Advances in the treatment of intrahepatic cholangiocarcinoma: An overview of the current and future therapeutic landscape for clinicians.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver tumor and remains a fatal malignancy in the majority of patients. Approximately 20%-30% of patients are eligible for resection, which is considered the only potentially curative treatment; and, after resection, a median survival of 53 months has been reported when sequenced with adjuvant capecitabine. For the 70%-80% of patients who present with locally unresectable or distant metastatic disease, systemic therapy may delay progression, but survival remains limited to approximately 1 year. For the past decade, doublet chemotherapy with gemcitabine and cisplatin has been considered the most effective first-line regimen, but results from the recent use of triplet regimens and even immunotherapy may shift the paradigm. More effective treatment strategies, including those that combine systemic therapy with locoregional therapies like radioembolization or hepatic artery infusion, have also been developed. Molecular therapies, including those that target fibroblast growth factor receptor and isocitrate dehydrogenase, have recently received US Food and Drug Administration approval for a defined role as second-line treatment for up to 40% of patients harboring these actionable genomic alterations, and whether they should be considered in the first-line setting is under investigation. Furthermore, as the oncology field seeks to expand indications for immunotherapy, recent data demonstrated that combining durvalumab with standard cytotoxic therapy improved survival in patients with ICC. This review focuses on the current and future strategies for ICC treatment, including a summary of the primary literature for each treatment modality and an algorithm that can be used to drive a personalized and multidisciplinary approach for patients with this challenging malignancy.

Futibatinib for FGFR2-Rearranged Intrahepatic Cholangiocarcinoma.

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.).

FGFR2 Inhibition in Cholangiocarcinoma.

Biliary tract cancer (BTC) is the second most common primary liver cancer after hepatocellular carcinoma and accounts for 2% of cancer-related deaths. BTCs are classified according to their anatomical origin into intrahepatic (iCCA), perihilar, or distal cholangiocarcinoma, as well as gall bladder carcinoma. While the mutational profiles in these anatomical BTC subtypes overlap to a large extent, iCCA is notable for the high frequency of IDH1/2 mutations (10-22%) and the nearly exclusive occurrence of FGFR2 fusions in 10-15% of patients. In recent years, FGFR2 fusions have become one of the most promising targets for precision oncology targeting BTC, with FGFR inhibitors already approved in Europe and the United States for patients with advanced, pretreated iCCA. While the therapeutic potential of nonfusion alterations is still under debate, it is expected that the field of FGFR2-directed therapies will be subject to rapid further evolution and optimization. The scope of this review is to provide an overview of oncogenic FGFR signaling in iCCA cells and highlight the pathophysiology, diagnostic testing strategies, and therapeutic promises and challenges associated with FGFR2-altered iCCA.

Chemotherapy with or without selective internal radiation therapy for intrahepatic cholangiocarcinoma: Data from clinical trials.

BACKGOUND AND AIMS: In advanced, liver-only intrahepatic cholangiocarcinoma (iCCA), selective internal radiation therapy (SIRT) has been suggested as promising in nonrandomized studies. We aimed to compare data from patients with advanced, liver-only iCCA treated in the first line in clinical trials with either chemotherapy alone or the combination with SIRT. APPROACH AND RESULTS: We collected individual patients' data from the ABC-01, ABC-02, ABC-03, BINGO, AMEBICA, and MISPHEC prospective trials. Data from patients with liver-only iCCA treated in chemotherapy-only arms of the first 5 trials were compared with data from patients treated with SIRT and chemotherapy in MISPHEC. Emulated target trial paradigm and Inverse Probability of Treatment Weighting (IPTW methods) using the propensity score were used to minimize biases. We compared 41 patients treated with the combination with 73 patients treated with chemotherapy alone, the main analysis being in 43 patients treated with cisplatin-gemcitabine or gemcitabine-oxaliplatin. After weighting, overall survival was significantly higher in patients treated with SIRT: median 21.7 months (95% CI: 14.1; not reached) versus 15.9 months(95% CI: 9.8; 18.9), HR = 0.59 (95% CI: 0.34; 0.99), p = 0.049. Progression-free survival was significantly improved: median 14.3 months (95% CI: 7.8; not reached) versus 8.4 months (95% CI: 5.9; 12.1), HR = 0.52 (95% CI: 0.31; 0.89), p < 0.001. Results were confirmed in most sensitivity analyses. CONCLUSIONS: This analysis derived from prospective clinical trials suggests that SIRT combined with chemotherapy might improve outcomes over chemotherapy alone in patients with advanced, liver-only iCCA. Randomized controlled evidence is needed to confirm these findings.

