Intrahepatic Cholangiocarcinoma Tumor Research Articles

CLK1 Activates YAP to Promote Intrahepatic Cholangiocarcinogenesis.

Cdc2-like kinase 1 (CLK1) has dual-specificity kinase ability to phosphorylate tyrosine and serine/threonine protein residues. CLK1 regulates many physiological processes and has been shown to contribute to multiple types of cancer. Here, we investigated the functional role of CLK1 during intrahepatic cholangiocarcinoma (ICC) development. The expression of CLK1 was elevated in ICC tumors, and patients with high expression of CLK1 demonstrated poor prognosis. In hydrodynamically transfected mouse models, CLK1 alone was insufficient to induce ICC, whereas CLK1 cooperated with AKT (AKT/CLK1) to trigger ICC initiation. In addition, overexpression of CLK1 in ICC cells facilitated proliferation in vitro and tumor growth in vivo, while loss of CLK1 elicited the opposite effects. Moreover, RNAseq analysis indicated that high levels of CLK1 corresponded with activation of the Hippo-YAP signaling pathway. Consistently, the AKT/CLK1 murine tumors displayed upregulation of YAP as well as its downstream targets. Furthermore, loss or pharmacological inhibition YAP in ICC cells inhibited CLK1-induced growth, and deletion of Yap completely retarded the induction of AKT/CLK1 tumors. Mechanistically, 4D-label free mass spectrometry and co-immunoprecipitation assays revealed WWC2 as a potential mediator of CLK1-YAP cascade. Collectively, the current findings identify a critical role for CLK1 in promoting ICC development and indicate that inhibiting YAP might be an effective approach for perturbing CLK1-mediated tumorigenesis.

Epstein-Bar Virus-Positive Lymphoepithelioma-Like Intrahepatic Cholangiocarcinoma: A Case Report and Literature Review.

Intrahepatic cholangiocarcinoma is a primary liver malignancy with poor prognosis and limited treatment options. Lymphoepithelioma-like intrahepatic cholangiocarcinoma is an exceedingly rare variant of intrahepatic cholangiocarcinoma that histologically resembles nasopharyngeal carcinoma, previously known as lymphoepithelioma. It was first reported by Hsu et al in 1996 and they were able to show the presence of Epstein-Barr virus in tumor cells. Even though several risk factors for the development of intrahepatic cholangiocarcinoma have been identified, the association between Epstein-Barr virus and intrahepatic cholangiocarcinoma is relatively rare. This article aims at discussing a female patient's lymphoepithelioma-like intrahepatic cholangiocarcinoma tumor and summarizing the existing literature on Epstein-Barr virus-positiveLymphoepithelioma-like intrahepatic cholangiocarcinoma, including the clinical features, pathogenesis, diagnostic challenges, treatment approaches, prognosis, and histopathologic description.

N-acetyltransferase 10 affects the proliferation of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating C-C motif chemokine ligand 2

BackgroundN-acetyltransferase 10 (NAT10) plays a crucial role in the occurrence and development of various tumors. However, the current regulatory mechanism of NAT10 in tumors is limited to its presence in tumor cells. Here, we aimed to reveal the role of NAT10 in intrahepatic cholangiocarcinoma (ICC) and investigate its effect on macrophage polarization in the tumor microenvironment (TME).MethodsThe correlation between NAT10 and ICC clinicopathology was analyzed using tissue microarray (TMA), while the effect of NAT10 on ICC proliferation was verified in vitro and in vivo. Additionally, the downstream target of NAT10, C-C motif chemokine ligand 2 (CCL2), was identified by Oxford Nanopore Technologies full-length transcriptome sequencing, RNA immunoprecipitation-quantitative polymerase chain reaction, and coimmunoprecipitation experiments. It was confirmed by co-culture that ICC cells could polarize macrophages towards M2 type through the influence of NAT10 on CCL2 protein expression level. Through RNA-sequencing, molecular docking, and surface plasmon resonance (SPR) assays, it was confirmed that berberine (BBR) can specifically bind CCL2 to inhibit ICC development.ResultsHigh expression level of NAT10 was associated with poor clinicopathological manifestations of ICC. In vitro, the knockdown of NAT10 inhibited the proliferative activity of ICC cells and tumor growth in vivo, while its overexpression promoted ICC proliferation. Mechanically, by binding to CCL2 messenger RNA, NAT10 increased CCL2 protein expression level in ICC and their extracellular matrix, thereby promoting the proliferation of ICC cells and M2-type polarization of macrophages. BBR can target CCL2, inhibit ICC proliferation, and reduce M2-type polarization of macrophages.ConclusionsNAT10 promotes ICC proliferation and M2-type polarization of macrophages by up-regulating CCL2, whereas BBR inhibits ICC proliferation and M2-type polarization of macrophages by inhibiting CCL2.

