875235), durvalumab + gemcitabine and cisplatin (D+GC) significantly improved overall survival (OS) versus placebo + GC (P+GC) in participants (pts) with advanced biliary tract cancer. Updated results have shown a clinically meaningful long-term OS benefit for D+GC versus P+GC at 3 years. This exploratory analysis tested circulating tumor DNA (ctDNA) in plasma samples for blood-based detection of clinically actionable alterations (CAAs) and investigated the potential of this method to guide treatment decisions. Methods: Baseline genomic alterations were retrospectively assessed in evaluable tumor (FMI biomarker evaluable population [BEP], n=441) and plasma samples (GH BEP, n=643) using FoundationOne (Foundation Medicine Inc., Cambridge, MA) and Guardant INFINITY (Guardant Health, Redwood City, CA) assays, respectively. Mutation prevalence and association with outcomes were compared in the FMI BEP and GH BEP. Positive and negative percent agreement of CAAs detected in tumor versus ctDNA were assessed in 419 pts with both tumor and plasma samples (FMI-GH BEP). Results: The FMI BEP and GH BEP represented 64% and 94% of the TOPAZ-1 final analysis set (685 pts), respectively. The relative prevalence and overall mutational landscape detected in plasma ctDNA was consistent with that observed by tumor profiling, with the notable exception that genes harboring complex alterations (e.g. gene amplification, rearrangements, or homozygous deletions) were less frequently detected in ctDNA (e.g. ERBB2 , FGFR2 , and CKDN2A/2B/MTAP ). The most common alterations (>15% prevalence in both BEPs) observed were in TP53 (49%/52%), KRAS (24%/17%), and ARID1A (21%/16%) (in the FMI/GH BEPs, respectively). The relative prevalence of alterations within geographic and anatomic subgroups was similar in tumor versus plasma for most CAAs. The overall percent agreement in CAAs was ≥93%, and negative percent agreement was ≥97%. However, positive percent agreement was notably low for ERBB2 amplification (52%) and FGFR2 fusions (47%). OS hazard ratios for D+GC versus P+GC in the GH BEP were <1 for both CAAs and wild-type, except for ERBB2 amplification, as previously reported in the FMI BEP. Conclusions: The overall concordance and relative prevalence of simple mutations (e.g. single nucleotide variants) were similar using the FoundationOne tumor assay and Guardant INFINITY ctDNA assay, suggesting that plasma ctDNA testing has potential utility in clinical practice. However, negative status by ctDNA for the complex alterations found in FGFR2 and ERBB2 would require further testing of tumors, based on their low detectability in plasma. Clinical trial information: NCT03875235 .

Background/Aim: This study aimed to identify mutation profile similarities between tissue and circulating tumor DNA (ctDNA) and to explore driver mutations as potential prognostic or predictive biomarkers or druggable targets in patients with advanced biliary tract cancer (BTC). Patients and Methods: We prospectively enrolled 18 patients with advanced BTC and analyzed next-generation sequencing data from 60 ctDNA samples using AlphaLiquid® 100. This assay screens up to 118 genes for single-nucleotide variants (SNVs) and insertion or deletions (INDELs), 27 genes for copy number alterations (CNAs), and 10 genes for fusions. We examined the intra-patient tissue-ctDNA concordance and studied the association between ctDNA variant allele frequency (VAF) and survival. Results: A total of seven gallbladder cancer cases, six intrahepatic cholangiocarcinoma cases, and five extrahepatic cholangiocarcinoma cases were observed. Among these cases, tumor tissues were available for 16 patients. Genetic alterations were detected in 88% (14/16) of tissue DNA samples and 89% (16/18) of samples with ctDNA at baseline. The most common genes altered in ctDNA were TP53 (n=11), ERBB3 (n=3), and KRAS (n=3). There was a 29% overlap in somatic SNVs/INDELs and a 60% overlap in CNAs between tissue DNA and ctDNA, while no fusion variant was detected. The sensitivity and positive predictive value of ctDNA for all types of somatic mutations were 47% and 43%, respectively. Among the 14 patients whose serial ctDNA was analyzed, 10 showed changes in ctDNA. A high pre-treatment VAF (>4.0%) was associated with poor overall survival. Conclusion: ctDNA sequencing can successfully identify molecular genetic alterations in patients with advanced BTC, providing insights into potential biomarkers and therapeutic targets.

Biliary tract cancer (BTC) is a rare but highly aggressive malignancy that includes intrahepatic cholangiocarcinoma (ICC), extrahepatic cholangiocarcinoma, and gallbladder cancer (GBC). While BTC has a low global incidence, its regional variations are notable. Among nations, Korea has the second-highest incidence of BTC globally, with the highest mortality rate worldwide, underscoring the need for a deeper understanding of this cancer. Liver fluke infection and hepatitis B virus infection are key risk factors unique to Korea, contributing to regional differences in BTC incidence. Additionally, genomic alterations in Korean patients with BTC differ from those in other populations, including lower frequencies of IDH1 mutations and FGFR2 fusions in ICC and a higher prevalence of ERBB2 amplification in GBC. Recognizing the clinical significance of these alterations, ivosidenib and pemigatinib have been approved in Korea for BTC patients with IDH1 mutations and FGFR2 fusions, respectively. This review explores the epidemiology, risk factors, and molecular features of BTC, along with corresponding targeted therapies. Furthermore, we compare the unique characteristics of BTC in Korea with global data to inform future research and clinical practice.

Effective second-line treatment for advanced biliary tract cancer (BTC) remains an unmet need. BTC often presents with homologous recombination repair (HRR) pathway deficiencies and IDH1/IDH2 mutations which suggest responsiveness to Poly (ADP-ribose) polymerase inhibitors. Thirteen patients were enrolled in our open-label, single-site phase II study of pembrolizumab and olaparib in the second-line setting and beyond for patients with advanced BTC. The objective response rate was 15.4% and the disease control rate was 53.8%. The median progression-free survival (PFS) was 5.45 months (95% CI 1.25-7.82), and the median overall survival was 7.21 months (95% CI 4.5-13.8). Both patients with IDH1 mutations and 2 of the 4 patients with HRR mutations achieved a PFS of at least 7.5 months. All BTC patients do not appear to benefit from pembrolizumab plus olaparib, but those with HRR deficiencies and/or IDH mutations may benefit although it would now represent a rechallenge with immunotherapy. Trial registration: NCT04306367, date of registration 3/10/2020.

Cholangiocarcinoma (CCA), recognized for its high malignancy, has been an enormous challenge due to lacking effective treatment therapy over the past decades. Recently, the targeted therapies, such as Pemigatinib and Ivosidenib, have provided new treatment options for patients carrying fibroblast growth factor receptor (FGFR) and isocitrate dehydrogenase 1/2 (IDH1/2) mutations, but only ~30% of patients harbor these mutants; it is urgent to explore novel targets and therapeutic therapies. The frequent downregulation of BAP1 has been observed in CCA, and the low expression of BAP1 is closely related to the poor prognosis of CCA. However, there are no effective interventions to re‐activate BAP1 protein; blocking its degradation may provide a feasible strategy for BAP1‐downregulation CCA treatment. In this study, we demonstrated the tumor‐suppressive roles of BAP1 in CCA and identified VCP functions as the key upstream regulator mediated by BAP1 protein homeostasis. Mechanistically, VCP binds to BAP1 and promotes the latter's ubiquitination degradation via the ubiquitin‐proteasome pathway, thus promoting cell proliferation and inhibiting cell apoptosis. Moreover, we found that VCP inhibitors inhibited CCA cell growth and promoted cell apoptosis by blocking BAP1 ubiquitination degradation. Collectively, our findings not only provided a novel mechanism underlying the aberrant low expression of BAP1 in CCA but also verified the anti‐tumor effect of VCP inhibitors in CCA, offering a novel therapeutic target for CCA treatment.

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Background Fibroblast growth factor receptor 2 (FGFR2) fusions and rearrangements are clinically actionable genomic alterations in cholangiocarcinoma (CCA). Pemigatinib is a selective, potent, oral inhibitor of FGFR1-3 and demonstrated efficacy in patients with previously treated, advanced/metastatic CCA with FGFR2 alterations in FIGHT-202 (NCT02924376). We report final outcomes from the extended follow-up period. Patients and methods The multicenter, open-label, single-arm, phase II FIGHT-202 study enrolled patients ≥18 years old with previously treated advanced/metastatic CCA with FGFR2 fusions or rearrangements (cohort A), other FGF/FGFR alterations (cohort B), or no FGF/FGFR alterations (cohort C). Patients received once-daily oral pemigatinib 13.5 mg in 21-day cycles (2 weeks on, 1 week off) until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) in cohort A assessed as per RECIST v1.1 by an independent review committee; secondary endpoints included duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety. Results FIGHT-202 enrolled 147 patients (cohort A, 108; cohort B, 20; cohort C, 17; unconfirmed FGF/FGFR alterations, 2). By final analysis, 145 (98.6%) had discontinued treatment due to progressive disease (71.4%), withdrawal by patient (8.2%), or adverse events (AEs; 6.8%). Median follow-up was 45.4 months. The ORR in cohort A was 37.0% (95% confidence interval 27.9% to 46.9%); complete and partial responses were observed in 3 and 37 patients, respectively. Median DOR was 9.1 (6.0-14.5) months; median PFS and OS were 7.0 (6.1-10.5) months and 17.5 (14.4-22.9) months, respectively. The most common treatment-emergent AEs (TEAEs) were hyperphosphatemia (58.5%), alopecia (49.7%), and diarrhea (47.6%). Overall, 15 (10.2%) patients experienced TEAEs leading to pemigatinib discontinuation; intestinal obstruction and acute kidney injury (n = 2 each) occurred most frequently. Conclusions Pemigatinib demonstrated durable response and prolonged OS with manageable AEs in patients with previously treated, advanced/metastatic CCA with FGFR2 alterations in the extended follow-up period of FIGHT-202.

Cholangiocarcinoma (CCA) is a highly aggressive cancer that arises from the bile duct and has an extremely poor prognosis. Pemigatinib is the only Food and Drug Administration (FDA)-approved CCA-targeted drug. The CCA treatment options are insufficient considering its poor prognosis and increasing morbidity. Recently, Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been reported to promote resistance to chemotherapy. In this study, we investigated the role that ROCK2 plays in the development of resistance of CCA cells to Pemigatinib. Here, we developed Pemigatinib-resistant CCA cells, performed mRNA sequencing, retrieved The Cancer Genome Atlas (TCGA) data, and analysed ROCK2 expression in a large CCA cohort. The expression level of ROCK2 in CCA cells was significantly higher than that in adjacent noncancerous tissues. Increased expression of ROCK2 in CCA was related to a late TNM stage and decreased overall survival. Functional experiments revealed that downregulating the expression of ROCK2 promotes the ferroptosis of CCA cells, and enhances sensitivity to Pemigatinib. Moreover, upregulation of ROCK2 increased the expression of Drp1 protein. The effect of downregulating ROCK2 was reversed by Drp1 overexpression, and Drp1 knockdown inhibited Ferroptosis driven by ROCK2 overexpression. Mechanistically, ROCK2 stabilized the expression of Drp1 by competing with UBA52 to bind Drp1 and inhibiting the ubiquitination-mediated degradation of Drp1. Blocking of the UBA52– Drp1 axis inhibited the antitumour effect of Pemigatinib in ROCK2-knockdown cells both in vitro and in vivo. In conclusion, the ROCK2/UBA52/Drp1 axis is a pivotal driver of Pemigatinib resistance in CCA cells. These results provide novel insights into Pemigatinib resistance in CCA cells, suggesting that ROCK2 is a promising therapeutic target for the treatment of CCA.

Background Fibroblast growth factor receptor-2 (FGFR-2) mutations are frequently observed in intrahepatic cholangiocarcinoma (ICC). While FGFR2-targeted therapies are primarily studied in advanced ICC, this report presents a rare case of locally recurrent ICC treated with systemic therapy, leading to significant tumor regression and successful R0 resection. Case presentation A 51-year-old female underwent right posterior hepatectomy and cholecystectomy in 2018 for ICC. In August 2022, postoperative MRI revealed tumor recurrence near the hepatic vein, accompanied by intrahepatic bile duct dilation and a tumor thrombus. Given the tumor’s proximity to critical structures and confirmed FGFR-2 fusion, systemic therapy with pemigatinib and sintilimab was initiated. After four cycles, the tumor showed partial remission, with a reduction in the bile duct tumor thrombus. In May 2023, the patient underwent successful right hemi-hepatectomy. Postoperatively, she continued combination therapy without recurrence or metastasis for 19 months. Conclusion This case highlights the efficacy of pemigatinib-based systemic therapy in achieving tumor regression and enabling curative resection in locally recurrent FGFR-2-positive ICC. The successful outcome underscores the potential of targeted therapies in managing recurrent ICC, warranting further investigation.

Simple Summary Cholangiocarcinoma is a rare and aggressive cancer of the bile ducts. For some patients, this cancer is linked to a genetic change in the FGFR2 protein. In 2021, Health Canada approved pemigatinib as a targeted therapy for patients with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma with an FGFR2 fusion or rearrangement. However, there is little real-world data on the use of pemigatinib in these patients in the Canadian setting. This study included 18 patients across six provinces who received pemigatinib through a patient support program. Most had advanced disease, and many had already received several lines of chemotherapy. After starting pemigatinib, over half showed a measurable response in their cancer, and nearly 90% had some level of disease control. On average, pemigatinib delayed disease progression for approximately one year. Importantly, none of the patients stopped treatment because of side effects. These results are comparable to those from earlier clinical trials, suggesting pemigatinib is effective and well tolerated in real-world settings. These findings reinforce the clinical value of pemigatinib for Canadian patients with cholangiocarcinoma and underscore the need for timely access to both targeted therapies and comprehensive genetic testing to ensure patients receive the most effective, personalized care.

4086 Background: IDH1 mutations occur in 13% of patients with intrahepatic cholangiocarcinoma. Ivosidenib is FDA approved for the treatment of advanced, previously treated, IDH1-mutated cholangiocarcinoma. In the ClarIDHy trial, ivosidenib led to an objective response rate of 2%, stable disease rate of 51%, median progression-free survival (PFS) of 2.7 months, and median overall survival (OS) of 10.3 months. Treatment-emergent adverse events resulted in study drug discontinuation in 7% of patients. Data on the real-world efficacy and safety of ivosidenib in cholangiocarcinoma remains limited. Methods: Patients with IDH1-mutated cholangiocarcinoma who were prescribed ivosidenib before December 1, 2024, were retrospectively identified from the national Veterans Affairs (VA) Corporate Data Warehouse. Demographic, clinical, and molecular data were abstracted from the National Precision Oncology database and electronic medical records. Response was assessed based on provider notes and radiology reports. Survival was assessed by the Kaplan-Meier method, and covariates evaluated by the Cox proportional hazards model. Results: Of 1094 veterans with cholangiocarcinoma who underwent molecular testing, 82 (7.5%) had an IDH1 mutation. 33 (40%) patients received ivosidenib at 27 VA medical centers. The median age was 74 years (range 46–82). 2 patients (6%) had a partial response (PR), 10 (30%) had stable disease (SD), 19 (58%) had progressive disease, and 2 were not assessed. 20 patients (60%) had received one and 5 patients (15%) received two prior lines of therapy. Of the 8 patients (24%) who received first-line ivosidenib, 2 (25%) had a PR and 3 (38%) had SD. Most patients (94%) started ivosidenib at the labeled dose (500 mg daily). Two patients who started ivosidenib at reduced dose (250 mg daily) had PR and SD as their best response. The median PFS from start of ivosidenib was 4.0 months, and the median OS was 10.5 months. In a multivariable analysis, PFS and OS were not significantly associated with age, line of therapy, IDH1 variant allele frequency, or IDH1 mutation (17 IDH1 R132C vs. 8 other). Patients with IDH1-mutated, advanced cholangiocarcinoma treated with ivosidenib had a median OS of 25.3 months from diagnosis, compared to 8.7 months for patients who did not receive ivosidenib. Toxicities leading to dose reduction, interruption, or discontinuation of ivosidenib occurred in 3 patients (9%). Conclusions: In this real-world cohort, patients with IDH1-mutated advanced cholangiocarcinoma treated with ivosidenib had similar response rate, PFS, and OS compared to ClarIDHy. Toxicities leading to dose reduction, interruption, or discontinuation were rare. The only two partial responses were observed in the first-line setting, including one with a reduced starting dose. This suggests that frontline ivosidenib may be a reasonable alternative for patients with advanced cholangiocarcinoma.