Bile exosomal miR-182/183-5p increases cholangiocarcinoma stemness and progression by targeting HPGD and increasing PGE2 generation.

BACKGROUND AIMS: Cholangiocarcinoma (CCA) is a highly lethal malignancy originating from the biliary ducts. Current CCA diagnostic and prognostic assessments cannot satisfy the clinical requirement. Bile detection is rarely performed, and herein, we aim to estimate the clinical significance of bile liquid biopsy by assessing bile exosomal concentrations and components. APPROACH RESULTS: Exosomes in bile and sera from CCA, pancreatic cancer, and common bile duct stone were identified and quantified by transmission electronmicroscopy, nanoparticle tracking analysis, and nanoFCM. Exosomal components were assessed by liquid chromatography with tandem mass spectrometry and microRNA sequencing (miRNA-seq). Bile exosomal concentration in different diseases had no significant difference, but miR-182-5p and miR-183-5p were ectopically upregulated in CCA bile exosomes. High miR-182/183-5p in both CCA tissues and bile indicates a poor prognosis. Bile exosomal miR-182/183-5p is secreted by CCA cells and can be absorbed by biliary epithelium or CCA cells. With xenografts in humanized mice, we showed that bile exosomal miR-182/183-5p promotes CCA proliferation, invasion, and epithelial-mesenchymal transition (EMT) by targeting hydroxyprostaglandin dehydrogenase in CCA cells and mast cells (MCs), and increasing prostaglandin E2 generation, which stimulates PTGER1 and increases CCA stemness. In single-cell mRNA-seq, hydroxyprostaglandin dehydrogenase is predominantly expressed in MCs. miR-182/183-5p prompts MC to release VEGF-A release from MC by increasing VEGF-A expression, which facilitates angiogenesis. CONCLUSIONS: CCA cells secret exosomal miR-182/183-5p into bile, which targets hydroxyprostaglandin dehydrogenase in CCA cells and MCs and increases prostaglandin E2 and VEGF-A release. Prostaglandin E2 promotes stemness by activating PTGER1. Our results reveal a type of CCA self-driven progression dependent on bile exosomal miR-182/183-5p and MCs, which is a new interplay pattern of CCA and bile.

Telomere length and risk of cirrhosis, hepatocellular carcinoma, and cholangiocarcinoma in 63,272 individuals from the general population.

BACKGROUND AND AIMS: Inherited short telomeres are associated with a risk of liver disease, whereas longer telomeres predispose to cancer. The association between telomere length and risk of HCC and cholangiocarcinoma remains unknown. APPROACH AND RESULTS: We measured leukocyte telomere length using multiplex PCR in 63,272 individuals from the Danish general population. Telomere length and plasma ALT concentration were not associated (ß = 4 ×10 -6 , p -value = 0.06) in a linear regression model, without any signs of a nonlinear relationship. We tested the association between telomere length and risk of cirrhosis, HCC, and cholangiocarcinoma using Cox regression. During a median follow-up of 11 years, 241, 76, and 112 individuals developed cirrhosis, HCC, and cholangiocarcinoma, respectively. Telomere length and risk of cirrhosis were inversely and linearly associated ( p -value = 0.004, p for nonlinearity = 0.27). Individuals with telomeres in the shortest vs. longest quartile had a 2.25-fold higher risk of cirrhosis. Telomere length and risk of HCC were nonlinearly associated ( p -value = 0.009, p -value for nonlinearity = 0.01). This relationship resembled an inverted J-shape, with the highest risk observed in individuals with short telomeres. Individuals with telomeres in the shortest versus longest quartile had a 2.29-fold higher risk of HCC. Telomere length was inversely and linearly associated with the risk of cholangiocarcinoma. Individuals with telomeres in the shortest versus longest quartile had a 1.86-fold higher risk of cholangiocarcinoma. CONCLUSIONS: Shorter telomere length is associated with a higher risk of cirrhosis, HCC, and cholangiocarcinoma.