Pan-lysyl oxidase inhibition disrupts fibroinflammatory tumor stroma, rendering cholangiocarcinoma susceptible to chemotherapy.

Cholangiocarcinoma (CCA) features highly desmoplastic stroma that promotes structural and functional resistance to therapy. Lysyl oxidases (LOX, LOXL1-4) catalyze collagen cross-linking, thereby increasing stromal rigidity and facilitating therapeutic resistance. Here, we evaluate the role of lysyl oxidases in stromal desmoplasia and the effects of pan-lysyl oxidase (pan-LOX) inhibition in CCA. Resected CCA and normal liver specimens were analyzed from archival tissues. Spontaneous and orthotopic murine models of intrahepatic CCA (iCCA) were used to assess the impact of the pan-LOX inhibitor PXS-5505 in treatment and correlative studies. The functional role of pan-LOX inhibition was interrogated through in vivo and ex vivo assays. All 5 lysyl oxidases are upregulated in CCA and reduced lysyl oxidase expression is correlated with an improved prognosis in resected patients with CCA. Spontaneous and orthotopic murine models of intrahepatic cholangiocarcinoma upregulate all 5 lysyl oxidase isoforms. Pan-LOX inhibition reversed mechanical compression of tumor vasculature, resulting in improved chemotherapeutic penetrance and cytotoxic efficacy. The combination of chemotherapy with pan-LOX inhibition increased damage-associated molecular pattern release, which was associated with improved antitumor T-cell responses. Pan-LOX inhibition downregulated macrophage invasive signatures in vitro, rendering tumor-associated macrophages more susceptible to chemotherapy. Mice bearing orthotopic and spontaneously occurring intrahepatic cholangiocarcinoma tumors exhibited delayed tumor growth and improved survival following a combination of pan-LOX inhibition with chemotherapy. CCA upregulates all 5 lysyl oxidase isoforms, and pan-LOX inhibition reverses tumor-induced mechanical forces associated with chemotherapy resistance to improve chemotherapeutic efficacy and reprogram antitumor immune responses. Thus, combination therapy with pan-LOX inhibition represents an innovative therapeutic strategy in CCA.

Characterization of the distinct immune microenvironments between hepatocellular carcinoma and intrahepatic cholangiocarcinoma

As two major types of primary liver cancers, the tumor immune microenvironment (TIME) of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) have been well studied separately. However, a systemic assessment of the similarities and differences between the TIME of HCC and ICC is still lacking. In this study, we pictured a landscape of combined TIME of HCC and ICC by sequencing and integrating 41 single-cell RNA-seq samples from four different tissue types of both malignancies. We found that T cells in HCC tumors generally exhibit higher levels of immunosuppression and exhaustion than those in ICC tumors. Myeloid cells in HCC and ICC tumors also exhibit distinct phenotypes and may serve as a key factor driving the differences between their TIMEs. Besides, we identified a cluster of EGR1+ macrophages specifically enriched in HCC tumors. Together, our study provides new insights into cellular composition, states and interactions in the TIMEs of HCC and ICC, which could pave the way for the development of future therapeutic targets for liver cancers.

Characterization of tumor microbiome and associations with prognosis in intrahepatic cholangiocarcinoma.

The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.