Background Cholangiocarcinoma (CCA) is a diverse group of aggressive liver tumors with up to 20% being intrahepatic CCA (iCCA). Up to 15% of patients with iCCA have fibroblast growth factor receptor 2 (FGFR2) fusions or rearrangements. Here we evaluated iCCA treatment with pemigatinib, a selective inhibitor of FGFR1–3, in two patients from Denmark and Finland. Patients We identified a total of two Nordic patients with iCCA in our clinics, who received first-line cisplatin/gemcitabine before initiating pemigatinib. Results Case 1 was a 34-year-old woman with aggressive, metastatic iCCA upon presentation, who progressed on cisplatin/gemcitabine. Pemigatinib was initiated after FGFR2 fusion detection by genomic testing. She had a partial response after three cycles (9 weeks) of pemigatinib but experienced disease progression after three more pemigatinib cycles. Adverse events were primarily managed by supportive care and dose reduction, except hyperphosphatemia, which was complicated by food allergies and required medication. She received subsequent chemotherapy but deteriorated rapidly and died 1 month later. Case 2 was an 81-year-old man with unresectable iCCA who achieved stable disease with first-line chemotherapy. He switched to pemigatinib after FGFR2 fusion detection by next-generation sequencing. The tumor shrank by 20% after three pemigatinib cycles and completely calcified with continued treatment. Adverse events were managed by two dose adjustments. Treatment has continued for 57 months and is ongoing. Interpretation CCA is an aggressive disease that requires early molecular testing of abundant biopsy tissue so not to delay second-line therapies, such as pemigatinib. Variability in treatment outcomes is expected.

ABSTRACT Cholangiocarcinoma (CCA) is a rare and aggressive cancer with a poor prognosis. Ivosidenib, an orally administered, first-in-class small-molecule inhibitor, targets the mutated isocitrate dehydrogenase 1 (IDH1) enzyme. Recently, ivosidenib has been approved for treating patients with locally advanced or metastatic IDH1 R132-mutated CCA following at least one prior systemic therapy. The pivotal phase 3 ClarIDHy trial revealed that ivosidenib nearly doubled the median progression-free survival and significantly improved median overall survival after adjusting for crossover. However, prospective real-world data on ivosidenib remain limited. The IDHIRA study is a prospective, multicenter, longitudinal, non-interventional study conducted in Germany. IDHIRA will enroll 100 patients with advanced IDH1 R132-mutated CCA, aiming to evaluate the real-world effectiveness, safety, and quality of life (QoL) associated with ivosidenib treatment in CCA patients. Clinical trial registration www.clinicaltrials.gov identifier is NCT06607302.

Cholangiocarcinoma (CCA) is a rare cancer with limited therapeutic options and a poor prognosis. While first‐line combination therapies have improved outcomes, second‐line treatment remains challenging. Ivosidenib, an IDH1 inhibitor, has shown promise in treating IDH1 mutant CCA, but real‐world data is limited. This study aims to evaluate ivosidenib's efficacy and safety in a large cohort of patients and compare it with second‐line chemotherapy.

Abstract Background There is limited evidence regarding the economic burden, treatment patterns, and overall survival (OS) of patients with cholangiocarcinoma (CCA) and cancer of unknown primary (CUP) who initiated the FGFR inhibitor pemigatinib. Patients and Methods We used the Komodo Healthcare Map to identify patients with CCA who initiated pemigatinib between 4/17/2020 and 5/31/2023. Follow-up began at initiation and lasted ≥ 1 month. Outcomes included health care resource utilization (HCRU), costs, treatment patterns, and OS. Results Two hundred twenty-one patients were included: 78 patients (35.3%) with CUP (median follow-up, 5.9 months) and 143 patients (64.7%) without CUP (median follow-up, 7.3 months). Pemigatinib was similarly well-tolerated in CUP vs non-CUP. Discontinuation was observed in 43.6% vs 49.0% (P = .445). Medication possession ratio ≥ 0.80 was achieved by 71.6% vs 67.2% (P = .504). CUP was associated with significantly higher prevalence of metastatic disease (100.0% vs 63.6%), per patient per month (PPPM) ambulatory HCRU (8.2 vs 5.5), and ambulatory costs ($8584 vs $5308). Medical costs averaged $13 444 vs $9881 PPPM for CUP and non-CUP, respectively (P = .066). Median OS was significantly shorter with CUP (10.2 vs 30.7 months). Conclusion Although pemigatinib was similarly well-tolerated regardless of CUP status, patients with CUP incurred greater ambulatory burden and had poorer OS. Patients with CUP were more likely to have evidence of metastatic disease at pemigatinib initiation, which may help explain these results. With the advent of targeted treatments for gene-altered CCA, reflexive genomic testing should be encouraged for all patients with CUP.

e16227 Background: The phase III ClarIDHy trial led to FDA approval of ivosidenib as a therapeutic option for locally advanced or metastatic cholangiocarcinoma (CCA) patients with isocitrate dehydrogenase 1 (IDH1) mutations. However, data regarding its efficacy and safety in Chinese population remains limited. Methods: We retrospectively analyzed survival outcomes of patients with locally advanced or metastatic IDH1-mutated intrahepatic cholangiocarcinoma (iCCA) treated with ivosidenib in clinical practice at our center from January 2019 to January 2025. Ivosidenib was administered at a standard dose of 500mg once daily in 28-days cycles. Molecular profiling was performed using next-generation sequencing. Results: As of January 20, 2025, five patients were included in the study. The cohort comprised predominantly men (60%) with a median age of 69 years (range: 54-78). At baseline, the overall Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of patients is 1-2, with 80% of patients having a score of 2. All patients were diagnosed with metastatic iCCA, with liver and lymph node metastases being most common (60% each). The IDH1 mutation type identified in all 5 patients was R132C missense mutation, and 3 patients reported traceable molecular analysis, highlighting TP53, ALK and ARID1A as the most common co-altered genes in these patients. Ivosidenib was administered as first-line therapy in 1 patient (20%), second-line therapy in 3 patients (60%) and third-line therapy in 1 patient (20%). At the data cutoff, two patients (40%) were long-term survivors (alive≥1 year) and their survival follow-up is ongoing. The median progression-free survival (PFS) from initiation of treatment with ivosidenib was 5.1 months (range: 2.0-28.5), and the median overall survival (OS) was 9.5 months (range: 3.2-30.5), regardless of the treatment line. One patient (20%) achieved a partial response (PR), while four patients (80%) maintained stable disease (SD) as their best response. Treatment-emergent adverse events (AEs) were reported in one patient, all of which were grade 1, including diarrhea, rash, and oral mucositis. Conclusions: This preliminary data suggested the clinical benefit of ivosidenib for metastatic IDH1-mutated iCCA in the Chinese population. Further multi-center studies with larger cohorts are warranted to validate these findings and explore the broader applicability of ivosidenib in this patient population. Start of ivosidenib regardless of treatment line. Patient ID Extent of disease Setting Best response Survival PFS (months) OS (months) IDH1 mutation Concomitant genetic alterations 1 IV II L PR Yes 4.6 15.3 R132C TP53 2 IV III L SD Yes 28.5 30.5 R132C ALK, ARID1A 3 IV I L SD No 2 3.2 R132C NA 4 IV II L SD No 5.1 9.5 R132C TP53 5 IV II L SD No 4.1 4.1 R132C NA

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Isocitrate dehydrogenase 1 (IDH1) mutations have gained interest because of their association with malignancies, including cholangiocarcinoma and acute myeloid leukemia. Ivosidenib, an inhibitor of IDH1 mutations, inhibits the formation of the oncometabolite D-2-HG, restoring normal cellular turnover and inhibiting tumorigenesis. In July 2024, a literature search was done using these databases: PubMed, Cochrane Library, and Embase. Studies were to show the safety and efficacy of ivosidenib using 95% confidence intervals (CIs). Preferred Reporting Items for Systematic reviews and Meta-Analyses flow guidelines were followed. Four articles involving 533 patients were included. The objective response rate (ORR) and progression-free survival (PFS) were significantly improved in the control group where risk ratio was 0.79, 95% CI: 0.71-0.89, Z = 4.05, a P value less than 0.001 for PFS, and odds ratio was 0.45, 95% CI: 0.30-0.68, Z value of 3.86, and P = 0.001 for ORR. The safety profile was favorable. Overall survival (OS) did not change significantly within the groups, as indicated by a P value of 0.78, risk ratio of 0.98, 95% CI: 0.83-1.15, and Z = 0.27. Ivosidenib demonstrated a PFS advantage and improved ORR with a favorable safety profile, but no effect on the OS. Evidence is suggestive of its plausibility for clinical usage as an adjunct therapy.

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We report a case of a 55-year-old male with intrahepatic cholangiocarcinoma (iCCA) who underwent living donor liver transplantation (LDLT) after complete radiographic response on second line pemigatinib. LDLT for iCCA is controversial, but recent reports have cited the potential benefit for patients with unresectable disease, especially those with disease stability after 6 months of systemic therapy. Concomitantly, genomic profiling has identified potentially treatable oncologic targets in iCCA. This patient's tumor genomic profile revealed a FGFR2 rearrangement and was treated with pemigatinib, a competitive inhibitor for FGFR1/2/3. This resulted in complete radiographic and metabolic response after two months of treatment. He was considered eligible for LDLT after 6 months of observation on treatment with sustained response. He underwent an uncomplicated LDLT (including an uncomplicated donor surgery) and at one year follow-up is without evidence of disease recurrence. We believe this is the first report of LDLT for this indication.

International guidelines recommend ivosidenib followed by modified FOLFOX (mFOLFOX) for advanced intrahepatic cholangiocarcinoma (ICC) with isocitrate dehydrogenase 1 (IDH1) mutations. Taiwan National Health Insurance covers only fluorouracil/leucovorin (5-FU/LV) chemotherapy for this ICC group, and there has been no prior economic evaluation of ivosidenib. Therefore, we aimed to assess ivosidenib’s cost-effectiveness in previously treated, advanced ICC-presenting IDH1 mutations compared with mFOLFOX or 5-FU/LV. A 3-state partitioned survival model was employed to assess ivosidenib’s cost-effectiveness over a 10-year horizon with a 3% discount rate, setting the willingness-to-pay threshold at 3 times the 2022 GDP per capita. Efficacy data for Ivosidenib, mFOLFOX, and 5-FU/LV were sourced from the ClarIDHy, ABC06, and NIFTY trials, respectively. Ivosidenib’s cost was assumed to be NT$10,402/500 mg. Primary outcomes included incremental cost-effectiveness ratios (ICERs) and net monetary benefit. Deterministic sensitivity analyses (DSA) and probabilistic sensitivity analyses (PSA) were employed to evaluate uncertainty and explore price reduction scenarios. Ivosidenib exhibited ICERs of NT$6,268,528 and NT$5,670,555 compared with mFOLFOX and 5-FU/LV, respectively, both exceeding the established threshold. PSA revealed that ivosidenib was unlikely to be cost-effective, except when it was reduced to NT$4,161 and NT$5,201/500 mg when compared with mFOLFOX and 5-FU/LV, respectively. DSA underscored the significant influence of ivosidenib’s cost and utility values on estimate uncertainty. At NT$10,402/500 mg, ivosidenib was not cost-effective for IDH1-mutant ICC patients compared with mFOLFOX or 5-FU/LV, indicating that a 50–60% price reduction is necessary for ivosidenib to be cost-effective in this patient group.

Abstract Background Pemigatinib demonstrated efficacy in fibroblast growth factor receptor (FGFR)-altered cholangiocarcinoma (CCA) in the FIGHT-202 trial. However, limited real-world evidence exists on treatment patterns and outcomes in this setting. Patients and Methods Patient characteristics, treatment patterns, and outcomes of US adults who received pemigatinib for unresectable, locally advanced or metastatic CCA were collected via retrospective physician-abstracted chart review. Results were summarized using descriptive statistics. Results Data from 120 patients (49.2% male; 55.0% White; 19.2% Hispanic; median age at initial pemigatinib prescription, 64.5 years) were collected from 18 physicians/practices. At the time of prescribing, 90.0% of patients had metastatic disease. FGFR2 testing was completed for 92.5% of patients; of those, all but one (result unknown) tested positive, and 95.5% were tested using next-generation sequencing. Pemigatinib was prescribed as second- and third-line therapy among 94.2% and 5.8% of patients, respectively. The most common starting dosage was 13.5 mg daily for 14 days of 21-day cycles (87.5% of patients). Among 60 patients (50.0% of the full cohort) who discontinued pemigatinib during the 6.5-month median study follow-up period, 68.3% discontinued due to disease progression. The median real-world progression-free survival (rwPFS) from the date of pemigatinib initiation was 7.4 months (95% CI: 6.4-8.6), and the real-world overall response rate (rwORR) was 59.2% (95% CI: 50.0%-68.4%). Conclusion This study complements the FIGHT-202 clinical trial by assessing the use of pemigatinib among a diverse population of patients with CCA under real-world conditions. Findings support the clinical benefit of pemigatinib demonstrated in FIGHT-202.

Report pharmacokinetic (PK)/pharmacodynamic (PD) findings from the phase III ClarIDHy study and any association between PK/PD parameters and treatment outcomes in this population. Patients with mutant isocitrate dehydrogenase 1 (mIDH1) advanced cholangiocarcinoma were randomized at a 2:1 ratio to receive ivosidenib or matched placebo. Crossover from placebo to ivosidenib was permitted at radiographic disease progression. Blood samples for PK/PD analyses, a secondary endpoint, were collected pre-dose and up to 4 h post-dose on day (D) 1 of cycles (C) 1 − 2, pre-dose and 2 h post-dose on D15 of C1 − 2, and pre-dose on D1 from C3 onwards. Plasma ivosidenib and D-2-hydroxyglutarate (2-HG) were measured using liquid chromatography-tandem mass spectrometry. All clinical responses were centrally reviewed previously. PK/PD analysis was available for samples from 156 ivosidenib-treated patients. Ivosidenib was absorbed rapidly following single and multiple oral doses (time of maximum observed plasma concentration [Tmax] of 2.63 and 2.07 h, respectively). Ivosidenib exposure was higher at C2D1 than after a single dose, with low accumulation. In ivosidenib-treated patients, mean plasma 2-HG concentration was reduced from 1108 ng/mL at baseline to 97.7 ng/mL at C2D1, close to levels previously observed in healthy individuals. An average 2-HG inhibition of 75.0% was observed at steady state. No plasma 2-HG decreases were seen with placebo. Plasma 2-HG reductions were observed in ivosidenib-treated patients irrespective of best overall response (progressive disease, or partial response and stable disease). Once-daily ivosidenib 500 mg has a favorable PK/PD profile, attesting the 2-HG reduction mechanism of action and, thus, positive outcomes in treated patients with advanced mIDH1 cholangiocarcinoma. NCT02989857 Registered February 20, 2017.