Integrated analyses of the genetic and clinicopathological features of cholangiolocarcinoma: cholangiolocarcinoma may be characterized by mismatch-repair deficiency.

Cholangiolocarcinoma (CLC) is a primary liver carcinoma that resembles the canals of Hering and that has been reported to be associated with stem cell features. Due to its rarity, the nature of CLC remains unclear, and its pathological classification remains controversial. To clarify the positioning of CLC in primary liver cancers and identify characteristics that could distinguish CLC from other liver cancers, we performed integrated analyses using whole-exome sequencing (WES), immunohistochemistry, and a retrospective review of clinical information on eight CLC cases and two cases of recurrent CLC. WES demonstrated that CLC includes IDH1 and BAP1 mutations, which are characteristic of intrahepatic cholangiocarcinoma (iCCA). A mutational signature analysis showed a pattern similar to that of iCCA, which was different from that of hepatocellular carcinoma (HCC). CLC cells, including CK7, CK19, and EpCAM, were positive for cholangiocytic differentiation markers. However, the hepatocytic differentiation marker AFP and stem cell marker SALL4 were completely negative. The immunostaining patterns of CLC with CD56 and epithelial membrane antigen were similar to those of the noncancerous bile ductules. In contrast, mutational signature cluster analyses revealed that CLC formed a cluster associated with mismatch-repair deficiency (dMMR), which was separate from iCCA. Therefore, to evaluate MMR status, we performed immunostaining of four MMR proteins (PMS2, MSH6, MLH1, and MSH2) and detected dMMR in almost all CLCs. In conclusion, CLC had highly similar characteristics to iCCA but not to HCC. CLC can be categorized as a subtype of iCCA. In contrast, CLC has characteristics of dMMR tumors that are not found in iCCA, suggesting that it should be treated distinctly from iCCA. © 2024 The Pathological Society of Great Britain and Ireland.

Proteome Landscapes of Human Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma.

Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types. In particular, we found an abnormal lipid metabolism in HCC and activated extracellular matrix-related pathways in CCA. We next developed a three-protein classifier to distinguish CCA from HCC, achieving an area under the curve (AUC) of 0.92, and an accuracy of 90% in an independent validation cohort of 51 patients. The distinct molecular characteristics of HCC and CCA presented in this study provide new insights into the tumor biology of these two major important primary liver cancers. Our findings may help develop more efficient diagnostic approaches and new targeted drug treatments.

Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Molecular portraits of patients with intrahepatic cholangiocarcinoma who diverge as rapid progressors or long survivors on chemotherapy.

OBJECTIVE: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. DESIGN: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. RESULTS: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. CONCLUSIONS: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

CircPCNXL2 promotes tumor growth and metastasis by interacting with STRAP to regulate ERK signaling in intrahepatic cholangiocarcinoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Effects of adoptive T-cell immunotherapy on immune cell profiles and prognosis of patients with unresectable or recurrent cholangiocarcinoma.

The identification of immune cell profiles (ICP) involved in anti-tumor immunity is crucial for immunotherapy. Therefore, we herein investigated cholangiocarcinoma patients (CCA) who received adoptive T-cell immunotherapy (ATI). Eighteen unresectable or recurrent CCA received ATI of αß T cells alone or combined with chemotherapy. ICP were evaluated by flow cytometry. There were 14 patients with intrahepatic cholangiocarcinoma (iCCA) and four with distal cholangiocarcinoma (dCCA). After one course of treatment, nine iCCA and four dCCA had progressive disease (PD), while five iCCA had stable disease (SD). Median overall survival (OS) was prolonged to 21.9 months. No significant differences were observed in OS between the PD and SD groups of iCCA. The frequency of helper T cells (HT) in iCCA decreased from 70.3% to 65.5% (P = .008), while that of killer T cells (KT) increased from 27.0% to 30.6% (P = .005). dCCA showed no significant changes of immune cells. OS was prolonged in iCCA with increased frequencies of CD3+ T cells (CD3) (P = .039) and αß T cells (αß) (P = .039). dCCA showed no immune cells associated with OS. The frequencies of CD3+ T cells and αß T cells in the PD group for iCCA decreased from 63.5% to 53% (P = .038) and from 61.6% to 52.2% (P = .028), respectively. In the SD group, the frequency of HT decreased from 65.8% to 56.9% (P = .043), whereas that of KT increased from 30.1% to 38.3% (P = .043). In conclusions, ATI affected ICP and prolonged OS. Immune cells involved in treatment effects differed according to the site of cholangiocarcinoma.