Molecular Profile of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a relatively uncommon but highly aggressive primary liver cancer that originates within the liver. The aim of this study is to review the molecular profile of intrahepatic cholangiocarcinoma and its implications for prognostication and decision-making. This comprehensive characterization of ICC tumors sheds light on the disease's underlying biology and offers a foundation for more personalized treatment strategies. This is a narrative review of the prognostic and therapeutic role of the molecular profile of ICC. Knowing the molecular profile of tumors helps determine prognosis and support certain target therapies. The molecular panel in ICC helps to select patients for specific therapies, predict treatment responses, and monitor treatment responses. Precision medicine in ICC can promote improvement in prognosis and reduce unnecessary toxicity and might have a significant role in the management of ICC in the following years. The main mutations in ICC are in tumor protein p53 (TP53), Kirsten rat sarcoma virus (KRAS), isocitrate dehydrogenase 1 (IDH1), and AT-rich interactive domain-containing protein 1A (ARID1A). The rate of mutations varies significantly for each population. Targeting TP53 and KRAS is challenging due to the natural characteristics of these genes. Different stages of clinical studies have shown encouraging results with inhibitors of mutated IDH1 and target therapy for ARID1A downstream effectors. Fibroblast growth factor receptor 2 (FGFR2) fusions are an important target in patients with ICC. Immune checkpoint blockade can be applied to a small percentage of ICC patients. Molecular profiling in ICC represents a groundbreaking approach to understanding and managing this complex liver cancer. As our comprehension of ICC's molecular intricacies continues to expand, so does the potential for offering patients more precise and effective treatments. The integration of molecular profiling into clinical practice signifies the dawn of a new era in ICC care, emphasizing personalized medicine in the ongoing battle against this malignancy.

Patlak Reconstruction Using Dynamic 18 F-FDG PET Imaging for Evaluation of Malignant Liver Tumors : A Comparison of HCC, ICC, and Metastatic Liver Tumors.

The aim of this study was to explore the different patterns of dynamic whole-body (D-WB) FDG PET/CT parameters among liver malignancy types as potential diagnostic clues and investigate the association between static and dynamic PET/CT parameters for each tumor histology. Seventy-one patients with intrahepatic cholangiocarcinoma (ICC), metastatic liver tumor (MLT), or hepatocellular carcinoma (HCC) who underwent D-WB and static dual-time-point FDG PET/CT were enrolled. We obtained Pearson correlation coefficients between the metabolic rate of FDG (MR FDG ; mg/min/ 100ml) or distribution volume of free FDG (DV FDG , %) and static PET/CT parameters. We compared MR FDG and DV FDG values by tumor type and performed receiver operating characteristic analyses for MR FDG and static images. A total of 12 ICC, 116 MLT, and 36 HCC lesions were analyzed. MR FDG and DV FDG showed excellent correlation with early (SUV e ) and delayed SUV max (SUV d ) ( r = 0.71~0.97), but DV FDG in the HCC lesions did not ( r = 0.62 and 0.69 for SUV e and SUV d , respectively) ( P < 0.001 for all). HCC lesions showed significantly lower MR FDG (2.43 ± 1.98) and DV FDG (139.95 ± 62.58) than ICC (5.02 ± 3.56, 207.06 ± 97.13) and MLT lesions (4.51 ± 2.47, 180.13 ± 75.58) ( P < 0.01 for all). The optimal MR FDG could differentiate HCC from ICC and MLT with areas under the curve of 0.84 and 0.80, respectively. Metastatic liver tumor lesions showed the widest distribution of MR FDG and DV FDG values but with no significant difference among most primary sites. MR FDG was strongly correlated with SUV max in the 3 malignancies and showed utility for differentiating HCC from ICC and MLT. Each tumor type has a different glucose metabolism, and D-WB FDG PET/CT imaging has the potential to visualize those differences.