PURPOSE This study aimed to investigate the clinicomolecular profiles and the efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapy in HER2-amplified biliary tract cancer (BTC). METHODS This study was an international collaboration that used combined data from the prospective SCRUM-Japan GOZILA and MONSTAR-SCREEN in Japan and retrospective reviews in the United States; patients with advanced BTC who had received systemic therapy were included. The clinicomolecular profiles were evaluated in an exploratory cohort, whereas the efficacy of HER2-targeted therapy was assessed in a biomarker-selected cohort. RESULTS Of the 439 patients in the exploratory cohort, 43 (10%) had HER2 amplification. The frequencies of coalterations were higher in patients with HER2 amplification versus patients without HER2 amplification including HER2 mutations (26% v 5%, P < .001), TP53 mutations (84% v 61%, P = .003), and BRAF amplification (9% v 2%, P = .030). There were no KRAS mutations identified in patients with HER2-amplified BTC. No significant difference in overall survival (OS) was observed between patients with and without HER2 amplification (median, 17.7 v 16.9 months; hazard ratio [HR], 0.95 [95% CI, 0.65 to 1.40]). Of the 60 patients with HER2-amplified BTC in the biomarker-selected cohort (43 from Japan and 17 from the United States), the OS was significantly longer in 29 patients who received HER2-targeted therapy than in those who did not receive HER2-targeted therapy (median, 24.3 v 12.1 months; HR, 0.39 [95% CI, 0.23 to 0.82]). Multivariate analysis identified HER2-targeted therapy as an independent prognostic factor for OS (HR, 0.29 [95% CI, 0.14 to 0.58]; P < .001). CONCLUSION HER2 amplification was found in 10% of advanced BTC and was not identified as an independent prognostic factor for OS. Patients with HER2-amplified BTC derive significant benefit from HER2-targeted therapy.

4085 Background: Cholangiocarcinoma (CC) is a rare disease with an increasing incidence among younger adults, which is poorly understood. Genomic profiling of tumors is both prognostic and predictive of benefit for targeted therapies. We investigated if there are clinical and molecular differences between younger vs older patients with CC. Methods: We collected TEMPUS genetic data via retrospective chart review from tumors in young vs old patients seen at our institution, defined as ≤50 vs > 50 years of age at time of diagnosis. We included patients diagnosed with CC between January 2008 and July 2024 with available clinical follow up and TEMPUS genetic sequencing data. We collected mutation data on the following actionable genes: FGFR2, IDH1/2, BRCA1, BRCA2, BRAF, ATM, ERBB2/3, and KRAS. Patient characteristics and gene expression variables were compared using Chi-square, Fisher’s exact and Wilcoxon rank-sum tests. Kaplan-Meier, log rank tests and a multivariable Cox model were used for survival analysis. This study was IRB exempt. Results: We included 410 patients, 84 in the young group with median age at diagnosis of 40.8 years, and 326 in the old group with median age 68.5 years. 91.5% of patients were white. There was no difference in BMI between groups, however the older group had higher rates of hypertension (15.5% vs 57.7%), hyperlipidemia (6.0% vs 49.7%), cardiovascular disease (1.2% vs 20.6%), and type 2 diabetes (6.0% vs 21.5%), (all p < 0.01). Primary sclerosing cholangitis was more common in the young group (26.2% vs 4.3%, p < 0.01). ECOG status of 0 at first treatment was seen in 65.3% of young vs 52.5% of old patients (p = 0.02). FGFR2 alterations were more common in the young group (17.9% vs 8.0%, p < 0.01), while ATM mutations were more common in old vs young (5.5% vs 0%, p = 0.03). There was no age difference seen for the other genetic alterations. Mean tumor mutational burden was higher in the old group (4.1 vs 3.8 mut/mb, p = 0.01). MSI-high was found in 2% of cases with no difference between groups. There was no significant difference in overall survival between age groups. There was a numeric difference in overall survival in stage IV patients, though not statistically significant (17.8 months vs. 16.3 months, p = 0.08). In a multivariable Cox analysis, female sex, earlier stage at diagnosis and clinical trial enrollment were associated with favorable prognostics. Conclusions: Our data highlight relatively low rates of comorbidities associated with metabolic dysfunction in younger adults with CC, suggesting alternative factors are likely to explain the increasing incidence of early-onset disease. FGRFR2 is a more common pathogenic alteration among the young and could inform targeted therapies. Younger patients with CC may not have improved survival outcomes compared to their older counterparts. This underscores the aggressive nature of CC and the need for more effective therapies to improve outcomes.

545 Background: Targeted therapy for actionable mutations in advanced cholangiocarcinoma (CCA) has revolutionized second and third-line treatment algorithms. Their application in early stage disease is not defined. The incidence and prognostic value of actionable mutations in pts with early stage resected disease is not known. Methods: The Cholangiocarcinoma Foundation and Ciitizen (a wholly owned subsidiary of Invitae Corporation) collaboratively launched a registry platform that directly consents pts and collects comprehensive medical records. Presently, de-identified data including clinical characteristics, molecular testing, interventions, and outcomes have been extracted and standardized for research use on 400 pts. The data is longitudinal with regularly planned updates. We identified pts who underwent resection of non-metastatic disease and underwent genetic testing. Actionable mutations recorded included FGFR2 fusion/rearrangement, IDH1 mutation, HER2 amplification, BRAF mutation, MSI-high, and TMB-high. Primary outcome was recurrence-free survival (RFS). Results: Of 400 pts, 137 underwent resection for CCA. When compared to pts with advanced disease, those who underwent resection had a similar frequency of genetic testing and presence of actionable mutations: 94.3% (n=248) vs 83.9% (n=115) and 38.7% (n=96) vs. 33.0% (n=38), respectively. When considering pts with non-metastatic, resected disease who underwent molecular testing (n=104), the mean age was 59.2 yrs, 66 (63.5%) were females, and 60 (57.7%) had intrahepatic cholangiocarcinoma (iCCA), 42 (40.4%) had extrahepatic disease, and 2 (1.9%) were not otherwise specified. The majority received molecular testing before or at the time of surgery (n=89, 85.6%). Of pts with non-metastatic resectable disease who received genetic testing, 34 pts (32.7%) had actionable mutations: FGFR2: n=7 (6.7%), IDH1: n=16 (15.4%), HER2: n=5 (4.8%), BRAF: n=0 (0%), MSI-high: n=3 (2.9%), TMB-high: n=8 (7.7%). FGFR2 fusion/rearrangement and IDH1 mutations were only identified in iCCA. Pts with an FGFR2 fusion/rearrangement were younger (49.9 vs 59.8 yrs; p=0.03) and all were female. Although not statistically significant, the presence of an FGFR2 fusion/rearrangement was associated with nearly a doubling of median RFS (32.0 vs 17.3 mos; p=0.19) for patients with iCCA. Conclusions: In this highly selected cohort of pts with resected, early stage, non-metastatic CCA, the majority underwent molecular profiling before or at the time of surgery. As expected, FGFR2 fusion/rearrangement and IDH1 mutations were only present in iCCA. An FGFR2 fusion/rearrangement was seen in young, female pts and may have a favorable prognostic value as suggested by a trend in increased RFS. Clinical trials are needed to assess the value of administering targeted therapy to this patient population in the adjuvant or neoadjuvant setting.

Background: Biliary tract cancer constitutes a heterogeneous group of diseases with a limited understanding of its molecular characteristics and prognostic/predictive biomarkers. We performed prespecified exploratory analyses of the STAMP trial, a multicenter randomized phase 2 trial of adjuvant capecitabine (CAP) or gemcitabine plus cisplatin (GemCis) for patients with resected extrahepatic cholangiocarcinoma (EH-CCA) with regional lymph node metastasis. Methods: Among 101 patients included in the intention-to-treat population of the STAMP trial, a total of 89 patients (45 in the GemCis group and 44 in the CAP group) were included in the current biomarker analysis after excluding 12 patients without available tissue and blood samples. Whole exome sequencing and proteomics were performed using surgical specimens. Results: TP53 (63%), SMAD4 (20%), and KRAS (18%) mutations were the most frequent in the pooled population. Somatic mutations in homologous recombination deficient (HRD) genes were present in 20% of patients, while one patient (1%) had a germline ATM mutation. The prevalence of microsatellite instability was 1%. Somatic mutations in known targetable genes were observed in 54 patients (60.7%) and proteogenomic integrative analysis revealed significant HER2 pathway alteration with HER2 amplification in 4 patients (7%). The characteristics of prognostic biomarkers varied by the adjuvant regimen used. In the adjuvant GemCis group, somatic mutations were associated with poor disease-free survival (DFS) and overall survival (OS), including KRAS (log-rank p=0.004 for DFS and p=0.003 for OS), FBXW7 (p=0.021 for DFS and p=0.011 for OS), and PIK3CA (p<0.001 for DFS and p=0.042 for OS) mutations. In the adjuvant CAP group, copy number alterations (CNAs) such as 8q24.21 amplification (p=0.006 for DFS and p=0.707 for OS) and 3q26.1 amplification (p=0.020 for DFS and p=0.026 for OS) were associated with poor survival, while 11q13.3 amplification (p=0.017 for DFS and p=0.099 for OS) was associated with improved survival. Additionally, a higher HRD score showed a tendency toward better DFS and OS in the GemCis group, but not in the CAP group, although it did not reach statistical significance in both groups (p>0.05). Non-negative matrix clustering of proteomics data classified patients into 4 clusters. Among those, cluster 2, which showed alterations in the metabolism pathway, was associated with higher CNA level, higher tendency of HRD scores, and favorable survival with GemCis (p=0.039 for DFS and p=0.100 for OS) but not with CAP. Conclusions: Comprehensive proteogenomic analyses enable the identification of prognostic biomarkers and may guide the selection of adjuvant chemotherapy in resected EH-CCA. Citation Format: Hyehyun Jeong, Ji-Hye Oh, Hee-Sung Ahn, Baek-Yeol Ryoo, Kyu-pyo Kim, Jae Ho Jeong, Inkeun Park, Seung-Mo Hong, Jinho Shin, Chae Won Park, Yoo Sook Cho, Kyunggon Kim, Chang Ohk Sung, Changhoon Yoo. Comprehensive proteogenomic profiling and biomarker analyses in resected extrahepatic cholangiocarcinoma: A prespecified exploratory analysis of the phase 2 STAMP trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6422.

PURPOSE BRAF mutations are rare in Biliary Tract Cancers (BTC), but of interest given the recent developments in targeted therapy for BTC. We investigated the clinical outcomes in a cohort of BRAF-mutant advanced BTC treated with first-line chemotherapy. Furthermore, we investigated the genomic landscape of BRAF Class I, II and III mutations in the intrahepatic cholangiocarcinoma (iCCA) subgroup of BTC. EXPERIMENTAL DESIGN We analyzed two, non-overlapping cohorts. We examined the genomic landscape of BRAF mutated iCCA in a 'genomic cohort' [187 Class I, 82 Class II, 113 Class III BRAF-mutants and 8,026 wildtype (WT)]. We also analyzed median progression free survival (PFS) and overall survival (OS) on first-line chemotherapy in a separate multi-institutional 'clinical cohort' of BTC (including intrahepatic and extrahepatic cholangiocarcinoma and gallbladder cancer) patients (41 Class I, 32 Class II+III BRAF-mutants and 1,042 WT). RESULTS In the entire BTC clinical cohort, the median PFS was shorter for class I [hazard ratio (HR) 2.11 (p<0.001)] and class II+III [HR 1.72 (p=0.007)] as compared with BRAF WT. OS was also shorter in Class I [HR 2.04 (p=0.011)] and Class II+III [HR 1.86 (p=0.002)] as compared with BRAF WT. In the iCCA subgroup, Class I alterations were mutually exclusive with FGFR2, IDH1/2, ERBB2 and KRAS mutations. Class II+III mutations appear to be mutually exclusive with FGFR2 and KRAS. CONCLUSIONS In BTC, all classes of BRAF mutations are associated with a worse prognosis. BRAF mutations occur in 5% of iCCA subgroup and may be mutually exclusive with other targetable mutations.

Liquid biopsy has already proven effective in aiding diagnosis, risk stratification and treatment personalization in several malignancies, and it could represent a practice-changing tool also in biliary tract cancer, even though clinical applications are currently still limited. It is promising for early diagnosis, especially in high-risk populations, and several studies on circulating free DNA (cfDNA), circulating tumour cells and differential microRNA (miRNA) profiles in this setting are ongoing. Circulating tumour DNA (ctDNA) also appears as a feasible noninvasive biomarker in the curative setting, in detecting minimal residual disease after resection and in monitoring disease recurrence. As of today, it can be particularly valuable in biliary tract cancer for genomic profiling, with a good concordance with tissue samples for most molecular alterations. CtDNA analysis may especially be considered in clinical practice when the tumour tissue is not sufficient for next-generation sequencing, or when urgent therapeutic decisions are needed. Moreover, it offers the possibility of providing a real-time picture to monitor treatment response and dynamically identify resistance mutations, potentially representing a way to optimize treatment strategies.

Biliary tract cancers, including intrahepatic, perihilar, and distal cholangiocarcinoma as well as gallbladder cancer, are low-incidence malignancies in most high-income countries, but represent a major health problem in endemic areas; moreover, the incidence of intrahepatic cholangiocarcinoma is rising globally. Surgery is the cornerstone of cure; the optimal approach depends on the anatomical site of the primary tumour and the best outcomes are achieved through management by specialist multidisciplinary teams. Unfortunately, most patients present with locally advanced or metastatic disease. Most studies in advanced disease have pooled the various subtypes of biliary tract cancer by necessity to achieve adequate sample sizes; however, differences in epidemiology, clinical presentation, natural history, surgical therapy, response to treatment, and prognosis have long been recognised. Additionally, the identification of distinct patient subgroups harbouring unique molecular alterations with corresponding targeted therapies (such as isocitrate dehydrogenase-1 mutations and fibroblast growth factor receptor-2 fusions in intrahepatic cholangiocarcinoma, among others) is changing the treatment paradigm. In this Seminar we present an update of the causes, diagnosis, molecular classification, and treatment of biliary tract cancer.