N-cadherin: A diagnostic marker to help discriminate primary liver carcinomas from extrahepatic carcinomas.

Distinguishing primary liver cancer (PLC), namely hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), from liver metastases is of crucial clinical importance. Histopathology remains the gold standard, but differential diagnosis may be challenging. While absent in most epithelial, the expression of the adherens junction glycoprotein N-cadherin is commonly restricted to neural and mesenchymal cells, or carcinoma cells that undergo the phenomenon of epithelial-to-mesenchymal transition (EMT). However, we recently established N- and E-cadherin expression as hallmarks of normal hepatocytes and cholangiocytes, which are also preserved in HCC and iCCA. Therefore, we hypothesized that E- and/or N-cadherin may distinguish between carcinoma derived from the liver vs carcinoma of other origins. We comprehensively evaluated E- and N-cadherin in 3359 different tumors in a multicenter study using immunohistochemistry and compared our results with previously published 882 cases of PLC, including 570 HCC and 312 iCCA. Most carcinomas showed strong positivity for E-cadherin. Strong N-cadherin positivity was present in HCC and iCCA. However, except for clear cell renal cell carcinoma (23.6% of cases) and thyroid cancer (29.2%), N-cadherin was only in some instances faintly expressed in adenocarcinomas of the gastrointestinal tract (0%-0.5%), lung (7.1%), pancreas (3.9%), gynecological organs (0%-7.4%), breast (2.2%) as well as in urothelial (9.4%) and squamous cell carcinoma (0%-5.6%). As expected, N-cadherin was detected in neuroendocrine tumors (25%-75%), malignant melanoma (46.2%) and malignant mesothelioma (41%). In conclusion, N-cadherin is a useful marker for the distinction of PLC vs liver metastases of extrahepatic carcinomas (P < .01).

Convergent MAPK pathway alterations mediate acquired resistance to FGFR inhibitors in FGFR2 fusion-positive cholangiocarcinoma.

BACKGROUND & AIMS: There is a knowledge gap in understanding mechanisms of resistance to fibroblast growth factor receptor (FGFR) inhibitors (FGFRi) and a need for novel therapeutic strategies to overcome it. We investigated mechanisms of acquired resistance to FGFRi in patients with FGFR2-fusion-positive cholangiocarcinoma (CCA). METHODS: A retrospective analysis of patients who received FGFRi therapy and underwent tumor and/or cell-free DNA analysis, before and after treatment, was performed. Longitudinal circulating tumor DNA samples from a cohort of patients in the phase I trial of futibatinib (NCT02052778) were assessed. FGFR2-BICC1 fusion cell lines were developed and secondary acquired resistance mutations in the mitogen-activated protein kinase (MAPK) pathway were introduced to assess their effect on sensitivity to FGFRi in vitro. RESULTS: On retrospective analysis of 17 patients with repeat sequencing following FGFRi treatment, new FGFR2 mutations were detected in 11 (64.7%) and new alterations in MAPK pathway genes in nine (52.9%) patients, with seven (41.2%) patients developing new alterations in both the FGFR2 and MAPK pathways. In serially collected plasma samples, a patient treated with an irreversible FGFRi tested positive for previously undetected BRAF V600E, NRAS Q61K, NRAS G12C, NRAS G13D and KRAS G12K mutations upon progression. Introduction of a FGFR2-BICC1 fusion into biliary tract cells in vitro sensitized the cells to FGFRi, while concomitant KRAS G12D or BRAF V600E conferred resistance. MEK inhibition was synergistic with FGFRi in vitro. In an in vivo animal model, the combination had antitumor activity in FGFR2 fusions but was not able to 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. CLINICAL TRIAL NUMBER: NCT02052778. 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. These findings have several implications for patients with FGFR2 fusion-positive tumors and provide mechanistic insight into emerging MAPK pathway alterations which may serve as a therapeutic vulnerability in the setting of acquired resistance to FGFRi.