A Novel Trojan Horse Nanotherapy Strategy Targeting the cPKM-STMN1/TGFB1 Axis for Effective Treatment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is characterized by its dense fibrotic microenvironment and highly malignant nature, which are associated with chemotherapy resistance and very poor prognosis. Although circRNAs have emerged as important regulators in cancer biology, their role in ICC remains largely unclear. Herein, a circular RNA, cPKM is identified, which is upregulated in ICC and associated with poor prognosis. Silencing cPKM in ICC cells reduces TGFB1 release and stromal fibrosis, inhibits STMN1 expression, and suppresses ICC growth and metastasis, moreover, it also leads to overcoming paclitaxel resistance. This is regulated by the interactions of cPKM with miR-199a-5p or IGF2BP2 and by the ability of cPKM to stabilize STMN1/TGFB1 mRNA. Based on these findings, a Trojan horse nanotherapy strategy with co-loading of siRNA against cPKM (si-cPKM) and paclitaxel (PTX) is developed. The siRNA/PTX co-loaded nanosystem (Trojan horse) efficiently penetrates tumor tissues, releases si-cPKM and paclitaxel (soldiers), promotes paclitaxel sensitization, and suppresses ICC proliferation and metastasis in vivo. Furthermore, it alleviates the fibrosis of ICC tumor stroma and reopens collapsed tumor vessels (opening the gates), thus enhancing the efficacy of the standard chemotherapy regimen (main force). This novel nanotherapy provides a promising new strategy for ICC treatment.

Radiomics nomogram based on optimal VOI of multi-sequence MRI for predicting microvascular invasion in intrahepatic cholangiocarcinoma.

Microvascular invasion (MVI) is a significant adverse prognostic indicator of intrahepatic cholangiocarcinoma (ICC) and affects the selection of individualized treatment regimens. This study sought to establish a radiomics nomogram based on the optimal VOI of multi-sequence MRI for predicting MVI in ICC tumors. 160 single ICC lesions with MRI scanning confirmed by postoperative pathology were randomly separated into training and validation cohorts (TC and VC). Multivariate analysis identified independent clinical and imaging MVI predictors. Radiomics features were obtained from images of 6 MRI sequences at 4 different VOIs. The least absolute shrinkage and selection operator algorithm was performed to enable the derivation of robust and effective radiomics features. Then, the best three sequences and the optimal VOI were obtained through comparison. The MVI prediction nomogram combined the independent predictors and optimal radiomics features, and its performance was evaluated via the receiver operating characteristics, calibration, and decision curves. Tumor size and intrahepatic ductal dilatation are independent MVI predictors. Radiomics features extracted from the best three sequences (T1WI-D, T1WI, DWI) with VOI10mm (including tumor and 10mm peritumoral region) showed the best predictive performance, with AUCTC = 0.987 and AUCVC = 0.859. The MVI prediction nomogram obtained excellent prediction efficacy in both TC (AUC = 0.995, 95%CI 0.987-1.000) and VC (AUC = 0.867, 95%CI 0.798-0.921) and its clinical significance was further confirmed by the decision curves. A nomogram combining tumor size, intrahepatic ductal dilatation, and the radiomics model of MRI multi-sequence fusion at VOI10mm may be a predictor of preoperative MVI status in ICC patients.

Unraveling the heterogeneity of intercellular crosstalk in intrahepatic and extrahepatic cholangiocarcinoma using single-cell and spatial transcriptome profiling.