The prognosis for patients with biliary tract cancers (cholangiocarcinoma and gallbladder cancer) is poor, while the incidence of these cancers is increasing. Most patients are diagnosed with advanced disease when treatment options are limited to palliative approaches, mainly focused on chemotherapy. In recent years, novel treatment targets of relevance to biliary tract cancers, mainly present in patients with intrahepatic cholangiocarcinoma, have been identified and are rapidly changing the field. These include fibroblast growth factor receptor (FGFR) fusions and isocitrate dehydrogenase (IDH)-1 and -2 mutations which are each present in around 10-20% of patients with intrahepatic cholangiocarcinoma. In addition, inhibition of other pathways/molecules is currently being explored, including human epidermal growth factor receptor (HER) family members, the Wnt pathway, neurotropic tyrosine kinase receptor (NTRK) fusions and BRAF mutations. The IDH1 inhibitor ivosidenib has already been tested in a phase III clinical trial in pretreated cholangiocarcinoma and showed benefit in terms of progression-free survival. Multiple FGFR inhibitors have consistently shown high response rates in phase II/III trials, especially for patients harbouring FGFR2 fusions. Herein, we provide an overview of the status of targeted therapies in biliary tract cancers, discussing the current clinical development of IDH and FGFR inhibitors in detail, as well as reviewing current caveats and future steps.

Management of biliary tract cancers (BTCs) is rapidly evolving. Curative management relies on surgical resection followed by adjuvant capecitabine for cholangiocarcinoma and gallbladder cancers. Unfortunately relapse rate remains high, and better adjuvant strategies are urgently required. A majority of patients are diagnosed with advanced disease, when chemotherapy with cisplatin and gemcitabine followed by second-line 5-FU and oxaliplatin /irinotecan is the cornerstone of treatment for most patients in the absence of targetable alterations. Targeted therapies, including therapies for tumours with fibroblast growth factor receptor-2 (FGFR-2) fusions, isocitrate dehydrogenase-1 (IDH-1) mutations, B-Raf proto-oncogene serine/threonine kinase (BRAF) V600E mutations, neurotrophic tyrosine receptor kinase (NTRK) fusions, Human epidermal growth factor-2 (HER-2) amplifications, and/or microsatellite instability are rapidly changing the treatment paradigm for many patients with advanced BTC, especially for patients with intrahepatic cholangiocarcinoma. Because of this, molecular profiling should be considered early on patients pathway to allow adequate planning of therapy. Ongoing research is likely to clarify the role of immunotherapy, liver-directed therapy, and liver transplant for BTCs in the future.

Systemic therapies for metastatic biliary tract cancers are few, and patients have a median overall survival of less than 1 year. MyPathway evaluates the activity of US Food and Drug Administration-approved therapies in non-indicated tumours with potentially actionable molecular alterations. In this study, we present an analysis of patients with metastatic biliary tract cancers with HER2 amplification, overexpression, or both treated with a dual anti-HER2 regimen, pertuzumab plus trastuzumab, from MyPathway. MyPathway is a non-randomised, multicentre, open-label, phase 2a, multiple basket study. Patients aged 18 years and older with previously treated metastatic biliary tract cancers with HER2 amplification, HER2 overexpression, or both and an Eastern Cooperative Oncology Group performance status of 0-2 were enrolled from 23 study sites in the USA and received intravenous pertuzumab (840 mg loading dose, then 420 mg every 3 weeks) plus trastuzumab (8 mg/kg loading dose, then 6 mg/kg every 3 weeks). The primary endpoint was investigator-assessed objective response rate according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The primary outcome and adverse events were analysed in all patients who received at least one dose of pertuzumab and trastuzumab. This trial is registered with ClinicalTrials.gov, NCT02091141, and is ongoing. 39 patients enrolled in the MyPathway HER2 biliary tract cancer cohort between Oct 28, 2014, and May 29, 2019, were evaluable for anti-tumour activity by the March 10, 2020, data cutoff date. Median follow-up was 8·1 months (IQR 2·7-15·7). Nine of 39 patients achieved a partial response (objective response rate 23% [95% CI 11-39]). Grade 3-4 treatment-emergent adverse events were reported in 18 (46%) of 39 patients, most commonly increased alanine aminotransferase and increased aspartate aminotransferase (each five [13%] of 39). Treatment-related grade 3 adverse events were reported in three (8%) of 39 patients, including increased alanine aminotransferase, aspartate aminotransferase, blood alkaline phosphatase, and blood bilirubin. Serious treatment-emergent adverse events were observed in ten (26%) of 39 patients, of which only abdominal pain occurred in more than one patient (two [5%] of 39). There were no treatment-related serious adverse events, treatment-related grade 4 events, or deaths. Treatment was well tolerated in patients with previously treated HER2-positive metastatic biliary tract cancer. The response rate is promising for the initiation of randomised, controlled trials of pertuzumab plus trastuzumab in this patient population. F Hoffmann-La Roche-Genentech.

Biliary tract cancers (BTC) are aggressive malignancies encompassing intrahepatic and extrahepatic cholangiocarcinoma, gallbladder carcinoma, and ampullary carcinoma. Here, we report integrative analysis of 63 BTC cell lines via multi-omics and genome-scale CRISPR screens. We identify widespread EGFR dependency in BTC, alongside dependencies selective to anatomic subtypes. Additionally, we delineate strategies to overcome therapeutic resistance, with combined EGFR inhibition potentiating targeting of KRAS-mutant and FGFR2 fusion-driven models and SHP2 inhibition effective in the latter context. Clustering RNA/protein expression and dependencies data revealed functional relationships transcending single-gene alterations, with biliary, squamous, or dual biliary/hepatocyte lineage signatures stratifying BTC models. These subtypes exhibit distinct dependency profiles-including cell fate transcription factors GRHL2, TP63, and HNF1B, respectively-and demonstrate prognostic significance in patient samples. Potential subtype-specific targetable vulnerabilities include integrinα3 and the detoxification enzyme UXS1. This cell line atlas reveals therapeutic targets in molecularly defined BTCs, unveils disease subtypes, and provides a resource for therapeutic development. This integrative analysis of BTC cell lines defines the landscape of vulnerabilities across BTCs, stratifying distinct subtypes, and provides a key resource for studying disease heterogeneity. The findings highlight strategies for targeting BTCs with specific genomic alterations, as well as broader approaches based on shared molecular programs and essential pathways.

Carcinoma of the biliary tree are rare tumors of the gastrointestinal tract with worldwide rising incidence for intrahepatic cholangiocarcinoma during the last years. Although complete surgical resection is the only curative approach, this can be accomplished in a minority of patients, since most of them present with advanced disease. In addition, those patients who have undergone complete surgical resection experience a high tumor recurrence rate. Non-resectable biliary tract cancer is associated with a poor prognosis due to wide resistance to chemotherapeutic agents and radiotherapy. It is therefore essential to search for new therapeutical approaches. After several years of preclinical research, the first clinical study data are now available for this tumor entity. Inhibitors of the epidermal growth factor receptor (EGFR) family, such as erlotinib, cetuximab, and lapatinib were recently investigated. Furthermore, bortezomib, an inhibitor of the proteasome, imatinib mesylate, an inhibitor of c-kit-R, bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF), and sorafenib (BAY 43-9006), a multiple kinase inhibitor that blocks not only receptor tyrosine kinases but also serine/threonine kinases along the RAS/RAF/MEK/ERK pathway, were studied, as well. Although early evidence of antitumor activity was seen, the results are still preliminary and require further investigations.

Biliary tract cancer (BTC) is a rare disease with a heterogeneous nomenclature. Carcinomas of the intra- and extrahepatic biliary tract as well as gallbladder cancer are oftentimes combined in clinical research and treatment algorithms. However, these different cancer types vary not only in their anatomical features, but also in the underlying molecular alterations.Surgery remains the only chance of cure. Adjuvant chemotherapy with capecitabine for 6 months should be recommended after curative intended surgery. In the palliative first-line treatment of advanced BTC, the combination chemotherapy gemcitabine and cisplatin remains the only evidence-based standard. For second-line treatment, the combination of 5-FU, folinic acid and oxaliplatin (FOLFOX) is a treatment option based on preliminary data from a randomized phase 3 study. Potential targeted therapies showing efficacy in prospective clinical studies are, for example, IDH-, BRAF-/MEK- and FGFR-inhibitors as well as immunotherapy. Therefore, in the era of personalized medicine, molecular testing should be offered to all patients with advanced disease and indication for systemic treatment. ÜBERBEGRIFF MALIGNE BILIäRE TUMOREN:  Unter dem Begriff maligne biliäre Tumoren (engl. biliary tract cancer) werden weiterhin Karzinome der intra- und extrahepatischen Gallengänge und der Gallenblase zusammengefasst, deren auch molekularpathologische Unterschiede immer besser verstanden werden Patienten nach einer kurativ intendierten Resektion sollte eine adjuvante Chemotherapie mit Capecitabin über 6 Monate angeboten werden. Die Kombination aus Gemcitabin und Cisplatin bleibt etablierter Standard in der palliativen Erstlinientherapie. Patienten in einem guten Allgemeinzustand kann aufgrund der positiven Ergebnisse der randomisierten ABC-06-Studie eine Zweitlinientherapie empfohlen werden. Mehr als 50 % aller Patienten mit malignen biliären Tumoren weisen therapierbare genetische Alterationen auf. Erste prospektive Daten belegen den Nutzen einer zielgerichteten Therapie bei diesen Patienten. Deswegen sollte möglichst allen Patienten, die eine palliative Systemtherapie benötigen, frühzeitig ein molekulares Profiling angeboten werden.

Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies. An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC. KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with ∼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFβ signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors. An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches. Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.

Biliary cancers are highly aggressive tumors that are often diagnosed an advanced disease stage and have a poor outcome with systemic therapy. Recent efforts towards molecular characterization have identified a subset of biliary patients that have HER2/neu amplification or mutation. HER2/neu amplification is associated with response to HER2/neu-directed therapy in breast and gastric cancers. However, the efficacy of HER2/neu-targeted therapy in biliary cancers is unknown. We retrospectively reviewed cases of advanced gallbladder cancer and cholangiocarcinoma with HER2/neu genetic aberrations or protein overexpression who received HER2/neu-directed therapy between 2007 and 2014. Clinical data were retrieved from medical records, and imaging studies were independently reviewed. Nine patients with gallbladder cancer and five patients with cholangiocarcinoma had received HER2/neu-directed therapy (trastuzumab, lapatinib, or pertuzumab) during the study period. In the gallbladder cancer group, HER2/neu gene amplification or overexpression was detected in eight cases. These patients experienced disease stability (n = 3), partial response (n = 4), or complete response (n = 1) with HER2/neu-directed therapy. One patient had HER2/neu mutation and experienced a mixed response after lapatinib therapy. The duration of response varied from 8+ to 168 weeks (median 40 weeks), and three patients are still on therapy. One patient developed HER2/neu amplification as a secondary event after FGFR-directed therapy for FGF3-TACC3 gene fusion. The cholangiocarcinoma cases treated in this series had a higher proportion of HER2/neu mutations, and no radiological responses were seen in these patients despite HER2/neu-directed therapy. HER2/neu blockade is a promising treatment strategy for gallbladder cancer patients with gene amplification and deserves further exploration in a multi-center study.

In recent years, intrahepatic cholangiocarcinoma (iCCA) has evolved as a "role model" for precision oncology in gastrointestinal cancers. However, its rarity, paired with its genomic heterogeneity, challenges the development and evolution of targeted therapies. Interrogating large datasets drives better understanding of the characteristics of molecular subgroups of rare cancers and enables the identification of genomic patterns that remain unrecognized in smaller cohorts. We performed a retrospective analysis of 6,130 patients diagnosed with iCCA from the FoundationCORE database who received diagnostic panel sequencing on the FoundationOne platform. Short variants/fusion-rearrangements and copy number alterations in >300 tumor-associated genes were evaluated, and the tumor mutational burden (TMB) as well as the microsatellite instability (MSI) status were available for the majority of the cohort. We provide a highly representative cartography of the genomic landscape of iCCA and outline the co-mutational spectra of seven therapeutically relevant oncogenic driver genes: IDH1/2, FGFR2, ERBB2, BRAF, MDM2, BRCA1/2, MET and KRAS Detailed knowledge of the most prevalent genomic constellations is key to the development of effective treatment strategies for iCCA. Our study provides a valuable resource that could be used to assess the feasibility of clinical trials and subgroup analyses, spurs the development of translationally relevant preclinical models, and serves as a knowledge base to predict potential mechanisms of resistance to targeted therapies in genomically defined subgroups. Due to the high frequency of targetable alterations, molecular diagnostics is recommended in patients with biliary tract cancers, and especially in those with iCCA. The identification of an actionable lesion, however, does not guarantee therapeutic success, and the co-mutational spectrum may act as a critical modifier of drug response. Using a large dataset of comprehensive panel sequencing results from 6,130 patients with iCCA, we provide a detailed analysis of the co-mutational spectrum of the most frequent druggable genetic alterations, which is meant to serve as a reference to establish genetically relevant preclinical models, develop hypothesis-driven combination therapies and identify recurrent genetic profiles.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in

Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy, primarily comprising intrahepatic (iCCA) and extrahepatic (eCCA) subtypes. Reconciling the variability between iCCAs and eCCAs in clinical trials remains a challenge, largely due to the inadequate understanding of their shared and subtype-specific cellular heterogeneity. We aim to address this issue using single-cell and spatially resolved transcriptomic approaches. We performed comprehensive single-cell RNA sequencing (scRNA-seq) by profiling 109,071 single cells from 28 samples, including chronic biliary inflammatory conditions (n = 7) and CCAs from different anatomical sites (n = 21). Findings were validated using external multi-omics datasets, tissue microarray cohort, spatial RNA in situ sequencing, CCA patient-derived organoids (PDOs), and mouse models. iCCAs and eCCAs exhibited distinct tumor ecosystems, with notable differences in cellular composition, diversity, and abundance across various cell types. Non-malignant epithelial cells displayed divergent precancer hallmarks from different biliary sites, with inflammatory extrahepatic bile ducts exhibiting early hijacking of the gastrointestinal metaplastic process. We identified seven meta-programs within cancer cells, mapped into four major subtypes. This subtyping was validated using external CCA cohorts and PDO models, distinguishing patients based on clinical outcomes and drug vulnerabilities. Specifically, iCCAs were associated with a senescent program, while eCCAs were enriched in an IFN-responsive program linked to adverse clinical outcomes and increased drug resistance. We identified a basal-like LY6D We present a comprehensive single-cell landscape of CCAs, uncovering the molecular heterogeneity between iCCA and eCCA subtypes. Transcriptomic subtyping of CCA cancer cells offers implications for clinical stratification and functional precision oncology. We identify basal-like epithelial progenitors and characterize their associated ISG15-enriched microenvironment in eCCAs. These findings hold significant promise for the development of novel prognostic biomarkers, therapeutic targets, and treatment strategies for CCAs. [Image: see text]

Cholangiocarcinoma (CCA) is a highly aggressive and lethal cancer that originates from the biliary epithelium. Systemic treatment options for CCA are currently limited, and the first targeted drug of CCA, pemigatinib, emerged in 2020 for CCA treatment by inhibiting FGFR2 phosphorylation. However, the regulatory mechanism of FGFR2 phosphorylation is not fully elucidated. Here we screened the FGFR2-interacting proteins and showed that protein tyrosine phosphatase (PTP) N9 interacts with FGFR2 and negatively regulates FGFR2 pY656/657 . Using phosphatase activity assays and modeling the FGFR2-PTPN9 complex structure, we identified FGFR2 pY656/657 as a substrate of PTPN9, and found that sec. 14p domain of PTPN9 interacts with FGFR2 through ACAP1 mediation. Coexpression of PTPN9 and ACAP1 indicates a favorable prognosis for CCA. In addition, we identified key amino acids and motifs involved in the sec. 14p-APCP1-FGFR2 interaction, including the "YRETRRKE" motif of sec. 14p, Y471 of PTPN9, as well as the PH and Arf-GAP domain of ACAP1. Moreover, we discovered that the FGFR2 I654V substitution can decrease PTPN9-FGFR2 interaction and thereby reduce the effectiveness of pemigatinib treatment. Using a series of in vitro and in vivo experiments including patient-derived xenografts (PDX), we showed that PTPN9 synergistically enhances pemigatinib effectiveness and suppresses CCA proliferation, migration, and invasion by inhibiting FGFR2 pY656/657 . Our study identifies PTPN9 as a negative regulator of FGFR2 phosphorylation and a synergistic factor for pemigatinib treatment. The molecular mechanism, oncogenic function, and clinical significance of the PTPN9-ACAP1-FGFR2 complex are revealed, providing more evidence for CCA precision treatment.