Revealing the role of necroptosis microenvironment: FCGBP + tumor-associated macrophages drive primary liver cancer differentiation towards cHCC-CCA or iCCA.

Previous research has demonstrated that the conversion of hepatocellular carcinoma (HCC) to intrahepatic cholangiocarcinoma (iCCA) can be stimulated by manipulating the tumor microenvironment linked with necroptosis. However, the specific cells regulating the necroptosis microenvironment have not yet been identified. Additionally, further inquiry into the mechanism of how the tumor microenvironment regulates necroptosis and its impact on primary liver cancer(PLC) progression may be beneficial for precision therapy. We recruited a single-cell RNA sequencing dataset (scRNA-seq) with 34 samples from 4 HCC patients and 3 iCCA patients, and a Spatial Transcriptomic (ST) dataset including one each of HCC, iCCA, and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Quality control, dimensionality reduction and clustering were based on Seurat software (v4.2.2) process and batch effects were removed by harmony (v0.1.1) software. The pseudotime analysis (also known as cell trajectory) in the single cell dataset was performed by monocle2 software (v2.24.0). Calculation of necroptosis fraction was performed by AUCell (v1.16.0) software. Switch gene analysis was performed by geneSwitches(v0.1.0) software. Dimensionality reduction, clustering, and spatial image in ST dataset were performed by Seurat (v4.0.2). Tumor cell identification, tumor subtype characterization, and cell type deconvolution in spot were performed by SpaCET (v1.0.0) software. Immunofluorescence and immunohistochemistry experiments were used to prove our conclusions. Analysis of intercellular communication was performed using CellChat software (v1.4.0). ScRNA-seq analysis of HCC and iCCA revealed that necroptosis predominantly occurred in the myeloid cell subset, particularly in FCGBP + SPP1 + tumor-associated macrophages (TAMs), which had the highest likelihood of undergoing necroptosis. The existence of macrophages undergoing necroptosis cell death was further confirmed by immunofluorescence. Regions of HCC with poor differentiation, cHCC-CCA with more cholangiocarcinoma features, and the tumor region of iCCA shared spatial colocalization with FCGBP + macrophages, as confirmed by spatial transcriptomics, immunohistochemistry and immunofluorescence. Pseudotime analysis showed that premalignant cells could progress into two directions, one towards HCC and the other towards iCCA and cHCC-CCA. Immunofluorescence and immunohistochemistry experiments demonstrated that the number of macrophages undergoing necroptosis in cHCC-CCA was higher than in iCCA and HCC, the number of macrophages undergoing necroptosis in cHCC-CCA with cholangiocarcinoma features was more than in cHCC-CCA with hepatocellular carcinoma features. Further investigation showed that myeloid cells with the highest necroptosis score were derived from the HCC_4 case, which had a severe inflammatory background on pathological histology and was likely to progress towards iCCA and cHCC-CCA. Switchgene analysis indicated that S100A6 may play a significant role in the progression of premalignant cells towards iCCA and cHCC-CCA. Immunohistochemistry confirmed the expression of S100A6 in PLC, the more severe inflammatory background of the tumor area, the more cholangiocellular carcinoma features of the tumor area, S100A6 expression was higher. The emergence of necroptosis microenvironment was found to be significantly associated with FCGBP + SPP1 + TAMs in PLC. In the presence of necroptosis microenvironment, premalignant cells appeared to transform into iCCA or cHCC-CCA. In contrast, without a necroptosis microenvironment, premalignant cells tended to develop into HCC, exhibiting amplified stemness-related genes (SRGs) and heightened malignancy.