50 Background: Extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) are two types of malignant tumors that arise from the bile duct epithelium at different locations. Currently, effective treatment options for these tumors are limited, and their microenvironmental differences remain poorly understood. Methods: We collected retrospective clinicopathological data of ECC (N=64) and ICC (N=153) patients from our hospital and integrated public database information to analyze the clinical characteristics, survival, and prognosis of ECC and ICC. We performed single-cell sequencing (scRNA-seq) and spatial transcriptomic techniques on three ECC and three ICC tumor tissues. Verification of scRNA-seq results was done using immunohistochemistry, flow cytometry, and polychromatic immunofluorescence. Additionally, we analyzed RNA and ATAC sequencing data for exhausted CD8+ T cells TEX obtained from ICC and ECC tissue samples. We used InferCNV to infer copy number variations (CNVs) of epithelial cells from ICC and ECC. Results: Our results showed that ECC and ICC differ in their clinical characteristics such as age, gender, bilirubin, CEA, and outcome. ECC had higher copy number amplification variation and exhibited inflammatory signaling and ongoing immunoediting, reflected in higher immune checkpoint expression such as PDCD1, CTLA4, and LAG3, and tumor infiltration with highly differentiated dysfunctional PDCD1+ LAG3+ CXCL13+ exhausted CD8+ T cells (TEX) and precursors of exhausted T (TPEX). RNA velocity analysis revealed different developmental trajectories of T cells in ECC and ICC, predominantly in naive/stem-like and memory T cells. We also identified a trefoil factor 3 (TFF3)-positive epithelial subpopulation in ECC. TFF3+ epithelial cells had closer interaction with CTHRC1+ fibroblasts, which induced more CD8+ T cells to TEX and TPEX. TFF3+ cells exhibited higher CNV scores and higher PD-L1 and PD-L2 expression, and co-localized with TEX and TPEX, providing one explanation for the immunosuppressive environment in ECC and suggesting a potential response to immune checkpoint therapy (ICT). Conclusions: Our study provides a comprehensive characterization of the cellular heterogeneity landscape of ECC and ICC, which can guide the optimization of personalized cancer immunotherapy. We identified potential biomarkers and targets for immunotherapies, and our findings shed light on the underlying mechanisms of tumor immune evasion and provide a foundation for further research in this area.

Hepatitis B virus-related intrahepatic cholangiocarcinoma originates from hepatocytes

BackgroundHepatitis B virus (HBV) infection is one of the most common risk factors for intrahepatic cholangiocarcinoma (ICC). However, there is no direct evidence of a causal relationship between HBV infection and ICC. In this study, we attempted to prove that ICC may originate from hepatocytes through a pathological study involving ICC tissue-derived organoids.MethodThe medical records and tumor tissue samples of 182 patients with ICC after hepatectomy were collected. The medical records of 182 patients with ICC were retrospectively analyzed to explore the prognostic factors. A microarray of 182 cases of ICC tumor tissue and 6 cases of normal liver tissue was made, and HBsAg was stained by immunohistochemistry (IHC) to explore the factors closely related to HBV infection. Fresh ICC tissues and corresponding adjacent tissues were collected to make paraffin sections and organoids. Immunofluorescence (IF) staining of factors including HBsAg, CK19, CK7, Hep-Par1 and Albumin (ALB) was performed on both fresh tissues and organoids. In addition, we collected adjacent nontumor tissues of 6 patients with HBV (+) ICC, from which biliary duct tissue and normal liver tissue were isolated and RNA was extracted respectively for quantitative PCR assay. In addition, the expression of HBV-DNA in organoid culture medium was detected by quantitative PCR and PCR electrophoresis.ResultsA total of 74 of 182 ICC patients were HBsAg positive (40.66%, 74/182). The disease-free survival (DFS) rate of HBsAg (+) ICC patients was significantly lower than that of HBsAg (−) ICC patients (p = 0.0137). IF and IHC showed that HBsAg staining was only visible in HBV (+) ICC fresh tissues and organoids, HBsAg expression was negative in bile duct cells in the portal area. Quantitative PCR assay has shown that the expression of HBs antigen and HBx in normal hepatocytes were significantly higher than that in bile duct epithelial cells. Combined with the IF and IHC staining, it was confirmed that HBV does not infect normal bile duct epithelial cells. In addition, IF also showed that the staining of bile duct markers CK19 and CK7 were only visible in ICC fresh tissue and organoids, and the staining of hepatocyte markers Hep-Par1 and ALB was only visible in normal liver tissue fresh tissue. Real-time PCR and WB had the same results. High levels of HBV-DNA were detected in the culture medium of HBV (+) organoids but not in the culture medium of HBV (−) organoids.ConclusionHBV-related ICC might be derived from hepatocytes. HBV (+) ICC patients had shorter DFS than HBV (−) ICC patients.