The complexity of cholangiocarcinoma (CCA) cellularity and the molecular perturbation mechanisms that underlie the diversity of growth patterns of this malignancy remain a clinical concern. Tumours of the biliary system display significant intrinsic chemoresistance, caused by significant stromal involvement and genome-wide tumour heterogeneity, hampering disease remission and palliation as well as promoting the metastatic behaviour. It is crucial to advance our present understanding of the risk and molecular pathogenesis of CCA. This will facilitate the delineation of patient subsets based on molecular perturbations and adjust for precision therapies.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is upregulated in many tumor types and is a promising target for cancer therapy. Herein, we elucidated the functional role of FAK in intrahepatic cholangiocarcinoma (iCCA) development and progression. Expression levels and activation status of FAK were determined in human iCCA samples. The functional contribution of FAK to Akt/YAP murine iCCA initiation and progression was investigated using conditional Fak knockout mice and constitutive Cre or inducible Cre mice, respectively. The oncogenic potential of FAK was further examined via overexpression of FAK in mice. In vitro cell line studies and in vivo drug treatment were applied to address the therapeutic potential of targeting FAK for iCCA treatment. FAK was ubiquitously upregulated and activated in iCCA lesions. Ablation of FAK strongly delayed Akt/YAP-driven mouse iCCA initiation. FAK overexpression synergized with activated AKT to promote iCCA development and accelerated Akt/Jag1-driven cholangiocarcinogenesis. Mechanistically, FAK was required for YAP(Y357) phosphorylation, supporting the role of FAK as a central YAP regulator in iCCA. Significantly, ablation of FAK after Akt/YAP-dependent iCCA formation strongly suppressed tumor progression in mice. Furthermore, a remarkable iCCA growth reduction was achieved when a FAK inhibitor and palbociclib, a CDK4/6 inhibitor, were administered simultaneously in human iCCA cell lines and Akt/YAP mice. FAK activation contributes to the initiation and progression of iCCA by inducing the YAP proto-oncogene. Targeting FAK, either alone or in combination with anti-CDK4/6 inhibitors, may be an effective strategy for iCCA treatment. We found that the protein FAK (focal adhesion kinase) is upregulated and activated in human and mouse intrahepatic cholangiocarcinoma samples. FAK promotes intrahepatic cholangiocarcinoma development, whereas deletion of FAK strongly suppresses its initiation and progression. Combined FAK and CDK4/6 inhibitor treatment had a strong anti-cancer effect in in vitro and in vivo models. This combination therapy might represent a valuable and novel treatment against human intrahepatic cholangiocarcinoma.

FGFR inhibitors are approved for the treatment of advanced cholangiocarcinoma harboring FGFR2 fusions. However, the response rate is moderate, and resistance emerges rapidly due to acquired secondary FGFR2 mutations or due to other less-defined mechanisms. Here, we conducted high-throughput combination drug screens, biochemical analysis, and therapeutic studies using patient-derived models of FGFR2 fusion-positive cholangiocarcinoma to gain insight into these clinical profiles and uncover improved treatment strategies. We found that feedback activation of EGFR signaling limits FGFR inhibitor efficacy, restricting cell death induction in sensitive models and causing resistance in insensitive models lacking secondary FGFR2 mutations. Inhibition of wild-type EGFR potentiated responses to FGFR inhibitors in both contexts, durably suppressing MEK/ERK and mTOR signaling, increasing apoptosis, and causing marked tumor regressions in vivo. Our findings reveal EGFR-dependent adaptive signaling as an important mechanism limiting FGFR inhibitor efficacy and driving resistance and support clinical testing of FGFR/EGFR inhibitor therapy for FGFR2 fusion-positive cholangiocarcinoma. We demonstrate that feedback activation of EGFR signaling limits the effectiveness of FGFR inhibitor therapy and drives adaptive resistance in patient-derived models of FGFR2 fusion-positive cholangiocarcinoma. These studies support the potential of combination treatment with FGFR and EGFR inhibitors as an improved treatment for patients with FGFR2-driven cholangiocarcinoma. This article is highlighted in the In This Issue feature, p. 1171.

Casein Kinase 2 (CK2) is a prosurvival protein kinase involved in cell growth/proliferation through the regulation of the cell cycle and apoptosis. CK2 is over-expressed in various cancers, which correlates with a poor prognosis. This study examined the anti-cancer effects of silmitasertib (CX-4945), a CK2 inhibitor, on cholangiocarcinoma (CCA) cells. The effects of CX-4945 on cell viability, cell cycle arrest, and apoptosis in the human cholangiocarcinoma cell lines TFK-1 and SSP-25 were evaluated. Alterations in posttranslational modifications and the levels of cell cycle regulators including p21, Polo-like kinase 1 (PLK1), andp53 were assessed by western blotting. Apoptotic responses were examined using Propidium iodine/Annexin V staining. TFK-1 and SSP-25 cells exposed to CX-4945 showed morphologic changes and a more than 50% decrease in cell viability (p<0.05). Cell cycle arrest at the G2 phase was detected following an increase in phosphorylated PLK1 and p21. Furthermore, phospho-PLK1 induced the degradation of p53, which led to the dissociation of Bax from Bcl-xL. The cleavage of Caspase3 and PARP were also induced by CX-4945 treatment. CX-4945 induces cell cycle arrest and cell death in cholangiocarcinoma cells via the regulation of PLK1 and p53. This may provide a novel therapeutic strategy for advanced cholangiocarcinoma.

Fibroblast growth factor receptor (FGFR) 2 gene alterations are involved in the pathogenesis of cholangiocarcinoma. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. This study evaluated the safety and antitumour activity of pemigatinib in patients with previously treated, locally advanced or metastatic cholangiocarcinoma with and without FGFR2 fusions or rearrangements. In this multicentre, open-label, single-arm, multicohort, phase 2 study (FIGHT-202), patients aged 18 years or older with disease progression following at least one previous treatment and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 recruited from 146 academic or community-based sites in the USA, Europe, the Middle East, and Asia were assigned to one of three cohorts: patients with FGFR2 fusions or rearrangements, patients with other FGF/FGFR alterations, or patients with no FGF/FGFR alterations. All enrolled patients received a starting dose of 13·5 mg oral pemigatinib once daily (21-day cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, withdrawal of consent, or physician decision. The primary endpoint was the proportion of patients who achieved an objective response among those with FGFR2 fusions or rearrangements, assessed centrally in all patients who received at least one dose of pemigatinib. This study is registered with ClinicalTrials.gov, NCT02924376, and enrolment is completed. Between Jan 17, 2017, and March 22, 2019, 146 patients were enrolled: 107 with FGFR2 fusions or rearrangements, 20 with other FGF/FGFR alterations, 18 with no FGF/FGFR alterations, and one with an undetermined FGF/FGFR alteration. The median follow-up was 17·8 months (IQR 11·6-21·3). 38 (35·5% [95% CI 26·5-45·4]) patients with FGFR2 fusions or rearrangements achieved an objective response (three complete responses and 35 partial responses). Overall, hyperphosphataemia was the most common all-grade adverse event irrespective of cause (88 [60%] of 146 patients). 93 (64%) patients had a grade 3 or worse adverse event (irrespective of cause); the most frequent were hypophosphataemia (18 [12%]), arthralgia (nine [6%]), stomatitis (eight [5%]), hyponatraemia (eight [5%]), abdominal pain (seven [5%]), and fatigue (seven [5%]). 65 (45%) patients had serious adverse events; the most frequent were abdominal pain (seven [5%]), pyrexia (seven [5%]), cholangitis (five [3%]), and pleural effusion (five [3%]). Overall, 71 (49%) patients died during the study, most frequently because of disease progression (61 [42%]); no deaths were deemed to be treatment related. These data support the therapeutic potential of pemigatinib in previously treated patients with cholangiocarcinoma who have FGFR2 fusions or rearrangements. Incyte Corporation.

No abstract

Berberine (BBR), a natural isoquinoline alkaloid, possesses diverse pharmacological properties and anti-cancer effects that have been demonstrated in many in vitro and in vivo studies. In this study, the inhibitory effects and molecular mechanism of low dose BBR on EMT-induced cell migration, and invasion capability of cholangiocarcinoma (CCA) cell lines were demonstrated. The commercially available BBR chloride powder with purity ≥ 95% was used in this study. Effects of BBR on cell growth of two human CCA cell lines, KKU-213A and KKU-213B were measured using MTT assay. The progressive phenotypes-cell adhesion, migration, and invasion were evaluated using cell adhesion, wound healing, and Boyden chamber assays. Molecular docking analysis was performed to assess the possible binding mode of BBR against EGFR, Erk, STAT3 and Akt. The effects of BBR on the activations of EGF/EGFR and its downstream effectors were demonstrated using Western blotting. BBR inhibited growth of CCA cells in a dose dependent manner. At sub-cytotoxic dose, BBR significantly inhibited cell adhesion, migration, invasion and decreased expression of vimentin, slug, and VEGFA of both CCA cell lines. Molecular docking suggested the simultaneous inhibitory activity of BBR on EGFR, Erk, STAT3 and Akt. The Western blot analyses revealed that upon the EGF/EGFR activation, BBR considerably attenuated the activations of EGFR, Erk, STAT3 and Akt. Low dose of BBR suppresses EMT and thus aggressiveness of CCA cells, in part by its multi-kinase inhibitor property on EGFR and its downstream pathways.  BBR might be beneficial for therapy of human CCA.

Infigratinib (TRUSELTIQ

Despite the overexpression and aberrant activation of epidermal growth factor receptor (EGFR) in intrahepatic cholangiocarcinoma (iCCA), the disease remains refractory to EGFR tyrosine kinase inhibitors (TKIs). Multiple clinical trials involving EGFR-targeting agents have been conducted; however, none have demonstrated clinically significant efficacy. The aim of this study was to elucidate the mechanisms underlying EGFR TKI resistance in iCCA. Genome-scale CRISPR-Cas9 chemical-genetic screens were conducted, identifying xanthine oxidoreductase (XOR) as a critical modulator. XOR expression was evaluated in surgical samples from patients with iCCA and matched peritumor tissue via immunohistochemistry, western blotting, and quantitative reverse-transcription PCR. iCCA models were established in Xdh CRISPR-Cas9 screening revealed that XOR, which is upregulated in iCCA compared to peritumor tissue and bile ducts, is required for EGFR TKI resistance. XOR knockdown inhibited iCCA cell proliferation, increased DNA damage, reduced tumor progression in iCCA models, and significantly enhanced the anti-tumor efficacy of gefitinib both in vitro and in vivo. CEBPβ was found to regulate XOR transcription. Mechanistically, XOR promoted EGFR stability via USP8-mediated deubiquitination and MUC1 upregulation, thereby enhancing signaling cascades and DNA damage repair - collectively driving resistance to TKIs. Targeting XOR-mediated EGFR stabilization and MUC1 upregulation represents a promising therapeutic strategy to inhibit iCCA progression and enhance sensitivity to EGFR TKIs in patients with iCCA. This study identifies XOR as a key driver of resistance to EGFR tyrosine kinase inhibitors (TKIs) in intrahepatic cholangiocarcinoma (iCCA), addressing a major limitation of current targeted therapies. By promoting EGFR stability through USP8-mediated deubiquitination and MUC1 upregulation, XOR enhances DNA repair and sustains oncogenic signaling. Inhibition of XOR sensitizes iCCA cells to EGFR TKIs, suppresses tumor progression, and exacerbates DNA damage. These findings suggest that targeting XOR could overcome TKI resistance and offer a novel therapeutic strategy for iCCA, potentially improving outcomes in a patient population with limited effective treatment options.

Cholangiocarcinoma (CCA) is a highly invasive cancer, diagnosed at an advanced stage, and refractory to surgical intervention and chemotherapy. Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcriptional processes, and are considered potential therapeutic targets for cancer. Dinaciclib is a small molecule multi-CDK inhibitor targeting CDK 2/5/9. In this study, the therapeutic efficacy of dinaciclib was assessed using patient-derived xenograft cells (PDXC) and CCA cell lines. Treatment with dinaciclib significantly suppressed cell proliferation, induced caspase 3/7 levels and apoptotic activity in PDXC and CCA cell lines. Dinaciclib suppressed expression of its molecular targets CDK2/5/9, and anti-apoptotic BCL-XL and BCL2 proteins. Despite the presence of cyclin D1 amplification in the PDXC line, palbociclib treatment had no effect on cell proliferation, cell cycle or apoptosis in the PDXC as well as other CCA cell lines. Importantly, dinaciclib, in combination with gemcitabine, produced a robust and sustained inhibition of tumor progression in vivo in a PDX mouse model, greater than either of the treatments alone. Expression levels of two proliferative markers, phospho-histone H3 and Ki-67, were substantially suppressed in samples treated with the combination regimen. Our results identify dinaciclib as a novel and potent therapeutic agent alone or in combination with gemcitabine for the treatment of CCA.

Understanding resistance to selective FGFR inhibitors is crucial to improve the clinical outcomes of patients with FGFR2-driven malignancies. We analyzed sequential ctDNA, ± whole-exome sequencing, or targeted next-generation sequencing on tissue biopsies from patients with tumors harboring activating FGFR2 alterations progressing on pan-FGFR-selective inhibitors, collected in the prospective UNLOCK program. FGFR2::BICC1 Ba/F3 and patient-derived xenograft models were used for functional studies. Thirty-six patients were included. In cholangiocarcinoma, at resistance to both reversible inhibitors (e.g., pemigatinib and erdafitinib) and the irreversible inhibitor futibatinib, polyclonal FGFR2 kinase domain mutations were frequent (14/27 patients). Tumors other than cholangiocarcinoma shared the same mutated FGFR2 residues, but polyclonality was rare (1/9 patients). At resistance to reversible inhibitors, 14 residues in the FGFR2 kinase domain were mutated-after futibatinib, only the molecular brake N550 and the gatekeeper V565. Off-target alterations in PI3K/mTOR and MAPK pathways were found in 11 patients, often together with on-target mutations. At progression to a first FGFR inhibitor, 12 patients received futibatinib or lirafugratinib (irreversible inhibitors), with variable clinical outcomes depending on previous resistance mechanisms. Two patients with TSC1 or PIK3CA mutations benefited from everolimus. In cell viability assays on Ba/F3 and in pharmacologic studies on patient-derived xenografts, irreversible inhibitors retained better activity against FGFR2 kinase domain mutations, with lirafugratinib active against the recalcitrant V565L/F/Y. At progression to FGFR inhibitors, FGFR2-driven malignancies are characterized by high intra- and interpatient molecular heterogeneity, particularly in cholangiocarcinoma. Resistance to FGFR inhibitors can be overcome by sequential, molecularly oriented treatment strategies across FGFR2-driven tumors.