Refining Classification of Cholangiocarcinoma Subtypes via Proteogenomic Integration Reveals New Therapeutic Prospects.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinomas (iCCs) are characterized by their rarity, difficult diagnosis, and overall poor prognosis. The iCC molecular classification for developing precision medicine strategies was investigated. METHODS: Comprehensive genomic, transcriptomic, proteomic, and phosphoproteomic analyses were performed on treatment-naïve tumor samples from 102 patients with iCC who underwent surgical resection with curative intent. An organoid model was constructed for testing therapeutic potential. RESULTS: Three clinically supported subtypes (stem-like, poorly immunogenic, and metabolism) were identified. NCT-501 (aldehyde dehydrogenase 1 family member A1 [ALDH1A1] inhibitor) exhibited synergism with nanoparticle albumin-bound-paclitaxel in the organoid model for the stem-like subtype. The oncometabolite dysregulations were associated with different clinical outcomes in the stem-like and metabolism subtypes. The poorly immunogenic subtype harbors the non-T-cell tumor infiltration. Integrated multiomics analysis not only reproduced the 3 subtypes but also showed heterogeneity in iCC. CONCLUSIONS: This large-scale proteogenomic analysis provides information beyond that obtained with genomic analysis, allowing the functional impact of genomic alterations to be discerned. These findings may assist in the stratification of patients with iCC and in developing rational therapeutic strategies.

Unveiling the immunogenomic landscape of cholangiocarcinoma: Identifying new prognostic markers and therapeutic targets based on CCL5 expression.

BACKGROUND: Cholangiocarcinoma (CCA) stands as an aggressive malignancy of the biliary tract. The interplay between the tumor and immune system plays a pivotal role in disease progression and treatment outcomes. Hence, the present study aimed to extensively explore the immunogenomic landscape of CCA, with the objective of unveiling unique molecular and immunological signatures that could guide personalized therapeutic approaches. METHODS: The study collected data from The Cancer Genome Atlas databases, performed gene set variation analysis for the chemokine ligand 5 (CCL5) high/low expression group, conducted principal component analysis, gene set enrichment analysis enrichment and mutation pattern analysis, generated a heatmap, and performed cox regression analysis. RESULTS: The two discrete subpopulations were found to exhibit contrasting mutational and immunogenomic characteristics, emphasizing the heterogeneity of CCA. These subsets also showed pronounced discrepancies in the infiltration of immune cells, indicating diverse interactions with the tumor immune microenvironment. Furthermore, the dissimilarities in mutational patterns were observed within the two CCA subgroups, with PBRM1 and BAP1 emerging as the most frequently mutated genes. In addition, a prognostic framework was formulated and validated utilizing the expression profiles of COX16 and RSAD2 genes, effectively segregating patients into high-risk and low-risk cohorts. Furthermore, the connections between immune-related parameters and these risk groups were identified, underscoring the potential significance of the immune microenvironment in patient prognosis. In vitro experiments have shown that COX16 promotes the proliferation and metastasis of CCA cells, whereas RSAD2 inhibits it. CONCLUSIONS: The present study provides an intricate depiction of the immunogenomic landscape of CCA based on CCL5 expression, thereby paving the way for novel immunotherapy strategies and prognostic assessment.

Liensinine inhibits progression of intrahepatic cholangiocarcinoma by regulating TGF-ß1 /P-smad3 signaling through HIF-1a.

Intrahepatic cholangiocarcinoma (ICC) is a high-grade malignant digestive system tumor with an insidious onset and unfavorable prognosis. Liensinine, a small molecule derived from plants, has been proven to have significant tumor suppressor activity in other cancers. However, there are no reports on whether liensinine can inhibit the proliferation or metastasis of ICC. This study aimed to explore the tumor-suppressive activity of liensinine in ICC and its underlying mechanisms. The phenotypic changes in ICC cells were monitored in vitro using cell function tests. Western blot and immunofluorescence analyses verified the efficacy of liensinine. Tumor-bearing nude mice were used to explore the effect of liensinine on tumors and its toxicity and side effects in vivo. Liensinine suppressed ICC cell proliferation and arrested the cell cycle at the G1 phase. The epithelial-mesenchymal transition (EMT) of ICC cells was also inhibited, thereby restraining their invasion and migration of tumor cells. In addition, this study found that the potential mechanism of liensinine inhibiting EMT may be via suppression of the TGF-ß1/P-smad3 signaling pathway through hypoxia-inducible factor 1 alpha (HIF-1a). In vivo experiments showed that liensinine inhibited the growth of Hucc-T1 transplanted tumors in nude mice. Liensinine restrained the proliferation of ICC cells and suppressed EMT in ICC via the HIF-1a-mediated TGF-ß1/P-smad3 signaling pathway.