MMP14 is a diagnostic gene of intrahepatic cholangiocarcinoma associated with immune cell infiltration.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor of the hepatobiliary system with concealed onset, strong invasiveness and poor prognosis. To explore the disease characteristic genes that may be helpful in the diagnosis of ICC and affect immune cell infiltration. We downloaded two ICC-related human gene expression profiles from GEO database as the training group (GSE26566 and GSE32958 datasets) for difference analysis, and performed enrichment analysis on differential genes. The least absolute shrinkage and selection operator (LASSO), support vector machine-recursive feature elimination (SVM-RFE) and random forest (RF), three machine learning algorithms, were used to screen the characteristic genes. Double verification was carried out on GSE107943 and The Cancer Genome Atlas, two verification groups. Receiver operating characteristic curve and area under the curve (AUC) were used to evaluate the diagnostic efficacy of genes for ICC. CIBERSORT and ssGSEA algorithms were used to evaluate the effect of characteristic genes on immune infiltration pattern. Human Protein Atlas (HPA) was used to analyze the protein expression level of the target gene. A total of 1091 differential genes were obtained in the training group. Enrichment analysis showed that the above genes were mainly enriched in small molecular catabolism, complement and coagulation cascade, bile secretion and other functions and pathways. Twenty-five characteristic genes were screened by LASSO regression, 19 by SVM-RFE algorithm, and 30 by RF algorithm. Three algorithms were used in combination to determine the characteristic gene of ICC: MMP14. The verification group confirmed that the genes had a high diagnostic accuracy (AUC values of the training group and the verification group were 0.960, 0.999, and 0.977, respectively). Comprehensive analysis of immune infiltration showed that MMP14 could affect the infiltration of monocytes, activated memory CD4 T cells, resting memory CD4 T cells, and other immune cells, and was closely related to the expression of CD200, cytotoxic T-lymphocyte-associated antigen 4, CD14, CD44, and other immune checkpoints. The results of immunohistochemistry in HPA database showed was indeed overexpressed in ICC. MMP14 can be used as a disease characteristic gene of ICC, and may regulate the distribution of immune-infiltrating cells in the ICC tumor microenvironment, which provides a new method for the determination of ICC diagnostic markers and screening of therapeutic targets.

Computerized Diagnosis of Liver Tumors From CT Scans Using a Deep Neural Network Approach.

The liver is a frequent site of benign and malignant, primary and metastatic tumors. Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common primary liver cancers, and colorectal liver metastasis (CRLM) is the most common secondary liver cancer. Although the imaging characteristic of these tumors is central to optimal clinical management, it relies on imaging features that are often non-specific, overlap, and are subject to inter-observer variability. Thus, in this study, we aimed to categorize liver tumors automatically from CT scans using a deep learning approach that objectively extracts discriminating features not visible to the naked eye. Specifically, we used a modified Inception v3 network-based classification model to classify HCC, ICC, CRLM, and benign tumors from pretreatment portal venous phase computed tomography (CT) scans. Using a multi-institutional dataset of 814 patients, this method achieved an overall accuracy rate of 96%, with sensitivity rates of 96%, 94%, 99%, and 86% for HCC, ICC, CRLM, and benign tumors, respectively, using an independent dataset. These results demonstrate the feasibility of the proposed computer-assisted system as a novel non-invasive diagnostic tool to classify the most common liver tumors objectively.

Convergent genomic diversity and novel BCAA metabolism in intrahepatic cholangiocarcinoma