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

Futibatinib, a highly selective, irreversible FGFR1-4 inhibitor, was evaluated in a large multihistology phase I dose-expansion trial that enrolled 197 patients with advanced solid tumors. Futibatinib demonstrated an objective response rate (ORR) of 13.7%, with responses in a broad spectrum of tumors (cholangiocarcinoma and gastric, urothelial, central nervous system, head and neck, and breast cancer) bearing both known and previously uncharacterized

Futibatinib (Lytgobi

Cholangiocarcinoma (CCA) displays enhanced glycolysis, pivotal for fulfilling the heightened energy demands intrinsic to its malignant progression. Recent research has indicated that endogenous glycogen rather than exogenous glucose acts as the major carbon source for glycolysis, highlighting the need to better understand the regulation of glycogen homeostasis in CCA. Here, through comprehensive integrative analysis, we identified that glycogen phosphorylase brain form (PYGB), the main enzyme involved in glycogen homeostasis, was markedly upregulated in CCA tissues, serving as an independent prognostic indicator for human patients with CCA. Moreover, elevated PYGB expression potentiated cholangiocarcinogenesis and augmented CCA cell proliferation in both organoid and xenograft models. Hypoxia stimulated PYGB activity in a phosphoglycerate kinase 1-dependent manner, leading to glycogenolysis and the subsequent release of glucose-6-phosphate (G6P) and thereby facilitating aerobic glycolysis. Notably, a virtual screening pinpointed the β-blocker carvedilol as a potent pharmacologic inhibitor of PYGB that could attenuate CCA progression. Collectively, these findings position PYGB as a promising prognostic biomarker and therapeutic target for CCA.  Significance: Cholangiocarcinoma cells exhibit high glycogen phosphorylase activity under hypoxic conditions that mediates metabolic reprograming to promote glycolysis and support tumor development.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive cancer characterized by a poor prognosis and resistance to chemotherapy. In this study, utilizing scRNA-seq, we discovered that the tetra-transmembrane protein mal, T cell differentiation protein 2 (MAL2), exhibited specific enrichment in ICC cancer cells and was strongly associated with a poor prognosis. The inhibition of MAL2 effectively suppressed cell proliferation, invasion, and migration. Transcriptomics and metabolomics analyses suggested that MAL2 promoted lipid accumulation in ICC by stabilizing EGFR membrane localization and activated the PI3K/AKT/SREBP-1 axis. Molecular docking and Co-IP proved that MAL2 interacted directly with EGFR. Based on constructed ICC organoids, the downregulation of MAL2 enhanced apoptosis and sensitized ICC cells to cisplatin. Lastly, we conducted a virtual screen to identify sarizotan, a small molecule inhibitor of MAL2, and successfully validated its ability to inhibit MAL2 function. Our findings highlight the tumorigenic role of MAL2 and its involvement in cisplatin sensitivity, suggesting the potential for novel combination therapeutic strategies in ICC.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.

Pemigatinib (PEMAZYRE™), a small molecule inhibitor of fibroblast growth factor receptor (FGFR) 1, FGFR2 and FGFR3, received accelerated approval in April 2020 in the USA for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test. Developed by Incyte Corporation, it is the first targeted treatment for cholangiocarcinoma in the USA. The recommended dosage of pemigatinib is 13.5 mg once daily, administered orally with or without food, on days 1-14 of a 21-day cycle until disease progression or unacceptable toxicity. Pemigatinib received orphan designation for the treatment of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2 in August 2019 in the USA. A regulatory assessment for pemigatinib as a treatment for adults with locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or rearrangement that is relapsed or refractory after ≥ 1 line of systemic therapy is underway in the EU. Pemigatinib is also undergoing clinical development in various countries worldwide for use in several other FGFR-driven malignancies (e.g. solid tumour, urothelial carcinoma). This article summarizes the milestones in the development of pemigatinib leading to this first approval for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test.

Oncogenic activation of fibroblast growth factor receptor 2 (FGFR2) drives multiple cancers and represents a broad therapeutic opportunity, yet selective targeting of FGFR2 has not been achieved. Although the clinical efficacy of pan-FGFR inhibitors (pan-FGFRi) validates FGFR2 driver status in FGFR2 fusion-positive intrahepatic cholangiocarcinoma, their benefit is limited by incomplete target coverage due to FGFR1- and FGFR4-mediated toxicities (hyperphosphatemia and diarrhea, respectively) and the emergence of FGFR2 resistance mutations. RLY-4008 is a highly selective, irreversible FGFR2 inhibitor designed to overcome these limitations. In vitro, RLY-4008 demonstrates >250- and >5,000-fold selectivity over FGFR1 and FGFR4, respectively, and targets primary alterations and resistance mutations. In vivo, RLY-4008 induces regression in multiple xenograft models-including models with FGFR2 resistance mutations that drive clinical progression on current pan-FGFRi-while sparing FGFR1 and FGFR4. In early clinical testing, RLY-4008 induced responses without clinically significant off-isoform FGFR toxicities, confirming the broad therapeutic potential of selective FGFR2 targeting. Patients with FGFR2-driven cancers derive limited benefit from pan-FGFRi due to multiple FGFR1-4-mediated toxicities and acquired FGFR2 resistance mutations. RLY-4008 is a highly selective FGFR2 inhibitor that targets primary alterations and resistance mutations and induces tumor regression while sparing other FGFRs, suggesting it may have broad therapeutic potential. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.

We performed proteogenomic characterization of intrahepatic cholangiocarcinoma (iCCA) using paired tumor and adjacent liver tissues from 262 patients. Integrated proteogenomic analyses prioritized genetic aberrations and revealed hallmarks of iCCA pathogenesis. Aflatoxin signature was associated with tumor initiation, proliferation, and immune suppression. Mutation-associated signaling profiles revealed that TP53 and KRAS co-mutations may contribute to iCCA metastasis via the integrin-FAK-SRC pathway. FGFR2 fusions activated the Rho GTPase pathway and could be a potential source of neoantigens. Proteomic profiling identified four patient subgroups (S1-S4) with subgroup-specific biomarkers. These proteomic subgroups had distinct features in prognosis, genetic alterations, microenvironment dysregulation, tumor microbiota composition, and potential therapeutics. SLC16A3 and HKDC1 were further identified as potential prognostic biomarkers associated with metabolic reprogramming of iCCA cells. This study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in iCCA.

About 15% of intrahepatic cholangiocarcinomas (iCCAs) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), usually alongside mutational inactivation of TP53, CDKN2A or BAP1. In FFs, FGFR2 residues 1-768 fuse to sequences encoded by a diverse array of partner genes (>60) causing oncogenic FF activation. While FGFR-specific tyrosine kinase inhibitors (F-TKI) provide clinical benefit in FF Four iCCA FFs carrying different fusion sequences were expressed in Tp53 Transplantation of FF-expressing liver organoids yielded tumors diagnosed as CCA based on histological, phenotypic and transcriptomic analyses. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or V565F mutation. Dual blockade of FF and the Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo. FF-driven iCCA pathogenesis was successfully modeled on a Tp53 Intrahepatic cholangiocarcinoma (iCCA) is a rare cancer that is difficult to treat. A subtype of iCCA is caused by genomic alterations that generate oncogenic drivers known as FGFR2 fusions. Patients with FGFR2 fusions respond to FGFR inhibitors, but clinical responses are often of modest duration. We used animal and cellular models to show that FGFR2 fusions require the activity of a downstream effector named Mek1/2. We found that dual blockade of FGFR2 fusions and Mek1/2 was more effective than isolated inhibition of FGFR2 fusions, pointing to the potential clinical utility of dual FGFR2-MEK1/2 blockade in patients with iCCA.

Cholangiocarcinoma (CCA) is a highly heterogeneous cancer with limited understanding and few effective therapeutic approaches. We aimed at providing a proteogenomic CCA characterization to inform biological processes and treatment vulnerabilities. Integrative genomic analysis with functional validation uncovered biological perturbations downstream of driver events including DPCR1 , RBM47 mutations, SH3BGRL2 copy number alterations, and FGFR2 fusions in CCA. Proteomic clustering identified three subtypes with distinct clinical outcomes, molecular features, and potential therapeutics. Phosphoproteomics characterized targetable kinases in CCA, suggesting strategies for effective treatment with CDK and MAPK inhibitors. Patients with CCA with HBV infection showed increased antigen processing and presentation (APC) and T cell infiltration, conferring a favorable prognosis compared with those without HBV infection. The characterization of extrahepatic CCA recommended the feasible application of vascular endothelial-derived growth factor inhibitors. Multiomics profiling presented distinctive molecular characteristics of the large bile duct and the small bile duct of intrahepatic CCA. The immune landscape further revealed diverse tumor immune microenvironments, suggesting immune subtypes C1 and C5 might benefit from immune checkpoint therapy. TCN1 was identified as a potential CCA prognostic biomarker, promoting cell growth by enhancing vitamin B12 metabolism. We characterized the proteogenomic landscape of 217 CCAs with 197 paired normal adjacent tissues and identified their subtypes and potential therapeutic targets. The multiomics analyses with other databases and some functional validations have indicated strategies regarding the clinical, biological, and therapeutic approaches to the management of CCA.

We describe a rapid target enrichment method for next-generation sequencing, termed anchored multiplex PCR (AMP), that is compatible with low nucleic acid input from formalin-fixed paraffin-embedded (FFPE) specimens. AMP is effective in detecting gene rearrangements (without prior knowledge of the fusion partners), single nucleotide variants, insertions, deletions and copy number changes. Validation of a gene rearrangement panel using 319 FFPE samples showed 100% sensitivity (95% confidence limit: 96.5-100%) and 100% specificity (95% confidence limit: 99.3-100%) compared with reference assays. On the basis of our experience with performing AMP on 986 clinical FFPE samples, we show its potential as both a robust clinical assay and a powerful discovery tool, which we used to identify new therapeutically important gene fusions: ARHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma, MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1 and RUFY2-RET in lung cancer, FGFR2-CREB5 in cholangiocarcinoma and PPL-NTRK1 in thyroid carcinoma. AMP is a scalable and efficient next-generation sequencing target enrichment method for research and clinical applications.

The incidence of biliary tract cancer (BTC), including intrahepatic (ICC) and extrahepatic (ECC) cholangiocarcinoma and gallbladder cancer, has increased globally; however, no effective targeted molecular therapies have been approved at the present time. Here we molecularly characterized 260 BTCs and uncovered spectra of genomic alterations that included new potential therapeutic targets. Gradient spectra of mutational signatures with a higher burden of the APOBEC-associated mutation signature were observed in gallbladder cancer and ECC. Thirty-two significantly altered genes, including ELF3, were identified, and nearly 40% of cases harbored targetable genetic alterations. Gene fusions involving FGFR2 and PRKACA or PRKACB preferentially occurred in ICC and ECC, respectively, and the subtype-associated prevalence of actionable growth factor-mediated signals was noteworthy. The subgroup with the poorest prognosis had significant enrichment of hypermutated tumors and a characteristic elevation in the expression of immune checkpoint molecules. Accordingly, immune-modulating therapies might also be potentially promising options for these patients.

Selective FGFR inhibitors are effective against cholangiocarcinomas that harbor gene alterations in FGFR2. Clinical trials suggest that expression of wild-type FGFR mRNA can predict sensitivity to FGFR inhibitors, but this biomarker has not been well characterized in cholangiocarcinoma. This study explores the prevalence of FGFR mRNA overexpression in cholangiocarcinoma, its role in predicting sensitivity to FGFR inhibitors, and its association with immune markers. Tissue microarrays of intrahepatic (ICC) and extrahepatic cholangiocarcinomas (ECC) resected between 2004 and 2015 were used to evaluate FGFR1-4 mRNA expression levels by RNA in situ hybridization (ISH). Expression levels of FGFR2 mRNA were correlated with FGFR2 fusion status and with patient outcomes. Immune markers expression was assessed by IHC and CSF1 and CSF1 receptor expression were examined by RNA ISH. Among 94 patients with resected cholangiocarcinoma, the majority had ICC (77%). FGFR2 fusions were identified in 23% of ICCs and 5% of ECCs. High levels of FGFR mRNA in FGFR2 fusion-negative ICC/ECC were seen for: FGFR1 (ICC/ECC: 15%/0%), FGFR2 (ICC/ECC: 57%/0%), FGFR3 (ICC/ECC: 53%/18%), and FGFR4 (ICC/ECC: 32%/0%). Overall, 62% of fusion-negative cholangiocarcinomas showed high levels of FGFR mRNA. In patients with advanced FGFR2 fusion-positive ICC, high levels of FGFR2 mRNA did not correlate with clinical benefit. FGFR2 fusion-positive tumors showed a paucity of PD-L1 on tumor cells. FGFR mRNA overexpression occurs frequently in cholangiocarcinoma in the absence of genetic alterations in FGFR. This study identifies a molecular subpopulation in cholangiocarcinoma for which further investigation of FGFR inhibitors is merited outside currently approved indications.

Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

Isocitrate dehydrogenase 1 (IDH1) mutations occur in approximately 13% of patients with intrahepatic cholangiocarcinoma, a relatively uncommon cancer with a poor clinical outcome. The aim of this international phase 3 study was to assess the efficacy and safety of ivosidenib (AG-120)-a small-molecule targeted inhibitor of mutated IDH1-in patients with previously treated IDH1-mutant cholangiocarcinoma. This multicentre, randomised, double-blind, placebo-controlled, phase 3 study included patients from 49 hospitals in six countries aged at least 18 years with histologically confirmed, advanced, IDH1-mutant cholangiocarcinoma who had progressed on previous therapy, and had up to two previous treatment regimens for advanced disease, an Eastern Cooperative Oncology Group performance status score of 0 or 1, and a measurable lesion as defined by Response Evaluation Criteria in Solid Tumors version 1.1. Patients were randomly assigned (2:1) with a block size of 6 and stratified by number of previous systemic treatment regimens for advanced disease to oral ivosidenib 500 mg or matched placebo once daily in continuous 28-day cycles, by means of an interactive web-based response system. Placebo to ivosidenib crossover was permitted on radiological progression per investigator assessment. The primary endpoint was progression-free survival by independent central review. The intention-to-treat population was used for the primary efficacy analyses. Safety was assessed in all patients who had received at least one dose of ivosidenib or placebo. Enrolment is complete; this study is registered with ClinicalTrials.gov, NCT02989857. Between Feb 20, 2017, and Jan 31, 2019, 230 patients were assessed for eligibility, and as of the Jan 31, 2019 data cutoff date, 185 patients were randomly assigned to ivosidenib (n=124) or placebo (n=61). Median follow-up for progression-free survival was 6·9 months (IQR 2·8-10·9). Progression-free survival was significantly improved with ivosidenib compared with placebo (median 2·7 months [95% CI 1·6-4·2] vs 1·4 months [1·4-1·6]; hazard ratio 0·37; 95% CI 0·25-0·54; one-sided p<0·0001). The most common grade 3 or worse adverse event in both treatment groups was ascites (four [7%] of 59 patients receiving placebo and nine [7%] of 121 patients receiving ivosidenib). Serious adverse events were reported in 36 (30%) of 121 patients receiving ivosidenib and 13 (22%) of 59 patients receiving placebo. There were no treatment-related deaths. Progression-free survival was significantly improved with ivosidenib compared with placebo, and ivosidenib was well tolerated. This study shows the clinical benefit of targeting IDH1 mutations in advanced, IDH1-mutant cholangiocarcinoma. Agios Pharmaceuticals.

