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Intrahepatic cholangiocarcinoma (iCCA) is an aggressive and lethal malignancy arising from the bile ducts. Patients with iCCA typically present with genetically heterogeneous tumours. There is an exception of a small minority of patients where actionable mutations are present, but the heterogeneity of this cancer has hampered the development of targeted therapeutics as the number of recurrent mutations between patients is limited. The majority of iCCA patients are not amenable to surgery, and standard of care chemotherapies act to extend life and are not considered as a curative treatment. As such, there is a significant requirement to explore the functional genetics of iCCA with the goal of developing more effective treatments and therapies. Deep sequencing studies have identified oncogenic mutations in KRAS in iCCA and these represent one of the most recurrent alterations in this cancer. Therefore I focussed on the role of KRAS mutations and their interactions with loss of function mutations. During this work, I identified and validated in vivo which loss of function mutations were capable of interacting with oncogenic KRASG12D mutations. To do this, I developed and carried out a CRISPR-spCas9 library screen informed by mutations that have been previously identified as pathogenic in patient iCCA. Using this approach, I identified the loss of cytoskeletal signalling protein Neurofibromin 2 (NF2) as a novel driver event in iCCA and that loss of this gene interacts with mutant KRAS to initiate cancer. To investigate the role of NF2 further, I explored the relationship between Nf2-loss, Trp53-loss and mutant KRAS, and I found that Nf2 loss cooperated with Trp53 loss to lead to cancer with a severely accelerated phenotype and increased lethality. Additionally, these tumours were representative of a rare and aggressive sarcomatoid subtype of iCCA. To understand how loss of both Trp53 and Nf2 leads to this aggressive phenotype, I used proteomic analysis of a number of signalling pathways, and RNA sequencing to define which mechanisms are altered and contribute to the aggressive cancer phenotype. These analyses identified the co-activation of Wnt/ β-catenin and Pi3K/AKT signalling as recurrently activated in iCCA. To test whether the aggressive nature of dual Trp53 and Nf2 loss was a result of changes in these signalling pathways, I inhibited them pharmacologically with inhibitors targeting Wnt/β-catenin and Pi3K/AKT signalling. Treatment with both of these inhibitors significantly improved survival of tumour bearing mice. Finally, I established a genetically engineered mouse model of iCCA not reliant on Nf2-loss for its progression to address whether inhibition of Wnt/β-catenin and Pi3K/AKT could represent a wide-spanning therapeutic and determine whether the treatment of a different pathological subtype of iCCA would be with these inhibitors would reduce disease progression. I showed this form of well-differentiated iCCA is also amenable to inhibition with this combination of therapies. These data demonstrate that Nf2 is a rare driver gene of iCCA that acts in a cooperative manner with oncogenic KRAS to accelerate tumourigenesis in vivo. Examination of these tumours highlighted a concurrent Wnt and PI3K signalling signature, and I demonstrate that pharmacological co-inhibition substantially impedes iCCA growth, highlighting the use of Wnt/β-catenin and Pi3K/AKT signalling inhibition as a broad treatment for iCCA patients.

Intrahepatic cholangiocarcinoma (iCCA) is a rare and universally lethal malignancy arising in the liver. Incidence has steadily been increasing globally yet effective therapeutics are lacking. Currently, gold standard treatment is complete resection for which a minority of patients are suitable and recurrence rates post resection are high. For the majority of patients unsuitable for surgery, standard chemotherapeutic options extend life by only 3-6 months on average. As such there is a pressing need to elucidate the genetic and molecular features of this malignancy so as to inform the development of more effective therapies. To date, a small but robust set of driver genes have been discovered and for some targeted therapies in development, with mixed results in clinical trials. This work aimed to extend the set of known driver genes by exploring the long tail of infrequently mutated genes in patient sequencing data. Through computational prediction and in vivo screening, a set of driver genes were uncovered which cooperate with the more commonly encountered oncogenic RASG12 isoforms of KRAS and NRAS. Validation studies in vivo confirmed that the membrane cytoskeleton adaptor protein Merlin, encoded by NF2, and the canonical transmembrane semaphorin receptor PLXNB2 encoded by PLXNB2 are tumour suppressors occurring at low frequency in the patient population. Mutation of these genes significantly accelerated disease progression and propagated aggressive, invasive tumour phenotypes.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with a dismal prognosis. Few patients present with disease amenable to resection and chemotherapy is not curative. The incidence of ICC is rising worldwide and new therapeutic approaches are urgently required. Notch signalling is critical for the embryological development and regeneration of the biliary tree in the mammalian liver. Dysregulation of Notch is known to drive tumorigenesis in a range of solid and haematological malignancies and the aim of this work was to define its contribution to the pathogenesis of ICC. Transgenic overexpression of Notch1 has been described to result in the formation of biliary lineage tumours in the liver. I have used resected human tissue, a chemically-induced model of ICC in rat and a novel transgenic murine model in which the tumour suppressor p53 is conditionally deleted from biliary epithelia, to demonstrate that endogenous Notch signalling is acting via the Notch3 receptor to drive tumorigenesis. I use multiple independent methods of Notch3 blockade to establish that Notch3 promotes epithelial cell survival and self-renewal in ICC and demonstrate that Notch3 inhibition significantly attenuates tumour growth in vivo. My data suggest that Notch3 promotes activity through the PI3K/AKT cell survival cascade via a mechanism independent of the effector of canonical Notch, RBPJκ. Given the significant toxicity associated with gamma-secretase inhibitors these findings offer a novel and specific target for further investigation and future therapeutic development in ICC.