BackgroundDriver alterations may represent novel candidates for driver gene-guided therapy; however, intrahepatic cholangiocarcinoma (ICC) with multiple genomic aberrations makes them intractable. Therefore, the pathogenesis and metabolic changes of ICC need to be understood to develop new treatment strategies. We aimed to unravel the evolution of ICC and identify ICC-specific metabolic characteristics to investigate the metabolic pathway associated with ICC development using multiregional sampling to encompass the intra- and inter-tumoral heterogeneity.MethodsWe performed the genomic, transcriptomic, proteomic and metabolomic analysis of 39–77 ICC tumour samples and eleven normal samples. Further, we analysed their cell proliferation and viability.ResultsWe demonstrated that intra-tumoral heterogeneity of ICCs with distinct driver genes per case exhibited neutral evolution, regardless of their tumour stage. Upregulation of BCAT1 and BCAT2 indicated the involvement of ‘Val Leu Ile degradation pathway’. ICCs exhibit the accumulation of ubiquitous metabolites, such as branched-chain amino acids including valine, leucine, and isoleucine, to negatively affect cancer prognosis. We revealed that this metabolic pathway was almost ubiquitously altered in all cases with genomic diversity and might play important roles in tumour progression and overall survival.ConclusionsWe propose a novel ICC onco-metabolic pathway that could enable the development of new therapeutic interventions.

Comprehensive transcriptomic analysis to identify biological and clinical differences in cholangiocarcinoma.

Cholangiocarcinoma (CC) is a rare and aggressive disease with limited therapeutic options and a poor prognosis. All available public records of cohorts reporting transcriptomic data on intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) were collected with the aim to provide a comprehensive gene expression-based classification with clinical relevance. A total of 543 patients with primary tumor tissues profiled by RNAseq and microarray platforms from seven public datasets were used as a discovery set to identify distinct biological subgroups. Group predictors developed on the discovery sets were applied to a single cohort of 131 patients profiled with RNAseq for validation and assessment of clinical relevance leveraging machine learning techniques. By unsupervised clustering analysis of gene expression data we identified both in the ICC and ECC discovery datasets four subgroups characterized by a distinct type of immune infiltrate and signaling pathways. We next developed class predictors using short gene list signatures and identified in an independent dataset subgroups of ICC tumors at different prognosis. The developed class-predictor allows identification of CC subgroups with specific biological features and clinical behavior at single-sample level. Such results represent the starting point for a complete molecular characterization of CC, including integration of genomics data to develop in clinical practice.

Development of Engineered CAR T Cells Targeting Tumor-Associated Glycoforms of MUC1 for the Treatment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a common malignancy arising from the liver with limited 5-year survival. Thus, there is an urgency to explore new treatment methods. Chimeric antigen receptor T (CAR T) cell therapy is a very promising cancer treatment. Though, several groups have investigated CAR T cells targeting MUC1 in solid cancer models, Tn-MUC1-targeted CAR T cells have not yet to be reported in ICC. In this study, we confirmed Tn-MUC1 as a potential therapeutic target for ICC and demonstrated that its expression level was positively correlated with the poor prognosis of ICC patients. More importantly, we successfully developed effective CAR T cells to target Tn-MUC1-positive ICC tumors and explored their antitumor activities. Our results suggest the CAR T cells could specifically eliminate Tn-MUC1-positive ICC cells, but not Tn-MUC1-negative ICC cells, in vitro and in vivo. Therefore, our study is expected to provide new therapeutic strategies and ideas for the treatment of ICC.

Genetic heterogeneity of intrahepatic cholangiocarcinoma: Implications for outcome.