Isocitrate dehydrogenase 1 (IDH1) variations occur in up to approximately 20% of patients with intrahepatic cholangiocarcinoma. In the ClarIDHy trial, progression-free survival as determined by central review was significantly improved with ivosidenib vs placebo. To report the final overall survival (OS) results from the ClarIDHy trial, which aimed to demonstrate the efficacy of ivosidenib (AG-120)-a first-in-class, oral, small-molecule inhibitor of mutant IDH1-vs placebo for patients with unresectable or metastatic cholangiocarcinoma with IDH1 mutation. This multicenter, randomized, double-blind, placebo-controlled, clinical phase 3 trial was conducted from February 20, 2017, to May 31, 2020, at 49 hospitals across 6 countries among patients aged 18 years or older with cholangiocarcinoma with IDH1 mutation whose disease progressed with prior therapy. Patients were randomized 2:1 to receive ivosidenib, 500 mg, once daily or matched placebo. Crossover from placebo to ivosidenib was permitted if patients had disease progression as determined by radiographic findings. The primary end point was progression-free survival as determined by blinded independent radiology center (reported previously). Overall survival was a key secondary end point. The primary analysis of OS followed the intent-to-treat principle. Other secondary end points included objective response rate, safety and tolerability, and quality of life. Overall, 187 patients (median age, 62 years [range, 33-83 years]) were randomly assigned to receive ivosidenib (n = 126; 82 women [65%]; median age, 61 years [range, 33-80 years]) or placebo (n = 61; 37 women [61%]; median age, 63 years [range, 40-83 years]); 43 patients crossed over from placebo to ivosidenib. The primary end point of progression-free survival was reported elsewhere. Median OS was 10.3 months (95% CI, 7.8-12.4 months) with ivosidenib vs 7.5 months (95% CI, 4.8-11.1 months) with placebo (hazard ratio, 0.79 [95% CI, 0.56-1.12]; 1-sided P = .09). When adjusted for crossover, median OS with placebo was 5.1 months (95% CI, 3.8-7.6 months; hazard ratio, 0.49 [95% CI, 0.34-0.70]; 1-sided P < .001). The most common grade 3 or higher treatment-emergent adverse event (≥5%) reported in both groups was ascites (11 patients [9%] receiving ivosidenib and 4 patients [7%] receiving placebo). Serious treatment-emergent adverse events considered ivosidenib related were reported in 3 patients (2%). There were no treatment-related deaths. Patients receiving ivosidenib reported no apparent decline in quality of life compared with placebo. This randomized clinical trial found that ivosidenib was well tolerated and resulted in a favorable OS benefit vs placebo, despite a high rate of crossover. These data, coupled with supportive quality of life data and a tolerable safety profile, demonstrate the clinical benefit of ivosidenib for patients with advanced cholangiocarcinoma with IDH1 mutation. ClinicalTrials.gov Identifier: NCT02989857.

Biliary tract cancer (BTC) is an uncommon but highly lethal malignancy with poor clinical outcomes. To promote the development of precision medicine for BTC, uncovering its genomic profile becomes particularly important. However, studies on the genomic feature of Chinese BTC patients remain insufficient. A total of 382 Chinese patients with BTC were enrolled in this study, including 71 with intrahepatic cholangiocarcinoma (ICC), 194 with extrahepatic cholangiocarcinoma (ECC), and 117 with gallbladder carcinoma (GBC). Genetic testing was performed by utilizing the next-generation sequencing (NGS) of 499 cancer-related genes and the results were compared to those of Western BTC patients (MSKCC cohorts). The most prevalent genes were Our study elaborated the distinct germline and somatic genomic characteristics of Chinese BTC patients and identified clinically actionable alterations, highlighting the possibility for the development and application of precision medicine.

Recent advances in molecular profiling have enabled the identification of potential therapeutic targets for biliary tract cancer (BTC). However, in patients with BTC, molecular profiling is hindered by challenges in obtaining adequate tissue samples. Circulating tumor DNA (ctDNA) may offer an alternative to tissue-based analysis. Herein, we aimed to assess the concordance between ctDNA and tissue genomic profiling in a large cohort of Asian patients with advanced BTC, and to evaluate the feasibility of liquid biopsy in BTC treatment. This study included patients with systemic treatment-naive advanced BTC, treated at CHA Bundang Medical Center between January 2019 and December 2022. We enrolled patients with available baseline tissue-based next-generation sequencing, and sufficient plasma samples for ctDNA analysis (AlphaLiquid®100 from IMBdx). Among 102 enrolled patients, 49.0% had intrahepatic cholangiocarcinoma, 26.5% extrahepatic cholangiocarcinoma, and 24.5% gallbladder cancer. The concordance between intra-patient ctDNA and tumor tissue mutations revealed a sensitivity of 84.8%, and positive predictive value of 79.4%. ctDNA revealed targetable alterations in 34.3% of patients - including FGFR2 fusions, IDH1 mutations, microsatellite instability-high, ERBB2 amplifications, PIK3CA mutations, BRCA1/2 mutations, and MET amplifications. Notably, a novel FGFR2-TNS1 fusion was identified in ctDNA, which was not targeted in the tissue NGS panel. A high maximum somatic variant allele frequency in ctDNA was associated with poor prognosis after gemcitabine/cisplatin-based chemotherapy, in terms of both overall survival (p = 6.9 × 10 Among patients with advanced BTC, ctDNA-based genotyping showed acceptable concordance with tissue genomic profiling. Liquid biopsy using ctDNA could be a valuable complement to tissue-based genomic analysis in BTC. Our study is the first large-scale investigation of the clinical utility of liquid biopsy, focusing on circulating tumor DNA (ctDNA), as an alternative to conventional tumor tissue analysis, among Asian patients with advanced biliary tract cancer. The results demonstrated acceptable concordance between analysis of ctDNA vs. tissue for identifying therapeutic targets and potentially actionable genetic alterations. This indicates that ctDNA analysis can provide critical insights regarding advanced biliary tract cancer treatment, particularly in cases where it is challenging to obtain or analyze tumor tissue.

This document is a summary of the French intergroup guidelines of the management of biliary tract cancers (BTC) (intrahepatic, perihilar and distal cholangiocarcinomas, and gallbladder carcinomas) published in September 2023, available on the website of the French Society of Gastroenterology (SNFGE) (www.tncd.org). This collaborative work was conducted under the auspices of French medical and surgical societies involved in the management of BTC. Recommendations were graded in three categories (A, B and C) according to the level of scientific evidence until August 2023. BTC diagnosis and staging is mainly based on enhanced computed tomography, magnetic resonance imaging and (endoscopic) ultrasound-guided biopsy. Treatment strategy depends on BTC subtype and disease stage. Surgery followed by adjuvant capecitabine is recommended for localised disease. No neoadjuvant treatment is validated to date. Cisplatin-gemcitabine chemotherapy combined to the anti-PD-L1 inhibitor durvalumab is the first-line standard of care for advanced disease. Early systematic tumour molecular profiling is recommended to screen for actionable alterations (IDH1 mutations, FGFR2 rearrangements, HER2 amplification, BRAF These guidelines are intended to provide a personalised therapeutic strategy for daily clinical practice. Each individual BTC case should be discussed by a multidisciplinary team.

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate-dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate-mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell-autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell-specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ-TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587.

Biliary tract cancers (BTCs) are a group of malignant neoplasms that have recently increased in incidence and have a poor prognosis. Surgery is the only curative therapy. However, most patients are only indicated for palliative therapy because of advanced-stage disease at diagnosis and rapid progression. The current first-line treatment for advanced BTC is gemcitabine and cisplatin chemotherapy. Nonetheless, many patients develop resistance to this regimen. Over the years, few chemotherapy regimens have managed to improve the overall survival of patients. Accordingly, novel therapies such as targeted therapy have been introduced to treat this patient population. Extensive research on tumorigenesis and the genetic profiling of BTC have revealed the heterogenicity and potential target pathways, such as EGFR, VEGF, MEK/ERK, PI3K and mTOR. Moreover, mutational analysis has documented the presence of

Intrahepatic cholangiocarcinoma (ICC) is the second-most lethal primary liver cancer. Little is known about intratumoral heterogeneity (ITH) and its impact on ICC progression. We aimed to investigate the ITH of ICC in the hope of helping to develop new therapeutic strategies. We obtained 69 spatially distinct regions from six operable ICCs. Patient-derived primary cancer cells (PDPCs) were established for each region, followed by whole-exome sequencing (WES) and multi-level validation. We observed widespread ITH for both somatic mutations and clonal architecture, shaped by multiple mechanisms, like clonal "illusion", parallel evolution and chromosome instability. A median of 60.3% of mutations were heterogeneous, among which 85% of the driver mutations were located on the branches of tumor phylogenetic trees. Many truncal and clonal driver mutations occurred in tumor suppressor genes, such as TP53, SMARCB1 and PBRM1 that are involved in DNA repair and chromatin-remodeling. Genome doubling occurred in most cases (5/6) after the accumulation of truncal mutations and was shared by all intratumoral sub-regions. In all cases, ongoing chromosomal instability is evident throughout the evolutionary trajectory of ICC. The recurrence of ICC1239 provided evidence to support the polyclonal metastatic seeding in ICC. The change of mutation landscape and internal diversity among subclones during metastasis, such as the loss of chemoresistance mediator, can be used for new treatment strategies. Targeted therapy against truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, was developed in 5/6 patients. Integrated investigations of spatial ITH and clonal evolution may provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ICC. We applied multiregional whole-exome sequencing to investigate the evolution of intrahepatic cholangiocarcinoma (ICC). The results revealed that many factors, such as parallel evolution and chromosome instability, may participate and promote the branch diversity of ICC. Interestingly, in one patient with primary and recurrent metastatic tumors, we found evidence of polyclonal metastatic seeding, indicating that symbiotic communities of multiple clones existed and were maintained during metastasis. More realistically, some truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, could be promising treatment targets in patients with ICC.

Most of the patients with cholangiocarcinoma (CCA) present with advanced (inoperable or metastatic) disease, and relapse rates are high in those undergoing potentially curative resection. Previous treatment nihilism of patients with advanced disease has been replaced by active clinical research with the advent of randomized clinical trials (RCTs) and a much greater effort at understanding molecular mechanisms underpinning CCA. Three RCTs have recently been reported evaluating adjuvant chemotherapy following curative resection; only one of these has the potential to change practice. The BILCAP study failed to meet its primary endpoint by intention-to-treat analysis; however, a survival benefit was seen in a preplanned sensitivity analysis (predominantly adjusting for lymph nodes status). This, along with the numerical difference in median overall survival has led to the uptake of adjuvant capecitabine by many clinicians. In patients with advanced disease, the only level 1 data available supports the use of cisplatin and gemcitabine for the first-line treatment of patients with advanced disease; there is no established second-line chemotherapy. Previous forays into targeted therapy have proven unfruitful (namely targeting the epithelial growth factor receptor and vascular endothelial growth factor pathways). An increasing number of genomic subtypes are being defined; for some of these on-target therapeutic options are under active investigation. The most developed are studies targeting IDH-1 (isocitrate dehydrogenase) mutations and FGFR-2 (fibroblast growth factor receptor) fusions, with promising early results. Several other pathways are under evaluation, along with early studies targeting the immune environment; these are too premature to change practice to date. These emerging treatments are discussed.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with a poor prognosis, predominantly CA19-9 positive. High CA19-9 levels correlate with increased aggressiveness and worse outcomes. This study employs multi-omics analysis to reveal molecular features and identify therapeutic targets of CA19-9 positive ICC, aiming to support individualized treatment. Data from seven clinical cohorts, two whole-exome sequencing cohorts, six RNA sequencing/microarray cohorts, one proteomic cohort, 20 single-cell RNA sequencing samples, and one spatial transcriptome sample were analyzed. Key findings were validated on tissue microarrays from 52 ICC samples. CA19-9 positive ICC exhibited poorer OS (median 24.1 v.s. 51.5 months) and RFS (median 11.7 v.s. 28.2 months) compared to negative group (all P < 0.05). Genomic analysis revealed a higher KRAS mutation frequency in the positive group and a greater prevalence of IDH1/2 mutations in the negative group (all P < 0.05). Transcriptomic analysis indicated upregulated glycolysis pathways in CA19-9 positive ICC. Single-cell analysis identified specific glycolysis-related cell subclusters associated with poor prognosis, including Epi_SLC2A1, CAF_VEGFA, and Mph_SPP1. Higher hypoxia in the CA19-9 positive group led to metabolic reprogramming and promoted these cells' formation. These cells formed interactive communities promoting epithelial-mesenchymal transition (EMT) and angiogenesis. Drug sensitivity analysis identified six potential therapeutic drugs. This study systematically elucidated the clinical, genomic, transcriptomic, and immune features of CA19-9 positive ICC. It reveals glycolysis-associated cellular communities and their cancer-promoting mechanisms, enhancing our understanding of ICC and laying the groundwork for individualized therapeutic strategies.

Advanced therapy-refractory biliary tract cancer (BTC) has poor prognosis and constitutes a major challenge for adequate treatment strategies. By mapping the molecular profiles of advanced BTC patients, precision cancer medicine may provide targeted therapies for these patients. In this analysis, we aimed to show the potential of PCM in metastatic BTC. In this single-center, real-world retrospective analysis of our PCM platform, we describe the molecular profiling of 30 patients diagnosed with different types of metastatic BTC. Tumor samples of the patients were examined using a 161-gene next-generation sequencing panel, immunohistochemistry (IHC), and fluorescence in situ hybridization for chromosomal translocations. In total, we identified 35 molecular aberrations in 30 patients. The predominant mutations were KRAS (n = 8), TP53 (n = 7), IDH2 (n = 4), and IDH1 (n = 3) that accounted for the majority of all molecular alterations (62.86%). BRAF mutations were observed in two patients. Less frequent alterations were noted in ARID1A, CTNNB1, ESR1, FBXW7, FGFR2, MET, NOTCH2, PIK3CA, PTCH1, SMAD4, and SRC1, each in one case. FGFR fusion gene was detected in one patient. No mutations were detected in eight patients. IHC revealed EGFR and p-mTOR expression in 28 patients. Applying these results to our patients, targeted therapy was recommended for 60% of the patients (n = 18). One patient achieved stable disease. PCM is a feasible treatment approach and may provide molecular-guided therapy recommendations for metastatic BTC.