Simple Summary Radioembolization is a locoregional therapy used in primary liver malignancies with different applications depending on the treatment goal. The aim of this retrospective study was to evaluate postoperative and long-term survival outcomes of patients with unresectable or high biological risk HCC and ICC treated with RE that were finally rescued to liver surgery with curative intent. In a cohort of 34 patients, we assessed that liver resection and transplantation after RE seem safe and feasible with adequate short-term outcomes. Moreover, long-term outcomes after RE and LR were optimal, with a 10-year OS rate greater than 50% for HCC and ICC patients. On the other hand, the 10-year OS rates from RE were also greater than 50% for patients with HCC downstaged or bridged to LT. Radioembolization (RE) may help local control and achieve tumor reduction while hypertrophies healthy liver and provides a test of time. For liver transplant (LT) candidates, it may attain downstaging for initially non-candidates and bridging during the waitlist. Methods: Patients diagnosed with HCC and ICC treated by RE with further liver resection (LR) or LT between 2005-2020 were included. All patients selected were discarded for the upfront surgical approach for not accomplishing oncological or surgical safety criteria after a multidisciplinary team assessment. Data for clinicopathological details, postoperative, and survival outcomes were retrospectively reviewed from a prospectively maintained database. Results: A total of 34 patients underwent surgery following RE (21 LR and 13 LT). Clavien-Dindo grade III-IV complications and mortality rates were 19.0% and 9.5% for LR and 7.7% and 0% for LT, respectively. After RE, for HCC and ICC patients in the LR group, 10-year OS rates were 57% and 60%, and 10-year DFS rates were 43.1% and 60%, respectively. For HCC patients in the LT group, 10-year OS and DFS rates from RE were 51.3% and 43.3%, respectively. Conclusion: Liver resection after RE is safe and feasible with optimal short-term outcomes. Patients diagnosed with unresectable or high biological risk HCC or ICC, treated with RE, and rescued by LR may achieve optimal global and DFS rates. On the other hand, bridging or downstaging strategies to LT with RE in HCC patients show adequate recurrence rates as well as long-term survival.

Cholangiocarcinomas (CCAs) are aggressive tumors arising along the biliary tract epithelium, whose incidence and mortality are increasing. CCAs are highly desmoplastic cancers characterized by a dense tumor microenvironment (TME), in which each single component plays a fundamental role in shaping CCA initiation, progression and resistance to therapies. The crosstalk between cancer cells and TME can affect the recruitment, infiltration and differentiation of immune cells. According to the stage of the disease and to intra- and inter-patient heterogeneity, TME may contribute to either protumoral or antitumoral activities. Therefore, a better understanding of the effect of each immune cell subtype may open the path to new personalized immune therapeutic strategies for the management of CCA. In this review, we describe the role of immune cells in CCA initiation and progression, and their crosstalk with both cancer-associated fibroblasts (CAFs) and the cancer-stem-cell-like (CSC) niche.