595 Background: Intrahepatic cholangiocarcinoma (IHC) is characterized by marked clinical heterogeneity, likely the result of multiple cells of origin and variable driver gene alterations. The hidden-genome classifier is a statistical algorithm that classifies tumors by integrating multi-level genomic features. In this study, we trained the hidden-genome classifier with extrahepatic cholangiocarcinoma (EHC), gallbladder cancer (GBC) and hepatocellular carcinoma (HCC) as extremes of a spectrum to quantify the genetic heterogeneity of IHC with a view toward improved tumor classification. Methods: An IRB approved retrospective review of patients with biopsy confirmed IHC, EHC, GBC and HCC was conducted. All tumors were subjected to MSK-IMPACT to determine the mutational profile. A two-class model was built and internally validated with the genomic data of EHC/GBC as one class and HCC as the other class. IHC tumors were analyzed in the model and classified into three groups based on their proportional genetic resemblance to EHC/GBC (Biliary Class) or HCC (HCC Class), with the remainder as Intermediate Class. The classification thresholds were 90% resemblance to EHC/GBC or HCC and were determined by the inflection point of predicted survival. The survivals of the three groups were analyzed and compared. Results: A total of 1497 patients were included: IHC (733), EHC (208), GBC (258) and HCC (298). 527 IHC tumors with complete metagenetic information were analyzed in the model, showing a continuous spectrum of alterations, ranging from Biliary Class (122 tumors), Intermediate Class (375 tumors) to HCC Class (30 tumor). The biliary-class IHC was characterized by frequent alterations of IDH1 R132C, KRAS, SMAD4, ERBB2 gain, MDM2 gain, and CKDN2A loss, while the HCC-class IHC was primarily characterized by TERT alterations. In patients with unresected IHCs, the median survival ranged from 1 year (CI 0.77, 1.5) in Biliary Class, 1.8 years (CI 1.5, 2.0) in Intermediate Class, to 2 years (CI 0.93, NR) in HCC Class. In patients subjected to resection, the median survival of Biliary Class (2.4 years, CI 2.1, NR) was lower than both the Intermediate Class (5.1 years, CI 4.8, 6.9) and the HCC Class (3.4 years, CI 2.7, NR). Conclusions: By integrating multi-level genomic features, we leveraged the mutational heterogeneity to classify IHC based on its resemblance to EHC/GBC or HCC tumors. We found that the survival in IHC patients appeared to decline with increasing genomic similarity to Biliary Class. The results support a genomic basis for IHC’s variable clinical behavior and point to a role of mutational testing to guide clinical intervention. [Table: see text]

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma

Despite the epidemiological association between intrahepatic cholangiocarcinoma (ICC) and hepatitis B virus (HBV) infection, little is known about the relevant oncogenic effects. A cohort of 32 HBV-infected ICC and 89 non-HBV-ICC patients were characterized using whole-exome sequencing, proteomic analysis, and single-cell RNA sequencing. Proteomic analysis revealed decreased cell-cell junction levels in HBV-ICC patients. The cell-cell junction level had an inverse relationship with the epithelial-mesenchymal transition (EMT) program in ICC patients. Analysis of the immune landscape found that more CD8 Tcells and Th2 cells were present in HBV-ICC patients. Single-cell analysis indicated that transforming growth factor beta signaling-related EMT program changes increased in tumor cells of HBV-ICC patients. Moreover, ICAM1+ tumor-associated macrophages are correlated with a poor prognosis and contributed to the EMT in HBV-ICC patients. Our findings provide new insights into the behavior of HBV-infected ICC driven by various pathogenic mechanisms involving decreased cell junction levels and increased progression of the EMT program.

Exosomal circ-PTPN22 and circ-ADAMTS6 mark T cell exhaustion and neutrophil extracellular traps in Asian intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is a liver tumor featured by challenges of non-invasive early diagnosis and a higher prevalence rate in Asian countries. These characteristics necessitate the development of liquid biopsy and immunotherapy methods to improve the prognosis of patients with ICC. Herein, we conducted a pilot study on the transcriptome of tumor tissues, adjacent normal tissues, and plasma exosomes of Asian patients with ICC from northern and southern China. We identified a subgroup of immunogenic Asian ICC, which is different from Caucasian ICC and is characterized by Tcell exhaustion and neutrophil extracellular traps. The levels ofcirc-PTPN22 (hsa_circ_0110529) and circ-ADAMTS6 (hsa_circ_0072688), potential circRNA biomarkers, were elevated in the ICC tumor tissues and plasma exosomes of this subgroup than in the other subgroups and normal controls. These circRNAs were derived from post-transcriptional backsplicing of PTPN22 and ADAMTS6 that were expressed in Tcells and endothelial cells, respectively, in the ICC microenvironment. Our results revealed a subgroup of Asian ICC characterized by Tcell exhaustion and neutrophil extracellular traps and marked by elevated levels of circ-PTPN22 and circ-ADAMTS6 in tumor tissues and plasma exosomes. This subgroup is potentially detectable by plasma exosomal circRNAs and treatable with immune checkpoint blockade.