The role of next-generation sequencing from either circulating tumor DNA (ctDNA) or formalin-fixed paraffin-embedded (FFPE) tissue to identify therapeutically targetable genomic alterations has been well established. Genomic profiling may also have untapped potential as a diagnostic tool in cases in which traditional immunohistochemistry assays cannot establish a clear histologic diagnosis. Expanding the number of histologies with unique genomic signatures or alterations is critical in this setting. Here we describe a case of a 73-year-old man who presented with a duodenal mass extending to the liver and peritoneal carcinomatosis, initially thought to be metastatic duodenal adenocarcinoma. Subsequent genomic profiling of ctDNA and FFPE tissue revealed an

Hepatocellular carcinoma (HCC) and biliary tract cancer are more frequent in East Asia including Japan than in Europe or North America. A compilation of 1340 multi-ethnic HCC genomes, the largest cohort ever reported, identified a comprehensive landscape of HCC driver genes, comprised of three core drivers (TP53, TERT, and WNT signaling) and combinations of infrequent alterations in various cancer pathways. In contrast, five core driver genes (TP53, ARID1A, KRAS, SMAD4, and BAP1) with characteristic molecular alterations including fusion transcripts involving fibroblast growth factor receptor 2 and the protein kinase A pathway, and IDH1/2 mutation constituted the biliary tract cancer genomes. Consistent with their heterogeneous epidemiological backgrounds, mutational signatures and combinations of non-core driver genes within these cancer genomes were found to be complex. Integrative analyses of multi-omics data identified molecular classifications of these tumors that are associated with clinical outcome and enrichments of potential therapeutic targets, including immune checkpoint molecules. Translating comprehensive molecular-genomic analysis together with further basic research and international collaborations are highly anticipated for developing precise and better treatments, diagnosis, and prevention of these tumor types.

Biliary tract cancers (BTCs) contain several actionable molecular alterations, including FGFR2, IDH1, ERBB2 (formerly HER2), and KRAS. KRAS allelic variants are found in 20% to 30% of BTCs, and multiple KRAS inhibitors are currently under clinical investigation. To describe the genomic landscape, co-sequence variations, immunophenotype, genomic ancestry, and survival outcomes of KRAS-mutated BTCs and to calculate the median overall survival (mOS) for the most common allelic variants. This retrospective, multicenter, pooled cohort study obtained clinical and next-generation sequencing data from multiple databases between January 1, 2017, and December 31, 2022. These databases included Princess Margaret Cancer Centre, MD Anderson Cancer Center, Foundation Medicine, American Association for Cancer Research Project GENIE, and cBioPortal for Cancer Genomics. The cohort comprised patients with BTCs who underwent genomic testing. The main outcome was mOS, defined as date of diagnosis to date of death, which was measured in months. A total of 7457 patients (n = 3773 males [50.6%]; mean [SD] age, 63 [5] years) with BTCs and genomic testing were included. Of these patients, 5813 had clinical outcome data available, in whom 1000 KRAS-mutated BTCs were identified. KRAS allelic variants were highly prevalent in perihilar cholangiocarcinoma (28.6%) and extrahepatic cholangiocarcinoma (36.1%). Thirty-six KRAS allelic variants were identified, and the prevalence rates in descending order were G12D (41%), G12V (23%), and Q61H (8%). The variant G12D had the highest mOS of 25.1 (95% CI, 22.0-33.0) months compared with 22.8 (95% CI, 19.6-31.4) months for Q61H and 17.8 (95% CI, 16.3-23.1) months for G12V variants. The majority of KRAS-mutated BTCs (98.9%) were not microsatellite instability-high and had low tumor mutational burden (ranging from a median [IQR] of 1.2 (1.2-2.5) to a mean [SD] of 3.3 [1.3]). Immune profiling through RNA sequencing of KRAS and NRAS-mutated samples showed a pattern toward a more immune-inflamed microenvironment with higher M1 macrophage activation (0.16 vs 0.12; P = .047) and interferon-γ expression compared with wild-type tumors. The G12D variant remained the most common KRAS allelic variant in all patient ancestries. Patients with admixed American ancestry had the highest proportion of G12D variant (45.0%). This cohort study found that KRAS allelic variants were relatively common and may be potentially actionable genomic alterations in patients with BTCs, especially perihilar cholangiocarcinoma and extrahepatic cholangiocarcinoma. The findings add to the growing data on genomic and immune landscapes of KRAS allelic variants in BTCs and are potentially of value to the planning of specific therapies for this heterogeneous patient group.

Intrahepatic cholangiocarcinoma (iCCA) is the second most prevalent primary liver cancer. Although the genetic characterization of iCCA has led to targeted therapies for treating tumors with FGFR2 alterations and IDH1/2 mutations, only a limited number of patients can benefit from these strategies. Epigenomic profiles have emerged as potential diagnostic and prognostic biomarkers for improving the treatment of cancers. In this study, we conducted whole-genome bisulfite sequencing on 331 iCCAs integrated with genetic, transcriptomic, and proteomic analyses, demonstrating the existence of four DNA methylation subtypes of iCCAs (S1-S4) that exhibited unique postoperative clinical outcomes. The S1 group was an IDH1/2 mutation-specific subtype with moderate survival. The S2 subtype was characterized by the lowest methylation level and the highest mutational burden among the four subtypes and displayed upregulation of a gene-expression pattern associated with cell cycle/DNA replication. The S3 group was distinguished by high interpatient heterogeneity of tumor immunity, a gene-expression pattern associated with carbohydrate metabolism, and an enrichment of KRAS alterations. Patients with the S2 and S3 subtypes had the shortest survival among the four subtypes. Tumors in the S4 subtype, which had the best prognosis, showed global methylation levels comparable to normal controls, increased FGFR2 fusions/BAP1 mutations, and the highest copy-number variant burdens. Further integrative and functional analyses identified GBP4 demethylation, which is highly prevalent in the S2 and S3 groups, as an epigenetic oncogenic factor that regulates iCCA proliferation, migration, and invasion. Together, this study identifies prognostic methylome alterations and epigenetic drivers in iCCA. Characterization of the DNA methylome of intrahepatic cholangiocarcinoma integrated with genomic, transcriptomic, and proteomic analyses uncovers molecular mechanisms affected by genome-wide DNA methylation alterations, providing a resource for identifying potential therapeutic targets.

Biliary tract cancers (BTCs) are a heterogeneous group of adenocarcinomas that originate from the epithelial lining of the biliary tree. BTCs are characterized by presentation with advanced disease precluding curative surgery, rising global incidence, and a poor prognosis. Chemotherapy is the mainstay of the current treatment, which results in a median overall survival of less than one year, underscoring the need for novel therapeutic agents and strategies. Next-generation sequencing-based molecular profiling has shed light on the underpinnings of the complex pathophysiology of BTC and has uncovered numerous actionable targets, leading to the discovery of new therapies tailored to the molecular targets. Therapies targeting fibroblast growth factor receptor (FGFR) fusion, isocitrate dehydrogenase (IDH) mutations, the human epidermal growth factor receptor (HER) family, DNA damage repair (DDR) pathways, and

Professor Arndt Vogel, a senior consultant and professor of gastrointestinal oncology in the Department of Gastroenterology, Hepatology and Endocrinology at Hannover Medical School, Germany, speaks with Rachel Jenkins (Senior Editor of Oncology Central) about cholangiocarcinoma, the challenges of the field and some promising avenues for therapy. To listen to the full podcast interview, visit Oncology Central (www.oncology-central.com/) now.

The molecular pathological examination of solid tumors is essential not only for supporting histological diagnosis but also for detecting hereditary variations and predictive biomarkers. Analyzing predictive markers is fundamental to personalized cancer therapy, directly affecting patient care through pathological testing. These analyses employ both traditional immunohistochemical staining methods and molecular genetic techniques. In both approaches, preanalytics is of critical importance, necessitating the adoption of standardized and reproducible processes. Molecular diagnostics in colorectal cancer focuses on detecting activating mutations in the MAPK pathway (KRAS, NRAS, BRAF), as well as evaluating microsatellite instability and HER2 amplification. Immunohistochemical methods can effectively identify biomarkers for gastric cancers, including the novel claudin18.2. The responsiveness of gastrointestinal stromal tumors to imatinib requires validation via molecular testing. Patients diagnosed with pancreatic cancer may see enhanced survival rates by targeted therapy addressing microsatellite instability and BRCA mutations. In bile duct malignancies, especially intrahepatic cholangiocarcinoma of the small duct variant, the analysis of IDH1 mutations and FGFR2 fusions presents new treatment prospects.

Surgical resection remains the mainstay of potentially curative treatment in the early stages of cholangiocarcinoma, whereas for the advanced stage, systemic chemotherapeutics and experimental targeted therapies are the primary treatment options. The molecular heterogeneity of the tumor is based on location, liver dysfunction, and relative rarity of the disease and confers challenges for clinical trial enrollment. The advancements in the understanding of molecular pathogenesis of cholangiocarcinoma have led to the development of targeted therapies that are currently being evaluated in the clinical trials. This review summarizes the current understanding and future directions of targeted therapeutic options in the management of advanced cholangiocarcinoma. Advanced cholangiocarcinoma has a dismal prognosis; improved understanding of the molecular pathogenesis and advancements in development of targeted therapy offers hope that we may improve outcomes in this rare, but highly lethal cancer. Among the newly discovered molecular alterations, targeting FGFR2 fusions, IDH1/2 mutations and HER2 receptors hold great promise for improving the future management of cholangiocarcinoma. Immunotherapy in combination with targeted agents and chemotherapy may improve outcomes. In addition, drugs targeting the MEK, EGFR, KRAS, BRAF, and ROS1 pathways and neo-angiogenesis may also provide new horizons in the management of cholangiocarcinoma.

The administration of many cancer drugs is tailored to genetic tests. Some genomic events, e.g., alterations of EGFR or BRAF oncogenes, result in the conformational change of the corresponding proteins and call for the use of mutation-specific compounds. Other genetic perturbations, e.g., HER2 amplifications, ALK translocations or MET exon 14 skipping mutations, cause overproduction of the entire protein or its kinase domain. There are multilocus assays that provide integrative characteristics of the tumor genome, such as the analysis of tumor mutation burden or deficiency of DNA repair. Treatment planning for non-small cell lung cancer requires testing for EGFR, ALK, ROS1, BRAF, MET, RET and KRAS gene alterations. Colorectal cancer patients need to undergo KRAS, NRAS, BRAF, HER2 and microsatellite instability analysis. The genomic examination of breast cancer includes testing for HER2 amplification and PIK3CA activation. Melanomas are currently subjected to BRAF and, in some instances, KIT genetic analysis. Predictive DNA assays have also been developed for thyroid cancers, cholangiocarcinomas and urinary bladder tumors. There is an increasing utilization of agnostic testing which involves the analysis of all potentially actionable genes across all tumor types. The invention of genomically tailored treatment has resulted in a spectacular improvement in disease outcomes for a significant portion of cancer patients.

Cholangiocarcinomas are an aggressive group of heterogeneous malignancies that affect over 210,000 individuals globally each year. Their incidence is rising, particularly in Western countries. Traditionally, cholangiocarcinomas are classified based on anatomic location of the tumor and are treated with similar cytotoxic chemotherapy despite significant molecular and genomic differences. With the rise of genetic and molecular sequencing, several driver mutations have been identified and targeted as novel therapeutic approaches. The most common genomic alterations include changes in

Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.

The use of immunohistochemistry (IHC) as a companion diagnostic is an increasingly important part of the case workup by pathologists and is often central to clinical decision making. New predictive molecular markers are constantly sought for to improve treatment stratification parallel to drug development. Unfortunately, official biomarker guidelines lag behind, and pathologists are often left hesitating when medical oncologists request off-labelled biomarker testing. We performed a literature review of five commonly requested off-label IHC predictive biomarkers in gastrointestinal tract (GIT) malignancies: HER2, mismatch repair (MMR), PD-L1, BRAF V600E and ROS1. We found that HER2 amplification is rare and poorly associated to IHC overexpression in extracolonic and extragastric GIT cancers; however in KRAS wild type colorectal cancers, which fail conventional treatment, HER2 IHC may be useful and should be considered. For MMR testing, more evidence is needed to recommend reflex testing in GIT cancers for treatment purposes. MMR testing should not be discouraged in patients considered for second line checkpoint inhibitor therapy. With the exception of gastric tumors, PD-L1 IHC is a weak predictor of checkpoint inhibitor response in the GIT and should be replaced by MMR in this context. BRAF inhibitors showed activity in BRAF V600E mutated cholangiocarcinomas and pancreatic carcinomas in non-first line settings. ROS1 translocation is extremely rare and poorly correlated to ROS1 IHC expression in the GIT; currently there is no role for ROS1 IHC testing in GIT cancers. Overall, the predictive biomarker literature has grown exponentially, and official guidelines need to be updated more regularly to support pathologists' testing decisions.

Cholangiocarcinoma is a highly aggressive and heterogenous group of biliary malignancies arising from any site in the biliary tree, comprising 15% of all primary liver cancers. The nature of the disease and nonspecific presentation leads to late diagnosis and ultimately poor outcomes for patients. Combination gemcitabine and cisplatin has been the standard of care for cholangiocarcinoma (CCA) since 2010, with a median overall survival of 11.7 months. The five-year survival for CCA remains 5-10%, revealing a clear need for improved treatment options. This targeted review highlights the role of next generation sequencing in CCA and the clinically relevant tumor biomarkers that have become the focus of therapeutic development. These tumor biomarkers or actionable mutations hold the potential to enable earlier diagnosis, provide prognostic information, and guide treatment decisions for patients with CCA. Specifically, the The discovery of these actionable mutations and identification of targeted therapies have challenged the notion of a "one-size fits all" for treatment of CCA, and generated optimism that these novel treatments will soon be available for patients with CCA.

This study explores genomic alterations in cholangiocarcinoma (CCC) tissues in Thai patients. We identified and reviewed the records of patients who had been diagnosed with CCC and for whom sufficient tumor samples for DNA and RNA extraction were available in our database. The specimens were explored for

The technique 3' rapid amplification of cDNA ends (3' RACE) allows for detection of translocations with unknown gene partners located at the 3' end of the chimeric transcript. We composed a 3' RACE-based RNA sequencing panel for the analysis of

The BRAF inhibitor encorafenib and anti-epidermal growth factor receptor (EGFR) antibody cetuximab modestly improve survival for patients with microsatellite stable (MSS) BRAF

Chronic liver diseases account for a considerable toll of incapacities, suffering, deaths, and resources of the nation's health systems. They can be prevented, treated or even cured when the diagnosis is made on time. Traditional liver biopsy remains the gold standard to diagnose liver diseases, but it has several limitations. Liquid biopsy is emerging as a superior alternative to surgical biopsy given that it surpasses the limitations: it is more convenient, readily and repeatedly accessible, safe, cheap, and provides a more detailed molecular and cellular representation of the individual patient's disease. Progress in understanding the molecular and cellular bases of diseased tissues and organs that normally release cells and cellular components into the bloodstream is catapulting liquid biopsy as a source of biomarkers for diagnosis, prognosis, and prediction of therapeutic response, thus supporting the realization of the promises of precision medicine. The review aims to summarize the evidence of the usefulness of liquid biopsy in liver diseases, including the presence of different biomarkers as circulating epithelial cells, cell-free nucleic acids, specific species of DNA and RNA, and the content of extracellular vesicles.