Metabolic reprogramming is a common feature of cancers with many showing specific alterations in both micro and macronutrient metabolism. In this study we evaluate the utility of bioluminescent technology in evaluating the metabolism of liver cancers in vivo. We assess both nutrient uptake and nutrient sensing probes to establish the handling and/or concentrations of lipids, glucose, nicotinamide riboside, iron, copper, and ROS in both healthy mice and tumor bearing mice. Our studies showed promise for the lipid, copper, iron, and ROS probes and were able to measure metabolic differences in copper and lipid metabolism between our different cancer models. We evaluated both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) models that were established through a hydrodynamic injection. Our bioluminescent study revealed a fatty acid uptake phenotype in our ICC model, but not our HCC models, which is a rare but increasingly documented phenotype. Classical cancer lipid metabolism focuses on the upregulation of de novo lipogenesis concomitant with a decreased dependence on exogenous fatty acids. Increased expression and activity of fatty acid synthase (FASN), the rate-limiting enzyme involved in de novo lipogenesis, is required for the survival and proliferation of many tumor cells, including hepatocellular carcinoma (HCC). Another study previously demonstrated that ICC development is insensitive to FASN deprivation. Our observation that ICC maintains robust fatty acid uptake rates suggests a role of exogenous fatty acids for the growth of ICC. Fatty acid transport proteins (FATPs), now classified as solute transporter family 27 (Slc27a1-6), are major transmembrane proteins for LCFA uptake and FATP2 and -5 are robustly expressed in the liver, FATP5 being liver specific. Using genetic manipulation and in vivo bio-imaging techniques, we measured the growth of ICC in an FATP5 knock out mouse model over time. Loss of FATP5 significantly impaired ICC growth indicating that tumor growth is dependent on exogenous fatty acid uptake. This was replicated using a knockdown system in both our ICC and HCC models, and only in ICC did we see a growth impairment. We also performed a survival experiment to see how long the growth effect in ICC was maintain. Over the course of 32 week, only three out of 7 mice grew robust tumor and when assayed for lipid uptake and metabolizing genes, two showed upregulation of FASN. Metabolomic were also performed to determine the fate and function of these fatty acids comparing ICC, HCC, and non-tumorous liver. Our data showed a robust increase in free fatty acids, structural, signaling lipids, and acyl-carnitines suggesting a differential handling of exogenous versus endogenous fatty acids. We attempted to follow up on the increased levels of acyl-carnitines, which are implicated in beta-oxidation and energy generation. We treated mice with CPT1 inhibitor etomoxir and saw no significant growth effect. While it is obvious ICC is dependent on fatty acid uptake, and the inhibition of uptake is able to prevent tumor growth, the differential handling of the downstream fatty acids is more complex. In sum, this study has identified FATP5 as a therapeutic target for the treatment of ICC and potentially other liver cancers dependent on protein mediated exogenous fatty acid uptake.

Background: Liver surgery after radioembolization (RE) entails highly demanding and challenging procedures due to the frequent combination of large tumors, severe RE-related adhesions, and the necessity of conducting major hepatectomies. Laparoscopic liver resection (LLR) and its associated advantages could provide benefits, as yet unreported, to these patients. The current study evaluated feasibility, morbidity, mortality, and survival outcomes for major laparoscopic liver resection after radioembolization. Material and methods: In this retrospective, single-center study patients diagnosed with hepatocellular carcinoma, intrahepatic cholangiocarcinoma or metastases from colorectal cancer undergoing major laparoscopic hepatectomy after RE were identified from institutional databases. They were matched (1:2) on several pre-operative characteristics to a group of patients that underwent major LLR for the same malignancies during the same period but without previous RE. Results: From March 2011 to November 2020, 9 patients underwent a major LLR after RE. No differences were observed in intraoperative blood loss (50 vs. 150 ml; p: 0.621), operative time (478 vs. 407 min; p: 0.135) or pedicle clamping time (90.5 vs 74 min; p: 0.133) between the post-RE LLR and the matched group. Similarly, no differences were observed on hospital stay (median 3 vs. 4 days; p = 0.300), Clavien-Dindo ≥ III complications (2 vs. 1 cases; p: 0.250), specific liver morbidity (1 vs. 1 case p: 1.000), or 90 day mortality (0 vs. 0; p: 1.000). Conclusion: The laparoscopic approach for post radioembolization patients may be a feasible and safe procedure with excellent surgical and oncological outcomes and meets the current standards for laparoscopic liver resections. Further studies with larger series are needed to confirm the results herein presented.

Chromosomal translocations have been a focus in human cancer research. Since the exponential advancement in sequencing technology, a number of actionable fusion proteins have emerged as oncogenic drivers, serving as potential targets for personalized treatments. As the second most common form of liver cancer, Intrahepatic Cholangiocarcinoma (ICC) is as a universally fatal disease with limited treatment options. Recent studies have revealed that translocation of Fibroblast Growth Factor Receptor 2 (FGFR2) to Periphilin 1 (PPHLN1) occurs in 16% ICC cases and FGFR2-PPHLN1 alone is sufficient in driving carcinogenesis. The clinically discovered FGFR2-PPHLN1 preserves the N-terminal structure of FGFR2 containing the extracellular domain, transmembrane domain, as well as an intact kinase domain; joined at the C-terminus of FGFR2 is the PPHLN1 moiety that contains a coiled-coil domain. In this study, we demonstrate that functions of FGFR2-PPHLN1 are achieved through constitutive receptor phosphorylation, thereby activating the canonical MAPK/ERK, JAK/STAT3 and PI3K/AKT pathways. Furthermore, we show that the introduction of an activating mutation in the kinase domain N549/550K on the fusion showed an even higher activation of these pathways, while the kinase dead K517/518R mutation completely abolished the signaling activities. These findings collectively reinforce the essential function of the kinase activity of the FGFR2 moiety. We also show that the oncogenicity of FGFR2-PPHLN1 is dependent upon the coiled-coil PPHLN1 domain. Finally, we determine the localization of this fusion protein and its entrance into the secretory pathway on its transforming ability by engineering a myristylation (Myr) signal, introducing a Myr G2A mutation, as well as deletion of the delta signal.

Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of low-molecular-weight metabolites by new high-throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19-9 (CA 19-9) and alpha-fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy-proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14-15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases.